RU2257904C2 - Immunomodulator preparation method - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: invention relates to food additives that can be used to prevent immunodeficiency conditions. Immunomodulator is prepared via homogenization of lymphatic nodes in sodium chloride solution, autolysis, centrifugation, heating of supernatant to 80°C, cooling, removal of precipitate, and drying of supernatant by cooling it and placing it into atmospheric sublimation installation followed by subsequent post-drying using IR drying technique.

EFFECT: simplified immunomodulator preparation process and preserved biological and food values of finished product without creation of special temperature regime.

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Description

The invention relates to the field of food industry, in particular to methods for producing food additives, and can be used for the prevention of immunodeficiency conditions.

Currently, the problem of regulation and restoration of the protective functions of the body in diseases and exposure to destabilizing environmental factors is one of the urgent in physiology and medicine. In recent years, there has been a trend towards the creation and use of medicines from raw materials of natural origin, including animals. One of the promising and cheap sources of biologically active substances is the organs of the immune system of farm animals (thymus, spleen, bone marrow, lymph nodes), which, it should be noted, are classified as waste from the main production.

A number of studies (Morozov V.G., Khavinson V.Kh., 1982-1985; Kuznik B.I., Tsybikov N.N. and others, 1979-1996) established the existence in humans and animals of a new class of regulatory peptides - cytomedines . The latter are a collection of polypeptides with a molecular weight of up to 10 kDa, possessing tissue specificity and organotropy, responsible for the transmission of information in a population of specialized cells. An experimental and clinical study of the biological activity of cytomedines shows that they provide restoration of the lost functions of those organs and systems from which they were obtained (Morozov V.G., Khavinson V.Kh., 1975-1985; Chipsns G.I., 1984, Denisov A.B., 1991, etc.). The first confirmations were obtained with complexes of polypeptides isolated from the central and peripheral organs of the immune system - the thymus, bone marrow, spleen, lymph nodes of cattle, as well as from the bag of Fabricius birds. Cytomedins from these organs have an immunomodulatory effect, while contributing to the normalization of nonspecific resistance of the body and the homeostasis system. The quick response of the body to the introduction of peptides, their high activity, the absence of side effects are increasingly attracting the attention of pharmacologists and clinicians to peptides of endogenous origin - potential drugs of a new class.

The main preventive measure in the development of diseases is the exogenous intake of these bioregulators that increase the protective functions of the body with nutritional support.

The problem of preserving for a sufficiently long time the initial properties of biological materials is extremely urgent. The successful practical use of technologies for preserving "living systems" by the method of freeze-drying is the most progressive. Therefore, the objective of the invention was to develop methods for preserving the immunomodulator by atmospheric freeze drying, followed by storage without creating any special temperature conditions.

The use of immunotropic preparations obtained from thymus (T-activin, thymalin) and spleen (splenin), the production of which requires a significant investment of time, reagents and the use of expensive equipment (see Auth. St. No. 997298, C. A 61 K 37/24, 1979).

A known method of obtaining an immunostimulant for medical purposes, having an immunostimulating effect from the lymph nodes of animals by treating them with acetone, freezing, grinding, extracting with a 3% solution of acetic acid and zinc chloride, centrifuging and removing the supernatant, removing ballast substances from the precipitate, adding acetic acid followed by filtration and lyophilization (see Aut. St. SU No. 1187824 A, class A 61 K 35/30, 1985).

A known method of obtaining funds with immunostimulating action from red bone marrow. All techniques and operations are similar to the above example (see RF Patent RU No. 1077089 C, class A 61 K 35/28, 1994).

There is a method for producing immunostimulating factors from gray whale thymus, in which the gray whale thymus salt homogenate is centrifuged in the cold, the supernatant is treated with chilled acetone, the acetone powder is dissolved in phosphate buffer with an alkaline pH value and gel chromatographed on Sephadex G-25 (see Auth. St. SU 1506668 A1, class A 61 K 35/26, 38/00, 1996).

A known method of obtaining immunomodulating peptides from the bone marrow of mammals, including homogenization, extraction with 3% trichloroacetic acid, 24-hour incubation at -4 ° C, subsequent dialysis, salting out with ammonium sulfate, treatment of the supernatant with cooled acetone, precipitation of the protein complex for 24 hours with its subsequent extraction by repeated extraction with double distillate and lyophilization (see Aut. St. SU 1836954 A1, class A 61 K 35/28, 1990).

However, the known methods are difficult to perform and inappropriate to obtain an immunostimulant in the form of a dietary supplement.

A known method of producing an immunostimulant as a food supplement, in which bone marrow cells are isolated from the bone marrow of small cattle, washed and incubated in an oncotic solution with a concentration of 0.85-0.90%, and then the supernatant is centrifuged, which is the target product . When choosing methods for preserving a food composition, cooling and freezing methods were considered (see patent RU 2120799 C1, A 61 K 35/28, 1998).

This method is similar in purpose and use of physiological solutions, but unacceptable for maintaining for a long time the biological value of the immunomodulator.

The closest method to the claimed invention in terms of features is a method for producing immunostimulatory polypeptides from animal feedstock, in which the reindeer thymus is homogenized in a sodium chloride solution, autolized, centrifuged, the supernatant is heated to 80 ° C, cooled, the precipitate is removed, treated with acetone , precipitate is formed, washed three times with acetone, dried, followed by dissolution in 0.01 M Tris-HCl buffer, centrifuged, ultrafiltered, chromed atografiyu on Sephadex followed by collection of the fractions containing the desired product were lyophilized, subjected to gel filtration, re-lyophilized (see. Ed. St. SU 1737798 A1, cl. A 61 K 38/00 // A 61 K 35/30, 1989).

