RU2418062C1 - Method for production of remedy with antineoplastic activity and remedy with antineoplastic activity - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method involves preparation of inoculation mycelium of basidiomycetes, preparation of productive nutrient medium and inoculation of the productive nutrient medium, containing carbohydrate and nitrogen source and mineral salts, with the prepared inoculation mycelium. Basidiomycetes cultivation with subsequent production of an immersion culture. The inoculation mycelium preparation is performed in a sterile nutrient medium containing glucose, soya flour, potassium dihydrogen phosphate, magnesium sulphate and arachidonic acid. The inoculation mycelium growing is performed at a temperature of 22-29°C during 2-6 days under aeration conditions with its subsequent homogenisation.

EFFECT: antineoplastic remedy produced from the basidiomycetes immersion culture is effective and the cultivation process duration is reduced from 2-3 weeks to one week; mycelium yield increases.

23 cl, 6 tbl, 12 ex

Description

The group of inventions relates to the field of biotechnology and can be used to obtain funds with antitumor activity by culturing basidiomycetes in an immersed culture.

The metabolites of many types of basidiomycetes are known to have healing properties, in particular, an antitumor effect, including a direct inhibitory effect on the proliferation and growth of tumor cells, an increase in antitumor immunity, anticarcinogenic effect, as well as antioxidant, antiviral, hepatoprotective and immunomodulating effects. In recent years, work has been ongoing in Russia, the United States, China, Japan, and Korea to obtain drugs based on basidiomycetes. The creation of new drugs is hampered by the complex and expensive technology for growing basidiomycetes and isolating from them substances with stable biological properties, in particular antitumor ones.

The formation of substances valuable for pharmacology by basidiomycetes depends both on the types of basidiomycetes used and on the technology of their cultivation (preparation of seed, cultivation conditions), as well as metabolite isolation processes.

For the treatment of cancer, chemicals are used, such as cytostatics, which exhibit high toxicity, which limits their use.

The toxicity of antitumor agents obtained from edible or inedible (non-toxic) basidiomycetes, according to published data, is either absent or very low (Chang & But, 1986, Su et al., 1987).

Currently, much attention is paid to the development of technology for the cultivation of basidiomycetes in order to obtain the maximum amount of mycelium during cultivation as soon as possible and methods for the isolation of antitumor agents.

A known method of obtaining funds with antitumor activity from the mycelium of the fungus Ganoderma lucidum. Mycelium is obtained by culturing Ganoderma lucidum in a submerged culture on a nutrient medium containing 50 g of glucose, 20 g of peptone, 0.87 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 10 g of iron (II) chloride, 7 g of manganese chloride, 10 g of zinc sulfate, 4 mg of zinc chloride per 1 liter of water, pH 5.5.

Pre-prepared inoculum mycelium. To do this, the specified fungus is cultivated at 180 rpm, 25 ° C for 10 days. The resulting culture is reseeded in fresh nutrient medium in an amount of 5% and re-cultured for 10 days. The mycelium is separated from the culture fluid and G009 proteoglycan is isolated from it - an agent with antitumor activity, it is purified and antitumor activity is determined against sarcoma 180 vaccinated in male mice (RU 2082755 C1, 08.19.1997).

The disadvantage of the described method is the long process of preparing seed mycelium (up to 20 days), the long process of cultivating the fungus (7 days) and the complex process of isolating an agent with antitumor activity.

A known method of obtaining a drug that affects tissue metabolism, including having antitumor activity, involving the deep cultivation of the fungus Pleurotus ostreatus 1137 (VKPM-F 819) in flasks on a medium containing beer wort 0.65 ° B, at 26-28 ° C on a rotary shaker at 200 rpm, the duration of the process is up to 24 days, followed by separation of the mycelium from the native solution by centrifugation and isolation of biologically active substances from the mycelium by extraction with ethanol at neutral or acidic pH values. The extracts are evaporated in vacuo to a dry residue. The dry residue is dissolved in an aqueous solution of ethanol. The obtained fractions are used for biological tests. The antitumor activity of the obtained agent is investigated in mice with transplanted ascites breast cancer with a single administration of chemical antitumor agents - cytostatics. The drug obtained by the above method is administered at a dose of 100 mg / kg 2 hours after tumor transplantation, and then at the same dose for 9 days daily. The average life expectancy of animals was 30.8 ± 0.8 days, and in the control, 18.0 ± 0.8 days, i.e. 71% more than in control (RU 2192873 C1, 11/20/2002).

The disadvantage of the described method is the duration of the process of cultivation of the fungus, as well as the study of antitumor activity against the background of cytostatics - toxic substances, and therefore it is impossible to judge the independent antitumor effectiveness of the funds obtained.

A known method of obtaining funds with antitumor activity from the mycelium of basidiomycetes selected from the group of Ganoderma lucidum, Hypsizygus ulmarius, Kuhneromyces mutabilis, Omphalotus olearis, Panus conchatus, Piptoporus betulinus, Pleurotus eryngii, Trametes z.

