RU2435848C1 - ENZYME PREPARATION OF THROMBOLYTIC AND FIBRINOLYTIC ACTION MADE OF BASIDIUM FUNGI OF Coprinus - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: preparation of thrombolytic and fibrinolytic action is produced by submerged cultivation on a liquid nutrient medium, of a strain of basidium fungus of Coprinus lagopides TI-12, or Coprinus lagopides TI-32, or Coprinus lagopides TI-33 stored in the high basidium fungi culture collection of the microbiological synthesis technology department of the St.-Petersburg State Technical University, separation of a mycelial biomass from a native solution. The prepared solution is concentrated by ultrafiltration with using a semipermeable membrane with a nominal molecular-mass rejection limit up to 20 kDa followed by cool dehumidification in the mode suitable for protein compounds. The invention has allowed detecting Coprinus lagopides from basidium fungi of Coprinus exhibiting high thrombolytic and fibrinolytic activity. ^ EFFECT: preparation in the low concentration shows a high level of thrombolytic and fibrinolytic activity in vivo. ^ 2 dwg, 4 tbl, 5 ex

Description

The invention relates to the biomedical biotechnology and relates to the use of higher basidiomycetes of the genus Coprinus, namely, the use of the macromycete Coprinus lagopides as a producer of enzymes of thrombolytic and fibrinolytic action, confirmed under experimental thrombosis.

Widespread medical and biological use of higher basidiomycetes with antitumor, immunostimulating, antiviral, antibacterial, antioxidant and other valuable medicinal properties is known, S.P. Wasser, A.L. Weis. Medicinal properties of substances occurring in higher basidiomycetes mushrooms: current perspectives (review) // Int. Journal of Medicinal Mushrooms, 1999, Vol. 1, pp. 31-62; A.Karmali. The role of mushroom nutrition as a delivery agent for enzyme therapy in cancer care? - Chemical and biological properties in mushroom nutrition Mycology News, 2002, Vol. 1 (6), p. 11-14; S-t. Chang. Development of the Culinary-Medicinal Mushrooms Industry in China: Past, Present, and Future, Int. Journal of Medicinal Mushrooms, 2006, Vol. 8, pp. 1-17; S-T Chang. Medicinal Mushrooms as a Good Source of Dietary Supplements for HIV / AIDS Patients, Int. Journal of Medicinal Mushrooms, 2007, Vol. 9, pp. 189-190; B.Štrukelj, J.Kac, D.Janeš, J.Kristl, A.Mlinarič et al. Medicinal Mushrooms and Drug Discovery: Identification of Potent Inhibitors of Thrombin, of Pancreatic Lipase, and Novel Antibiotic and Antigenotoxic Compounds, Int. Journal of Medicinal Mushrooms, 2007, Vol. 9, p. 195; S.M. Badalyan, K.W. Hughes and U. Kües. Biology, genetic variability and biotechnological application of several medicinal mushrooms. International Conference "Biotechnology and Health - 2", Yerevan, Armenia, April 21-25, 2008, p.21-27.

Known medical mushrooms are Ganoderma lucidum (Reishi), Grifola frondosa (Maitake), Lentinula edodes (Shiitake), which are widely used in official and traditional medicine of the countries of the Eastern region in the treatment of various human diseases and are considered in Eastern medicine as a panacea, Terry Willard, Kenneth Jones, Reishi Mushroom, Herb of Spiritual Potency and Medical Wonder, Sylvan Press, 1990, 167 p .; Kenneth Jones, Shiitake, The Healing Mushroom, Healing Arts Press, Rochester, Vermont, 1995, 120 p .; Ch.Hobbs. Medicinal mushrooms. 3-rd ed., 1996, 252 p .; N.P. Denisova. The healing properties of mushrooms, 1998, St. Petersburg State Medical University, St. Petersburg, 59 s .; Alice W. Chen, John Seleen, Potential Benefits of Ling Zhi or Reishi Mushroom Ganoderma lucidum (W. Curt: Fr.) P. Karst. (Aphyllophoromycetideae) to Breast Cancer Patients, Int. Journal of Medicinal Mushrooms, 2007, Vol. 9, p. 29-38; Bojana Boh and Marin Berovic, Grifola frondosa (Dicks .: Fr.) S.F. Gray (Maitake Mushroom): Medicinal Properties, Active Compounds and Biotechnological Cultivation, Int. Journal of Medicinal Mushrooms, 2007, Vol. 9, p. 89-108.

Known higher basidiomycetes are widely used in medicine.

