RU2560845C1 - Method for producing low-molecular activated embryonic complex (nika-em) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves selecting fresh non-infected chicken eggs, incubating them, exposing to laser light generated by a laser apparatus and to ultraviolet light generated by a lamp, cooling the eggs and placing them into a deep freeze store and defrosting; the embryonic and extraembryonic tissues are uncracked and homogenised; the embryo-egg mass is diluted with sodium chloride and added with formalin solution, homogenised, centrifuged; the supernatant is separated, bottled into sterile flasks, sealed and placed into a thermostat; the produced preparation is kept in a fridge in the certain environment.

EFFECT: method enables producing the more stable and effective immunotropic agent.

1 tbl, 3 ex

Description

FIELD OF THE INVENTION

The invention relates to biotechnology and veterinary medicine, in particular to a method for producing a biologically active preparation of immunotropic action. The development of effective immunomodulatory agents for veterinary medicine is of high relevance due to the wide spread in animals of immunodeficiency diseases of various etiologies. At the same time, immune disorders can act both as primary and aggravated in the dynamics of the pathological process in various infectious and non-infectious diseases, which sharply reduce the profitability of animal husbandry. The situation with increased immune status is aggravated by the uncontrolled use of vaccines, antibiotic, hormonal and various synthetic pharmacological agents, which, although aimed at increasing the number of livestock products, has significantly worsened its quality due to metabolic and morphological homeostasis in animals that are directly controlled by immune processes . These violations of immunity, as a rule, are of a combined complex nature, in which it is difficult to distinguish the pathogenetically leading one, since several links of immunity are involved at once. Thus, the immune imbalance, which serves as the basis for the formation of numerous pathological processes, accompanied by profound disorders of the system level, is also the result of systemic disorders in the body.

The developed low molecular weight activated embryonic complex (NIKA-EM) includes a wide range of biologically active substances that act on all parts of the immune system and can be used by agricultural and domestic animals to increase the level of specific and nonspecific resistance of the body both as part of complex therapy and separately.

State of the art

A known method of obtaining a biologically active tissue preparation, including washing the placental membrane, coarse grinding, fine grinding to obtain particles with a size of 0.01-1.0 mm and hemolysis of the placental membrane, which is carried out for 10-18 hours in purified water with a temperature of 8- 10 ° C, with a water flow rate of not more than 3 l per 1 kg of tissues, their treatment with an oxidizing solution is carried out with a 3% hydrogen peroxide solution, acting on the suspension for 50-60 minutes with constant stirring, is subjected to fine grinding twice for 4.5 -5 mi n and filter it, obtaining the finished product (see patent for the invention of the Russian Federation 2148408, class A61K 35/00).

The disadvantage of this method of preparation of the drug is the complexity of the selection of raw materials, the complexity of the technology, low yield and high cost.

A known method of obtaining a biologically active substance from chicken blood to activate the proliferation of undifferentiated human bone marrow cells, including electrical stimulation of chicken heads in the mode of 100-120 V, a current of 3-5 A for 3-5 seconds, sampling and incubation of blood. Then the serum is separated. A polypeptide fraction with a molecular weight of 150-170 kDa is isolated from it. The resulting peptide fraction is lyophilized and sterilized by irradiation in a linear electron accelerator with a dose of 28-32 kGy (see patent for the invention of the Russian Federation 2508297, class A61K 35/14).

The disadvantage of this method of preparation of the drug is the complexity of the technology, low yield and high cost.

A known method of obtaining funds for non-specific immunotherapy. The invention relates to the field of pharmacology and medicine. A non-specific immunotherapy agent is claimed comprising an embryonic antitumor modulator (EPOM) as an active agent and a pharmaceutically acceptable carrier. EPOM is obtained from embryonic substances by cryodestruction, extraction and precipitation in alcohols of increasing concentration and contains a complex of hyaluronic acid, tumor-associated antigens of normal embryogenesis. The tool can be made in the form of a solution, liniment, cream, toothpaste (see patent for the invention of the Russian Federation 2341272, class A61K 35/54).