Basidiomycetes are first grown on agar medium at 27 ° C, and then subcultured on liquid medium and cultured in an immersed culture at 27 ° C, 180 rpm for 2-3 weeks. The medium contains 2% glucose, 0.1% peptone, 0.1% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate and trace elements (iron (II) sulfate, manganese sulfate, zinc sulfate, copper sulfate) . Mycelium is isolated from a submerged culture, dried, and an agent having antitumor activity is extracted from dry mycelium. The extractant used is methanol, ethanol, acetonitrile, ethyl acetate, chloroform, dichloromethane or mixtures thereof (US 20060057157, March 16, 2006).

A disadvantage of the described method for producing an agent having antitumor activity is the long-term cultivation of basidiomycetes (2-3 weeks), the use of an antitumor agent only separated from the culture fluid and dried mycelium and the low efficiency of the obtained agent in the in vitro system. In an in vivo system, the resulting preparation has not been tested.

The task to which the group of inventions is directed is to reduce the duration of the process of culturing basidiomycetes, increase the yield of mycelium and obtain a more effective antitumor agent.

The problem is solved due to the fact that in the method of obtaining funds with antitumor activity, including the preparation of seed mycelium of basidiomycete, preparation of a sterile production nutrient medium, inoculation of a production nutrient medium containing sources of carbon, nitrogen, mineral salts, prepared inoculum mycelium, cultivation of basidiomycete with the subsequent receipt of an immersed culture, according to the invention, the preparation of seed mycelium is carried out on a sterile liquid nutrient medium containing (g / l of water): glucose - 18.0-22.0, soy flour - 8.0-12.0, potassium dihydrogen phosphate - 2.0-3.0, magnesium sulfate - 0.2 -0.4 and arachidonic acid - 1.0-5.0 × 10 ^-5 , seed cultivation is carried out at a temperature of 22-29 ° C for 2-6 days during aeration, while the finished seed mycelium is homogenized.

Soy flour can be used both genetically modified and unmodified soybean varieties.

Homogenization of the finished seed can be carried out both before sowing the production nutrient medium in the homogenizer for 2-5 minutes, and in the process of sowing directly in the bioreactor with the stirrer turned on at 300-500 rpm for 0.5-6 minutes.

For cultivation, a basidiomycete selected from the group Armillaria mellea (Fr.) Karst., Flammulina velutipes (Curt.:Fr.) Singer, Hericium erinaceus (Bull.:Fr.) Pers., Hypsizygus marmareus (Peck) HEBigelow, Hypsizygus should be used tessulatus (Bulliard: Fr.) Singer, Lentinus edodes (Berk.) Singer, Lyophyllum shimeji (Kawam.) Hongo, Pleurotus djamor (Rumphius ex Fries) Boedjin, Pleurotus ostreatus (Jacg.:Fr.) Kumm.

When cultivating Armillaria mellea basidiomycete into the production medium, it is necessary to introduce (g / l of water): vegetable oil in the amount of 15.0-18.0, soy flour - 18.0-20.0, potassium dihydrogen phosphate - 0.5-2.5, magnesium sulfate - 0.05-0.26, cultivation is carried out at a temperature of 24-28 ° C for 4.5-5.5 days.

When cultivating the basidiomycete Flammulina velutipes into the production medium, it is necessary to introduce (g / l water): vegetable oil in an amount of 19.0-27.0, soy flour in an amount of 19.0-27.0, potassium dihydrogen phosphate - 1.4-3.0 , magnesium sulfate - 0.14-0.30, cultivation is carried out at a temperature of 20-28 ° C for 5.5-6.5 days.

When cultivating Hericium erinaceus basidiomycete, it is recommended to introduce (g / l water) into the production nutrient medium: vegetable oil and soy flour in amounts of 10.0-27.0 and 20.0-25.0, respectively, to introduce potassium dihydrogen phosphate in an amount of 2.0-3 , 3, magnesium sulfate - 0.2-0.3, cultivation is carried out at a temperature of 24-32 ° C for 5.5-6.5 days. Molasses in the amount of 1-40 g / l of medium can be added to the indicated production medium for cultivation of Hericium erinaceus.

When cultivating the basidiomycete Hypsizygus marmareus, it is advisable to introduce (g / l water) into the production medium: vegetable oil in an amount of 20.0-24.0, soy flour in an amount of 16.0-22.0, potassium dihydrogen phosphate - 1.5-3 , 0, magnesium sulfate - 0.2-0.3, cultivation is carried out at a temperature of 24-31 ° C for 5.5-6.5 days.

When cultivating the basidiomycete Hypsizygus tessulatus, it is necessary to introduce (g / l water) into the production medium: vegetable oil in the amount of 18.0-25.0, soy flour - 20.0-28.0, potassium dihydrogen phosphate - 1.6-2.6, sulfate magnesium - 0.2-0.3, cultivation is carried out at a temperature of 24-33 ° C for 6.5-7.5 days.