The presence of antithrombin compounds in the species Laetiporus sulphurous, Hypsizygus marmoreus, Inonotus obliquus (chaga), T. Okamura, T. Takeno, M. Dohi et al., Development of mushrooms for thrombosis prevention by protoplast fusion, J. Biosci is known. Bioengineering, 2000, V. 89, No. 5, p. 474-478; K. Akahane, K.Satoh, O. Inoue, et al. Extracts of an edible mushroom with antithrombin III-independent anticoagulant activity. J. Thromb. Haemost. 2007, V. 5, Suppl. 2: Abstracts XXI ISTH Congress, P-W-050.

The appearance of anticoagulant properties in soy sauce is known due to the fermentation of its soybeans by the mycelium of the winter mushroom Flammulina velutipes, T. Okamura-Matsui, H. Izuta, T. Tomoda, et al., Fermented soybean with thrombosis preventing activity using mushroom mycelia as microbial source. Food Sci. Technol. Res., 2003, V.9 (3), p. 227-230.

The presence of fibrinolytic and thrombolytic properties in cultures of various basidiomycetes is known, including the species Flammulina velutipes, N.P. Denisova, N.N. Falina // Mikol. and phytopathol, 1981, v.15, p.123-125; N.P. Denisova, I.A. Alekhine, ibid. 1987, v.21, p. 471-477, NP Denisova, Proteolytic enzymes of basidiomycetes, taxonomic and environmental aspects of their study. Abstract of diss. Doctor of Biological Sciences, Leningrad, 1991, 31 pp .; K. Takao, S. Kumiko. Thrombolytic enzyme activity in Grifola frondosa. Bioactive substances in Mushrooms. Part I. Jin'ai Joshi Tanki Daigaku Kenkyu Kiyo, V.32, 2000, P.65-71; J.H. Kim, Y.S. Kim. Characterization of a metalloenzyme from a wild mushroom Tricholoma saponaceum. Biosci. Biotechnol. Biochem. 2001, V.65, P.356-362.

Known is the possibility of using for medical purposes basidiomycetes of the genus Panus, which reduce platelet aggregation, Lorenzen K., Anke T. et al., 1994 - Two inhibitors of platelet aggregation from a Panus species (Basidiomycetes). // Zeitschrift fur Naturforschung 49, pp. 132-138.

The thrombolytic activity of fungal proteinases from cultures of various wood-destroying fungi is known, Nina P. Denisova, Vladimir N. Mikhailov, and Nickolay N. Petrishchev, Thrombolytic Activity of Proteineses from Coprinus domesticus, C.cinereus, and Cerrena unicolor. // Int. Journal of Medicinal Mushrooms, 1999, Vol. 1, No. 2, pp. 187-190.

Known higher basidiomycetes have fibrinolytic and thrombolytic properties, however, the species Coprinus lagopides was not found among them.

A known method of producing a milk-clotting enzyme by cultivating agaric macromycete Coprinus lagopides on a liquid nutrient medium containing carbon sources, nitrogen and mineral salts, RU No. 2354698 C2, C12N 9/58, 05/10/2009.

Known agaric macromycete Coprinus lagopides is used as a producer of milk-clotting enzyme, the technology for producing this enzyme is limited in purpose and does not provide for the possibility of using it in any other quality.

Known strain of the basidiomycete fungus Coprinus domesticus (Fr.) Gray BKM F 2459 D - producer of fibrinolytic and thrombolytic enzymes, SU No. 1137762 A1, C12N 15/00, C12N 9/50, 07.08.1991.

An enzyme preparation with thrombolytic and fibrinolytic activity is obtained from the well-known fungus Coprinus domesticus by deep cultivation of the producer.

To isolate a known enzyme preparation, depigmentation and desalination of the native solution is carried out by ion-exchange chromatography on FAF resin using two-stage fractional ethanol precipitation at -20 ° C in a ratio of 1: 1, followed by centrifugation, centrifugation precipitation at an ethanol ratio of 1: 3, followed by centrifugation, dissolution of the precipitate, freeze drying.

In addition, the chemical composition of the mineral background of the nutrient medium includes 8 mineral salts and malt-sprout extract, which requires laborious preparation, is used in the composition.

An insufficiently effective producer cultivation technology and a low-cost method for producing a known enzyme preparation (a complex isolation scheme and nutrient composition) do not provide a finished product with a high level of thrombolytic activity, which in vivo leads to the need to use an enzyme preparation in sufficiently high concentrations.

The thrombolytic effect of the known enzyme preparation from the agaric maromycete Coprinus domesticus is manifested at a concentration of 3.0 mg / kg.

