Antiviral preparation obtaining method and antiviral preparation

FIELD: chemistry.

SUBSTANCE: method of obtaining an antiviral preparation is carried out by preparation of an inoculation mycelium of basidiomycete Enokitake Flammulina velutipes (Curtis) Singer, preparation of a liquid nutritional medium, which contains water, vegetable oil and molasses as a carbon source, as a nitrogen source - corn flour, as mineral salts - potassium dihydrophosphate and magnesium sulphate, its sterilisation, inoculation of the sterile nutritional medium with the prepared inoculation mycelium, cultivation of basidiomycete in it in an aerobic conditions; the obtained submerged culture is divided into basidiomycete biomass and a culture liquid, from which a clot is separated by addition to the latter of ethyl alcohol, which is pressed, dried and crushed with obtaining the antiviral preparation under specified conditions.

EFFECT: method makes it possible to simplify technological process, increase the output of the antiviral preparation, possessing higher activity.

2 cl, 1 tbl, 4 ex

 

Group of inventions relates to biotechnology, in particular to a method of receiving antiviral agents by culturing strains of the basidiomycete, in particular Flammulina limestone (Curtis) Singer, liquid nutrient medium in submerged culture and antiviral agent.

It is known that basidiomycete xylotrophic fungus Armillaria F. winter limestone capable of synthesis of biologically active compounds found in fruit bodies and vegetative mycelium. Selected active metabolites belonging to different classes of compounds, polysaccharides, triterpenes, fatty acids and other Metabolites F. limestone can have a pronounced antiviral effect (X-h, et al., 2008, M. Gong et al., 2009, V.P. Sharma et al., 2009, N.K. Sinha, 2011).

A method of obtaining antiviral substance from the fruit bodies and substrate mycelium grown on solid nutrient substrate on the basis of waste sugar cane (US 4629627, 16.12.1986). The disadvantages of this process are the length of the process of cultivation of the fungus and the complexity of the technology selection antiviral substance. The exact duration of the process in this patent is not specified. However, it is known that the timing of the formation of the fruiting bodies of F. limestone ranges from 1 to 4 weeks, the duration of the process of developing nutrient substrate vegetative mycelium at least 3 weeks is. In addition, in the flowsheet are energy-intensive operations grinding sprouted mushroom mycelium blocks of the substrate and the extraction of the target product with hot water followed by extraction and direct drying of an aqueous extract.

Closer to describe a group of inventions is a method of obtaining antiviral agents using edible mushroom and antiviral agent (RU 2004130188, 2004). The method of obtaining antiviral agents containing as active substance a modified water-soluble lignin-polysaccharide complex that includes decomposition and modification of lignins and polysaccharides from plant waste edible mushroom Pleurotus ostreatus in solid-phase conditions of cultivation, extraction, separation of the liquid phase, acidification, separation of the precipitate, wash. When this peptide component of the sediment hydrolyzing an aqueous solution of hydrochloric acid at 70°C for several hours, the residual precipitate is washed until neutral pH, dried, mixed with NaOH in the ratio by dry weight of 100:21, dissolve when heated, add HCl to pH 7.0.

Antiviral agent with a wide spectrum of action for the treatment of infectious diseases, isolated from an aqueous extract of the plant waste subjected to decomposition edible mushroom P. ostreatus, contains as an asset of the CSOs substances modified water-soluble lignin-polysaccharide complex does not contain peptides, the content of lignin in the lignin-polysaccharide complex mass is at least 90%.

The disadvantage of this method of obtaining antiviral agents is time consuming process of solid-phase cultivation of P. ostreatus, complexity and mnogostadiinost technology selection of the target product, in low concentrations in the target product polysaccharides responsible unlike lignin for the antiviral properties of the resulting complex. The antiviral agent does not have a sufficiently high activity.

The task of the group of inventions in the part of the method is a simplification of the process of obtaining the target means at its high output, in terms of substance - increasing antiviral activity funds.

