RU2480227C2 - Melanine antiviral agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry, namely an antiviral agent. The soluble melanine antiviral agent prepared by extraction of Inonotus obliquus basidium fungus and possessing antiviral activity on viruses of influenza, type 2 herpes simplex, immunodeficiency (HIV-1) and variolovaccine.

EFFECT: agent possess a wide spectrum of antiviral action.

6 cl, 4 tbl, 13 ex

Description

The invention relates to an antiviral agent based on melanin obtained from the higher basidiomycete Inonotus obliquus (chaga), and can be used in biotechnology, pharmaceutical industry and medicine.

All types of melanin pigments are long chain polymers with a giant molecular weight and complex liquid crystal structure. There are reports of the practical use of synthetic, semi-synthetic and isolated from biological sources of melanins in medicine, cosmetology, and due to their semiconductor properties in technology and electronics.

Melanins isolated from microorganisms show similar properties to animal melanins. These are pharmacologically active substances with antioxidant, antitoxic, anti-inflammatory, immunostimulating properties, which protect against photodynamic damage, etc.

Melanins - amorphous macromolecular substances insoluble in water, mineral acids, organic solvents; well soluble in alkalis, and then precipitate upon acidification of solutions, which is used to isolate them.

According to their predecessors, melanins are divided into eumelanins, pheomelanins and allomelanins. Eumelanins (black) and pheomelanins (yellow, red and brown) are common in animals, allomelanins (black) - in plants, fungi, bacteria. The eumelanin precursor is tyrosine, from which the body produces pigments containing C, H, N, and O. The precursors of allomelanins are diphenols (pyrocatechol, etc.), and nitrogen-free melanins are formed from them.

Melanin pigments contained in mushrooms are the most powerful bioprotectors that protect a living cell from adverse external and internal influences. They are also the most powerful natural antioxidants. Melanins are able to neutralize various free radicals that occur in a living cell under the influence of penetrating radiation, ultraviolet radiation, various toxins and enzymes of pathogenic bacteria. Many medicinal mushrooms, especially tinder fungi, have a high melanin content and can reach 30% dry weight and even more. Some fungal melanins are able to pass into solution and can be absorbed in the gastrointestinal tract, spread by blood throughout the body and protect living cells from the damaging effects of free radicals (http://www.amrita.net.ua/pi/products_id/329).

It was established that melanin from the chaga Inonotus obliquus and some other fungi has photo- and radioprotective, antioxidant and genoprotective properties [Scherba VV, Babitskaya VG, Kurchenko VP, Ikonnikova NV, Kukulyanskaya T .BUT. Antioxidant properties of melanin pigments of fungal origin // Applied Biochemistry and Microbiology. 2000. - T.36. - No. 5. - S.569-574; Sushinskaya N.V., Kurchenko V.P., Gorovoy L.F., Senyuk O.F. Obtaining and use in medicine of melanins from tinder fungi // Successes of medical mycology. - 2005. - T.6. - S.255-259].

The closest analogue (prototype) is an antiviral agent based on water-soluble melanins obtained by chemical synthesis from the following components: L-deoxydopamine, L-deoxydopamine, cysteine, L-deoxydopamine / glutathione, L-tyrosine, serotonin, dopamine, adrenaline and norepinephrine (Patent US No. 5057325, IPC A61K 31/195, publ. 1991). The specified antiviral agent can fully or partially protect in vitro human lymphocytes from the immunodeficiency virus (HIV-1 and HIV-2).

However, the publication does not contain information about antiviral activity

the claimed funds in relation to other viruses, in particular influenza viruses, herpes simplex, vaccinia.

The technical result of the invention is to obtain an antiviral agent based on melanin, which has a wider spectrum of antiviral activity.

The specified technical result is achieved in that the antiviral agent based on melanin, according to the invention, as melanin it contains water-soluble melanin obtained by extraction from the basidiomycete Inonotus obliquus and having antiviral activity against influenza viruses, herpes simplex type 2, immunodeficiency (HIV -1) and smallpox vaccines.

