RU2485964C1 - Immunostimulating composition for animals - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry and veterinary science, and represents an immunomodulatory composition for animals which contains lactoferrin milk whey protein as active substance, and distilled water as a solvent. In addition to lactoferrin, the active substance contains lactalbumin and lactoglobulin milk whey proteins, wherein the active substance represents an aqueous solution of mixed milk whey proteins of the total protein concentration of 10 g/l, containing 10-20% of lactoferrin, 20-30% of lactalbumin, 60-70% of lactoglobulin; moreover, the active substance further comprises selenium nanoparticles in the following proportions, wt %: aqueous solution of mixed milk whey proteins of the total protein concentration of 10 g/l containing 10-20% of lactoferrin, 20-30% of lactalbumin, 60-70% of lactoglobulin, 0.1-100 selenium nanoparticles 00001-10, distilled water up to 100.

EFFECT: invention provides a higher immunostimulating effects on the cell and humoral immunity.

3 ex, 3 tbl

Description

The invention relates to veterinary medicine, namely to pharmacology, and can be used as an immunostimulating agent.

Currently, the treatment and prevention schemes for diseases of both infectious and non-infectious etiologies include a number of immunostimulating drugs to increase the general resistance of the body or its nonspecific immunity, as well as the effect on specific immune reactions. There are several groups of immunostimulating drugs. The first group of immunostimulants are synthetic immunostimulants. It includes: levamisole (decaris), leacadine, diuciphone, etc. The second group is represented by endogenous immunostimulants and their synthetic analogues. It includes preparations of the thymus, red bone marrow, spleen and their synthetic analogues (thymalin, thymogen, taktivin (T-activin), thymoptin, thymactide, thymostimulin, imunofan, myelopid, splenin), immunoglobulins (human polyvalent immunoglobulin (interferon) (human immune interferon-gamma, recombinant interferon-gamma (gammaferon, imukin)), interleukins (recombinant interleukin-2 (aldesleukin, proleukin, roncoleukin), recombinant interleukin 1-beta (betaleukin), etc.). The third group includes preparations of microbial origin and their synthetic analogues (pyrogenal, prodigiosan, ribomunil, imudon, lycopid, etc.). The fourth group is represented by drugs of various pharmacological classes with immunostimulating activity. It includes adaptogens and herbal preparations (Dibazole, Bemitil, Echinacea, Eleutherococcus, Ginseng, Rhodiola rosea, Tonsilgon N, etc.), as well as vitamins (ascorbic acid (Vitamin C), tocopherol acetate (Vitamin E), retinol acetate (vitamin A)).

Closest to the claimed invention is an injectable therapeutic veterinary drug "Polyferrin-A" containing the active substance lactoferrin isolated from animal colostrum (http://www.narvac.com/art_poliferrin.htm, http://www.webvet.ru /equipment.asp?e_id=1954; http://www.veterinarka.ru/content/view/1142). Lactoferrin is a natural mammalian glycoprotein, it belongs to the iron-containing proteins responsible for the primary defense of the body as an immunostimulating substance (http://www.narvac.com/art_laktoferrin3.htm: Lactoferrin as a modulator of the immune and inflammatory processes. D. Legrand, E. Elass, M. Carpentier and J. Mazurier). However, polyferrin-A does not actively stimulate cellular immunity in animals, and in some cases causes allergic reactions.

The technical task is to increase the immunostimulating effect on cellular and humoral immunity.

The technical result that can be obtained from the use of the claimed composition is to enhance the functional activity of the drug, including increasing the stimulating effect not only on cellular, but also on humoral immunity, by directly presenting the active components of the drug to the cells of the reticuloendothelial system.

