Genotoxic and Anti-Genotoxic Potential of Hydrosols from Water-Steam Distillation of Oil-Bearing Roses Rosa centifolia L. and Rosa gallica L. from Bulgaria.

Rosa centifolia L. and Rosa gallica L. (Rosaceae) are grown as raw materials for valuable essential oils and hydrosols. There are scarce data about the biological activities and the genoprotective potential of the hydrosols of these roses. The aim of the study was to provide information on their cytotoxic/genotoxic activity and anti-cytotoxic/anti-genotoxic capacity against mutagenic N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The evaluation was performed using classical tests for chromosomal aberrations and micronuclei in the higher plant Hordeum vulgare and human lymphocyte test systems. The experimental schemes included combined hydrosol and mutagen treatment. Both hydrosols (6, 14, 20%) had no cytotoxic effect on barley and showed low genotoxicity in both test systems as the injuries were enhanced to a lesser extent compared to the controls. Lymphocytes were more susceptible than H. vulgare. Under the conditions of combined treatment, it was found that the two hydrosols possessed good anti-cytotoxic and anti-genotoxic potential against MNNG. Both rose products exerted genoprotective potential to a similar extent, decreasing the frequencies of aberrations in chromosomes and micronuclei to a significant degree in both types of cells when non-toxic concentrations of hydrosols were applied before MNNG. This was performed both with and without any inter-treatment time. The observed cytoprotective/genoprotective potential suggests that these hydrosols are promising for further application in phytotherapy and medicine.

Cannabinoid and Serotonergic Systems: Unraveling the Pathogenetic Mechanisms of Stress-Induced Analgesia.

The perception of "stress" triggers many physiological and behavioral responses, collectively called the stress response. Such a complex process allows for coping with stress and also triggers severe pathology. Because of the multidirectional effect of stress on the body, multiple systems participate in its pathogenesis, with the endogenous cannabinoid and the serotoninergic ones among them. These two systems also take part in the pain perception decrease, known as stress-induced analgesia (SIA), which can then be taken as an indirect indicator of the stress response. The aim of our study was to study the changes in cold SIA (c-SIA) resulting from the exogenous activation of cannabinoid receptor type 1 (CB1) and 5-hydroxytryptamine (5-HT, serotonin) receptor type 1A (5-HT1A). Various combinations of agonists and/or antagonists of CB1 and 5-HT1A, before or after 1 h of cold exposure, were applied, since we presumed that the exogenous activation of the receptors before the cold exposure would influence the pathogenesis of the stress response, while their activation after the stressful trigger would influence the later development. Our results show that the serotonergic system "maintained" c-SIA in the pre-stress treatment, while the cannabinoids' modulative effect was more prominent in the post-stress treatment. Here, we show the interactions of the two systems in the stress response. The interpretation and understanding of the mechanisms of interaction between CB1 and 5-HT1A may provide information for the prevention and control of adverse stress effects, as well as suggest interesting directions for the development of targeted interventions for the control of specific body responses.

Remdesivir-Loaded Nanoliposomes Stabilized by Chitosan/Hyaluronic Acid Film with a Potential Application in the Treatment of Coronavirus Infection.

An object of the present study was the development of liposomes loaded with the medicine Veklury® (remdesivir) stabilized by electrostatic adsorption of polysaccharide film formed from chitosans with different physicochemical characteristics and hyaluronic acid. The functionalization of the structures was achieved through the inclusion of an aptamer (oligonucleotide sequence) with specific affinity to the spike protein of the human coronavirus HCoV-OC43. The hydrodynamic size, electrokinetic potential and stability of the structures were evaluated at each step in the procedure. The encapsulation efficiency and loaded amount of remdesivir (99% and 299 µg/mL) were estimated by UV-vis spectroscopy. Our investigations showed manifestation of promising tendencies for prolonged periods of the drug release and increased effectiveness of its antiviral action. Among all studied versions of the delivery system, the most distinguished and suitable in a model coronavirus therapy are the liposomes formed from chitosan oligosaccharides. The cytotoxicity of the liposomes was determined against the HCT-8 cell line. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the compounds. The virucidal activity and the effect on the viral adsorption of the samples were reported by the end-point dilution method, and the alteration in viral titer was determined as Δlgs compared to untreated controls. The redox-modulating properties of the nanoparticles were studied in vitro in certain/several/a few chemical model systems. Our investigations showed a manifestation of promising tendencies for a prolonged effect of the drug release and increased effectiveness of its antiviral action.

