RU2470648C1 - Preparation for stimulating animal metabolism and nonspecific resistance - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary medicine, particularly producing biologically active preparations. A preparation for stimulating animal metabolism and nonspecific resistance contains sodium nucleinate, biological waste of quail fibroblast culture on a primary culture medium containing a synthetic modified Eagle's medium, a synthetic medium 199 and bovine blood serum; besides, the preparation additionally contains antiseptic Dorogov's stimulator F-2 (ADS F-2) in the following proportions, wt %: sodium nucleinate 2.5; ADS-F2 3.5; biological waste of quail fibroblast culture 94.

EFFECT: invention provides higher biological activity of the preparation.

5 tbl, 4 ex

Description

The invention relates to veterinary medicine, in particular to the production of biologically active drugs that stimulate metabolism, growth and increase natural resistance in animals.

A method of obtaining a preparation for increasing the resistance of the animal organism, comprising freezing tissue parenchymatous organs, thawing, homogenization and amendment succinic acid extract followed by hydrolysis of biomass, decanting of the extract with the addition of sodium hydroxide (Patent RF № 2237485, cl. A61K _^3/00, 10.10 .2004).

The disadvantage of this method is that it is multi-stage and complex in its technical performance.

There is also the drug “Amber biostimulant” to increase the resistance of the animal organism, which involves mixing succinic acid with the tissue preparation ASD F-2 (Dorogov's antiseptic stimulator, fraction-2), novocaine and distilled water (RF Patent No. 2303979, class A61K 3/00 from 05/23/2005).

The disadvantage of this method is that it does not have a stimulating effect on the metabolism in animals.

As a prototype, “Complex drug for the prevention and treatment of metabolic disorders, microelements, increase the resistance of the animal organism” was selected. The preparation of the drug involves mixing the following components from the calculation, g / l: succinic acid (10.0), ferrous sulfate (2.0), copper sulfate (0.1), cobalt sulfate (1.0), zinc sulfate (0, 1), sodium nucleinate (10.0), methionine (10.0), water for injection (up to 1000 ml) (RF Patent No. 2404761, CL A61K 3/00, dated March 24, 2009).

The disadvantage of this drug is that the resulting drug does not have a stimulating effect on protein-amino acid and vitamin metabolism, and also has a relatively low immunostimulating effectiveness. Moreover, the use of the components that make up the drug, as well as the use of sterilization by autoclaving, makes the drug relatively expensive.

The technical result of the invention is to provide a stimulating effect of the drug on protein-amino acid, mineral and vitamin metabolism, increase immunostimulating effectiveness, reduce the cost of the drug.

The specified technical result is achieved by the fact that the known preparation containing sodium nucleinate according to the invention additionally contains waste from the biological industry obtained by culturing quail fibroblasts on a primary culture medium, including synthetic MEM medium (minimal Needle medium), synthetic medium 199 and bovine serum livestock, as well as the drug additionally contains an antiseptic stimulator Dorogova F-2 (ASD F-2) in the following ratio of components, wt.%:

sodium nucleinate 2.5 ASD-F2 3,5 biological industry waste during the cultivation of quail fibroblasts 94

Biological industry waste is obtained after culturing cells, in particular quail fibroblasts, in the technological conditions of biofactories, for example, the Kursk Biofactory. When culturing cells, a primary culture medium is used, including MEM synthetic medium (Eagle's minimal medium), 199 synthetic medium, and cattle blood serum. These media contain a complex of amino acids, mineral and vitamin components in ratios that ensure optimal growth of cell culture. After the process of cultivation in liquid waste, these substances remain in almost the same amount and proportions. In addition to the components listed above, cultural waste products include biological waste products of cultured cells that act as natural biostimulants.

An analysis of the biochemical composition of the waste obtained after culturing quail fibroblast cells in the production of a liquid vaccine against Marek’s disease under the conditions of the Kursk Biofactory showed that the waste contains a complex of biochemical components that correspond to the components of the primary culture medium. Moreover, the content of some substances was greater than in the primary culture medium. This confirms that during the cultivation of cells they produce biochemical substances that enter the environment, and thereby increase its biological value.