The technical result is achieved by the fact that the immunomodulatory composition for animals contains as an active substance milk whey protein lactoferrin, as a solvent distilled water, according to the invention, the active substance includes, in addition to lactoferrin, milk whey proteins lactoalbumin and lactoglobulin, while the active substance represents an aqueous solution of a mixture of milk whey proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% varnish toalbumin, 60-70% of lactoglobulin, in addition, the specified active substance additionally contains selenium nanoparticles, in the following ratio of components, wt.%:

aqueous solution of a mixture of whey protein proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin 0.1-10.0 selenium nanoparticles 0.0001-1.0 distilled water up to 100.

What is new is that the combined use of 3 milk whey proteins - lactoferrin, lactoalbumin and lactoglobulin in the active substance, and the introduction of selenium nanoparticles into the active substance for the first time have been proposed for use as an immunostimulating drug. Three of these proteins in combination with each other increase the stimulating effect of the active substance. Nanoparticles of selenium, included in the active substance, give the composition an additional antioxidant effect.

An immunostimulating composition for animals is prepared as follows. From whole cow's milk, an acidic whey is obtained, consisting of lactoglobulin, lactoalbumin, immunoglobulins, lactoferrin and other proteins, by curdling with citric acid. Proteins from this serum are recovered by precipitation with ammonium sulfate. The resulting precipitate is dissolved in distilled water to obtain a protein suspension, from which a mixture of lactoferrin, lactoalbumin, lactoglobulin is separated: for this, the protein suspension is dialyzed using dialysis membranes with a pore diameter of 12000-14000 Da in order to remove protein substances with a molecular weight of less than 12000 Da. At the end of dialysis, an aqueous solution of a mixture of three proteins is obtained, which contains 10-20% lactoferrin (molecular weight 75000-80000 Da), 20-30% lactoalbumin (molecular weight 18000 Da), 60-70% lactoglobulin (molecular weight 67000 Da) . With this method of obtaining a mixture, the presence of the three indicated proteins and their percentage are constant, which is confirmed by the inventors by conducting studies of an aqueous solution of a mixture of three proteins by native polyacrylamide gel electrophoresis using standards (http://molbiol.ru/protocol/17_01. html). The data from the studies were statistically processed using student criteria. By the same electrophoresis method, the above molecular weight indices of the proteins of lactoferrin, lactoalbumin, lactoglobulin are confirmed.

In the resulting aqueous solution of a mixture of three proteins (lactoferrin, lactoalbumin, lactoglobulin), the concentration of the total protein is determined. If the concentration of total protein is more than 10 g / l, then this solution is diluted with distilled water to a concentration of 10 g / l. Then, selenium nanoparticles are introduced into the composition as follows. A 1 M solution of hydrazine hydrochloric acid and 1 M sodium selenite NaSeO ₄ solution are added to an aqueous solution of a mixture of three proteins, then the volume of the resulting mixture is adjusted with distilled water to 100 wt.%. Stop the reaction by adjusting the pH of the solution to 7.2 with a 1 M sodium hydroxide solution and free of low molecular weight compounds by dialysis against distilled water. The result is an immunostimulating composition for animals containing:

aqueous solution of a mixture of whey protein proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin 0.1-10.0 selenium nanoparticles 0.0001-1.0 distilled water up to 100

Selenium in the immunostimulating composition for animals is in a nanoscale state: the particle size of colloidal selenium in the preparation is from 60 to 140 nm, which is confirmed by electron microscopy using a LIBRA 120 electron microscope (Carl Zeiss, Germany).

Example 1

An aqueous solution of a mixture of whey protein proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin 0.1 selenium nanoparticles 0.0001 distilled water up to 100

Example 2

An aqueous solution of a mixture of whey protein proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin 5,0 selenium nanoparticles 0.01 distilled water up to 100

Example 3

An aqueous solution of a mixture of whey protein proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin 10.0 selenium nanoparticles 1,0 distilled water up to 100

To establish an increase in the immunostimulating properties of the claimed composition in comparison with analogues, it was studied:

- the effect of the immunostimulating composition on the cellular immune response (studies have been conducted on the effect of the immunostimulating composition on the survival of staphylococcus in the presence of animal peritoneal cells);

- the effect of the immunostimulating composition on the humoral immune response (studies have been conducted on the effect of the immunostimulating composition on the effectiveness of immunization of animals);

- the effect of the immunostimulating composition on the nonspecific resistance of the body (studies have been conducted on the effect of the immunostimulating composition on the redox activity of cells).