When cultivating Lentinus edodes basidiomycete, it is advisable to introduce (g / l water) into the production medium: vegetable oil and soy flour in amounts equal to 12.0-16.0 and 18.0-24.0, respectively, and additionally introduce a carbon source in the form glucose in an amount of 8-12, inject potassium dihydrogen phosphate in an amount of 1.3-2.6, magnesium sulfate - 0.2-0.3, cultivation is carried out at a temperature of 24-30 ° C for 5.5-6.5 days .

When cultivating the basidiomycete Lyophyllum shimeji into the production medium, it is necessary to introduce (g / l of water): vegetable oil in the amount of 16.0-22.0, soy flour - 18.0-25.0, potassium dihydrogen phosphate - 2.0-3, 0, magnesium sulfate - 0.2-0.3, cultivation is carried out at a temperature of 24-34 ° C for 3.5-4.5 days.

When cultivating Pleurotus djamor basidiomycete into the production environment, it is necessary to introduce (g / l water): vegetable oil in an amount of 15.0-20.0, soy flour - 15.0-21.0, potassium dihydrogen phosphate - 2.0-3.3, sulfate magnesium - 0.2-0.3, cultivation is carried out at a temperature of 26-35 ° C for 2.5-3.5 days.

When cultivating Pleurotus ostreatus basidiomycete, it is advisable to introduce (g / l water) into the production nutrient medium: vegetable oil in an amount of 14.0-20.0, soy flour - 11.0-19.0, potassium dihydrogen phosphate - 2.0-3.0, magnesium sulfate - 0.2-0.3, cultivation is carried out at a temperature of 24-28 ° C for 2.5-3.5 days.

As a vegetable oil in the production culture media for cultivation of Armillaria mellea, Flammulina velutipes, Hericium erinaceus, Hypsizygus marmareus, Hypsizygus tessulatus, Lentinus edodes, Lyophyllum shimeji, Pleurotus djamor, olive oil, sunflower oil, sunflower oil, sunflower, sunflower, mixtures thereof.

The introduction of vegetable oil into the composition of the production nutrient media in the declared amount helps not only to reduce the duration of the cultivation process and increase the yield of mycelium, but also reduces the foaming process in the cultivation process.

After basidiomycete cultivation, the resulting submerged culture should be freeze-dried, then chopped dry mycelium and used as an antitumor agent.

After basidiomycete cultivation, the resulting submerged culture can be autoclaved at 1.2-1.5 atm for 1.5-3 hours, the liquid phase can be separated by filtration and used as an antitumor agent.

To obtain an antitumor agent after cultivation of basidiomycete, mycelium can be separated from the submerged culture by filtration, extracted with distilled water in the ratio of 90-360 g / l of water during autoclaving at 1.2-1.5 atm for 1.5-2.5 hours The separated aqueous extract is used as a target.

After cultivation, it is advisable to separate the mycelium of basidiomycete from the submerged culture and carry out extraction with ethanol in a ratio of 90-360 g / 100 ml of extractant for 0.5-1.5 hours with stirring, separate the resulting ethanol extract from the mycelium residues, evaporate to a dry residue on an evaporator and fill the remainder of mycelium with distilled water in a ratio of 90-360 g / l, autoclave at 1, 2-1.5 atm for 1.5-2.5 hours, separate the resulting aqueous extract and combine with the dry residue of the ethanol extract and the resulting mixture isp use as an antitumor agent.

After cultivation, the basidiomycete mycelium can be separated from the submerged culture, dried at a temperature of 35-80 ° C, ground and used as an antitumor agent.

Dry crushed mycelium of basidiomycete should be extracted with boiling distilled water in a ratio of 18-72 g / l at 1.2-1.5 atm for 1.5-2.5 hours, then the aqueous extract should be separated from the remains of the mycelium and used as an antitumor facilities.

To increase the shelf life without decreasing biological activity, the aqueous extract of basidiomycete mycelium can be frozen at a temperature of -18-70 ° C. Before use as an antitumor agent, the aqueous extract should be thawed.

For the same purpose, the aqueous extract of basidiomycete mycelium can be dried on a spray dryer. It is possible to use both the dry extract itself and its solution in distilled water.

2-6 volumes of ethanol can be added to the aqueous extract of mycelium of basidiomycete, the precipitate should be separated, dried and used as an antitumor agent.

As follows from the foregoing, the antitumor agent can be used both in dry powder form and in liquid. Various pharmaceutical additives, such as vitamins, trace elements, antioxidants, compatible antitumor agents, can be added to the resulting antitumor agent.

The antitumor agent can be made in the form of tablets, capsules, a drink and can be used both orally and in the form of microclysters.

The dosage of the antitumor agent depends on the disease, the age of the patient, his weight, and the composition of the agent. In terms of the content of polysaccharides, the preferred dosage for laboratory animals can be 1-3 mg of polysaccharides per kg of weight, for humans - 0.08-0.25 mg / kg.