The product contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of from 0.002 mg / ml to 25 mg / ml.

The product contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of from 0.002 mg / ml to 0.2 mg / ml, which has activity against the immunodeficiency virus (HIV-1).

The product contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of 0.125 mg / ml to 1.56 mg / ml, which has activity against the vaccinia virus.

The product contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of 0.02 mg / ml to 0.25 mg / ml, which has activity against the influenza virus.

The product contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of from 0.006 mg / ml to 25.0 mg / ml, which has activity against herpes simplex virus type 2.

Known sources of information lack information on natural melanins of fungal origin, including in water-soluble form, with antiviral activity. In the presence of antiviral properties in chemically synthesized melanins (US patent No. 5057325) it is not obvious that these properties will appear in natural melanins in a water-soluble form, which confirms the novelty and inventive step of the claimed technical solution.

As the raw material containing melanin, the claimed technical solution uses the basidiomycete Inonotus obliquus (chaga), growing on birches of the forest zone of the south of Western Siberia. In the Russian Federation, this fungus develops on the warty birch and white birch (Betula pendula Roth and Betula alba L.), has a wide distribution area. Inonotus obliquus mushroom preparations are widely used in medicine.

The following are examples of antiviral formulations:

Example 1. The tool contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 0.002 mg / ml, having activity against HIV-1.

Example 2. The tool contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 0.2 mg / ml, having activity against HIV-1.

Example 3. The tool contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 0.125 mg / ml, having activity against the vaccinia virus.

Example 4. The tool contains an aqueous solution of melanin from the basidiomycetes Inonotus obliquus at a concentration of 1.56 mg / ml, having activity against the vaccinia virus.

Example 5. The agent contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 0.006 mg / ml, which has activity against the herpes simplex virus type 2.

Example 6. The tool contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 25.0 mg / ml, which has activity against the herpes simplex virus type 2.

Example 7. The tool contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 0.02 mg / ml, having activity against the influenza virus.

Example. 8. The product contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus at a concentration of 0.25 mg / ml, which has activity against the influenza virus.

Example 9. The technology of preparation of a water-soluble extract of melanin from basidiomycete fungus

First, the mushroom mass is ground in a known manner, followed by extraction of the target product by hydrolysis at pH 12-14 under a pressure of 0.5-07 atm for 30 minutes or at 50 ° C for 1-2 days; acidification of the centrate after centrifugation to pH 2.0-3.0, precipitation of the pigment precipitate by centrifugation at 10,000 rpm for 20 min, washing and drying the product at a temperature of 50 ° C. The resulting preparation does not dissolve in water, but dissolves well in alkalis. A water-soluble form is obtained by thoroughly dissolving the resulting pigment in a 10% ammonia solution, followed by gentle evaporation of the residual ammonia and water. The resulting preparation is completely soluble in water.

In the finished form, melanin is a black crystal with a bright flickering brilliance. The obtained pigments were identified using a qualitative reaction with KMnO ₄ , H ₂ O ₂ , FeCl ₃ . Melanin discolors under the influence of Н ₂ О ₂ , when interacting with FeCl ₃ , a flocculent precipitate precipitates, which dissolves when an excess of iron chloride is added, and when pigment KMnO _{4 is} added to the solution, the color changes to green, followed by precipitation and decolorization of the solutions [Kukulyanskaya T .A., Kurchenko N.V., Kurchenko V.P., Babitskaya V.G. Physico-chemical properties of melanins formed by chaga under natural conditions and during cultivation. // Applied biochemistry and microbiology. 2002. - T. 38. - No. 1. - S.68-72]. Absorption of UV and visible light by aqueous and alkaline pigment solutions was recorded on a spectrophotometer (Genesys 5 Spectrophotometers, United States). The absorption spectrum of the melanin solution was in the form of an oblique straight line, characteristic of fungal origin melanins [Babitskaya VG, Shcherba VV, Ikonnikova NV Melanin complex of the fungus Inonotus obliquus. // Applied biochemistry and microbiology. - 2000. - No. 4. - T.36. - S.439-444].