The disadvantage of this method of preparation of the drug is the complexity of the technology, low yield and high cost.

A known method of obtaining biologically active raw materials from reindeer embryos. The method consists in the fact that the collection of raw materials is carried out until the embryos are covered with wool, frozen at a temperature below minus 20 ° C, then canning is carried out at + 35 ° C and a vacuum of 0.096-0.098 MPa for 12 hours, then it is actively cooled and ventilated in air flow at + 20 ° C with an air speed of 7-12 m / s. When the raw material reaches 13-17% moisture, it is crushed and packaged in vacuum packaging. The powder is treated with ultraviolet rays and / or an electromagnetic field and / or autoclaving. From the finished powder by phased extraction, an aqueous and aqueous-alcoholic extract is obtained (see patent for the invention of the Russian Federation 2300386, class A61K 35/54).

The disadvantage of this method of preparation of the drug is the complexity of the selection of raw materials, the complexity of the technology, low yield and high cost.

A known method of obtaining funds that normalize the metabolism in the tissues of the skin. The method consists in the fact that to obtain an nRNA fraction, 100 g of a mixture of embryonic tissues are finely ground in 500 ml of 50 mm sodium citrate pH 7.5 and then homogenized in a knife homogenizer for 2 minutes at 4000 rpm to open cells and nuclei. An equal volume of a phenol / chloroform mixture (1: 1) is then added to the homogenate and incubated in a water bath at 40 ° C. for 30 minutes, stirring regularly. At this stage, the separation of cellular proteins and DNA from RNA and the transition of the latter into the aqueous phase are achieved. After cooling for 15 minutes at 4 ° C, the resulting mixture was centrifuged for 20 minutes at 3000 rpm at room temperature. The aqueous phase was collected and 2.5 volumes of chilled 96% ethanol were added to it and kept at minus 20 ° C for at least 24 hours to form an RNA precipitate. To increase the yield of the drug, the intermediate layer separated from the phenol / chloroform phase is mixed and centrifuged again under the same conditions. The supernatant is collected and processed as described above. To enrich the preparation with low molecular weight RNA, 50 ml of 50 mM sodium citrate pH 7.5, an equal volume of phenol / chloroform are added to the remaining interphase and the mixture is incubated at 70 ° C for 30 min, stirring regularly. After incubation, the mixture is cooled for 30 minutes and then centrifuged at room temperature for 20 minutes at 3000 rpm. Supernatant is administered as described above. Alcohol mixtures of supernatants containing various temperature fractions of mRNA are mixed. MRNA is precipitated from alcohol by centrifugation (20 min, 3000 rpm, room temperature), dried in air and dissolved in distilled water. To liberate phenol residues, re-precipitation from alcohol is carried out. The preparation thus obtained contains low molecular weight RNAs associated with proteins from nuclei and cytoplasm of cells (see RF Patent 2229886, class A61K 38/00).

The disadvantage of this method of preparation of the drug is the complexity of the technology, low yield and high cost.

There is a method of producing a tissue preparation for normalizing the course of lambing and tearing, growth rate, prevention and treatment of acute and chronic diseases of animals of non-infectious etiology. A tissue preparation is prepared using the ovaries of cows without corpus luteum, thyroid and parathyroid glands, thymus, pancreas, cattle liver up to 4 years. The organs are kept separately for 9-10 days in the refrigerator, separately chopped 3 times with a meat grinder, diluted with fresh tap water in a ratio of 1: 2, extracted for 4 hours in a glass dish at room temperature, thoroughly mixed every 10-15 minutes, treated for one hour in water bath at 58-59 ° C, separately filtered through 2 layers of sterile gauze, the filtrate 4 times with a daily interval is kept in a water bath at 58-59 ° C for an hour. The components are mixed so that their ratio in the preparation is, weight. %: ovarian tissue emulsion - 10.3, thyroid and parathyroid gland emulsion - 10.3, thymus gland - 10.3, pancreas - 17.2, liver - 20.6, ASD f-2 - 10.3, formalin - 0.4, natural bee honey - 20.6. Before injection, the drug is mixed and 1/2 dose is injected into the dorsal muscles of the lower back at the rate of 0.02-0.04 ml / kg body weight of the animal (see patent for the invention of the Russian Federation 2259195, class A61K 31/115).