The technical result of the claimed group of inventions is to increase the yield of mycelium due to the development of the composition of the production environment, cultivation conditions, the selection of the composition of the medium to obtain seed, to reduce the duration of the cultivation of basidiomycetes from 2-3 weeks to 7.5 days. The inclusion of vegetable oil in the production environment not only increases the yield of basidiomycete biomass, but also prevents foaming of the medium during the cultivation of basidiomycete. Together, the developed method for cultivating basidiomycete and the method for isolating an antitumor agent made it possible to obtain an effective agent inhibiting tumor growth in experiments in vivo to 94%.

The invention is illustrated by the following examples, which do not cover the entire scope of the claims, but do not limit it.

Example 1. Preparation of seed mycelium of basidiomycetes.

Sterile liquid culture media were prepared, the composition of which is shown in Table 1, and sowed with mycelium of basidiomycetes grown on an agar culture medium.

The cultivation of seed mycelium was carried out on a rotary shaker at a temperature of 22-29 ° C, the rotation speed of the shaker 180-220 rpm for 2-6 days. The cultivation conditions of basidiomycetes are shown in table 2.

The prepared seed mycelium of Hericium erinaceus strain He-4, Lentinus edodes strain Le-14, Pleurotus ostreatus strain 447 was homogenized during inoculation of the production medium in the bioreactor with the stirrer at 500 rpm for 0.5 minutes.

Table 1. Compositions of media for obtaining seed mycelium of basidiomycetes. Type of basidiomycete Power sources, g / l of water Glucose Soya flour KN ₂ PO ₄ MgSO ₄ Arachidonic acid A.mellea eighteen 12 2,4 0.20 1.0 × 10 ^-5 F.velutipes 22 8 2.5 0.24 3.0 × 10 ^-5 H. erinaceus 19 10 3.0 0.4 5.0 × 10 ^-5 H. marmareus 21 9 2,4 0.25 2.0 × 10 ^-5 H. tessulatus 22 8.5 2.0 0.4 4,5 × 10 ^-5 L.edodes twenty 9 3.0 0.3 4.0 × 10 ^-5 L.shimeji 21 9.5 2.5 0.24 3,5 × 10 ^-5 P.djamor 19 10 2.0 0.3 2.0 × 10 ^-5 P.ostreatus 22 9 2.5 0.4 1.0 × 10 ^-5

Table 2. The growing conditions of the seed mycelium of basidiomycetes. Type of basidiomycete Cultivation parameters Mixer rotation speed, rpm Temperature ° C The duration of the process, days A.mellea 220 25 four F.velutipes 180 22 6 H.erinaceus 200 28 6 H. marmareus 220 25 3 H. tessulatus 220 25 3 L.edodes 220 25 6 L.shimeji 200 28 3 P.djamor 200 29th 2 P.ostreatus 220 25 2

Example 2. Submerged cultivation of basidiomycetes.

For submerged cultivation of basidiomycetes, production media were prepared, the composition of which is shown in Table 3. Production media was sterilized at 1.2 atm for 30 minutes, cooled and seeded with seed mycelium of basidiomycetes obtained in Example 1.

The prepared seed mycelium Armillaria mellea strain Am-4, Flammulina velutipes strain Fv-7, Hypsizygus marmareus strain Hm-1, Hypsizygus tessulatus strain Ht-1, Lyophyllum shimeji strain Ls-2, Pleurotus djamor genome strain 4 minutes before inoculation of the production medium, the seed mycelium Hericium erinaceus, Lentinus edodes, Pleurotus ostreatus were homogenized during the inoculation of the production medium in the bioreactor with the mixer turned on at 500 rpm for 0.5 minutes.

Table 3. Compositions of media for submerged cultivation of basidiomycetes. View Power sources, g / l of water Carbon sources Nitrogen sources Mineral salts oil rust. glucose molasses soy flour KN ₂ PO ₄ MgSO ₄ A.mellea 17 - - twenty 0.5 0.05 F.velutipes 27 - - 27 2,4 0.24 H.erinaceus 10 - thirty 22 2,8 0.28 H.erinaceus 27 - - 25 3.3 0.30 H.marmareus 22 - - 19 2.0 0.2 H. tessulatus 24 - - 28 1,6 0.24 L.edodes fifteen 8 - 21 1,5 0.24 L.edodes 12 eleven - 24 2.6 0.3 L.shimeji twenty - - 23 2.6 0.26 P.djamor eighteen - - eighteen 2.6 0.26 P.ostreatus fifteen - - eleven 2,4 0.24

The cultivation of basidiomycetes Armillaria mellea, Flammulina velutipes, Hypsizygus marmareus, Hypsizygus tessulatus, Lyophyllum shimeji, Pleurotus djamor was carried out in flasks on a rotary shaker at 200 rpm. Bazidiomycetes Hericium erinaceus, Lentinus edodes, Pleurotus ostreatus were grown in bioreactors with a constantly working stirrer (200 rpm) and an air supply of 1.5 rpm. per minute.