2.5% solutions in distilled water are prepared from water-soluble dry melanin to study antiviral properties. The solubility of melanin was 94-98%.

Example 10. Determination of the antiviral activity of the extract of melanin against influenza virus in cell culture

This work was carried out on a transplantable MDCK cell culture. First, when various dilutions of the preparations were added to the monolayer of MDCK cells, the concentrations of extracts having a toxic effect on the cells were determined using an inverted microscope. To study antiviral activity, those dilutions of extracts were taken that did not have a toxic effect on MDCK cells. The antiviral activity of the preparations was evaluated in relation to the strain of avian influenza virus A / chicken / Kurgan / 05/2005 (H5N1). The presence of the virus in the medium of cultivation of MDCK cells was determined 2 days after their infection by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

Cell culture. To test the antiviral activity of the preparations, a transplantable culture of MDCK cells obtained from the collection of cell cultures of the Federal State Pedagogical Research Center of the World Bank “Vector” was used. An MDCK cell culture was obtained from the laboratory as a suspension with the indicated concentration. In a sterile place, the suspension was diluted with Axcevir - MDCK medium preheated to a temperature of + 37 ° C to a concentration of 1 × 10 ⁵ cells / ml. 100 μl of a suspension of MDCK cells was dispensed into 96-well plates with a 12-channel automatic pipette. Tablets with cells were placed in a thermostat at a temperature of + 37 ° C, 5% CO ₂ and 100% humidity for 2-3 days before the formation of a cell monolayer.

To determine the toxic doses of the preparations, the extracts were diluted several times and the presence of toxic effects in monolayers of the MDCK cell culture was evaluated using an inverted microscope. To do this, dilutions of the initial preparation were done 2 times, 10, 50, 100, 500, 1000, 10,000 times with Axcevir medium - MDCK, 150 μl were added to the corresponding wells of the plate and placed in a thermostat at a temperature of + 37 ° C, 5% CO ₂ and 100% humidity for 2 days. After 2 days, the presence of a toxic effect in monolayers of MDCK cells incubated with different concentrations of fungal extracts was evaluated using an inverted microscope. At the next stage, the maximum tolerated concentrations (MIC) and lower were taken to determine the antiviral activity of the fungal extracts. The toxic dose for MDCK cells (1: 100 dilution) was 0.25 mg / ml.

Virus. We used the bird flu strain A / chicken / Kurgan / 05/2005 (H5N1) and the human influenza virus strain A / Aichi / 2/68 (H3N2) from the collection of the Federal State Institution of Science and Science of the World Bank "Vector", developed on 10-day-old chicken embryos (CE). The virus concentration in the test samples was determined by titration on CE or on MDCK cells, calculated and expressed in logEID ₅₀ / ml (decimal logarithms of 50% of embryonic infectious doses in ml) or in logTCD ₅₀ / ml (decimal logarithms of 50% tissue cytopathic doses in ml ), respectively, according to the Spearman-Kerber method. A series of virus-allantoic fluid (IMPORTANT), accumulated and used in the work, with influenza virus strains was stored at -70 ° C.

The concentration of the virus in the virus-allantoic fluid (IMPORTANT) was in different experiments from 6.3 to 8.3 lg EID ₅₀ / ml (50% of embryonic infectious doses in ml).

An IMPORTANCE dilution of 1 to 8 was prepared in tenfold increments using Axcevir-MDCK medium containing 2 μg / ml trypsin. To determine the antiviral activity of the preparations, 50 μl of the selected dilution of the extract and 100 μl of the IMPORTANT dilution were added to the monolayer of the MDCK cell culture. Infection of the cell monolayer was carried out according to the generally accepted method, and Axcevir support medium — MDCK (Stem Alpha, France) containing 2 μg / ml trypsin TRPC (Sigma, USA) was used for virus cultivation. Cells were incubated for 2 days at a temperature of + 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% chicken erythrocytes.