As follows from table 1, waste cultural production include nitrogenous components: proteins and amino acids. Among amino acids, the highest content falls on lysine, arginine, threonine, glutamic acid, isoleucine, leucine, tyrosine and phenylalanine. The mineral part of the waste is represented by calcium, phosphorus, magnesium, potassium, sodium and chlorides. The waste contains a lot of carbohydrates in the form of glucose, as well as vitamins: retinol, thiamine, calciferol, ascorbic acid. At the same time, cultural wastes represent a pink sterile liquid with a pH of 6.7-7.0.

Cultural production wastes are not used in further technological processes and, for example, at the Kursk Biofactory, are disposed of.

Table 1 The content of biochemical components in the waste obtained after the cultivation of quail fibroblast cells Test components Units measurements Content Albumin % 52.0 Alpha globulins % 20,0 Beta globulins % 18.0 Gamma globulins % 10.0 Lysine mg / l 135.7 Histidine mg / l 67.8 Arginine mg / l 158.4 Aspartic acid mg / l 73,4 Threonine mg / l 111.8 Serine mg / l 63.7 Glutamic acid mg / l 144.8 Proline mg / l 53,2 Glycine mg / l 88.5 Alanine mg / l 74.7 Valine mg / l 98.8 Methionine mg / l 63.3 Isoleucine mg / l 105.6 Leucine mg / l 208.7 Tyrosine mg / l 103.0 Phenylalanine mg / l 107.7 Calcium mg / l 388.4 Phosphorus mg / l 225.6 Magnesium mg / l 199.4 Chlorides mg / l 955.7 Potassium mg / l 108.3 Sodium mg / l 211.8 Glucose mg / l 1988.0 Pyridoxine mg / l 1.47 Retinol mg / l 0.48 Thiamine mg / l 0.12 Calciferol mg / l 2.05 Vitamin C mg / l 1.88 Riboflavin mg / l 0.97 Pantothenic acid mg / l 1.17

Sodium Nucleinate is a synthetic immunostimulant, it is used to prevent and treat infectious diseases that occur against the background of immunodeficiency states. Thermostable. The inclusion of sodium nucleinate in the preparation increases its immunomodulating properties, since sodium nucleinate stimulates bone marrow activity, intracellular metabolism and nucleic acid metabolism, has the activity of a polyclonal immunomodulator, stimulates the phagocytic activity of macrophages and generally increases the nonspecific defense of the body, especially in immunodeficiencies.

The addition of F-2 ASD (Dorogov's antiseptic stimulator, fraction-2) to the complex preparation enhances the activity of tissue enzymes that are involved in the active transport of ions and nutrients through cell membranes, in phosphorylation processes, improves tissue trophism, and increases the level of metabolic processes in a healthy body and restores metabolism to normal in various dystrophic conditions.

The proposed drug is prepared as follows.

To obtain 1 l of the proposed drug, 25 g of sodium nucleinate, 35 ml of F-2 ASD (Dorogov's antiseptic stimulator, fraction-2) are successively added to a sterile volumetric flask and the volume is adjusted to 1000 ml by adding biological waste obtained by culturing cells in liquid form .

The indicated amounts of sodium nucleinate and ASD F-2 included in the proposed drug were determined empirically and this is justified by the fact that with a decrease in these quantities, the effectiveness of the drug decreases, and with an increase it remains at a constant level.

The resulting preparation is a pink liquid with a faint specific odor. The drug is poured into bottles with a capacity of 50-100 ml, which are closed with rubber stoppers and rolled in aluminum caps.

General properties and stability of the drug are determined according to the provisions of the State Pharmacopoeia. Criteria are such parameters as appearance, transparency, color, toxicity, lack of mechanical impurities.