Of the known basidiomycetes of the genus Coprinus, the use of the macromycete Coprinus lagopides as a producer of enzymes of thrombolytic and fibrinolytic action was not found.

The present invention is based on the solution of the problem, allowing to identify from basidiomycetes of the genus Coprinus a type of fungus that produces enzymes of thrombolytic and fibrinolytic action.

EFFECT: identified species of the fungus Coprinus lagopides, which has high thrombolytic and fibrinolytic activity and the manifestation in vivo of a high level of thrombolytic and fibrinolytic action at different concentrations of the enzyme preparation, including at low levels, characterized by technological methods for its preparation.

According to the invention, this problem is solved due to the fact that the enzyme preparation of thrombolytic and fibrinolytic action from a basidiomycetes of the genus Coprinus is characterized by the fact that it is obtained by deep cultivation of macromycetes Coprinus lagopides TI-12 or Coprinus lagopides TI-32, or Coprinus lagopides TI-33 on liquid nutrient medium, separation of mycelial biomass from the native solution and concentration by ultrafiltration followed by drying of the ultrafiltrate.

Applicants have not identified sources containing information on technical solutions identical to the present invention, which allows us to conclude that it meets the criterion of "novelty."

Due to the implementation of the distinguishing features of the invention (in conjunction with the features indicated in the restrictive part of the formula), important new properties of the object are achieved.

Macromycete Coprinus lagopides was detected from a basidiomycetes of the genus Coprinus.

Macromycete Coprinus lagopides TI-12 or Coprinus lagopides TI-32, or Coprinus lagopides TI-33 - an enzyme producer with thrombolytic and fibrinolytic activity is characterized by technological methods for its preparation.

Applicants are not aware of any publications that would contain information on the influence of the distinguishing features of the invention on the achieved technical result. In this regard, according to the applicants, it can be concluded that the claimed technical solution meets the criterion of "inventive step".

An enzyme preparation of thrombolytic and fibrinolytic action was obtained by deep cultivation of Macrinomycete Coprinus lagopides in a liquid nutrient medium.

Agrinic macromycete Coprinus lagopides is a xylotrophic basidiomycete, growing on rich lignin soil, buried or charred wood, fine sawdust, etc. This species has its ecological peculiarity - unlike many other species of the genus Coprinus, it does not occur under natural conditions on dung-type substrates. Young fruiting bodies are edible.

The culture of the fungus Coprinus lagopides was isolated from the fruiting bodies of the species Coprinus lagopides collected in the northeastern suburban forest zone of St. Petersburg. Fruit bodies (basidiomas) are collected and induced into a pure culture at the Department of Microbiological Synthesis Technology of St. Petersburg State Technical University.

To obtain the enzyme preparation, strains of agaric macromycete Coprinus lagopides TI-12, Coprinus lagopides TI-32 and Coprinus lagopides TI-33 from the culture collection of higher basidiomycetes of the Department of Microbiological Synthesis Technology of St. Petersburg State Technical University are used.

In the collection, the indicated strains of macromycete Coprinus lagopides TI-12, TI-32 and TI-33 are stored in glass tubes on jambs of must-agar (sugar content 4 ° Balling, 20% agar) in a refrigerator at a temperature of 4-6 ° C ± 2 ° C.

The system of the vegetative mycelium of Macromycete Coprinus lagopides is monomitic: hyphae of two types are undifferentiated generative 3-8 μm in diameter, long-celled, with regular small buckles, hyaline and sack-shaped hyphae differentiated from them, and identical to those of the cuticle surface of fruiting bodies.

When storing Macrinomycete Coprinus lagopides in a laboratory, small fruiting bodies may appear in test tubes. Morphological characters of the resulting basidiomas confirm their belonging to the species Coprinus lagopides P. Karst. (Watling R. and Gregory N.M., 1987. In: British Fungus Flora. Agarics and Boleti: Part 5. Royal Botanic Garden, Edinburgh, 121 pp), which serves as a complete guarantee of classifying the crops of the claimed producer to the indicated type of agaric macromycete.

In the deep conditions of the biotechnological process, Coprinus lagopides macromycetes develop, like most basidi um macromycetes cultures, in the form of fluffy pellets (balls), the average diameter of which is 5-7 mm. The color change of the culture fluid in the fermentation process does not occur.

The deep cultivation of agaric macromycete Coprinus lagopides is as follows.

The cultivation is carried out on a liquid semi-synthetic glucose peptone medium.

As a nutrient medium, a natural medium containing beer wort (4 ° Balling) and soy flour (1%), pH 5.8-6.0 can also be used.