The problem is solved by the described method of obtaining antiviral agents by making seed mycelium of the basidiomycete Armillaria winter Flammulina limestone (Curtis) Singer, cooking liquid nutrient medium containing water as sources of carbon and vegetable oil in the amount 19,0-to 27.0 g/l of water and molasses in the amount of 15-40 g/l of water as a source of nitrogen in corn flour 7-15 g/l of water, mineral salts of potassium dihydrophosphate in the amount of 2.0-2.4 g/l of water and magnesium sulfate in the amount of 0.18 to 0.20 g/l of water, its CTE is waste utilization technologies, planting cooked seed mycelium with a sterile liquid nutrient medium, the cultivation of basidiomycete in aerobic conditions with getting immersed in the culture, followed by separation of the submerged culture for biomass-degrading basidiomycete and the culture fluid and the release of the latter by adding thereto at a temperature of 4°C 96%ethyl alcohol, when the ratio of the volume of the culture fluid and ethanol 1:2-1:4, clot, which squeezed, dried at 40°C until the moisture content of 6% and pulverized to obtain antivirals.

Preferably liquid medium additionally contains zeatin in the number 0,0010-0.0015 g/l of water, vitamin B3the number 0,0060-0,0065 g/l of water, and yeast extract in the amount 0,050-to 0.055 g/l of water.

The problem is solved also described antiviral obtained in the above way.

Technical results the described group of inventions are the simplification of the process, obtaining a high yield of antiviral agents with improved activity.

The following examples illustrate the invention but do not limit them.

Example 1.

Prepare the growing mycelium of the basidiomycete Armillaria winter Flammulina limestone (Curtis) Singer. To do this, prepare sterile sowing fluid is Yu nutrient medium, composition, g/l: molasses - 30,0, soy flour to 6.0, potassium dihydrophosphate - 2.0, magnesium phosphate - 0,28, arachidonic acid of-1.0×10-5and sow it with mycelium of F. limestone grown on agar nutrient medium. The process of obtaining seed mycelium is carried out in flasks on a rotary shaker at 200 rpm at 22°C for 4 days with aeration.

Then prepare a liquid nutrient medium, composition, g/l water: vegetable oil - 19,00, molasses - 40,00, corn flour - 7,00, potassium dihydrophosphate KH2PO4- 2,40, magnesium sulfate MgSO4- 0,18, water - to 1.00 L.

This environment is poured into the flask, sterilized and inoculated with mycelium obtained in the above way.

Submerged cultivation is carried out in flasks on a rotary shaker under aerobic conditions at 25°C for 4 days with getting immersed in the culture. Submerged culture share of biomass-degrading basidiomycete and the culture fluid. The culture fluid is cooled to a temperature of 4°C, then with continuous stirring to the last add of 96% ethyl alcohol the same temperature when the ratio of the volume of the culture fluid and ethanol 1:2. Formed during the deposition of the clot squeezed, dried at 40°C until the moisture content of 6%, ground and receive antiviral agent. Cleaning antiviral agents carry out the ay triple reconstitution of exopolysaccharide in distilled water followed by precipitation with alcohol by the method described above. Output antivirals - 0.65 g/l

Example 2.

The process of obtaining antiviral agents carried out as in example 1.

Using liquid nutrient medium of the following composition, g/l water: vegetable oil - 27,00, molasses - 20,00, corn flour -15,00, potassium dihydrophosphate - 2,00, sulfate MgSO4- 0,20, water - to 1.00 L.

Submerged cultivation is carried out in flasks on a rotary shaker under aerobic conditions at 25°C for 4 days. The process of allocation of antiviral agents is carried out at the ratio of the volume of the culture fluid and ethanol 1:4.

Output antivirals - 0.87 g/L.

Example 3.

The process of obtaining carried out as in example 1, the composition of the liquid nutrient medium has the following ratio of components, g/l water: vegetable oil - 24,00, molasses - 15,00, corn flour - 12,00, KH2PO4- 2,20, MgSO4- 0,19, zeatin - 0,0010 g/l of water, vitamin B3- 0,0065 g/l water, yeast extract, 0.05 g/l of water, water - to 1.00 L.

The components of setain, vitamin B3that yeast extract is administered as follows.

Prepare a solution of zeatin, to which 100 mg of the substance are dissolved in 100 ml of distilled water and sterilized at 112°C for 30 minutes. Contribute at the rate of 1 ml per 1 liter of production medium.

Prepare rest the p vitamin B 3why 100 mg (or 1 tablet of calcium Pantothenate in pharmaceutical packaging) is dissolved in 50 ml of distilled water and sterilized at 112°C for 30 minutes. Contribute at the rate of 3 ml per 1 liter of production medium.

Prepare a solution of yeast extract, to which 5 g dry product is dissolved in 100 ml of distilled water and sterilized at 112°C for 30 minutes. Contribute at the rate of 1 ml per 1 liter of production medium.