To determine the shelf life of the drug at the preliminary stage of research, the “accelerated aging” method was used, and to obtain more accurate results, experimental samples of the drug were stored at room temperature, as well as in a refrigerator at 4-8 ° C. Every 3 months, the drug was tested for bacterial contamination using Endo medium and MPA.

During the audit, it was found that during storage, the initial parameters remain without significant changes for 12 months.

The drug was tested for pyrogenicity in 6 healthy rabbits of different sexes with a body weight of 2.5 ± 0.14 kg. Each experimental rabbit was kept in a separate cage and received a diet identical for all animals.

Before and after a single injection of the drug into the ear vein at a dose of 1 ml / kg, the body temperature was measured in rabbits, and blood was also taken after 1, 3, and 6 hours. ESR, hematocrit and total leukocyte count were determined in the blood.

The results of testing the drug for pyrogenicity showed that after administration of the drug, an increase in the total body temperature in 6 experimental rabbits was within the physiological norms of 38.6-39.3 ° C. ESR and hematocrit, respectively, were 1.5-2.0 mm / h and 37.0-38.0%. The leukocyte count after administration of the drug increased, however, this increase (7.0 ± 0.25-7.6 ± 0.27 × 10 ¹² / L) compared to background values (6.8 ± 0.34 × 10 ¹² / L ) slightly.

The results obtained served as the basis for evaluating the manufactured drug as pyrogen-free.

To study the local effect of the proposed drug on the mucous membranes, it was introduced once into the conjunctival sac of rabbits in an amount of 1-2 drops. When making the drug, the inner corner of the conjunctival sac was pulled, then the lacrimal-nasal canal was clamped for 1 minute and the behavior of the rabbits was monitored. In the first 1-2 hours, only mild conjunctival hyperemia was observed, changes in the vessels of the sclera, cornea and pupil in experimental animals were not observed.

Acute toxicity was assessed on white outbred mice and rabbits of the Soviet chinchilla breed, which were kept in the vivarium of the Kursk State Agricultural Academy. The drug was administered intraperitoneally: mice - 0.5; 1.0 and 1.5 ml / goal., For rabbits - 5.0 and 10.0 ml / goal.

The research results showed that the selected doses of the drug did not have a negative effect on the body of white mice and rabbits. With the slaughter of experimental animals, no visible changes in organs and tissues were found.

The anaphylactogenic properties of the drug were studied in 10 guinea pigs, which were intraperitoneally injected with waste from the biological industry obtained after culturing cells in a dose of 1.0 ml, and after 15 days, 1.0 ml of the proposed drug. After administration of the drug, no experimental animal observed reactions characteristic of anaphylactic shock, which indicates the absence of a heterogeneous protein in the manufactured preparation.

Scientific and production tests of the drug were carried out on three types of domestic animals: rabbits, pigs and cattle.

Example 1. In order to assess the general condition, hematological and immunological parameters after the administration of different doses of the drug, an experiment was conducted on rabbits of the breed Soviet chinchilla, which were kept in the vivarium of the Kursk State Agricultural Agricultural Academy.

Three groups of 2-year-old analog rabbits were formed, 5 animals each.

Rabbits of the 1st group were control, instead of the proposed drug they were injected intramuscularly with 1.0 ml / goal. sterile isotonic sodium chloride solution (saline) once a day for three consecutive days.

Rabbits of group 2 were intramuscularly injected with the proposed drug at a dose of 0.5 ml / goal. once a day for three consecutive days.

Group 3 rabbits were injected with 1.0 ml / goal. the proposed drug according to the same scheme as the animals of the second group.

During the experiment, rabbits were monitored and blood was taken for laboratory analysis before drug administration, 48 and 72 hours after the last drug administration. In the blood, the erythrocyte sedimentation rate (ESR), hematocrit, and the content of erythrocytes, leukocytes, and hemoglobin were determined by conventional methods. The content of total immunoglobulins was determined by the zinc sulfate method, the phagocytic activity of leukocytes (FAL) by the reaction of phagocytosis with latex, the bacterial activity of blood serum (BASK) using a culture of Staphylococcus aureus, the lysozyme activity of blood serum (LASK) was evaluated using a daily culture of Micrococcus lisodeus.