The disadvantage of this method of preparation of the drug is the complexity of the selection of raw materials, the complexity of the technology, low yield and high cost.

A known method of producing nucleoprotein complexes from bird embryos with immunotropic and radioprotective activity. 100 eggs with bird embryos (chickens, quail, etc.) obtained from the incubator are wiped with alcohol, the embryos are removed and washed three times with 250 ml of 0.15 M NaCl. An equal volume of 0.15 M NaCl (usually 60-65 ml) is added to the washed embryos and homogenized in a tissue grinder for 1 min at a knife speed of 8000 rpm. The homogenate is centrifuged in the cold at 4000 g for 15-20 minutes. A 120-130 ml supernatant is passed through polyamide hollow fibers with an upper selectivity of 50 kDa. To do this, use the installation UPL-0.1 manufactured in Kirishi or similar. Pressure and flow rate are set according to the installation data sheet. Ultrafiltrate in a volume of 100-110 ml is collected and passed through hollow polyamide fibers with an upper selectivity of 5 kDa. The concentrate, which is the target fraction in a volume of 25 ml, is centrifuged to separate the denatured proteins and freeze-dried. To obtain a dosage form, the drug is subjected to sterilizing filtration, filling according to the dose and drying by standard methods provided for drugs. From 100 embryos, 350 doses of an immunostimulating radioprotective and antitumor drug are obtained (see RF patent 2110267, class A61K 35/00).

The disadvantage of this method of preparation of the drug is the complexity of the technology, low yield and high cost.

A known method of producing extracts of chicken embryos. Chicken eggs are taken after 12 days of incubation. The entire contents of the eggs are homogenized, freeze-dried, pulverized and stored at 20 ° C until use. This egg powder was first dissolved in water (5% w / v) and then subjected to sequential in vitro cleavage. The pH of the samples was adjusted to pH 2.0 using 1 N. HCl, and the mixture was incubated with pepsin at 800 U / ml in a shaking bath for 2 hours at 37 ° C with shaking at 100 rpm. Then the sample was neutralized with 1 M NaOH to pH 7.0 before adding pancreatin at a dose of 2 mg / ml and bile extract 0.3 mg / ml, then incubated for 2 hours. Finally, digestion was completed by boiling for 10 minutes (Xi Li, Yujie Su, Jun Sun, Yanjun Yang, 2012).

The disadvantage of this method of preparing the extract is the complexity and high cost.

The closest in technical essence and the achieved positive effect and adopted by the authors for the prototype is a method of preparing an embryonic biostimulator, including the selection of fresh, uninfected chicken eggs, then their incubation for 9 days, while for 5, 6, 7 days the eggs are irradiated for 30 s at a modulation frequency of 9 Hz and a radiation power of 9 mW, then the eggs are cooled for 6-7 days, homogenized, diluted the egg mass with 1:10 saline, mix thoroughly, add 5% to the resulting solution th phenol solution so that the phenol concentration is 0.3%, filter the resulting solution and pour into sterile vials, close and store in a refrigerator at a temperature of 2-6 ° C (see patent for the invention of the Russian Federation 2197251, class A61K 35/12 )

The disadvantage of this method of preparing a biostimulant is low stability.

Disclosure of invention

The technical result that can be achieved using the proposed method is reduced to immunomodulation of the body, obtaining a more stable and effective immunotropic agent.

