Inhibitor of influenza a virus reproduction base on extract of basidial fungus phallus impudicus

FIELD: medicine, pharmaceutics.

SUBSTANCE: inhibitor of influenza A virus reproduction represents water extract of basidial fungus Phallus impudicus, obtained by extraction of crushed fungus with water at ratio 1:5 with further removal of insoluble deposit from extract.

EFFECT: inhibitor possesses high activity against human and avian influenza of A type.

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The invention relates to antiviral agents, in particular inhibitor of reproduction of the virus of human influenza and birds and can be used in medicine, Virology and pharmacology.

The influenza virus is an infectious disease of the upper respiratory tract. Annual influenza epidemics in the world lead to the 3.5 million cases of severe illness and to 300-500 thousand deaths [CDC 2005. Centers for Disease Control. Prevention and control of influenza recommendations of the advisory committee on immunization practices (ACIP) // MMWR. - 2005. - V.54 (RR08). - P.1-40. Available from URL: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5408a1.html].

New epidemique strains of influenza virus And occur every 1-2 years as the result of point mutations in the two surface glycoproteins - hemagglutinin and the neuraminidase (NA). Using the variety and variability of antigenic structure of the virion, the influenza virus is able to bypass the security mechanisms of human immunity and, therefore, long-term immunity against the virus no after natural infection or after vaccination. Up to the present time there is not enough effective means of preventing the spread of this infection. Currently, control of infection caused by influenza virus, to treat the flu with the use of causal, pathogenetic and symptomatic means. The search for new effective against DOF is and drugs is one of the priority health problems.

Veselka ordinary (Phallus impudicus L.: Pers.) is representative of gasteromycetes refers to the order Veselkova (Phallales), family Veselkova (Phallaceae), the genus Veselka (Phallus). The popular name "the devil's egg", "thunderbolt", etc. reflect the ability of the fungus from the egg stage is rapidly developing in the fruit body, resembling a phallus. Using a mathematical model and analysis, scientists have calculated that the force pushing the mushroom up, is that three of the fungus can lift a weight of approximately 400 kg [Niksic M., Hadzic I. and M. Glisic Is Phallus impudicus a mycological giant? // Micologist, 2004. - Vol.18. - P.21-22].

In folk medicine, the mushroom has long been used as a remedy for rheumatism and gout. In traditional medicine the fungus is used as an antitumor agent, for example, is used together with chemical drugs in breast cancer [Kuznecova G., Kuznecovs I., Legina K., Kuznecovs S. Lymphederma prevention and management with Phallus impudicus: the six survey years after breast cancer treatment // European Journal of Cancer Supplements, 2007. - Vol. 5. - P. 457; Kuznecova G., Kuznecovs I., Legina K., Kuznecovs S. Phallus impudicus in tromboprophylaxis in breast cancer patients undergoing chemotherapy and hormonal treatment // The Breast. Prima. ry therapy of early breast cancer. 10thInternational conference, 2007. - Vol.16. - S56]. Phallus impudicus antioxidant activity [Babicka VG, szczerba V.V., Filimonov T.V., Rozhkova Z.A., Bout D., Trojanovic CENTURIES, Osadchaya O.V. Phallus impudicus (L.: Pers.), Hericium erinaceus (Bull.: Fr.) Pers. and Trametes versicolor (Fr.) Quel. - The future development of the main objects of biotechnology // Advances in medical Mycology. Edited by academician of the Russian Academy Y.V. Sergeev. The fourth all-Russian Congress on medical Mycology. - M., 2006. - V.7. - S-222].

Known natural compounds that provide inhibitory effect on influenza virus. For example, it is known the use of an aqueous extract of the mushroom Rozites caperata as antiviral drugs protein nature, preventing the replication of influenza virus type A [Piraino F. F. The Development of The Antiviral Drug RC 28 from Rozites caperata (Pers.: Fr.) P. Karst. (Agaricomycetideae) // International Journal of Medicinal Mushrooms. Vol.7 (2005), p.356].

Known antiviral activity of an extract of Ganoderma pfeifferi against influenza virus a and herpes simplex virus type 1. The main anti-virus component of the extract were triterpenoids: Ganoderma, elucidation, aproximada acid G [Mothana R.A.A., Awadh li N.A., Jansen R., Wegner, U., Mentel R, Lindequist U. Antiviral lanostanoid triterpenes from the fungus Ganoderma preifferi // Fitoterapia. - 74 (2003).-P. 177-180].