The research results (Table 2) showed that in all experimental rabbits, the general hematological and immunological parameters were within the physiological norms. Moreover, the erythrocyte content in rabbits of the experimental groups after administration of the drug was significantly higher (p <0.05) than before administration of the drug and compared to control animals. An increase in the concentration of hemoglobin in the blood of experimental animals was also noted, however, this increase was statistically unreliable (p> 0.05).

table 2 General hematological and immunological parameters in rabbits after administration of the proposed drug Indicators Before drug administration 48 hours after drug administration 72 hours after drug administration 1 group (control) ESR, mm / hour 1.1 ± 0.09 1.3 ± 0.10 1.2 ± 0.07 Hematocrit% 37.7 ± 0.44 36.9 ± 0.36 37.4 ± 0.47 Red blood cells, 10 ¹² / L 5.9 ± 0.05 5.7 ± 0.07 5.9 ± 0.09 White blood cells, 10 ⁹ / L 7.0 ± 0.11 6.9 ± 0.14 7.0 ± 0.08 Hemoglobin, g / l 109.5 ± 5.59 107.5 ± 4.18 105.5 ± 5.07 General immunoglobulins, g / l 1.19 ± 0.07 1.16 ± 0.08 1.16 ± 0.09 BASK,% 69.4 ± 2.00 68.3 ± 1.16 67.7 ± 1.11 FAL,% 18.4 ± 0.25 18.0 ± 0.54 17.8 ± 0.24 LASK,% 41.5 ± 0.46 42.0 ± 0.58 41.6 ± 0.49 2 group (0.5 ml / goal.) ESR, mm / hour 1.2 ± 0.10 1.2 ± 0.09 1.3 ± 0.08 Hematocrit% 37.0 ± 0.24 38.8 ± 0.11 ^• 39.5 ± 0.10 ^• Red blood cells, 10 ¹² / L 5.8 ± 0.07 6.2 ± 0.06 * ^• 6.4 ± 0.05 * White blood cells, 10 ⁹ / L 6.9 ± 0.08 6.9 ± 0.05 7.3 ± 0.04 Hemoglobin, g / l 109.5 ± 5.86 114.0 ± 7.04 118.5 ± 6.00 General immunoglobulins, g / l 1.15 ± 0.03 1.23 ± 0.05 1.35 ± 0.04 * BASK,% 68.8 ± 2.16 70.0 ± 3.04 74.4 ± 2.08 ^• FAL,% 18.8 ± 0.24 21.1 ± 0.15 * ^• 22.0 ± 0.14 * ^• LASK,% 41.7 ± 0.31 44.9 ± 0.44 48.8 ± 0.56 3 group (1.0 ml / goal.) ESR, mm / hour 1.3 ± 0.07 1.2 ± 0.08 1.4 ± 0.08 Hematocrit% 37.4 ± 0.34 39.5 ± 0.22 ^• 39.8 ± 0.18 ^• Red blood cells, 10 ¹² / L 6.0 ± 0.06 6.5 ± 0.05 * ^• 6.7 ± 0.04 * White blood cells, 10 ⁹ / L 6.8 ± 0.07 7.0 ± 0.09 7.4 ± 0.08 * Hemoglobin, g / l 104.5 ± 6.17 119.4 ± 5.05 121.0 ± 5.11 ^• General immunoglobulins, g / l 1.17 ± 0.05 1.31 ± 0.04 * 1.38 ± 0.04 * ^• BASK,% 67.0 ± 3.17 72.5 ± 4.15 ^• 80.7 ± 3.00 * ^• FAL,% 17.5 ± 0.34 21.7 ± 0.25 * ^• 22.4 ± 0.18 * ^• LASK,% 40.8 ± 0.56 48.7 ± 0.64 51.4 ± 0.25 Note: * - at P <0.05 in comparison with the corresponding indicators obtained before drug administration; ^• - at P <0.05 compared with control animals.