The technical result is achieved using a known method of preparing a low molecular weight activated embryonic complex, including selection of eggs, cooling at a temperature of 2-4 ° C, freezing, thawing, homogenization, dilution, homogenization under high pressure, centrifugation, selection of a low molecular weight supernatant fraction, placement in a thermostat moreover, before homogenization, the egg is incubated for 10 days, and on the 4th, 5th, 6th, 7th day the egg is irradiated with the AL-01 Semicon laser apparatus for 30 s at a frequency modulation of 9 Hz and a radiation power of 9 mW and irradiated for 30 s under an ultraviolet lamp at a distance of 50 cm and cooled for 5-6 days at a temperature of 2-6 ° C, frozen at a temperature of minus 38-40 ° C for 24 hours, thawed for 24 hours at a temperature of 2-6 ° C, while 0.9% sodium chloride in a ratio of 1:10 with the addition of a 40% formalin solution to a concentration of 0.2% is used as a diluent.

The essence of the method of preparation of the low molecular weight complex of the activated embryonic is as follows.

Fresh, uninfected chicken eggs are selected, then they are incubated for 10 days, while on the 4th, 5th, 6th, 7th day the eggs are irradiated with the AL-01 Semicon laser apparatus for 30 s at a modulation frequency of 9 Hz and a radiation power of 9 mW and irradiated for 30 s under an ultraviolet lamp at a distance of 50 cm, then the eggs are cooled for 5-6 days at a temperature of 2-6 ° C, then placed for 24 hours in a low-temperature refrigerator at a temperature of minus 38-40 ° C and thawed at a temperature 2-6 ° C for 24 hours, embryonic and extraembryonic tissues are separated from the shell, etc. homogenize them, dilute the embryo-egg mass with 0.9% sodium chloride 1:10, mix thoroughly, add a 40% formalin solution to the resulting mixture so that the formalin concentration is 0.2%) homogenize using a high pressure homogenizer at 600 bar four times, centrifuged the resulting solution at 4800 g for 30 minutes at a temperature of 2-6 ° C, separated the supernatant and poured into sterile vials, closed, placed in a thermostat at a temperature of 37 ° C for 7 days, stored preparation refrigerator at 2-6 ° C.

Examples of specific experiments on the preparation of a low molecular weight complex of activated embryonic.

Experience 1. A selection of fresh, uninfected chicken eggs is carried out, then they are incubated for 10 days, during incubation on days 4, 5, 6, 7, the eggs are irradiated with the AL-01 Semicon laser apparatus for 30 s at a modulation frequency of 9 Hz and a radiation power of 9 mW and irradiated for 30 s under an ultraviolet lamp at a distance of 50 cm, then the eggs are cooled for 5-6 days at a temperature of 2-6 ° C, placed for 24 hours in a low-temperature refrigerator at a temperature of minus 38-40 ° C and thaw at a temperature of 2-6 ° C for 24 hours, the embryonic and extraembryonic tissues they are dried from the shell and homogenized to a homogeneous mass, the egg mass is diluted with 0.9% sodium chloride 1:10, add 5% phenol to its concentration of 0.3%, mix thoroughly, pour into sterile vials, close, the resulting preparation is stored in the refrigerator at temperature 2-6 ° C.

The resulting preparation has disadvantages: the drug is not stable, a significant precipitate falls out, when applied to animals it does not give a pronounced immunomodulating effect, cases of local allergic reaction are detected.

Experiment 2. Conducted similarly to experiment 1, but to stabilize after homogenization, instead of phenol, a 40% formalin solution to a concentration of 0.2% is used, and after filtration, the drug is placed in a thermostat at 37 ° C for 7 days for sterilization.

The resulting preparation is not stable, a significant precipitate.

Experiment 3. Conducted similarly to experiment 2, but after adding formalin, the resulting suspension is crushed in a high-pressure homogenizer at 600 bar four times, the resulting solution is centrifuged at 4800 g for 30 minutes at a temperature of 2-6 ° C, the supernatant is separated and poured into sterile vials , close, placed in a thermostat at a temperature of 37 ° C for 7 days, the resulting preparation is stored in a refrigerator at a temperature of 2-6 ° C.