When conducting studies on the effect of the immunostimulating composition on the survival of staphylococcus in the presence of animal peritoneal cells, mouse peritoneal cells (PCM) were obtained by a standard method at a concentration of 3 million cells / ml. In the experiment, cell cultures of Staphylococcus aureus 209-P microorganisms were used in the logarithmic growth phase on meat and peptone broth to obtain a staphylococcus concentration of 200 million CFU / ml and 20 million CFU / ml.

In the 1st research option (table 1) were mixed:

I. mouse peritoneal cells at a concentration of 3 million cells / ml, cell cultures of these microorganisms at a concentration of 200 million CFU / ml and an immunostimulating composition.

II. mouse peritoneal cells at a concentration of 3 million cells / ml, cell cultures of these microorganisms at a concentration of 20 million CFU / ml and an immunostimulating composition.

In the 2nd research option (table 1) were mixed:

I. mouse peritoneal cells at a concentration of 3 million cells / ml, cell culture of these microorganisms at a concentration of 200 million CFU / ml.

II. mouse peritoneal cells at a concentration of 3 million cells / ml, cell culture of these microorganisms at a concentration of 20 million CFU / ml.

In the 3rd research option (table 1) were mixed:

I. cell culture of these microorganisms at a concentration of 200 million CFU / ml and an immunostimulating composition.

II. cell cultures of these microorganisms at a concentration of 20 million CFU / ml and an immunostimulating composition.

In the 4th research option (table 1) studied:

I. cell cultures of these microorganisms at a concentration of 200 million CFU / ml.

II. cell cultures of these microorganisms at a concentration of 20 million CFU / ml.

In all variants of the study, the resulting cultures were incubated for 40 min at 37 ° C, the number of CFU of staphylococcus was determined by seeding dilutions of the initial samples on salt meat and peptone agar and by counting the number of grown colonies. PCM phagocytosis activity was calculated by the formula:

AF = 100 × (a-b) / a,

where a is the concentration of staphylococcus in the 4th variant, b is the concentration of staphylococcus in the 1st variant, or 2nd variant, or 3rd variant of the study.

The research results are shown in columns 7 and 8 of table 1.

When evaluating the results of studies (table 1):

- compared the concentration of staphylococcus cells after incubation of the 4th research option with the same indicators of the 1st, 2nd and 3rd options: in the 1st embodiment, staphylococcus survival is markedly reduced, in the 2nd embodiment there is a slight suppression of staphylococcus growth and in 3- m variant, it was found that the immunostimulating composition does not have a bacteriostatic effect against staphylococcus, which indicates the absence of negative effects of the immunostimulating composition on cells of a living organism;

- PCM phagocytosis activity indicators were analyzed: in the 1st embodiment, as a result of the exposure of the immunostimulating composition to the PCM, cellular immunity is activated, which is manifested in a staphylococcus growth retardation due to an increase in the phagocytic activity of PCM by 2-8 times in comparison with the 2nd variant.

When conducting studies on the effect of the immunostimulating composition on the humoral immune response, a study was conducted of the effect of the immunostimulating composition on the effectiveness of immunization. In the experiment used 3 groups of mice with 3 animals each. The mice of the first group (control) during the entire immunization were injected intraperitoneally with the biomass of the field isolate Salmonella thyphimurium, killed by heat. The second group of mice was injected intraperitoneally with a mixture of Salmonella biomass and an immunostimulating composition, the dose of which was 0.4 ml per 20 g of live weight. The mice of the third group received an intraperitoneal immunostimulating composition in the above dose and a day later salmonella biomass. The dose of salmonella wet biomass in all groups was 0.2 mg per 20 g of live weight. Immunization was carried out with an interval of 2 weeks - only 4 introductions. Before slaughter, salmonella biomass was boosted. The titer of agglutinating antibodies in the sera of experimental animals was determined using the method of radial immunodiffusion in 1.5% agar gel. Table 2 presents the results of determining the titer of agglutinating AT in the sera of immunized mice.