Postbiotics Production of Candidate-Probiotic Lactiplantibacillus plantarum AC131 with Renewable Bio Resources.

Lactiplantibacillus plantarum is a versatile specie, well known as a producer of lactic acid (LA) and other metabolites with biotechnological significance. The present work characterizes growth and lactic acid production of the candidate-probiotic strain L. plantarum AC131, from Bulgarian white brined cheeses. Different nutritional media with ingredients from renewable resources-reduced sugars from dried distillers' grains with soluble (DDGS) and waste waters from the water-vapor distillation of Bulgarian Rosa alba L. and Rosa damascena Mill. essential oil-were assessed. The results obtained showed significant LA production (up to 95% conversion) in modified MRS broth with reducing sugars from DDGS hydrolysates. The addition of R. alba L. and R. damascena Mill. distillation effluents stimulated the growth and biological activity of postbiotics produced by L. plantarum AC131. In both experimental approaches, a statistically significant inhibition (from 20 to 60%) of E. coli HB 101 growth was found during 24 h exposure and a variable effect on the biofilm formed. In conclusion, reducing sugars from DDGS hydrolysates can be successfully used as a carbon source for lactic acid production. In the case of fermentation without pH control, the process is product inhibited, while with pH control, nearly full conversion was achieved. Postbiotics produced during the process of fermentation showed a variety of biological activity and inhibitory effects on the growth of Escherichia coli HB 101.

Evaluation of Acute and Sub-Acute Toxicity, Oxidative Stress and Molecular Docking of Two Nitrofuranyl Amides as Promising Anti-Tuberculosis Agents.

Tuberculosis (TB) remains a widespread infectious disease and one of the top 10 causes of death worldwide. Nevertheless, despite significant advances in the development of new drugs against tuberculosis, many therapies and preventive measures do not lead to the expected favorable health results for various reasons. The aim of this study was to evaluate the acute and sub-acute toxicity and oxidative stress of two selected nitrofuranyl amides with high in vitro antimycobacterial activity. In addition, molecular docking studies were performed on both compounds to elucidate the possibilities for further development of new anti-tuberculosis candidates with improved efficacy, selectivity, and pharmacological parameters. Acute toxicity tests showed that no changes were observed in the skin, coat, eyes, mucous membranes, secretions, and vegetative activity in mice. The histological findings include features consistent with normal histological architecture without being associated with concomitant pathological conditions. The observed oxidative stress markers indicated that the studied compounds disturbed the oxidative balance in the mouse liver. Based on the molecular docking, compound DO-190 showed preferable binding energies compared to DO-209 in three out of four targets, while both compounds showed promising protein-ligand interactions. Thus, both studied compounds displayed promising activity with low toxicity and can be considered for further evaluation and/or lead optimization.

Cytogenetic Studies on Genoprotective Effect of Rosa damascena Mill. Hydrosol in Plant and Lymphocyte Test Systems.

Bulgarian Rosa damascena Mill. is has been known since ancient times for its high-quality oil, hydrosol, and other aromatic products. Rose hydrosol has various biological activities, but no research on its anticytotoxic/antigenotoxic effects exists. This study aimed to assess its defense potential against the genotoxin N-methyl-N'-nitro-N-nitrosoguanidine and to test its cytotoxic/genotoxic activity in plant and human lymphocyte test systems. Endpoints for cytotoxicity (mitotic index and nuclear division index) and genotoxicity (chromosome aberration and micronuclei) were used. Hydrosol was applied as a single treatment in concentrations ranging from 3% to 20% (4 h) to assess its cytotoxic and genotoxic effects. Its protective potential against MNNG was tested by applying an experimental scheme involving (i) conditioning treatment with non-toxic or slightly toxic concentrations of hydrosol, followed by genotoxin challenge (50 µg/mL) with a 4 h intertreatment time and (ii) treatment with hydrosol and mutagen with no time between the treatments. Hydrosol induces low cytotoxicity and clastogenicity, demonstrating cytoprotective/genoprotective effects against the mutagen in both applied test systems. The hydrosol defense potential was expressed by a more than twofold reduction in both chromosomal aberrations and micronuclei and by enhancing the mitotic activity compared with that of the mutagen, regardless of the experimental conditions. The results are promising for further hydrosol applications in pharmaceutical and medical practice.