The temperature regime and the duration of the processes of submerged cultivation, as well as the yield of air-dried biomass (humidity 5-6%) are presented in table 4. The purity of the cultures was monitored using light microscopy. The presence of buckles on the mycelium of fungi, which are a specific morphological structure of basidiomycetes, and the absence of extraneous micro- and microbiota were recorded.

Table 4. The temperature regime, the duration of the processes of submerged cultivation and the yield of air-dry biomass of basidiomycetes. View Cultivation temperature, ⁰ C The duration of the cultivation process, day The output of air-dry biomass, g / l A.mellea 24-26 4,5 20-22 F.velutipes 20-22 6.0 36-40 H.erinaceus 25-27 6.5 24-25 H. marmareus 24-26 5.5 27-29 H. tessulatus 24-26 7.5 18-21 L.edodes 24-26 6.0 20-21 L.shimeji 28-30 4.0 26-28 P.djamor 29-31 3.0 23-24 P.ostreatus 25-27 3.0 26-27

Example 3. Obtaining funds with antitumor properties.

Received an immersed culture of Hericium erinaceus strain He-4 according to example 2 in a bioreactor with a volume of 15 l (the volume of the culture medium is 8 l). At the end of the cultivation process, the submerged basidiomycete culture was freeze-dried at a profile temperature change, including a multiple drop in temperature to minus 30 ° С, crushed, and the obtained dry powder was used as an antitumor agent. The results of the evaluation of the antitumor effect of the drug are presented in table 5.

Example 4. Obtaining funds with antitumor properties.

An immersed culture of Hypsizygus marmareus strain Hm-1 was prepared according to Example 2. At the end of the process, the immersed culture of basidiomycete was autoclaved at 1.2-1.5 atm for 2 hours, the liquid phase was separated and used as an antitumor agent. The results of the evaluation of the antitumor effect of the drug are presented in table 5.

Example 5. Obtaining funds with antitumor properties.

An immersed culture of Lentinus edodes strain Le-14 in Example 2 was prepared by growing basidiomycete in a 15 L bioreactor with 8 L loading of the production medium. At the end of the submerged cultivation, the mycelium was separated by filtration. Crude mycelium was poured with distilled water in a ratio of 100 g / l, autoclaved at 1.5 atm for 2 hours. The resulting aqueous extract was separated by filtration and used as an antitumor agent. The results of the evaluation of the antitumor effect of the agent are presented in table 6.

Example 6. Obtaining funds with antitumor properties.

Immersed cultures of Hericium erinaceus strain He-4 and Lentinus edodes strain Le-14 according to Example 2 were prepared by growing basidiomycetes in 15 L bioreactors containing 9 L of production medium. At the end of the cultivation processes, the mycelium was separated by filtration. Weighed portions of crude biomass Hericium erinaceus and Lentinus edodes in the amount of 150 and 300 g, respectively, were poured into 100 ml of ethanol, extraction was carried out for 1 hour with stirring. The ethanol extracts were separated by filtration and evaporated to dryness on a vacuum evaporator. The portions of the mycelium Hericium erinaceus and Lentinus edodes remaining after ethanol extraction were poured into 1 liter of distilled water and autoclaved at 1.2 atm for 2.5 hours. The aqueous extracts were separated from the mycelium by filtration, the aqueous extract of the mycelium Hericium erinaceus was combined with the dry residue of the ethanolic extract of the mycelium Hericium erinaceus. Lentinus edodes mycelium water extract - with Lentinus edodes ethanol mycelium extract. Thus, 2 antitumor agents were obtained. The results of the evaluation of the antitumor effect of the agents are presented in table 6.

Example 7. Obtaining funds with antitumor properties.

Received submerged cultures of Flammulina velutipes strain Fv-7 and Pleurotus ostreatus strain 447 according to example 2, growing the first basidiomycete in flasks on a rocking chair, and the second in a bioreactor with a total volume of 15 l from 9 l of production medium. At the end of the submerged cultivation processes, the mycelium of basidiomycetes was separated by filtration, dried at 60 ° C, ground and used as antitumor agents. The results of the evaluation of the antitumor effect of the funds are presented in table 5.

Example 8. Obtaining funds with antitumor properties.

Immersed cultures of Armillaria mellea strain Am-4, Flammulina velutipes strain Fv-7, Hypsizygus tessulatus strain Ht-1, Lyophyllum shimeji strain Ls-2, Pleurotus djamor strain Pd-3 and Pleurotus ostreatus strain 447 were grown in Example 2, cultivated on a rocking chair. After the processes of submerged cultivation of the mycelium, the basidiomycetes were separated by filtration, dried at 65 ° C and ground. Weighed out dry powdered mycelium of basidiomycetes: Armillaria mellea - 6 g, Flammulina velutipes - 6 g, Hypsizygus tessulatus - 5 g, Lyophyllum shimeji - 10 g, Pleurotus djamor - 1.8 g, Pleurotus ostreatus - 3.6 g. Weighed portions 100 g. ml of distilled water and was autoclaved at 1.2 atm for 2 hours. The aqueous extracts were separated by filtration and used as antitumor agents. The evaluation results of the antitumor effect of the funds obtained are presented in table 5.