As a nutrient medium can be used and other known nutrient medium.

As a carbon source, glucose, beer wort, maltose and other di- and oligosaccharides can be used.

The use of peptone and potassium nitrate is preferred as a source of nitrogen; ammonium salts and soy flour are also possible.

As mineral salts, KH ₂ PO ₄ , ZnSO ₄ , FeSO ₄ , MgSO ₄ * 7H ₂ O, CaCl ₂ , NaCl are used.

The mycelial biomass is separated from the native solution by vacuum filtration.

In the native solution, fibrinolytic activity is determined.

The determination of fibrinolytic activity is carried out by the method of quantifying the content of plasminogen activator using the ChromoTech-Plasminogen reagent kit.

Concentrate the native solution.

Purification of the concentrated native solution is carried out by ultrafiltration.

For ultrafiltration, a MIFIL PA-20 brand semipermeable membrane with a nominal molecular weight retention limit of up to 20 kDa (molecular weight of globular proteins retained by the membrane) is used.

The ultrafiltrate is freeze-dried in the mode intended for protein compounds.

The amount of protein in dry ultrafiltrate is determined by the standard Lowry method.

The fibrinolytic activity of the dry enzyme preparation is determined.

As producers of enzyme preparations, strains of agaric macromycete Coprinus lagopides TI-12, Coprinus lagopides TI-32 and Coprinus lagopides TI-33 from the culture collection of higher basidiomycetes of the Department of Microbiological Synthesis Technology of St. Petersburg State Technical University are used.

Example 1

Obtaining an enzyme preparation by cultivation of agrinic macromycete Coprinus lagopides TI-12 in a liquid nutrient medium.

The enzyme preparation is obtained from the culture fluid (native solution) of agaric macromycete Coprinus lagopides TI-12, grown under deep cultivation.

Deep cultivation is carried out on a rotary shaker at 28-30 ° C in Erlenmeyer flasks (0.75 L) containing 250 ml of culture medium.

Use a semi-synthetic medium of the following composition (g / l): peptone - 2.5; glucose - 10; KH ₂ PO ₄ - 1.0; MgSO ₄ * 7H ₂ O - 0.5; FeSO ₄ - 0.005; ZnSO ₄ - 0.001; CaCl ₂ - 0.05; NaCl - 0.5; dry yeast extract - 2.0; pH of the medium before sterilization 5.8-6.0.

Cultivation is carried out for 4-9 days.

The dependence of the fibrinolytic activity of the native solution on the duration of cultivation of agaric macromycete Coprinus lagopides TI-12 is presented in table 1.

From table 1 it is seen that the highest values of the fibrinolytic activity of the native solution are observed on the 7th day of cultivation.

The mycelial biomass (after 7 days of cultivation) is separated from the native solution by vacuum filtration.

In the native solution, fibrinolytic activity is determined.

The concentration of the native solution is 2 times and the concentrate is purified by ultrafiltration.

Ultrafiltration is carried out on a semi-permeable membrane of the MIFIL PA-20 brand with a nominal molecular weight retention limit of 20 kDa.

The concentrated ultrafiltrate is freeze-dried.

Freeze drying is carried out on a Hita installation (USA), the freezing temperature of the material was -40 ° C, and the condenser temperature was -35 ° C.

An enzyme preparation is obtained in the form of a dry powder, and its fibrinolytic and thrombolytic activity is tested.

Example 2

Obtaining an enzyme preparation by cultivation of agrinic macromycete Coprinus lagopides TI-32 on a liquid nutrient medium.

The enzyme preparation is obtained from the culture fluid (native solution) of the agaric macromycete Coprinus lagopides TI-32 grown under deep cultivation.

Deep cultivation is carried out according to example 1.

The dependence of the fibrinolytic activity of the native solution on the duration of cultivation of agaric macromycete Coprinus lagopides TI-32 is presented in table 2.

From table 2 it is seen that the highest values of the fibrinolytic activity of the native solution are observed on the 7th day of cultivation.

The mycelial biomass (after 7 days of cultivation) is separated from the native solution by vacuum filtration.

In the native solution, fibrinolytic activity is determined.

The concentration of the native solution and the purification of the concentrate is carried out according to example 1.

An enzyme preparation is obtained in the form of a dry powder, and its fibrinolytic and thrombolytic activity is tested.

Example 3

Obtaining an enzyme preparation by culturing the agrinic macromycete Coprinus lagopides TI-33 on a liquid nutrient medium.