The resulting preparation with these parameters has stability, a wide list of biologically active substances and a pronounced immunomodulating effect (table. 1, Fig. 1, 2, 3, 4).

The physico-chemical characteristics of the new version of the NIKA-EM preparation were tested, including the zeta potential, the size of the particles forming the preparation, the total amount of protein, RNA, DNA and the spectrophotometric profile in comparison with the STEMB preparation.

To determine the zeta potential and particle size, the Malvern Zetasizer Nano ZS analyzer was used (Malvern, UK).

To determine the chemical composition was carried out:

1) a quantitative assessment of the protein content in the sample by specific fluorescence (Qubit 2.0 fluorimeter, Life Technologies, USA);

2) a quantitative assessment of the content of ribonucleic acids (RNA) in the sample by the method of specific fluorescence (Qubit 2.0 fluorimeter, Life Technologies, USA);

3) a quantitative assessment of the content of deoxyribonucleic acids (DNA) in the sample by specific fluorescence (Qubit 2.0 fluorimeter, Life Technologies, USA);

4) an assessment of the characteristic absorption spectra of the samples under study in the wavelength range 190–840 nm (NanoDrop 2000C spectrophotometer, Thermo Fisher Scientific, USA) (Table 1).

The research results indicate that the biologically active drug "NIKA-EM" in comparison with the drug "STEMB" has acquired signs of greater physical stability. In addition, it was found that most of the particles in the preparation have a diameter below 100 nm and thus relate to low molecular weight compounds. In the NIKA-EM preparation, the amount of total protein, DNA, and especially RNA, significantly increased by 71 μg / ml (by 27.3%), 0.34 μg / ml (by 9.7%) and 13.2 μg, respectively / ml (by 194.1%), which indicates a better destruction of cell structures and the release of biologically active active substances into the solution, which determine the biological activity of the drug.

In addition, the expansion of the range of characteristic spectra to the right indicates a qualitative increase in the list of biologically active substances in the drug "NIKA-EM" (Fig. 1, 2). In FIG. 1 shows the characteristic absorption spectrum in the wavelength range of 190-840 nm, tissue preparation "STEMB" (max 219 nm, 229 nm). In FIG. 2. - the characteristic absorption spectrum in the wavelength range of 190-840 nm, tissue preparation "NIKA-EM" (max 215 nm, 228 nm, 275 nm).

In addition, studies have been conducted on the effect of the NIKA-EM preparation on the development of post-vaccination immunity. According to the normative documents STO 00482861-0005-2006 (FGUP Stavropol Biofactory), which included testing the vaccine for immunogenicity, two groups of clinically healthy animals were taken, without abscesses and lesions, normal nutrition of rabbits with a live weight of 3-3.5 kg, 5 animals each each. The vaccine was administered to the animals of the first group in the ear vein; the animals of the second group, in addition to the vaccine, were injected subcutaneously with NIKA-EM at a dose of 0.1 ml per kg of live weight once. 25 days after the introduction of the vaccine in rabbits from the ear vein, blood was collected in an amount of 5-8 cm ³ in glass tubes, which were then kept in an thermostat at 37 ° C for 30 minutes. At the end of time, the clot was circled and placed in the refrigerator for clot retraction for 1 day. Settled serum was examined in a PMA (microagglutination reaction) with all 5-10-day-old leptospira strains that are part of the vaccine, with 60-70 motile leptospira accumulated in the microscope's field of vision. Each serum sample was diluted with saline to 1: 1600, antigen - to 1: 3200. PMA was placed on plexiglass plates, which after the manipulations were placed in a thermostat for 1 hour. The reaction was taken into account under a microscope with a dark-field condenser at a magnification of 20 × 1.5 × 10. Agglutination was manifested in the bonding of leptospira and the formation of “spiders”. The free ends of the leptospira remained mobile. Serum titer was considered the largest dilution in which at least 50% leptospira was agglutinated.