As a control, we used:

1) control of MDCK cells cultured in Axcevir nutrient medium - MDCK (Stem Alpha, France) containing 2 μg / ml trypsin TRPC (Sigma, USA), without virus and without drugs;

2) control of the reproduction of influenza virus in dilutions from 1 to 8 with a ten-fold step in MDCK cells cultured in Axcevir-MDCK nutrient medium (Stem Alpha, France) containing 2 μg / ml trypsin TRSK (Sigma, USA), without introducing dilution of fungal extracts.

Table 1 presents the data on the determination of the antiviral activity of melanin against avian influenza virus A / chicken / Kurgan / 05/2005 (H5N1) on MDCK cells.

Table 1 The results of the antiviral activity of the melanin extract in the culture of MDCK cells infected with the influenza virus strain A / chicken / Kurgan / 05/2005 (H5N1) Melanin solution and its dilution Effective dose of melanin solution, mg / ml Virus infectivity (VIA titer) in MDCK cells (ID ₅₀ in lgTCD ₅₀ / ml) after 2 days ID neutralization index ₅₀ cont - ID ₅₀ experience (lg) after 2 days Virus control 6.7 0 Example 1. Melanin (1: 500) 0.02 4,5 2.2

As can be seen from table 1, the index of neutralization of the virus with a solution of melanin was 2.2 lg, the effective dose of an aqueous solution of melanin was 0.02 mg / ml.

Example 11. Determination of the antiviral activity of the extract of melanin against herpes simplex virus type 2 (HSV-2)

Type 2 herpes simplex virus MS strain (HSV-2) was obtained from the American Type Culture Collection (ATCC). At the SSC VB "Vector" the viral strain passed 1 passage on a Vero cell culture. Before starting work, the initial suspension containing the virus was stored in liquid nitrogen. Strain Egypt 101 (Eg101) West Nile virus (WNV) was obtained from the State collection of viral strains of the Institute of Virology named after DI. Ivanovo RAMS (Moscow, Russia).

Determination of toxicity of samples for cells. To determine the toxicity to cells, experimental samples were added to 96-well plates with Vero cell culture, which were previously diluted on DMEM nutrient medium (SSC WB “Vector”) containing 5% serum. The exposure was carried out for 1 hour at 37 ° C in an atmosphere of 5% CO ₂ . Then the cells were washed three times with serum-free DMEM and filled with DMEM with 2% cattle serum (BioloT LLC, Moscow), heated to 56 ° C. Monitoring the state of cells treated with drugs and in the control, untreated rows was carried out for seven days using light microscopy. The initial concentration of 25.0 mg / ml was nontoxic for Vero cells by this technique.

Experimental samples were sterilized by filtration through a 0.2 μm filter (Millipore, USA).

Determination of antiviral activity. The study of the antiviral activity of fungal samples was carried out, guided by the methods presented in the Guide to the experimental (preclinical) study of new pharmacological substances and the article of the State Pharmacopoeia (State Pharmacopoeia of the USSR. General methods of analysis. Medicinal raw materials. - M .: Medicine, 1990. - II ed. - Issue 2. - Volume 2. - S.187-209). The decrease in the infectious titer of the virus was evaluated after incubation of the viral suspension with experimental preparations for one hour at 37 ° C. Further, these suspensions of viral preparations were added to the wells of 96-well cell culture plates. After adsorption for 1 hour at 37 ° C, the cell monolayer was washed with serum-free medium and the support medium was poured. The decrease in the infectious titer of the virus, processed or inactivated by samples, was expressed for the herpes simplex virus type 2 (HSV-2) in plaque forming units (PFU). The results were taken into account visually during daily observation under a light microscope. The protocols were completed for each day of observation. On the day when the CPD was detected in the wells of the control series of the titration of the virus (for HSV-2 usually 2–3 days), the result of titration of HSV-2 in PFU and virus inactivation with the studied drugs was taken into account, that is, less or no 10-100 PFU for HSV compared to control. The results are presented in table.2.

table 2 Antiviral activity of melanin extract against herpes simplex virus type 2 (HSV-2) The initial solution of melanin,% Dilution of the drug, 100% inhibiting the adsorption of the virus on Vero cells Virus infection multiplicity 0.1 PFU / cell 0.01 PFU / cell Melanin (2.5% solution) 1: 4046 1: 4046

As can be seen from the results obtained (Table 2), a complete suppression of the development of HSV-2 virus in Vero cell culture occurred at very low melanin concentrations. The effective dose was 0.006 mg / ml (example 5).