The enzyme preparation is obtained from the culture fluid (native solution) of the agrinic macromycete Coprinus lagopides TI-33 grown under deep cultivation.

Deep cultivation is carried out according to examples 1,2.

The dependence of the fibrinolytic activity of the native solution on the duration of cultivation of agaric macromycete Coprinus lagopides TI-33 is presented in table 3.

From table 3 it is seen that the highest values of the fibrinolytic activity of the native solution are observed on the 7th day of cultivation.

The mycelial biomass (after 7 days of cultivation) is separated from the native solution by vacuum filtration.

In the native solution, fibrinolytic activity is determined.

Concentration of the native solution and purification of the concentrate is carried out according to examples 1, 2.

An enzyme preparation is obtained in the form of a dry powder, and its fibrinolytic and thrombolytic activity is tested.

Examples 1, 2, 3 showed that the maximum values of fibrinolytic activity in native solutions of agaric macromycetes Coprinus lagopides TI-12, TI-32, TI-33 are achieved by all strains on the 7th day of cultivation on glucose-peptone medium.

Obtaining an enzyme preparation by culturing the agrinic macromycete Coprinus lagopides, according to examples 1, 2, 3, on a liquid nutrient medium in comparison with the closest analogue provides:

- modification of the composition of the nutrient medium by reducing the species composition of the mineral background from 8 to 6 mineral salts and replacing the malt-sprout extract, which requires a labor-intensive preparation, with commercial yeast extract;

- simplification and improvement of the scheme for isolating the enzyme preparation by replacing the multi-stage scheme (depigmentation and desalting of the native solution on a column with FAF anion exchange resin, two-stage fractional precipitation with ethanol at -20 ° C in a ratio of 1: 1, centrifugation, precipitation of a centrifugate with an ethanol ratio of 1: 3, centrifugation, dissolution of the precipitate, freeze drying) to the modern scheme of ultrafiltration of the native solution through a semi-permeable membrane of the brand "MIFIL PA-20", allowing to remove low molecular weight components and concentrate the solution, followed by freeze-drying.

All of these strains of agrinic macromycete Coprinus lagopides were cultured on various nutrient media at various degrees of concentration of native solutions with the aim of further purification by ultrafiltration on a MIFIL PA-20 membrane. As a result, the strain Coprinus lagopides TI-32, which has the highest level of fibrinolytic activity in the original native solution, was selected as the producer of the fibrinolytic enzyme for further testing the activity of the drug in vivo.

The thrombolytic activity of the obtained enzyme preparations was tested in vivo using models of venous and arterial thrombosis in experimental animals (rats).

A 24-hour occlusion of the posterior vena cava was used as a model of venous thrombosis.

A photothrombosis model was used as a model of arterial thrombosis (30-minute irradiation with a diode laser at a wavelength of 532 nm and a power of 60 mW with the introduction of a photosensitizer - Bengal pink A at the rate of 1.7 mg per 100 g of weight).

In the case of venous thrombosis, the thrombolytic effect of enzyme preparations was evaluated by the wet and dry mass of blood clots formed in the vena cava of animals.

In the case of arterial thrombosis, the thrombolytic effect was evaluated by the dynamics of the linear blood flow velocity (by high-frequency ultrasonic Doppler ultrasonography on a Minimax-Doppler-K device).

The studies were performed on experimental animals (male Wistar rats, 18 weeks old, animal weight 270-300 g) from the laboratory animal nursery of the Institute of Physiology named after I.P. Pavlova RAS, Koltushi, St. Petersburg.

The experimental animals were kept on unlimited feed and water consumption (standard diet for laboratory rats K-120, Inform-feed, Russia).

Experimental animals were divided into control and experimental groups. The control group included 6 animals, the experimental group - 9 animals.

Example 4

Testing fibrinolytic activity in experimental venous thrombosis.

The technique of thrombosis of the inferior vena cava.

Experimental animals under anesthesia (sodium thiopental 50 mg / kg, intraperitoneally) perform median laparotomy (5 cm). A section of the inferior vena cava, 3 cm long in the caudal direction from the place where the left renal vein flows, is released from the connective tissue, a ligature is placed under the inferior vena cava, and it is tied, completely covering the lumen of the vessel [Henke PK et al, 2007]. The wound is sutured.

Immediately before the operation, the animals of the experimental group (t = 9) were pre-weighed and a single intravenous enzyme preparation of agaric macromycete Coprinus lagopides TI-32 at a dose of 12.5 mg / kg was administered. The animals in the control group (n = 6) are injected with an appropriate amount of sterile 0.9% NaCl solution.