Known substances histidin and Espolon having isoprenoid nature and found in the ethanol extract of the fungus Inonotus hispidus, which exhibit antiviral activity against influenza virus type a and type C. Antiviral activity was expressed as the extracts of the fruit bodies and mycelial extracts [Ali N.A.A., Jansen R., Pilgrim H., Liberra K, Lindequist U. Hispolon, a yellow pigment from Inonotus hispidus // Phytochemistry. 1996. - V.41, I.3, P.927-929; Awadh Ali N.A., Mothana R.A.A., Lesnau A., Pilgrim H., Lindequist. Antiviral activity of Inonotus ispidus // Fitoterapia. - 74 (2003). - P.483-485].

The closest analogue (prototype) is the drug of vodka infusions of Veselka that the authors and volunteers were used as a preventive measure before the epidemics of flu, colds, causes almost 100%effect of protection [Babayants O.V., Busulfan M.A., Zalogin M.A. Phallus impudicus L.: Pers. - Prospects for the use in medicine // Advances in medical Mycology edited by academician of the Russian Academy Y.V. Sergeev. Materials of the third all-Russian Congress on medical Mycology. M., 2005. - V.5. - S-242].

However, in the above analogue is not given dosage (concentration of extract from the fruit bodies of Veselka in solution), as well as the scheme of the drug based on the results of scientific studies supporting anti-influenza activity of the extract of the mushroom Phallus impudicus, depending on the concentration of extractives mushroom Phallus impudicus.

The technical result of the claimed technical solution is the possibility of obtaining funds on the basis of an aqueous extract of basidiomycete Phallus impudicus or polysaccharide from the specified extract has inhibitory activity against influenza virus type A.

This technical result is achieved in that the inhibitor reproduction of influenza virus And representing water extrac the basidiomycete Phallus impudicus, obtained by extraction of powdered mushroom water at a ratio of 1:5 with subsequent removal from the extract insoluble precipitate.

Crushed basidiomycete mushroom Phallus impudicus is obtained from the fruit bodies specified mushroom or from mycelium specified by fungus cultivation of biomass from fruiting bodies of the fungus, which is in the egg stage, in a liquid nutrient medium on the basis of the oat-corn broth.

Below are examples 1-2 obtaining of extractive substances from fruit bodies and mycelium of the fungus Phallus impudicus.

Example 1. Obtaining the extract of the fruit phone 12 g of chopped frozen fruit bodies suspended in 60 ml of sterile distilled water and treated in an ultrasonic disintegrator (MSE, UK) for 20 min at an amplitude of 24 μm. The precipitate was separated by centrifugation in a centrifuge J-2-21 (Beckman, USA) for 20 min at 10000 rpm Prepared extract was frozen at -20°C. the solids Content was 5 mg/ml (sample 09-90).

Example 2. Obtaining the extract from the mycelium. Phallus impudicus was isolated in pure culture from the fruiting bodies of the fungus, which is in the egg stage. Biomass of mycelium obtained by cultivation on oat-corn broth for 30 days in a hospital. 5 g of chopped frozen biomass suspended in 25 ml of sterile.

Example 3. The definition of protivovirusnoi efficiency samples of mushroom Phallus impudicus in cell culture

Cell culture. For testing antiviral drugs used transplantable cell culture MDCK obtained from the collection of cell cultures fsri SRC VB "Vector". Cell culture MDCK comes from the laboratory in the form of a suspension with a specified concentration. Under sterile conditions, the suspension was diluted with pre-warmed to a temperature of +37°C medium RPMI-1640 containing 5% serum fruits cows, to a concentration of 1.0 to 1.5×105cells/ml 100 ál of cell suspension MDCK were made in 96-well tablets 12-channel automatic pipette. Tablets with cells was placed in a thermostat at a temperature of +37°C, 5% CO2and 100% humidity for 2-3 days before the formation of the cell monolayer.

Determination of the toxicity of the sample. For the detection of toxic doses of drugs extracts were diluted several times and evaluated the available toxicity in culture monolayers of MDCK cells using an inverted microscope. This was done by dilution of the initial drug 5 times, 10, 100, 1000, 10000, 100000, 1000000 once the medium RPMI-1640 containing 5% serum fruits cows, made of 150 μl in appropriate wells and placed in a thermostat at a temperature of +37°C, 5% CO2and 100% humidity for 2 days. After 2 days using an inverted microscope to assess the presence of toxic effect in monolayers of cells MCK, incubated with different concentrations of fungal drugs.

In the experiments for the determination of antiviral activity of fungal drugs used predefined maximum tolerated concentration (MBC).

Determination of the antiviral activity of the sample. In this work, we used the virus of avian influenza (strain A/chicken/Kurgan/05/2005 (H5N1)and human (strain A/Aichi/2/68 (H3N2))obtained from the Department of "Collection of microorganisms" fsri SRC VB "Vector". The production and titration of viruses produced in 10-day-old developing chicken embryos (RCA). The concentration of virus in virusbulletin liquid (IMPORTANT) was different experiments from 6.5 to 9.5 lg EID50/ml (50% embryo infectious doses per ml).