Example 9. Obtaining funds with antitumor properties.

The aqueous extract of dry powdered mycelium Armillaria mellea strain Am-4 obtained in example 8 was frozen at a temperature of -18 ° C, the aqueous extract of dry powdered mycelium Pleurotus djamor strain Pd-3 obtained in example 8 was frozen at a temperature of -70 ° C. The extracts were stored at freezing temperature for 4 months, after which they were thawed at room temperature and used as antitumor agents. The results of a study of the antitumor activity of the agents are presented in table 5.

Example 10. Obtaining funds with antitumor properties.

Hericium erinaceus immersed mycelium extract He-4 strain obtained in example 6 and Hypsizygus tessulatus Ht-1 dry ground immersed mycelium extract obtained in example 8 were dried using a spray dryer. The resulting preparations were dissolved in distilled water so that the concentration of the resulting solutions corresponded to the concentration of the solutions subjected to drying, and was used to evaluate their antitumor properties. The results are shown in table 5.

Example 11. Obtaining funds with antitumor properties.

We used an aqueous extract of raw mycelium Lentinus edodes strain Le-14 obtained in Example 5 and aqueous extracts of dry minced mycelium Flammulina velutipes strain Fv-7 and Pleurotus ostreatus strain 447 obtained in example 8. 96% ethanol was added to the extracts. The ratio of the volumes of ethanol and Lentinus edodes mycelium extract was 4: 1, the ratio of the volumes of ethanol and Flammulina velutipes extract was 2: 1, the ratio of the volumes of ethanol and Pleurotus ostreatus extract was 6: 1. Precipitated precipitates, representing the total fractions of water-soluble mycelium polysaccharides, were separated by centrifugation and subjected to freeze drying. Dry sediments were used as antitumor agents. The results of the study of their activity are presented in table 5.

Example 12. Evaluation of the antitumor effect of the obtained antitumor agents.

The antitumor effect of preparations from the submerged culture of the studied species of fungi was studied in vivo on two transplantable tumor strains: T-lymphoma EL-4 and T-cell lymphocytic leukemia P388. Tumors were inoculated with hybrid mice (C57B1 / 6 × DBA / 2) F ₁ (hereinafter B6D2F ₁ ), T-lymphoma EL-4 was inoculated in males, and T-cell lymphocytic leukemia P388 - in females. Mice were obtained from the nursery RAMS "Kryukovo". After admission, the mice were quarantined for 21 days. Tumor strains were stored in a nitrogen bank at a temperature of -196 ° C. After thawing, the tumor strains were maintained under syngeneic conditions in ascites form by serial intraperitoneal passages on DBA / 2 mice. In the experiment on day 0, EL-4 T-lymphoma was inoculated with 10 ⁵ cells under the skin of the right side. T-cell lymphocytic leukemia P388 - 10 ⁶ cells. Treatment was started 10 days after vaccination with T-lymphoma EL-4 and 2 days after vaccination with T-cell lymphocytic leukemia P388. The effect of the studied drugs was evaluated by oral administration. The drugs were administered using a probe intragastric daily 1 time per day for 10 days. Dry crushed mushroom biomass and sublimated submerged fungal cultures were tested at a dose of 50 mg / kg / day, preparations were prepared by adding crushed mushroom material to starch paste so that the daily dose for each animal was contained in 0.3 ml of paste. Polysaccharide fractions were used at a dose of 2 mg / kg / day in the form of aqueous solutions; the daily dose of the solution was 0.3 ml / mouse. The dose of aqueous extracts of mycelium of basidiomycetes was 0.25-0.3 ml / kg / day, the solids content in aqueous extracts varied in the range of 13-19 mg / ml. The spray dried aqueous extracts of fungal mycelium were tested at a dose of 4.8 mg / kg / day, the weighed portions of dry extracts were dissolved in water, the volume of the injected solution was 0.3 ml / mouse. The number of mice in the control and experimental groups was 8-10 animals. During the experiments, the general condition of the mice, changes in body weight, tumor grafting, and tumor growth dynamics were monitored. Tumor growth inhibition (TPO) was calculated by the formula: TPO (%) = (M _k -M _o ) / (M _k ) × 100, where M _k and M _o are the average calculated tumor mass in the control and experiment, respectively. Tumor mass was calculated according to the formula M (mg) = (a × b × s) / 2, where M is the calculated mass of the tumor, and a, b, c are the 3 largest mutually perpedicular diameters of the tumor node in mm. The significance of differences in the average values of tumor mass was determined by t-student test. For reliable, differences were taken at P≤0.05. The antitumor efficacy of the funds obtained is presented in tables 5 and 6.