The Role of Renewable Resources for Ecology and Human Health.

Scientists are increasingly asking very serious and topical questions: what do we throw away as waste from industrial production [...].

The Endogenous Cannabinoid and the Nitricoxidergic Systems in the Modulation of Stress Responses.

The effects on stress-induced analgesia (SIA) from endogenous cannabinoid system (ECS) and nitric oxide (NO) interaction after 1 h of restraint stress were evaluated in male Wistar rats. The animals were subjected to 1 h of restraint and then injected with different combinations of cannabinoid receptor type 1 agonist anandamide (AEA) or antagonist AM251 along with an NO donor, NO precursor, or inhibitor of NO synthase. Nociception was evaluated using paw pressure (PP) or hot plate (HP) tests. AEA was administered immediately after the end of restraint-SIA (r-SIA). Administration of NO precursor reversed the pronociceptive effect of the CB1 agonist on r-SIA. Both the CB1 antagonist and the NOS inhibitor neutralized the pro-analgesic effect of L-arginine (L-arg). Administration of an NO donor, instead, increased r-SIA. Our experiments confirmed that the endogenous cannabinoid and the NO-ergic systems interact in the modulation of r-SIA. This interaction probably implies NO as a second messenger of the ECS.

Study on Cytotoxic and Genotoxic Potential of Bulgarian Rosa damascena Mill. and Rosa alba L. Hydrosols-In Vivo and In Vitro.

The Rosa alba L. and Rosa damascena Mill. growing in Bulgaria are known for their extremely fine essential oil and valuable hydrosols. Irrespectively of its wide use in human life, little research exists on the cytotoxic and genotoxic activity of the hydrosols. This set our goal to conduct cytogenetic analyses to study these effects. A complex of classical cytogenetic methods was applied in three types of experimental test systems-higher plant in vivo, ICR mice in vivo, and human lymphocytes in vitro. Mitotic index, PCE/(PCE + NCE) ratio, and nuclear division index were used as endpoints for cytotoxicity and for genotoxicity-induction of chromosome aberrations and micronuclei. Rose hydrosol treatments range in concentrations from 6% to 20%. It was obtained that both hydrosols did not show considerable cytotoxic and genotoxic effects. These effects depend on the type of the tested rose hydrosols, the concentrations applied in the experiments, and the sensitivity and specificity of the test systems used. Human lymphocytes in vitro were the most sensitive to hydrosols, followed by higher plant and animal cells. Chromosomal aberrations and micronucleus assays suggested that R. damascena and R. alba hydrosols at applied concentrations possess low genotoxic risk. Due to the overall low values in terms of cytotoxic and/or genotoxic effects in all test systems, hydrosols are promising for further use in various areas of human life.

Anti-Coronavirus Efficiency and Redox-Modulating Capacity of Polyphenol-Rich Extracts from Traditional Bulgarian Medicinal Plants.