In the experiments for the determination of antiviral activity of mushroom preparations were prepared by dilution of the IMPORTANCE of each virus from 1 to 8 with a tenfold step using RPMI-1640 medium containing 2 μg/ml trypsin. To determine the antiviral activity of drugs in monolayer cultures of MDCK cells were introduced into 50 ál of the selected dilution of the extract in the medium RPMI-1640 containing 2 μg/ml of trypsin, and 100 μl of the diluted from 1 to 8 or undiluted IMPORTANT. In the case of undiluted mushroom drugs or IMPORTANT trypsin was made up to a final concentration of 2 μg/ml supportive environment in well 96-well plate. Zara is giving the cell monolayer was carried out according to the standard technique, for cultivation of viruses used supporting environment RPMI-1640 company LLC Biolot", St. Petersburg, containing 2 μg/ml trypsin TRNC (Sigma, USA). Cells were incubated 2 days at a temperature of +37°C in an atmosphere of 5% CO2in thermostat TC-1/80 SDA (Russia). 2 days in each well using an inverted microscope recorded the JRC in the monolayer of cells and determined the presence of the virus in the environment of the cultivation by the reaction of haemagglutination (DSA) with 1% chicken erythrocytes.

As control was used:

1. Control MDCK cells cultivated in a nutrient medium RPMI-1640 company LLC Biolot", St. Petersburg, containing 2 μg/ml trypsin TRNC (Sigma, USA).

2. Control of reproduction of influenza virus A/Aichi/2/68 (H3N2) and A/chicken/Kurgan/05/2005 (H5N1) from 1 to 8 with a tenfold dilution step without making preparations mushroom extracts.

In table 1 presents the results of determining the antiviral activity of samples in cell culture against strain A/Aichi/2/68 (H3N2) virus of human influenza.

As can be seen from table 1, the extract of the fruit body Veselka (09-90) showed high antiviral effect against the virus of human influenza A/Aichi/2/68 (H3N2). Index neutralize this strain was 6.3 lg. When this was observed in 100%inhibition of virus reproduction. Extract from the biomass of the fungus, resulting from cultivation is nutritional in the th environment (09-84), expressed in the same strain less neutralizing effect - 2,3 lg. This is due to a lower content of dry substances in the extract from the mycelium, compared with an extract of the fruit bodies. According to the results of the experiments, the effective concentration of the extract of the fruit of the body for complete neutralization of influenza virus (strain A/Aichi/2/68 (H3N2)) was 0.05 mg/ml, while the effective concentration of the extract of the mycelium for neutralization of the virus by 2.3 lg amounted to 0.02 mg/ml

Table 1
Antiviral activity of extracts from the fruiting bodies and mycelium Phallus impudicus in respect of the virus of human influenza A/Aichi/2/68 (H3N2)
The drug and its breedingThe content of dry matter, mg/mlThe final concentration of dry substances of the drug in the culture liquid, mg/mlVirus infectivity (titer IMPORTANT) in MDCK cells (EID50in lg50/50 ál)The neutralization index EID50pin-EID50experience (lg)
09-90 extract from the fruit. body 1:305,00,050,0 6,3
09-84 extract from mycelium 1:302,00,024,02,3
The control virus--6,30,0

At all dilutions complete suppression of virus was observed only in the extract of the mycelium of Veselka (sample 09-84). Even in a dilution of 1:10, when the effective concentration of the fungus was only 0,067 mg/ml, there was a 100%virpiniemi effect that persisted at a dilution of 1:30. In other samples, except sample 09-90, starting with a dilution of 1:5 there was a fall of inhibitory capacity.

Thus, the antiviral activity against influenza virus type And exhibits a complex of biologically active substances (BAS)present in the fruit body and in cultured mycelium of the fungus.

1. Inhibitor reproduction of the influenza a virus, which is an aqueous extract of a basidiomycete Phallus impudicus, obtained by extraction of powdered mushroom water at a ratio of 1:5 with subsequent removal from the extract insoluble precipitate.

2. Inhibitor reproduction of the influenza a virus according to claim 1, characterized in that the crushed basidiomycete Phallus impudicus is obtained from the fruit bodies specified mushroom.

3. Inhibitor reproduction of the influenza a virus according to claim 1, characterized in that the crushed basidiomycete mushroom Phallus impudicus is obtained from mycelium specified by fungus cultivation of biomass from fruiting bodies of the fungus, which is in the egg stage, in a liquid nutrient medium on the basis of the oat-corn broth.



 

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