Table 5. Antitumor effect of the funds received. Model: T-cell lymphocytic leukemia P388, accounting for 14 days of experience. Example No. A drug SRW,% one 2 3 3 Hericium erinaceus, sublimated submerged culture 75 four Hypsizygus marmareus, liquid extract obtained from submerged culture 56 7 Flammulina velutipes, dry shredded submerged mycelium 52 Pleurotus ostreatus, dry shredded submerged mycelium 56 8 Armillaria mellea, dry submerged mycelium aqueous extract 69 Flammulina velutipes, dry submerged mycelium aqueous extract 87 Hypsizygus tessulatus, dry submerged mycelium aqueous extract 59 Lyophyllum shimeji, dry submerged mycelium aqueous extract 72 Pleurotus djamor, dry submerged mycelium aqueous extract 44 Pleurotus ostreatus, aqueous extract of dry submerged mycelium 75 9 Armillaria mellea, dry submerged mycelium aqueous extract stored at -18 ° C 66 Pleurotus djamor, dry submerged mycelium aqueous extract stored at -70 ° C 45

one 2 3 10 Hericium erinaceus, dry extract of dry submerged mycelium (after spray drying) 65 Hypsizygus tessulatus, dry extract of dry submerged mycelium (after spray drying) 63 eleven Lentinus edodes, polysaccharide fraction of submerged mycelium 81 Flammulina velutipes, polysaccharide fraction of submerged mycelium 94 Pleurotus ostreatus, polysaccharide fraction of submerged mycelium 74

Table 6. Antitumor effect of the funds received. Model: T-lymphoma EL-4 accounting for 18 days of experience. No. A drug SRW, an example % 5 Lentinus edodes, crude submerged aqueous extract 62 mycelium 6 Hericium erinaceus, combined aquatic and 58 ethanol extract of crude submerged mycelium Lentinus edodes, combined water and ethanol 59 raw submerged mycelium extract

Thus, the task is solved. The duration of the cultivation process is reduced to 2-6 days while maintaining the effectiveness of the obtained funds with antitumor activity.

Claims (23)