The drug had a positive effect on the nonspecific protective properties of the body, in particular, they had higher blood levels of total immunoglobulins, BASK, LASK and FAL blood activity. In turn, it was noted that in rabbits that were injected with 1.0 ml / goal. of the preparation, non-specific protective factors were higher compared to rabbits receiving a dose of 0.5 ml / goal. Thus, the used dosages of the manufactured preparation had a positive effect on the rabbit organism and had an immunostimulating effect.

Given that the proposed drug contains a complex of interchangeable and essential amino acids, studies have been conducted on the content of free amino acids before and after administration of the drug.

Example 2. For this purpose, in a pig farm educational and experimental farm Kursk State Agricultural Academy were selected three groups of gilts of 4 months of age (10 animals each) in compliance with the principle of analogues.

Pigs of the 1st group were control, they were injected intramuscularly with 3.0 ml / goal. isotonic sodium chloride solution three times daily after 24 hours.

The pigs of group 2 were injected with the well-known drug "Comprehensive drug for the prevention and treatment of metabolic disorders, microelements, increase the body's resistance" at a dose of 3.0 ml / goal. daily three times in 24 hours.

The pigs of group 3 were intramuscularly injected with the proposed preparation at a dose of 3.0 ml / head, daily three times every 24 hours in the same sequence as the animals of the second group.

Blood from pigs was taken from the vessels of the tail before drug administration and 10 days after the last drug administration. Plasma levels of free amino acids were determined on a Hitachi analyzer in the research laboratory of the Kursk Biofactory.

Comparison of the content of free amino acids (Table 3) showed that their total content in the blood of pigs that were administered the proposed drug was significantly greater (p <0.05) than in animals of groups 1 and 2. Moreover, the increase in the amount of amino acids occurred due to both essential and non-essential amino acids.

The relatively high amino acid content in the blood of pigs of group 3 can be explained by the stimulating properties of the metabolites secreted by cells during cultivation on the digestibility of the diet protein, as well as the presence of a complex of amino acids contained in the manufactured product in optimal proportions, which contributes to their absorption in the intestines of pigs.

To study the effect of the proposed drug on the biochemical and immunological status, as well as on sperm production in bulls, a scientific-production experiment was conducted under the conditions of Kursk OJSC for breeding.

Example 3. The experiment was carried out on two groups of bulls.

Bulls of group 1 (control) were injected intramuscularly with isotonic sodium chloride at a dose of 10.0 ml / goal. once a day 3 times in 24 hours.

Bulls of group 2 (experimental) were injected intramuscularly with the proposed drug at a dose of 10.0 ml / goal. once a day 3 times in 24 hours.

The conditions for keeping and feeding the bulls of both groups were stereotypical. All animals were bled prior to drug administration and on the seventh day after its last administration. Biochemical and immunological parameters were determined in the blood.

The research results are presented in table 4, the data of which indicate that after the administration of the proposed drug in the bulls, the content of biochemical blood components increased, as well as the indicators of the body's natural resistance were higher. In control animals, the studied parameters were at the level of the initial data or slightly lower.

Table 4 Biochemical and immunological parameters in bulls after the introduction of the proposed drug Indicators 1 group (control) 2 group (experimental), Record Hicks Rax Myth Start Skipper Romer Hannibal Hemoglobin, g / l

Total protein, g / l

Total calcium, mmol / l

Inorganic phosphorus, mmol / l

Total magnesium, mmol / l

Glucose, mmol / L

Vitamin A, μmol / L

Vitamin E, mg / L

Vitamin C; mmol / l

General immunoglobulins, mg / l

LASK,%

BASK,%

FAL,%

Note: 1 group: numerator - before the introduction of an isotonic sodium chloride solution; denominator - after the introduction of isotonic sodium chloride solution; 2 group: numerator - before the introduction of the proposed drug; denominator - after the introduction of the proposed drug.