30 days after the start of the experiment, which included testing the vaccine for immunogenicity, all experimental animals were slaughtered, followed by autopsy and selection of organs for histological examination.

Histological examination of organs was carried out according to the generally accepted method. The organs were placed in 10% neutral formalin for 3 days, then washed in running water for 24 hours. The cutting was carried out on the Tekhnom MZP-01 microtome and using the OMT 0228 freezing device at a temperature of minus 12 ° C. Sections were glued onto glass and stained with hematoxylin-eosin without dewaxing according to the standard technique. The area of red and white pulp of the spleen was determined micromorphometrically using an eyepiece micrometer × 16 and a Mikmed-2 microscope. All calculations were carried out under a fixed increase in 10 different fields of the histological preparation of the spleen with the calculation of the average value. The experimental results were subjected to variational-statistical processing using the Primer of Biostatistics program (Version 4.03).

When studying the development of post-vaccination immunity against leptospirosis in both groups of animals, it was found that in relation to the Pomona serogroup in the control, an increase in titers was higher than the norm in two rabbits 1: 800 and 1: 3200, in the experiment - in four from 1: 800 to 1: 1600 ; in relation to the Tarassovi serogroup, in control and experiment, all rabbits had almost maximum titers; in relation to the serogroup of Influenza typhoid in the control, an increase in titers is higher than the norm - in one rabbit 1: 800, in the experiment in four from 1: 800 to 1: 3200; in relation to serogroup Seiro (serovar Khardzhio) in the control, all rabbits had minimal normative titers of 1: 400, and in the experiment, an increase in titers higher than normative was in three rabbits from 1: 800 to 1: 1600.

An analysis of the results of the study of specific immunity using the NIKA-EM preparation showed that antibody titers in most animals are aligned with the Pomona serogroup, in contrast to controls, where antibody titers ranged from minimum in most animals (1: 400) to maximum values (1: 3200). In relation to the Tarassovi serogroup, the introduction of the NIKA-EM preparation did not affect the antibody titers, both in the control and experimental groups the titers were maximum. In relation to the serogroups of Flu and Typhoid (Serovar Khardzhio), post-vaccination immunity was stimulated - in the control, almost all animals had a minimal titer level (1: 400), and when using the NIKA-EM preparation, in most animals the antibody titers ranged from 1 : 800 to 1: 1600.

To study the effect of the administration of the multivalent VGNKI vaccine against animal leptospirosis in combination with the NIKA-EM immunomodulator on the morphology of the internal organs 30 days after the start of the experiment, including testing the vaccine for immunogenicity, all experimental animals were slaughtered, followed by autopsy and selection pieces of organs for histological examination.

As a result of postmortem autopsy, in all rabbits that were given the vaccine together with the NIKA-EM preparation, an enlargement of the spleen was detected, its tuberosity and structural severity were noted in the section, which corresponds to the pathomorphological signs of parenchymal organ hyperplasia, other organs and tissues were without macroscopic changes.

A histological examination of the spleen of animals that were jointly administered with the vaccine and the NIKA-EM preparation revealed the development of a large number of secondary splenic follicles with a germinal center, arterial hyperemia, intense infiltration of the spleen parenchyma by white blood cells, especially macrophages and plasma cells. In a comparative assessment of the area of white and red pulp of the spleen of animals of the first and second groups, it was determined that the area of white pulp of the spleen in rabbits to which the drug NIKA-EM was administered against the vaccine was 62% ± 5.6 at P≤0.05, which is 30% more than in rabbits of the control group, where the reactivity of the splenic follicles was relatively weak (Fig. 3, 4). In FIG. Figure 3 shows a hundredfold enlarged spleen of rabbits in the control group: weak reactivity of secondary splenic follicles; hematoxylin and eosin staining. In FIG. 4 shows a hundred-fold enlarged spleen of rabbits in the control group: weak reactivity of secondary splenic follicles; hematoxylin and eosin staining.