Example 12. Evaluation of the antiviral effect of melanin extract on the reproduction of human immunodeficiency virus in cell culture MT-4

The toxicity of a sample of an aqueous melanin solution was investigated using a human cell lymphoid culture MT-4. For this, a suspension of MT-4 cells diluted to a seed concentration of 0.5 × 10 ⁶ cells per milliliter with RPMI-1640 nutrient medium supplemented with 10% fetal serum, previously inactivated by heating at 56 ° C for 30 minutes, 300 mg / ml L β-glutamine, 80 μg / ml gentamicin and 30 mg / ml lincomycin, were placed in the wells of 96-well plates, 180 μl per well. A solution of melanin (20 μl per well) is added to the cells, until the final dilutions are 1:10, 1:20, 1:40, 1: 80 ... 1: 1280. The maximum investigated concentration of melanin in the MT-4 cell culture in the toxicity test was 300 mg / ml. Used 3 wells per dose. The control was cells in which instead of the test compound the same amount of solvent was added (RPMI-1640 medium without serum and antibiotics). The plates were incubated in an incubator at 37 ° C. On the 5th day of cultivation, the concentration and cell viability were calculated by the method of intravital staining of MTT cells (3- (4,5-dimethylthiazol-2-y1) -2,5-diphenyltetrazolium bromide). The result was taken into account on an Anthos spectrophotometer at 570-620 nm. Dilution, which has a 50% toxic effect on MT-4 cells (in which the cells retained 50% viability, TC ₅₀ ), was determined from the graph of the dependence of optical density on cell concentration and viability. As a result of the experiment, a 50% toxic concentration of melanin (TC ₅₀ ) was determined for the MT-4 cell culture, which was 200 μg / ml.

The antiviral effect of melanin was evaluated by the suppression of virus production in MT-4 cells. Virus-containing material (HIV-1, strain HBV 4046) was added to the MT-4 cell suspension, and the cells were spread into wells of 96-well plates. At the same time, a melanin sample diluted in RPMI-1640 medium was added to the wells with a step of 3-fold sequential dilutions of the preparation (1: 3, 1: 9, 1:27, etc.), three wells per dose. The plates were incubated for one hour at 37 ° C to adsorb the virus, then the cells were diluted to inoculum with RPMI-1640 culture medium supplemented with 10% fetal serum, previously inactivated by heating at 56 ° C for 30 minutes, 300 mg / ml L-glutamine , 80 μg / ml gentamicin and 30 mg / ml lincomycin containing the appropriate dose of melanin. The controls were HIV-1 infected MT-4 cells without the addition of melanin. The plates are incubated at 37 ° C in an atmosphere of 5% CO ₂ for 5 days.

The anti-HIV activity of melanin (IC ₅₀ ) was assessed by the MTT method (a comparative assessment of the viability of HIV-1 infected and non-infected MT-4 cells) and by suppressing the accumulation of p24 viral protein in HIV-1 infected cells using p24 quantification by direct enzyme immunoassay . 50% inhibitory concentration of melanin (IC ₅₀ ) against HIV-1 in experiments using MTT-4 human cell culture by MTT was 1.3 μg / ml. The inhibition of the growth of the virus-specific protein p24 by various dilutions of melanin was determined using the test system produced by Vector-Best. The result was taken into account on a BioRad spectrophotometer at 450 nm. The baseline p24 protein level at the time of infection was 6.67 ng / ml (log 6.67 = 0.8241). The concentration of p24 protein after 5 days during the cultivation of HIV-1 on MT-4 cells without the addition of melanin was 1.03 × 10 ⁴ ng / ml (log 1.03 × 10 ⁴ = 4.0128), with 50% inhibition of the growth of virus-specific p24 protein occurred with the addition of melanin at a concentration of 2.6 μg / ml. Averaged data on two independent methods for assessing the antiviral efficacy of the drug showed that melanin is capable of 50% suppressing the reproduction of HIV-1 at a concentration of 1.95 ± 0.65 μg / ml, the selectivity index (TC ₅₀ / IC ₅₀ ) for melanin exceeds 100 .