In a series of specially conducted experiments, it was confirmed that the formation of a thrombus takes place already 1 hour after vein ligation.

The observation period was one day.

After a day after the administration of the enzyme preparation, the degree of thrombolytic effect of the enzyme preparation from the agrinic macromycete Coprinus lagopides TI-32 by the weight of blood clots in the control and experimental animals is evaluated.

All animals survived.

24 hours after ligation of the vessel, the animals were removed from the experiment (sodium thiopental 150 mg / kg, intraperitoneally), after which the animals' abdominal cavity was opened, a longitudinal section of the wall of the aforementioned section of the inferior vena cava was made, and the thrombus formed.

Weighing is carried out on an analytical balance VLR-200.

The mass of the wet thrombus is determined immediately after removal.

The mass of a dry thrombus is after 7 days of drying at 37 ° C.

The results of the study:

24 hours after surgery, free blood clots were found below the ligation site in all animals of the control group (n = 6). In rats of the experimental batch (n = 9), which received an injection of the enzyme preparation from agaric macromycete Coprinus lagopides TI-32 before vein ligation, thrombi were found only in 6 animals, thrombi were completely absent in 3 animals.

In animals of the control group (n = 6), the average weight of the wet thrombus was 30.4 mg, and the total weight of the thrombotic mass in animals of the control group was 182.4 mg.

In animals of the experimental group having thrombi (n = 6), the average weight of the wet thrombus was 10.7 mg, and the total weight of the thrombotic mass in animals of the experimental group was 64.2 mg.

The research data presented in example 4 indicate that the enzyme preparation from agrinic macromycete Coprinus lagopides TI-32 has both a pronounced thrombolytic effect and a protective effect (thrombosis is reduced by 76% compared with the control).

Example 5

Testing fibrinolytic activity in experimental arterial thrombosis.

Blood flow dynamics in experimental animals is determined by high-frequency ultrasound Doppler ultrasound.

The experimental stages preceding the direct testing of fibrinolytic activity in arterial thrombosis include:

The introduction of a photosensitizer, for which

- produce under anesthesia (sodium thiopental 50 mg / kg, intraperitoneally) an incision of about 2 cm long on the inner surface of the left thigh of the animal;

- release a portion of the femoral vein from connective tissue;

- a Bengal pink A photosensitizer is punctured and slowly injected at the rate of 1.7 mg per 100 g of weight in 1 ml of a 0.9% sodium chloride solution (Boselli C. et al, 2006);

- impose on the puncture site a piece of hemostatic sponge for hemostasis;

- the wound is sutured.

Laser irradiation, for what

- make an incision about 2 cm long on the inner surface of the right thigh of the animal immediately after the introduction of the photosensitizer;

- release a portion of the femoral artery about 4 mm long from the connective tissue and secrete from the neurovascular bundle;

- bring a strip of opaque black plastic 3 mm wide under this section of the artery, thereby isolating the vein and surrounding tissue from radiation;

- irradiation is carried out using a diode laser "DPSS-laser" (Diode Pumpd Solid State Laser) (wavelength 532 nm, power 60 mW) for 30 minutes;

- remove the plastic after the termination of exposure;

- sutured the wound.

Blood flow velocity control in the irradiated vessel:

Blood flow in the femoral artery of animals is examined using high-frequency ultrasound Doppler ultrasound (Minimax-Doppler-K device, sensor frequency 20 MHz).

Linear blood flow velocities are evaluated immediately before irradiation, every 5 minutes after irradiation and 24 hours after the irradiation is stopped.

The results of testing fibrinolytic activity in arterial thrombosis:

In all experimental animals (n = 17), 15-20 minutes after the start of irradiation a. femoralis against the background of Bengal pink A, there was a decrease and subsequent absence of blood flow, which indicated thrombosis of the vessel, as shown in the graph (figure 1).

24 hours after the formation of a blood clot, blood flow did not recover.

This model of photochemical thrombosis was used to evaluate the fibrinolytic activity of enzyme preparations obtained from the agrinic macromycete Coprinus lagopides TI-32.

To prepare a solution for infusion, 10 mg of the studied enzyme preparation from agaric macromycete Coprinus lagopides TI-32 was dissolved in 10 ml of a sterile 0.9% NaCl solution.