Background: The use of various herbal therapists as part of traditional medicine in different parts of the world, including Bulgaria, is due to the knowledge accumulated over the centuries by people about their valuable biological activities. In this study, we investigate extracts from widely used Bulgarian medicinal plants for their ability to prevent the coronavirus infection of cells by testing different mechanisms of antiviral protection, their polyphenol content, and redox-modulating capacity. Methods: The influence on the stage of viral adsorption, the inhibition of extracellular virions, and the protective effect on uninfected cells of the plant's extracts were reported by the end-point dilution method, and virus titer (in Δ lgs) was determined as compared to the untreated controls. The total content of polyphenols and flavonoids was also determined. We tested the antioxidant power of the extracts by their ability to inhibit the generation of superoxide anionic radicals and to scavenge DPPH radicals. We determined their iron-reducing, copper-reducing, and metal-chelating antioxidant powers. Results: Most of the extracts tested suppress the extracellular virions of HCov. They also inhibit the stage of viral adsorption to the host cell to varying degrees and have a protective effect on healthy cells before being subjected to viral invasion. The examined extracts contained significant levels of polyphenols and quercetin-like flavonoids and showed remarkable antioxidant, radical, and redox-modulating effects. Conclusions: All of these 13 extracts from Bulgarian medicinal plants tested can act as antioxidants and antiviral and symptomatic drugs for the management of coronavirus infection.

Chromatographic Profile and Redox-Modulating Capacity of Methanol Extract from Seeds of Ginkgo biloba L. Originating from Plovdiv Region in Bulgaria.

Oxidative stress underlies the pathogenesis of many diseases, which determines the interest in natural substances with antioxidant properties. Ginkgo biloba L. leaves are well known and widely used in the pharmaceutical industry, but the therapeutic properties of the seeds are less studied. This study aimed to identify the chromatographic profile and to evaluate the antioxidant properties of methanol extract from seeds of G. biloba (GBSE). In the GBSE, flavonoids and terpenes were found as terpenes predominated. The GBSE antioxidant capacity determined by 2,2 azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and 1-diphenyl-2-picrylhydrazyl (DPPH) methods were equal to 1.34% and 0.58% of the activity of reference substance Trolox, respectively. The results of the ferric reducing antioxidant power method showed that the effect of concentration 1 mg/mL (w/v) GBSE was equal to 7.418 mM FeSO4 used as a standard. The cupric reducing antioxidant capacity activity of the GBSE was found to be 215.39 µmol Trolox/g GBSE and is presented as Trolox equivalent. The metal chelation effect of 1 mg/mL (w/v) GBSE was equal to that obtained for 0.018 mM EDTA. In conclusion, GBSE showed a good ability to neutralize ABTS and DPPH radicals and could have a beneficial effect in pathological conditions with oxidative stress etiology.

In Vitro Study of the Biological Potential of Wastewater Obtained after the Distillation of Four Bulgarian Oil-Bearing Roses.

The wastewater after rose oil distillation is usually discharged into the drainage systems and it represents a serious environmental problem. While being rich in polyphenols, which have beneficial biological activity and application in the pharmaceutical industry, limited research has been carried out about the biological activity of the specific wastewaters per se. Wastewaters after distillation of the four Bulgarian oil-bearing roses Rosa damascena Mill., R. alba L., R. centifolia L., and R. gallica L. exerted significant antioxidant activity and good antiherpes simplex virus type-1 (HSV-1) activity while maintaining a good toxicological safety profile (low cytotoxic effect) towards normal cell lines. More precisely, the non-tumorigenic cells were a human (HEK-293 embryonic kidney cells) and a mouse cell line (CCL-1 fibroblasts, which are recommended as a standard for cytotoxicity evaluation in Annex C of ISO 10993-5). The concentrations that achieved antioxidant and radical scavenging effects (0.04-0.92% v/v) were much lower than most of the maximum tolerated concentrations for the tissue culture cells (0.2-3.4% v/v). The wastewaters had a weak antiproliferative effect against Staphylococcus aureus. None of the wastewaters had activity against Gram-negative bacteria or a bactericidal or antifungal effect. We can conclude that these four species, which are the most preferred species worldwide for producing high-quality rose oil, have the potential to be developed as promising antioxidant and antiherpesvirus nutraceuticals.

The Anti-Genotoxic Activity of Wastewaters Produced after Water-Steam Distillation of Bulgarian Rosa damascena Mill. and Rosa alba L. Essential Oils.