Table 3 shows the results of a study of the toxic and antiviral effects of melanin against HIV-1.

Evaluation of the effectiveness of the antiviral effect of melanin on a human cell culture of MT-4 infected with HIV-1 showed that, with low toxicity (TS ₅₀ is 200 μg / ml), melanin extract has the ability to 50% suppress HIV-1 reproduction at a concentration of 1.95 ± 0.65 μg / ml (example 1), the selectivity index (TC ₅₀ / IC ₅₀ ) for melanin exceeds 100. The example confirms that melanin has a pronounced inhibitory effect against human immunodeficiency virus type I.

Table 3 The study of the toxic and antiviral effects of melanin extract against HIV-1 (strain ГКВ 4046) in experiments on MT-4 cells The initial solution of melanin,% Method for assessing anti-HIV activity TC ₅₀ , μg / ml IC ₅₀ μg / ml Selectivity index Melanin (2.5% solution) MTT 200,0 2.6 76.9 Melanin (2.5% solution) Quantitative analysis of p24 HIV-1 200,0 1.3 153.8 Melanin (2.5% solution) Average data for two independent methods 200,0 1.95 ± 0.65 102.6

Example 13. Evaluation of the antiviral effect of melanin extract on the reproduction of vaccinia virus in Vero cell culture

Virus. Smallpox vaccine virus (BOB), strain L-IVP was obtained from the collection of FGUN SSC VB "Vector" and developed on a Vero cell culture with a titer of 5 × 10 ⁵ PFU / ml.

Cell culture. The Vero cell culture was obtained from the collection of cell cultures of the Federal State Institution Scientific Center of the World Bank “Vector”. To obtain a monolayer, cells were grown for 1-3 days in 96-well plastic plates in a nutrient medium (100 μl / well): DMEM medium containing 125 μg / ml kanamycin, 150 μg / ml L-glutamine and 2% fetal fetal serum cows (Fetal Bovine Serum, HyClone, Lot. No. CSC0518) inactivated at 56 ° C for 30 minutes).

Antiviral effect of melanin extract. The antiviral effect of the drug was evaluated using the method (Paragas J., Whitehouse CA, Endy T.R., Bray M. A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus // Antiviral Research. - 2004. - V 62. P.21-25).

Melanin with an initial concentration of 25 mg / ml was diluted in a nutrient medium seven times in triplicate. To the 100 μl culture medium contained in wells with a Vero cell monolayer, 50 μl of the drug dilution was added, including the drug without dilution (zero dilution). The concentration of melanin in the wells of the tablet was 6.25; 2.08; 0.69; 0.23; 0.08; 0.025; 0.0086 and 0.0029 mg / ml. Each dilution of the drug was added to six wells. In three of them, a virus diluted in culture medium was added in a volume of 50 μl at a dose of 1000 PFU, and in three 50 μl of culture medium was added - these wells were used to determine the toxicity of the drug. Six wells each were used to control the virus and control the cells.

The plates were incubated at 37 ° C in a CO ₂ incubator for 5 days, daily using an inverted microscope, observing the state of the cell monolayer and making sure that after 5 days in wells with the virus, but without the drug (virus control), a complete 100% cytopathic effect is observed the virus.