2 hours after the termination of irradiation under anesthesia (animals, as a rule, do not have time to get out of anesthesia, and if necessary additionally inject sodium thiopental up to 20 mg / kg intraperitoneally):

- make an incision about 2 cm long on the front surface of the neck of the animal;

- release a section of the jugular vein from connective tissue;

- the test drug is punctured and slowly administered at a dose of 12.5 mg / kg of the animal’s weight, in the control group, the corresponding amount of sterile 0.9% physiological saline (NaCl) (the use of this dose for initial testing of the activity of the enzyme preparation in vivo was due to the orientation to the therapeutic daily the dose of streptokinase used in medical practice for human thrombosis);

- impose on the puncture site a piece of hemostatic sponge for hemostasis;

- the wound is sutured.

The observation period was one day.

24 hours after ligation, the vessels of the animals were anesthetized (sodium thiopental 50 mg / kg, intraperitoneally), after which the surgical wound on the right thigh was opened (sutures were removed) and a Doppler blood flow test was performed in the same area as on the day of irradiation.

In all experimental animals (n = 17) 15-20 minutes after irradiation a. femoralis against the background of the introduction of Bengal pink A a blood clot formed and blood flow was not registered. In control rats (n = 12), which were injected with 0.9% NaCl solution, blood flow was not restored 24 hours after the formation of a blood clot.

In the experimental group of animals that were injected with the enzyme preparation (n = 5), blood flow was restored in all animals, as shown in the drawing (figure 2).

Additionally, the following observation was carried out.

The animals of the control group after re-suturing the wounds were divided into 2 groups. The first group (n = 6) was left for further observation. The rats of the second group (n = 6) were injected intravenously, using the method described above, the studied enzyme preparation from agrinic macromycete Coprinus lagopides TI-32. After 24 hours (i.e., 48 hours after the end of irradiation), a second Doppler study of blood flow velocity was performed in the same area as on the day of irradiation. In control animals of the first group (n = 6), blood flow was not restored, and in animals that received an injection of the enzyme preparation from agrinic macromycete Coprinus lagopides TI-32 (n = 6), restoration of the initial blood flow was observed. These data also confirm that the studied enzyme preparation from agrinic macromycete Coprinus lagopides TI-32 has a pronounced thrombolytic activity.

In addition, in addition to the model of arterial thrombosis, fibrinolytic activity of the studied enzyme preparation was tested at various concentrations.

The thrombolytic effect of various doses of the enzyme preparation from Coprinus lagopides TI-32 is presented in table 4.

The results of table 4 show that in all animals of the experimental group, almost complete restoration of blood flow is observed, which indicates the preservation of the thrombolytic activity of the enzyme preparation from the agrinic macromycete Coprinus lagopides TI-32 even when its dose is reduced to a minimum amount of 0.5 mg / kg.

The possibilities of using an enzyme preparation from agrinic macromycete Coprinus lagopides TI-32 are confirmed on two different models of experimental thrombosis and at different periods of its development.

It was found that intravenous administration of the proposed enzyme preparation from agrinic macromycete Coprinus lagopides TI-32 can be an effective and safe way to treat arterial and venous thrombosis.

The thrombolytic and fibrinolytic activity of the enzyme preparation of the agrinic macromycete Coprinus lagopides TI-32, according to examples 4, 5, was compared with the closest analogue on modern models of experimental venous and arterial thrombosis and at different stages of its development, the thrombolytic effect on these models was preserved at the dose of the drug 0.5 mg / kg (unlike the closest analogue 3.0 mg / kg).

During the experiments, thrombolytic enzyme preparations from the agrinic macromycete Coprinus lagopides were administered to more than 100 experimental animals in doses from 12.5 mg / kg to 0.5 mg / kg. Moreover, no changes in the general condition were observed in any of the animals, either during the experiment or a month later.

The enzyme preparation from coprinus lagopides agaric macromycete has a prophylactic effect, significantly reducing thrombosis at the sites of damage to the vascular wall, and may be promising for exploring the possibility of its use for late-stage systemic thrombolytic therapy.

The proposed enzyme preparation was revealed during the cultivation of a well-known agaric macromycete from the genus Coprinus from the culture collection of higher basidiomycetes of the Department of Microbiological Synthesis Technology of St. Petersburg State Technical University, and its thrombolytic and fibrinolytic activity was confirmed in an experiment conducted at St. Petersburg State Medical University. Acad. IP Pavlova, which determines, according to the applicants, the compliance with his criterion of "industrial applicability".