The steam distillation of valuable rose essential oil from R. damascena Mill. and R. alba L. generates large volumes of wastewaters. Although such wastewaters are bio-pollutants, they contain valuable bioactive compounds. In this study we investigated the cytotoxic/genotoxic and anti-cytotoxic/anti-genotoxic potential of these products. We used cytogenetic methods for induction of chromosome aberrations and micronuclei in two different experimental test-systems: ahigher plant and human lymphocyte cultures. Different experimental schemes of treatment with the waste products showed that the genotoxic activity of wastewater from the distillation of oils from R. alba and R. damascena was low in both test-systems. Human lymphocytes showed a higher sensitivity to the products than plant cells. Both types of waste products manifested anti-genotoxic effect against N-methyl-N'-nitro-N-nitrosoguanidine, a direct mutagen. The wastewaters obtained from steam distillation of rose essential oil have cytoprotective/genoprotective effect and could decrease DNA damage. Data are promising for further use of these products in pharmacy and other areas of human life.

Antimicrobial and Antioxidant Potential of Scenedesmus obliquus Microalgae in the Context of Integral Biorefinery Concept.

Small-scale photobioreactors (PBRs) in the inoculum stage were designed with internal (red or green) and external white LED light as an initial step of a larger-scale installation aimed at fulfilling the integral biorefinery concept for maximum utilization of microalgal biomass in a multifunctional laboratory. The specific growth rate of Scenedesmus obliquus (Turpin) Kützing biomass for given cultural conditions was analyzed by using MAPLE software. For the determination of total polyphenols, flavonoids, chlorophyll "a" and "b", carotenoids and lipids, UHPLC-HRMS, ISO-20776/1, ISO-10993-5 and CUPRAC tests were carried out. Under red light growing, a higher content of polyphenols was found, while the green light favoured the flavonoid accumulation in the biomass. Chlorophylls, carotenoids and lipids were in the same order of magnitude in both samples. The dichloromethane extracts obtained from the biomass of each PBR synergistically potentiated at low concentrations (0.01-0.05 mg/mL) the antibacterial activity of penicillin, fluoroquinolones or oregano essential oil against the selected food-borne pathogens (Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) without showing any in vitro cytotoxicity. Both extracts exhibited good cupric ion-reducing antioxidant capacity at concentrations above 0.042-0.08 mg/mL. The UHPLC-HRMS analysis revealed that both extracts contained long chain fatty acids and carotenoids thus explaining their antibacterial and antioxidant potential. The applied engineering approach showed a great potential to modify microalgae metabolism for the synthesis of target compounds by S. obliquus with capacity for the development of health-promoting nutraceuticals for poultry farming.

In Vivo Toxicity, Redox-Modulating Capacity and Intestinal Permeability of Novel Aroylhydrazone Derivatives as Anti-Tuberculosis Agents.

The emergence and spread of Mycobacterium tuberculosis strains resistant to many or all anti-tuberculosis (TB) drugs require the development of new compounds both efficient and with minimal side effects. Structure-activity-toxicity relationships of such novel, structurally diverse compounds must be thoroughly elucidated before further development. Here, we present the aroylhydrazone compounds (3a and 3b) regarding their: (i) acute and subacute toxicity in mice; (ii) redox-modulating in vivo and in vitro capacity; (iii) pathomorphology in the liver, kidney, and small intestine tissue specimens; and (iv) intestinal permeability. The acute toxicity test showed that the two investigated compounds exhibited low toxicity by oral and intraperitoneal administration. Changes in behavior, food amount, and water intake were not observed during 14 days of the oral administration at two doses of 1/10 and 1/20 of the LD50. The histological examination of the different tissue specimens did not show toxic changes. The in vitro antioxidant assays confirmed the ex vivo results. High gastrointestinal tract permeability at all tested pH values were demonstrated for both compounds. To conclude, both compounds 3a and 3b are highly permeable with low toxicity and can be considered for further evaluation and/or lead optimization.

Redox-Modulating Capacity and Antineoplastic Activity of Wastewater Obtained from the Distillation of the Essential Oils of Four Bulgarian Oil-Bearing Roses.