The results obtained indicate that the drug "NIKA-EM" had an immunomodulatory effect on the indicators of post-vaccination immunity with the introduction of the polyvalent vaccine "VGNKI" against leptospirosis of animals. The immunomodulating effect consists in the adequate dynamics of antibody formation as a result of antigen-dependent differentiation of spleen B-lymphocytes depending on the specific reaction of the animal organism to a certain leptospira serogroup.

Literature

1. Patent No. 2341272 of the Russian Federation. Means for non-specific immunotherapy / L.N. Mkrtchyan. - Publ. 02/27/2014.

2. Patent No. 2229886 of the Russian Federation. A tool that normalizes the metabolism in skin tissues / T.P. Klyushnik, A.A. Zozulya, R.V. Korneev. - Publ. 06/10/2004.

3. Patent No. 2197251 of the Russian Federation. A method of preparing a fetal biostimulator / L.D. Timchenko, I.V. Rzhepakovsky, V.V. Mikhailenko and others. - Publ. 01/27/2003. - Bull. Number 3. - Part 3.

4. Patent No. 2148408 of the Russian Federation. A method of obtaining a biologically active tissue preparation / P.M. Colonels. - Publ. 05/10/2000.

5. Patent No. 2508297 of the Russian Federation. A method of obtaining a biologically active substance from the blood of chickens to activate the proliferation of undifferentiated human bone marrow cells / A.G. Rumyantsev, V.A. Shestakov, S.A. Rumyantsev, A.P. Ivanov. - Publ. 02/27/2014.

6. Patent No. 2300386 of the Russian Federation. A method of obtaining biologically active raw materials from reindeer embryos / A.A. Kaiser, Kasim A.L., A.A. Gnedov, I.N. Rings. - Publ. 06/10/2007.

7. Patent No. 2110267 of the Russian Federation. A method of producing nucleoprotein complexes from bird embryos with immunotropic and radioprotective activity / V.I. Masycheva, V.V. Khomov, A.A. Sizov, V.A. Razvorotnev, V.A. Fadina. - Publ. 05/10/1998.

8. Patent No. 2259195 of the Russian Federation. A method for the prevention and treatment of animal diseases with biostimulants and hormones of a tissue preparation of complex action / O.N. Moiseev, K.S. Savenkov, A.I. Klimenko, Yu.D. Drobin, G.A. Tichenko. - Publ. 08/27/2005.

9. Xi, Li Chicken embryo extracts enhance spleen lymphocyte and peritoneal macrophages function / Li Xi, Su Yujie, Sun Jun, Yang Yanjun // Journal of Ethnopharmacology. - 2012. - No. 144. - P. 255-260.

Claims (1)

A method of preparing a low molecular weight complex, characterized in that they select fresh, uninfected chicken eggs, then they are incubated for 10 days, while on days 4, 5, 6, 7, the eggs are irradiated with a laser machine AL-01 "Semicon" for 30 s at a modulation frequency of 9 Hz and a radiation power of 9 mW and irradiated for 30 s under an ultraviolet lamp at a distance of 50 cm, then the eggs are cooled for 5-6 days at a temperature of 2-6 ° C, then placed for 24 hours in a low-temperature refrigerator at a temperature of minus 38-40 ° C and thawed at a temperature 2-6 ° C for 24 hours, the embryonic and extraembryonic tissues are separated from the shell and homogenized, diluted with 0.9% sodium chloride 1:10 in the embryonic egg mass, 40% formalin solution is added to the resulting mixture, so that the concentration formalin was 0.2%, homogenized using a high-pressure homogenizer at 600 bar four times, the resulting solution was centrifuged at 4800 g for 30 minutes at a temperature of 2-6 ° C, the supernatant was separated and poured into sterile vials, closed, placed in a thermostat etc at 37 ° C for 7 days, the resulting preparation is stored in a refrigerator at 2-6 ° C.

Images