1. The method of obtaining funds with antitumor activity, including the preparation of inoculum seed mycelium of basidiomycete, preparation of the production medium, inoculation of the production medium containing sources of carbon, nitrogen, mineral salts and water prepared by inoculum mycelium, cultivation of basidiomycete, followed by obtaining a submerged culture, characterized the fact that the preparation of seed mycelium is carried out on a sterile liquid nutrient medium containing, g / l of water: glucose - 1 8.0-22.0, soy flour - 8.0-12.0, potassium dihydrogen phosphate - 2.0-3.0, magnesium sulfate - 0.2-0.4 and arachidonic acid - 1.0-5, 0 × 10 ^-5 , the cultivation of seed mycelium is carried out at a temperature of 22-29 ° C for 2-6 days during aeration, while the finished seed mycelium is homogenized. 2. The method according to claim 1, characterized in that the homogenization of the finished seed mycelium before sowing the production nutrient medium is carried out in the homogenizer for 2-5 minutes, and in the process of sowing in the bioreactor with the stirrer turned on at 300-500 rpm for 0.5-6 minutes 3. The method according to claim 1, characterized in that the cultivated basidiomycetes selected from the group Armillaria mellea (Fr.) Karst., Flammulina velutipes (Curt.:Fr.) Singer, Hericium erinaceus (Bull.:Fr.) Pers., Hypsizygus marmareus 1611 (Peck) HEBigelow, Hypsizygus tessulatus (Bulliard: Fr.) Singer, Lentinus edodes (Berk.) Singer, Lyophyllum shimeji (Kawam.) Hongo, Pleurotus djamor (Rumphius ex Fries) Boedjin, Pleus ostre, Fr.) Kumm. 4. The method according to claim 3, characterized in that the cultivation of basidiomycete Armillaria mellea is carried out in a production medium containing, g / l of water: vegetable oil - 15.0-18.0, soy flour -18.0-20.0 , potassium dihydrogen phosphate - 0.5-2.5, magnesium sulfate - 0.05-0.26, at a temperature of 24-28 ° C for 4.5-5.5 days. 5. The method according to claim 3, characterized in that the cultivation of the basidiomycete Flammulina velutipes is carried out in a production medium containing, g / l water: vegetable oil - 19.0-27.0, soy flour 19.0-27.0, potassium dihydrogen phosphate - 1.4-3.0, magnesium sulfate - 0.14-0.30, at a temperature of 20-28 ° C for 5.5-6.5 days. 6. The method according to claim 3, characterized in that the cultivation of basidiomycetes Hericium erinaceus is carried out in a production medium containing, g / l of water: vegetable oil - 10.0-27.0, soy flour - 20.0-25.0 , potassium dihydrogen phosphate - 2.0-3.3, magnesium sulfate - 0.2-0.3, at a temperature of 24-30 ° C for 5.5-6.5 days. 7. The method according to claim 6, characterized in that molasses is additionally introduced into the production medium in an amount of 1-40 g / l of medium. 8. The method according to claim 3, characterized in that the cultivation of the basidiomycete Hypsizygus marmareus is carried out in a production nutrient medium containing, g / l of water: vegetable oil - 20.0-24.0, soy flour - 16.0-22.0 , potassium dihydrogen phosphate - 1.5-3.0, magnesium sulfate - 0.2-0.3, at a temperature of 24-31 ° C for 5.5-6.5 days. 9. The method according to claim 3, characterized in that the cultivation of the basidiomycete Hypsizygus tessulatus is carried out in a production medium containing, g / l of water: vegetable oil - 18.0-25.0, soy flour - 20.0-28.0 , potassium dihydrogen phosphate - 1.6-2.6, magnesium sulfate - 0.2-0.3, at a temperature of 24-33 ° C for 6.5-7.5 days. 10. The method according to claim 3, characterized in that the cultivation of Lentinus edodes basidiomycetes is carried out in a production medium containing, g / l water: vegetable oil - 12.0-16.0, soy flour - 18.0-24.0 glucose - 8-12, potassium dihydrogen phosphate - 1.3-2.6, magnesium sulfate - 0.2-0.3, at a temperature of 24-30 ° C for 5.5-6.5 days. 11. The method according to claim 3, characterized in that the cultivation of basidiomycete Lyophyllum shimeji is carried out in a production medium containing, g / l water: vegetable oil - 16.0-22.0, soy flour - 18.0-25.0 , potassium dihydrogen phosphate - 2.0-3.0, magnesium sulfate - 0.2-0.3, at a temperature of 24-34 ° C for 3.5-4.5 days. 12. The method according to claim 3, characterized in that the cultivation of the basidiomycete Pleurotus djamor is carried out in a production medium containing, g / l water: vegetable oil - 15.0-20.0, soy flour - 15.0-21.0 , potassium dihydrogen phosphate - 2.0-3.3, magnesium sulfate - 0.2-0.3, at a temperature of 26-35 ° C for 2.5-3.5 days. 13. The method according to claim 3, characterized in that the cultivation of basidiomycete Pleurotus ostreatus is carried out in a production nutrient medium containing, g / l of water: vegetable oil - 14.0-20.0, soy flour - 11.0-19.0 , potassium dihydrogen phosphate - 2.0-3.0, magnesium sulfate - 0.2-0.3, at a temperature of 24-28 ° C for 2.5-3.5 days. 14. The method according to claim 1, characterized in that after the cultivation of basidiomycete, the resulting submerged culture is subjected to freeze-drying, then the dried culture is ground and the resulting dry powder is used as an agent with antitumor activity. 15. The method according to claim 1, characterized in that after culturing the basidiomycete, the resulting submerged culture is autoclaved at 1.2-1.5 atm for 1.5-3 hours, the liquid phase is separated and used as an agent with antitumor activity . 16. The method according to claim 1, characterized in that after cultivation, the mycelium of basidiomycete is separated from the submerged culture, filled with distilled water in an amount of 90-360 g / l, autoclaved at 1.2-1.5 atm for 1.5- 2.5 hours, the resulting aqueous extract is separated and used as an agent having antitumor activity. 17. The method according to claim 1, characterized in that after cultivation, the mycelium of basidiomycete is separated from the submerged culture and extraction is carried out with ethanol in an amount of 90-360 g / 100 ml of extractant for 0.5-1.5 hours with stirring, the obtained ethanol extract separated from the remains of the mycelium, evaporated to a dry residue on the evaporator, and the remainder of the mycelium is poured with distilled water in an amount of 90-360 g / l, autoclaved at 1.2-1.5 atm for 1.5-2.5 hours, the resulting aqueous the extract is separated from the residue of the mycelium and combined with a dry residue of ethanol extra one and the resulting mixture was used as an agent having antitumoral activity. 18. The method according to claim 1, characterized in that after cultivation, the mycelium of basidiomycete is separated from the submerged culture, dried at a temperature of 35-80 ° C, ground, and dry mycelium powder is used as an agent with antitumor activity. 19. The method according to p. 18, characterized in that the dry ground mycelium of basidiomycete is subjected to extraction with distilled water in an amount of 18-72 g / l at 1.2-1.5 atm for 1.5-2.5 hours, then water the extract is separated from the remains of the mycelium and the resulting aqueous extract of mycelium is used as an agent with antitumor activity. 20. The method according to claim 19, characterized in that the obtained antitumor agent is frozen at a temperature of -18 ... -70 ° C and is used as an agent having antitumor activity. 21. The method according to claim 19, characterized in that the obtained liquid antitumor agent is dried and used as an agent having antitumor activity. 22. The method according to claim 19, characterized in that 2-6 volumes of ethanol are added to the aqueous extract of mycelium of basidiomycete, the precipitated precipitate is separated and used as an agent with antitumor activity. 23. An agent having antitumor activity, characterized in that it is obtained according to any one of claims 1 to 22.