The wastewater from the distillation of rose oils is discharged directly into the soil because it has a limited potential for future applications. The aim of the present study was to determine in vitro the chromatographic profile, redox-modulating capacity, and antineoplastic activity of wastewater obtained by distillation of essential oils from the Bulgarian Rosa alba L., Rosa damascena Mill., Rosa gallica L., and Rosa centifolia L. We applied UHPLC-HRMS for chromatographic analysis of rose wastewaters, studied their metal-chelating and Fe(III)-reducing ability, and performed MTT assay for the evaluation of cytotoxic potential against three tumorigenic (HEPG2-hepatocellular adenocarcinoma, A-375-malignant melanoma, A-431-non-melanoma epidermoid squamous skin carcinoma) and one non-tumorigenic human cell lines (HaCaT-immortalized keratinocytes). The median inhibitory concentrations (IC50) were calculated with nonlinear modeling using the MAPLE® platform. The potential of the wastewaters to induce apoptosis was also examined. Mono-, di-, and acylated glycosides of quercetin and kaempferol, ellagic acid and its derivatives as main chemical components, and gallic acid and its derivatives-such as catechin and epicatechin-were identified. The redox-modulating capacity of the samples (TPTZ test) showed that all four wastewaters exhibited the properties of excellent heavy metal cleaners, but did not exert very strong cytotoxic effects. The lowest IC50 rate was provided in wastewater from R. centifolia (34-35 µg/mL of gallic acid equivalents after a 72 h period for all cell lines). At 24 and 48 hours, the most resistant cell line was HEPG2, followed by HaCaT. After 72 h of exposure, the IC50 values were similar for tumor and normal cells. Still, R. damascena had a selectivity index over 2.0 regarding A-431 non-melanoma skin cancer cells, showing a good toxicological safety profile in addition to moderate activity-IC50 of 35 µg/mL polyphenols. The obtained results related to wastewaters acquired after the distillation of essential oils from the Bulgarian R. alba, R. damascena, R. gallica, and R. centifolia direct our attention to further studies for in-depth elucidation of their application as detoxifying agents under oxidative damage conditions in other experimental datasets.

Antiviral Activity of Rosa damascena Mill. and Rosa alba L. Essential Oils against the Multiplication of Herpes Simplex Virus Type 1 Strains Sensitive and Resistant to Acyclovir.

BACKGROUND: The specific chemotherapeutics against herpes simplex virus type 1 (HSV) are nucleoside analogues such as acyclovir (ACV), but the most important problem is the formation of resistant mutants. The search for new therapeutic alternatives leads us to the purpose of investigating the effects of Rosa damascena Mill. and Rosa alba L. essential oils on the viral reproduction of susceptible (Victoria) and acyclovir-resistant (R-100) strains of HSV-1 replication in vitro, individually and in combination with acyclovir. METHODS: Cytopathic effect inhibition test was used for assessment of antiviral activity of the oils, and the three-dimensional model of Prichard and Shipman was applied to evaluate the combined effect of oils with ACV on HSV-1 replication. RESULTS: Both oils do not affect the replication of viral strains; they are able to influence only viral adsorption and extracellular virions and protect healthy cells from subsequent infection. In combination with lower doses of acyclovir, both oils demonstrate a significant synergistic effect on the replication of HSV-1, which is more contagious than the Victoria strain. CONCLUSIONS: The nonspecific mechanism of the reduction in viral reproduction caused by rose oils and the synergistic effect of their co-administration with the lower doses of specific inhibitor ACV makes them suitable therapeutics for overcoming viral resistance to HSV-1 infections.

Rose Flowers-A Delicate Perfume or a Natural Healer?