The tablets are stained using a neutral red vital dye and is based on the absorption of the dye by living cells. To prepare the main dye solution, 3.33 g of dry dye was dissolved in 1 L of distilled water, the solution was filtered through a paper filter and stored + 4 ° C. Before use, 1 part of the dye solution was mixed with 2 parts of a nutrient medium, the resulting solution was heated to + 37 ° C and in a volume of 50 μl was added to the wells of the plate. The plates were returned to the CO ₂ incubator for 1.5 hours. After incubation, the medium from the plate was removed, and the wells were washed twice with physiological saline (300 μl / well). The neutral red dye remaining in the wells was dissolved by adding 100 μl of a solution of 50% ethyl alcohol and 50% 0.1 M NH ₄ H ₂ PO ₄ (pH 3.5) to the wells. The plates were held for 30 minutes at room temperature and the optical density (OD) of the solution in the wells was measured at a wavelength of 490 nm using a tablet spectrophotometer connected to a computer.

Data analysis. The antiviral effect of the melanin extract was evaluated by the increase in OD in the wells containing the drug and the virus compared with the control of the virus. To plot the dependence of OD on the concentration of the drug in the wells, the “4-Parameter” option of SOFTmax PRO, version 4.0 was used. Using this program, we determined the inhibitory 50% concentration of the drug (IC ₅₀ ) - the concentration of the drug at which 50% of the cell monolayer remains protected from the virus, and the toxic 50% concentration of the drug (TC ₅₀ ) - the concentration of the drug, at which 50% of the cell the monolayer is damaged due to the toxic effect of the drug itself. TC ₅₀ and IC ₅₀ were calculated as average values from three values obtained during the experiment on one tablet. The selectivity index (SI) was calculated as the ratio of TC ₅₀ to IC ₅₀ (Duraffour S, Snoeck R, de Vos R, van Den Oord JJ, Crance JM, Garin D, Hmby DE, Jordan R, De Clercq E, Andrei G. Activity of the anti-orthopoxvirus compound ST-246 against vaccinia, cowpox and camelpox viruses in cell monolayers and organotypic raft cultures // Antivir Ther. 2007; 12 (8): 1205-16).

The results of a study of the effectiveness of the antiviral effect of melanin extracts in relation to smallpox vaccine are presented in table 4.

Table 4 The results of a study of the toxic and antiviral effects of chaga melanin extract in relation to vaccinia virus (strain L-IVP) in experiments on Vero cells The initial solution of melanin,% Range of studied concentrations Antiviral concentration range TC ₅₀ , mg / ml IC ₅₀ mg / ml SI Melanin (2.5% solution from 0.0029 to 6.25 mg / ml from 0.125 to 1.56 mg / ml (examples 3-4) 1,56 0.125 12.5

Based on the data presented in Table 4, it can be concluded that the melanin extract obtained from Inonotus obliquus has antiviral activity against vaccinia virus (selectivity index SI = 12.5).

Claims (5)

1. Antiviral agent based on melanin, characterized in that as melanin it contains water-soluble melanin in a concentration of from 0.002 mg / ml to 25 mg / ml, obtained by extraction from the basidiomycetes Inonotus obliquus and having antiviral activity against influenza viruses, herpes simplex Type 2 immunodeficiency (HIV-1) and smallpox vaccines. 2. The tool according to claim 1, characterized in that it contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of from 0.002 mg / ml to 0.2 mg / ml, having activity against the immunodeficiency virus (HIV-1). 3. The tool according to claim 1, characterized in that it contains an aqueous solution of melanin from the basidiomycete Inonotus obliquus in a concentration of from 0.125 mg / ml to 1.56 mg / ml, having activity against the vaccinia virus. 4. The tool according to claim 1, characterized in that it contains an aqueous solution of melanin from the basidiomycetes Innonotus obliquus in a concentration of from 0.02 mg / ml to 0.25 mg / ml, having activity against the influenza virus. 5. The tool according to claim 1, characterized in that it contains an aqueous solution of melanin from the basidiomycetes Innonotus obliquus in a concentration of from 0.006 mg / ml to 25.0 mg / ml, having activity against herpes simplex virus type 2.