The proposed enzyme preparation is characterized by:

- high fibrinolytic and thrombolytic activity when used as a producer of agaric macromycete Coprinus lagopides TI-12, Coprinus lagopides TI-32, Coprinus lagopides TI-33;

- manifestation of a thrombolytic effect at low concentrations of the enzyme preparation (0.5 mg / kg);

- testing on modern models of experimental venous and arterial thrombosis;

- selection by simplified technology when modifying the composition of the nutrient medium by reducing the chemical composition of the mineral background;

- separation by simplified technology due to ultrafiltration through a semi-permeable membrane, which allows you to simultaneously remove low molecular weight ballast components and concentrate the solution, followed by freeze drying.

Enzyme preparation of thrombolytic and fibrinolytic action from a basidiomycete of the genus Coprinus

The dependence of the fibrinolytic activity of the native solution on the duration of cultivation of agaric macromycete Coprinus lagopides TI-12

Example 1

Table 1 The cultivation time, day The protein content, mg / ml Activity, units / ml Specific activity, units / mg protein four 0.3 ± 0.02 23.0 ± 1.8 69.7 ± 4.0 5 0.4 ± 0.02 31.0 ± 1.7 73.8 ± 4.0 6 0.5 ± 0.02 43.2 ± 2.0 91.9 ± 4.3 7 0.5 ± 0.03 53.5 ± 2.9 109.2 ± 5.7 8 0.5 ± 0.03 35.3 ± 1.9 69.2 ± 3.2 9 0.4 ± 0.02 20.7 ± 2.2 46.0 ± 4.5

Enzyme preparation of thrombolytic and fibrinolytic action from a basidiomycete of the genus Coprinus

The dependence of the fibrinolytic activity of the native solution on the duration of the cultivation of agaric macromycete Coprinus lagopides TI-32

Example 2

table 2 The cultivation time, day The protein content, mg / ml Activity, units / ml Specific activity, units / mg protein four 0.30 ± 0.02 25.0 ± 1.8 73.5 ± 4.1 5 0.42 ± 0.02 34.0 ± 1.7 81.0 ± 4.0 6 0.48 ± 0.02 42.0 ± 2.0 86.7 ± 4.3 7 0.52 ± 0.03 58.0 ± 2.9 115.6 ± 5.7 8 0.50 ± 0.03 33.6 ± 1.7 67.2 ± 3.3 9 0.43 ± 0.02 21.0 ± 2.0 48.8 ± 4.3

Enzyme preparation of thrombolytic and fibrinolytic action from a basidiomycete of the genus Coprinus

The dependence of the fibrinolytic activity of the native solution on the duration of the cultivation of agaric macromycete Coprinus lagopides TI-33

Example 3

Table 3 The cultivation time, day The protein content, mg / ml Activity, units / ml Specific activity, units / mg four 0.31 ± 0.02 27.0 ± 1.7 87.1 ± 4.2 5 0.40 ± 0.03 32.0 ± 1.7 80.0 ± 4.5 6 0.45 ± 0.02 41.3 ± 2.0 91.8 ± 4.3 7 0.53 ± 0.02 55.0 ± 2.9 103.8 ± 4.9 8 0.48 ± 0.03 38.6 ± 1.7 80.4 ± 3.3 9 0.42 ± 0.02 24.0 ± 2.0 57.1 ± 4.3

Enzyme preparation of thrombolytic and fibrinolytic action from a basidiomycete of the genus Coprinus

Thrombolytic effect of different doses of the enzyme preparation from Coprinus lagopides TI-32

Table 4 Preparations The concentration of the drug for infusion, mg / ml The studied dose of the drug, mg / kg Blood flow Background, ml / min, mm ³ 15 min after irradiation 20 min after irradiation 24 hours after drug administration ml / min, mm ³ % ml / min, mm ³ ml / min, mm ³ % Control, 0.9% NaCl - 8.2 ± 0.8 2.1 ± 0.7 27 0 0 0 Experience. Coprinus lagopides 1.0 5.0 5.2 ± 1.2 2.1 ± 0.9 47 0 5.3 ± 0.8 102 0.5 2.5 8.8 ± 1.4 2.8 ± 0.8 34 0 8.1 ± 1.0 93 0.1 0.5 4.7 ± 0.9 1.3 ± 0.6 22 0 4.7 ± 0.8 102

Claims (1)

An enzyme preparation of thrombolytic and fibrinolytic action from a basidiomycete of the genus Coprinus, characterized in that it is obtained by deep cultivation of macromycete Coprinus lagopides TI-12, or Coprinus lagopides TI-32, or Coprinus lagopides TI-33 on a liquid nutrient medium, separating mycelial biomass native solution, concentration of the native solution 2 times, its purification by ultrafiltration using a semi-permeable membrane with a nominal molecular weight retention limit of up to 20 kDa and lyophilic drying in the mode intended for protein compounds.

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