Submerged cultivation is carried out in flasks on a rotary shaker at 25°C for 4 days, making 5 servings of molasses 5 g in 24, 36, 48, 60 and 72 hours of cultivation.

The allocation of antiviral agents is carried out according to example 1.

Output antivirals - 1.2 g/l

Example 4.

To evaluate the antiviral properties of the obtained funds on the basis of medicinal basidiomycete F. limestone above described test is done to evaluate the induction of interferon by leukocytes of donor human blood.

For research use dry sample of exopolysaccharides medicinal basidiomycete F. limestone, obtained in example 1. Prepare the initial solution of the obtained sample of exopolysaccharides with a concentration of 800 μg/ml With this purpose from the sample take sample with a mass of 4 mg of the sample add 5 ml disti the reach of the water. Then by the method of dilutions prepare solutions of exopolysaccharides with concentration 400, 200, 100, 50, 10, 5, 1 and 0.1 µg/ml of These samples are used as inducers of interferon.

Cells producers are leukocytes donated human blood. The concentration of cells was 1×106/ml. as the environment used the medium RPMI 1640, the volume of medium in each sample was 1.0 ml. of the suspension of cells in each sample was added 0.1 ml of the appropriate dilution analyzed the total fraction. As a control, use the Newcastle disease virus (VBN) as inducer of α-interferon and phytohemagglutinin P (PHA) as the inductor γ-interferon, as well as a suspension of cells in medium without addition of inducer. The interferon synthesis is carried out at a temperature of 37°C. the Samples taken after 24, 48 and 72 hours. The activity of interferon is determined by titration in diploid culture fireblasts of a human embryo against 10 and 1 JRC50virus encephalomyocarditis mice. The titer of interferon take his last dilution in which there is a 50% protection of the cell monolayer from the cytopathic effect of the virus.

The test results on the ability of exopolysaccharides from the culture fluid F. limestone, obtained in example 1, to induce in blood leukocytes of human production of interferon in the table.

Table
The test results on the ability of exopolysaccharides culture fluid F. limestone, obtained in example 1, to induce in blood leukocytes of human production of interferon (IU/ml)
The drug concentration, mg/mlTime after induction
24 hours48 hours72 hours
50<8<8<8
5884
0,5884
0,18168-16
0,05163216
0,01163216
0,0058 168
0,001<2168

0,0001<288
control cells<2<2<2
control VBN160808
control PHA161616

When testing interferon biological method in the culture of diploid human fibroblasts by suppressing the reproduction indicator virus described antiviral agent in a concentration of from 0.0001 mg/ml to 5 mg/ml induces in the human leukocyte production of interferon. The highest activity of interferon is 16-32 units/ml and detected using the drug at a concentration of 0.005-0.1 mg/ml the Optimal time to identify the highest titer of interferon is 48 hours. The tool induces interferon-level control inducer of interferon-gamma f the A.

The obtained results allow to conclude about the presence of sufficiently high antiviral activity of exopolysaccharides F. limestone, obtained by the described method.

1. The method of obtaining antiviral agents by making seed mycelium of the basidiomycete Armillaria winter Flammulina limestone (Curtis) Singer, cooking liquid nutrient medium containing water as sources of carbon and vegetable oil in the amount 19,0-to 27.0 g/l of water and molasses in the amount of 15-40 g/l of water as a source of nitrogen in corn flour 7-15 g/l of water, mineral salts of potassium dihydrophosphate in the amount of 2.0-2.4 g/l of water and magnesium sulfate in the amount of 0.18 to 0.20 g/l of water, sterilizing, seeding prepared cultivated mycelia sterile liquid nutrient medium, the cultivation of basidiomycete in aerobic conditions with getting immersed in the culture, with subsequent separation of the submerged culture for biomass-degrading basidiomycete and the culture fluid and the release of the latter by adding thereto at a temperature of 4°C 96%ethyl alcohol, when the ratio of the volume of the culture fluid and ethanol 1:2-1:4, clot, which squeezed, dried at 40°C until the moisture content of 6% and pulverized to obtain antivirals.

2. The method according to claim 1, characterized in that the liquid is th culture medium additionally contains zeatin in the number 0,0010-0.0015 g/l of water, vitamin B3the number 0,0060-0,0065 g/l of water, and yeast extract in the amount 0,050-to 0.055 g/l of water.

3. Antiviral agent obtained according to any one of claims 1 to 2.



 

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