table 2 Agglutinating AT titers against salmonella in sera of mice immunized with salmonella biomass in conjunction or in parallel with an immunostimulating composition No. p / p The degree of dilution of serum Precision Zone 1 group 2 group 3 group one one 56.25 64 52,5625 2 2 49 56.25 49 3 four 36 49 36 four 8 30.25 42.25 30.25 5 16 20.25 36 27.5625 6 32 20.25 30.25 20.25 7 64 12.25 20.25 12.25 8 128 0 12.25 0 9 256 0 9 0 10 512 0 0 0

As can be seen from table 2, the highest titer of AT was found in the serum of mice immunized with a mixture of salmonella and immunostimulating composition. In mice immunized with biomass only and mice immunized with biomass one day after injection of the immunostimulating composition, the AT titers are the same and significantly lower than in animals receiving the immunostimulating composition and antigen simultaneously. Immunization with Salmonella biomass in conjunction with the immunostimulating composition is more effective, while the preliminary injection of the immunostimulating composition a day before the immunization does not affect the serum AT level compared to the control. Conclusion: the immunostimulating composition during immunization stimulates phagocytosis of the corpuscular antigen, thereby contributing to a more complete and effective presentation of the immune system of the macroorganism.

When studying the effect of the immunostimulating composition on the nonspecific resistance of an organism, we studied the effect of the immunostimulating composition on the redox activity of cells. In one embodiment of the study, the prototype preparation was introduced into a monolayer cell culture at a dose of 0.5 ml per 10 ml of medium; in the second study variant, the preparation according to the proposed composition was introduced into a monolayer cell culture at a dose of 0.5 ml per 10 ml of medium. Studies were performed on the cell line SPEV-2. The cultivation was carried out for 48 hours, after which the cells were removed by trypsinization and they determined the intensity of respiration in the MTT test. Table 3 shows comparative indicators of changes in respiratory activity in the cell population when they are cultured in the presence of the prototype preparation and the proposed immunostimulating composition (P <= 0.05).

Table 3 Comparative indicators of changes in respiratory activity in the cell population when they are cultured in the presence of the drug according to the prototype and the proposed immunostimulating composition (P <= 0.05) Indicator Monolayer culture of the cell line SPEV-2 (Control) The preparation of the prototype + monolayer culture of the cell line SPEV-2 Immunostimulating composition + monolayer culture of cell line SPEV-2 The concentration of formazan in 1 cell, Ng / ml 0.015217 ± 0.02545 ± 0,071245 ± 0,003381 0.005653 0.012305

As can be seen from table 3, when the cells are cultured in the presence of an immunostimulating composition, respiratory activity increases by 4 times, compared with the control, and also more than 2 times compared with the prototype preparation, which indicates the ability of the proposed immunostimulating composition for animals to activate redox processes of cellular metabolism.

Claims (1)

An immunomodulating composition for animals containing, as an active ingredient, milk whey protein lactoferrin, as a solvent distilled water, characterized in that the active substance includes, in addition to lactoferrin, milk whey proteins lactoalbumin and lactoglobulin, wherein the active substance is an aqueous solution of a mixture of whey proteins milk with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin, except o, the specified active substance additionally contains selenium nanoparticles in the following ratio of components, wt.%:
an aqueous solution of a mixture of milk whey proteins with a total protein concentration of 10 g / l, containing 10-20% lactoferrin, 20-30% lactoalbumin, 60-70% lactoglobulin 0.1-100, selenium particles 0.0001-1.0, distilled water up to 100.