Plants from the Rosacea family are rich in natural molecules with beneficial biological properties, and they are widely appreciated and used in the food industry, perfumery, and cosmetics. In this review, we are considering Rosa damascena Mill., Rosa alba L., Rosa centifolia L., and Rosa gallica L. as raw materials important for producing commercial products, analyzing and comparing the main biological activities of their essential oils, hydrolates, and extracts. A literature search was performed to find materials describing (i) botanical characteristics; (ii) the phytochemical profile; and (iii) biological properties of the essential oil sand extracts of these so called "old roses" that are cultivated in Bulgaria, Turkey, India, and the Middle East. The information used is from databases PubMed, Science Direct, and Google Scholar. Roses have beneficial healing properties due to their richness of beneficial components, the secondary metabolites as flavonoids (e.g., flavones, flavonols, anthocyanins), fragrant components (essential oils, e.g., monoterpenes, sesquiterpenes), and hydrolysable and condensed tannins. Rose essential oils and extracts with their therapeutic properties-as respiratory antiseptics, anti-inflammatories, mucolytics, expectorants, decongestants, and antioxidants-are able to act as symptomatic prophylactics and drugs, and in this way alleviate dramatic sufferings during severe diseases.

Oseltamivir and S-Adenosyl-L-Methionine Combination as Effective Therapeutic Strategy for Suppression of Oxidative Damage in Lung Caused by Influenza Virus Infection in Mice.

BACKGROUND AND OBJECTIVES: The pathogenesis of influenza infection is associated with two general processes in the body: (a) lung damage based on virus replication; (b) overproduction of free radicals, antioxidant deficiency, and development of oxidative stress. To attack these aspects of flu pathogenesis, we explored the combined effect of the antiviral agent oseltamivir, and s-adenosyl-l-methionine (SAM) as a precursor of the endogenous antioxidant glutathione, in mice infected with influenza virus. METHODS: After inoculation of albino mice with 10 MLD50 of influenza virus A/Aichi/2/68 (H3N2), oseltamivir was applied twice a day, for five days post-infection in doses of 1.25 and 2.5 mg/kg. SAM was administered once a day for 10 days, starting 5 days before infection in doses of 50, 100 and 150 mg/kg. RESULTS: Monotherapy with SAM did not influence the markers of oxidative stress in the lung. Combination of SAM 50 mg/kg and oseltamivir 2.5 mg/kg affected best the virological parameters - viral titer, protection index, and mean survival time, as well as the biochemical markers of oxidative stress. INTERPRETATION AND CONCLUSIONS: Combining of SAM and oseltamivir in a dose of 1/4 of optimal therapeutic could be considered as a perspective therapy of influenza viral infection.

Combination activity of neuraminidase inhibitor oseltamivir and α-tocopherol in influenza virus A (H3N2) infection in mice.

BACKGROUND: Influenza is a highly contagious viral infection of the respiratory system. To attack two processes involved in flu pathogenesis-viral replication in the infected body and oxidative damages, we studied the combination effect of neuraminidase inhibitor oseltamivir and antioxidant α-tocopherol in experimental model of influenza. METHODS: After inoculation of albino mice with 10 MLD50 (50% mouse lethal dose) of influenza virus A/Aichi/2/68 (H3N2), oseltamivir was applied orally at three doses, 2.5 mg/kg, 1.25 mg/kg, and 0.625 mg/kg, for five days post infection. α-Tocopherol (120 mg/kg, in sunflower oil) was administered intraperitoneally. Three schemes of α-tocopherol five-day course were tested: onset five or two days before infection, or on the virus inoculation day. RESULTS: Strongly dose-dependent augmented antiviral effect of the combination α-tocopherol and 0.625 mg/kg oseltamivir was demonstrated when α-tocopherol was administered simultaneously with oseltamivir: a pronounced decrease in mortality rate (a 78% protection), and a lengthening of mean survival time by 3.2-4 days. Lung parameters showed a substantial decrease in infectious virus content (Δ logs = 3.8/4.1) and a marked diminishment of lung index and pathology. Combination α-tocopherol with 1.25 mg/kg oseltamivir manifested a marked protective effect, but the effect on lung parameters was less. The combination effect of α-tocopherol with 2.5 mg/kg oseltamivir did not surpass the monotherapeutic effect of oseltamivir. When α-tocopherol was applied in courses starting five or two days before infection, its combination with oseltamivir was ineffective. CONCLUSIONS: Evidently, α-tocopherol could be considered as prospective component of influenza therapy in combination with oseltamivir.