In turn, the proposed drug had a positive effect on the sperm production of bulls (table 5). In the bulls of the experimental group where the proposed drug was used, the sperm concentration, survival and mobility were higher.

Table 5 Indicators of sperm production in bulls after the introduction of the proposed drug Indicators 1 group (control) 2 group (experimental) Record Hicks Rax Myth Start Skipper Romer Hannibal The volume of ejaculate, ml

The concentration of sperm, billion / ml

The percentage of live sperm

Sperm motility, score

The total number of sperm in the ejaculate, billion

Note: 1 group: numerator - before the introduction of an isotonic sodium chloride solution; denominator - after the introduction of isotonic sodium chloride solution; 2 group: numerator - before the introduction of the proposed drug; denominator - after the introduction of the proposed drug.

The proposed drug was tested in order to increase metabolic processes and the body's defenses in calves hypotrophic.

Example 4. In the conditions of the livestock farm of the Znamenskoye educational and experimental farm of the Kursk State Agricultural Academy, three groups of hypotrophic calves were selected.

Group 1 calves were control, they were injected intramuscularly with isotonic sodium chloride at a dose of 5 ml / goal. once a day 3 times in 24 hours.

Calves of group 2 were injected intramuscularly with the already known drug at a dose of 5 ml / goal. once a day 3 times in 24 hours.

Calves of group 3 were intramuscularly injected with the proposed drug at a dose of 5 ml / goal. in the same way as the animals of the second group.

All calves were bled prior to administration and 10 days after the last administration. Biochemical and immunological parameters were determined in the blood.

In the course of the studies, it was found that in calves that were injected with drugs (group 2, 3), the content of the studied biochemical blood components increased and was higher compared to control animals of group 1 (Table 6). In turn, in calves of the 3rd experimental group, the content of glucose, vitamins A, E and C was significantly higher (p <0.05) than in calves of the 2nd experimental group.

Clinical observations of experimental animals showed that the calves that were injected with the proposed drug recovered faster, they had higher average daily gain, among them there were no sick calves. In animals of the 2 experimental group, the gain was less; in two calves, an upset gastrointestinal tract with clinical signs of dyspepsia was observed. The calves of the 1st control group were stunted in growth; during the experiment, two calves fell.

Conducted scientific and industrial experiments indicate that the manufactured drug has a more pronounced biological effect compared to the prototype drug. The complex of biochemical components contained in the manufactured product in optimal proportions facilitates their active entry into the animal organism and has a beneficial effect on metabolism. Moreover, metabolic products secreted by cells during cultivation are biological stimulants and have a stimulating effect on cellular metabolism and non-specific animal protection factors.

Thus, the proposed drug for stimulating the metabolism and non-specific resistance of animals has at the same time a stimulating effect on protein-amino acid, mineral and vitamin metabolism, and increases the non-specific resistance of the animal organism. This can have a positive value both in the prevention and complex treatment of infectious diseases, and in the prevention of other diseases, which are based on metabolic disorders and immunological deficiency of the body. In addition, upon receipt of the proposed drug does not apply autoclaving, but uses the waste of the biological industry obtained by culturing cells that are disposed of at the Kursk Biofactory.

Therefore, the method of obtaining the proposed drug is technologically simple, reduces the economic costs and time to obtain the drug in comparison with known analogues.

Claims (1)

A preparation for stimulating the metabolism and non-specific resistance of animals, including sodium nucleinate, characterized in that it additionally contains waste from the biological industry obtained by culturing quail fibroblasts on a primary culture medium, including synthetic MEM medium (minimal Needle medium), synthetic medium 199 and serum blood of cattle, as well as the drug additionally contains an antiseptic stimulator Dorogova F-2 (ASD F-2) in the following ratio of components, wt. %:
sodium nucleinate 2,5 ASD F-2 3,5 biological industry waste obtained by culturing cells 94