However, the known method is time consuming, because requires a significant investment of time and the use of expensive equipment, as well as the use of acetone, the use of which does not allow its use for food purposes.

Thus, the problem solved in the present invention is to obtain an immunomodulator as a component of food products. The technical result obtained by the implementation of this method is the simplification of the process, the modification of canning methods, the expansion of the range of finished products for functional use.

The specified technical result is achieved by the fact that in the method for producing an immunostimulant, including the isolation of polypeptides from animal feedstock, homogenization in a sodium chloride solution, autolysis, centrifugation, heating of the supernatant, cooling, sediment removal, drying of the supernatant, lymph nodes are used as raw materials according to the invention animals, while the drying of the supernatant is carried out by freezing it and placing it in an atmospheric freeze-drying unit, followed by suturing using infrared drying.

Distinctive features of the proposed method are the use of animal lymph nodes as a raw material and new methods of preserving the product, namely, by freezing and atmospheric freeze-drying.

The prior art is the use of lymph nodes to obtain an immunostimulant (see Aut. St. SU No. 1187824 A, class A 61 K 35/30, 1985), but it is intended for medical purposes, and the method itself is complex, expensive, and the proposed method of obtaining an immunostimulant from lymph nodes is simple to perform, it allows you to store the finished product without creating any special temperature conditions, while maintaining its biological and nutritional value.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the claimed invention, allowed to establish that the applicant did not find a source characterized by features identical to all the essential features of the claimed invention. Comparison of the proposed method with the prototype, the closest in the totality of the features of the analogue, allowed to identify the set of essential distinguishing features set forth in the claims to the technical result perceived by the applicant.

Therefore, the claimed invention meets the conditions of "novelty" and "inventive step".

The inventive method of obtaining an immunomodulator is as follows:

1 kg of lymph nodes are cleaned of connective tissue, homogenized in 0.15 M NaCl in a ratio of 1: 3. The resulting homogenate is autolysed at 4-6 ° C for 12-16 hours, centrifuged at 4000 rpm for 10 minutes. Then the supernatant is heated with stirring in a water bath to 80 ° C and maintained at this temperature for 10 minutes. After cooling, a copious amount of denatured proteins is removed by centrifugation at 5000 rpm for 30 minutes. In the supernatant, the amount of protein is determined by Lowry. Its content is 8.9 mg / ml. The resulting product (the active fraction of the lymph nodes - AFL) is subjected to canning by atmospheric freeze-drying, which includes the processes of freezing, sublimation and drying. Previously, the product is poured into 20 ml and frozen in a special chamber (-10-12 ° C). The frozen product is placed in an atmospheric sublimation unit for 30-32 hours at a temperature of -8-12 ° C. To determine the degree of drying, determine the moisture content (moisture content of at least 10-12%). Sublimation product requires drying to 4-5% moisture content, which is a prerequisite for long-term storage of dry product. The drying is carried out using infrared drying at a temperature of 30-45 ° C for 20 minutes. The finished product - lymph node polypeptides - a yellowish-white powder.

Recovery of the freeze-dried product: 20 ml of distilled water are added to the dried product. Determine the amount of protein according to Lowry. Its content is 8.7 mg / ml.

The inventive method is confirmed by the following examples of specific performance:

Example 1

1 kg of lymph nodes are cleaned of connective tissue, homogenized in 0.15 M NaCl in a ratio of 1: 3. The resulting homogenate is autolysed at 4-6 ° C for 14 hours, centrifuged at 4000 rpm for 10 minutes. Then the supernatant is heated with stirring in a water bath to 80 ° C and maintained at this temperature for 10 minutes. After cooling, a copious amount of denatured proteins is removed by centrifugation at 5000 rpm for 30 minutes. In the supernatant, the amount of protein is determined by Lowry. Its content is 7.9 mg / ml. The product is poured into 20 ml and frozen in a special chamber (-12 ° C). The frozen product is placed in an atmospheric sublimation unit for 32 hours at a temperature of -8 ° C. The drying is carried out using infrared drying at a temperature of 30 ° C for 20 minutes. The finished product - lymph node polypeptides - a yellowish-white powder.

Recovery of the freeze-dried product: 20 ml of distilled water are added to the dried product. Determine the amount of protein according to Lowry. Its content is 7.7 mg / ml.

The study of the biological activity of the polypeptides obtained by the proposed method was carried out on mice of both sexes of the F1 line (CBAxC57B1 / 6) with an average body weight of 20-22 grams in the reactions of Staph phagocytosis. aureus peritoneal macrophages in vitro and in vivo according to the method of I. S. Freidlin [I. S. Freidlin. Using culture of mouse peritoneal macrophages as models for studying cells of the mononuclear phagocytic system of the body and their use under the influence of biologically active substances // Method. recommended - L., 1976]. In phagocytosis reactions, two indicators were calculated: activity (percentage of phagocytic cells of the total number of macrophages counted) and intensity (average number of microorganisms absorbed by one cell). The suppression of macrophage functions was modeled by the introduction of the classical immunosuppressant azathioprine (at a concentration of 500 μg / ml into the culture medium in an in vitro experiment and orally for 5 days at a dose of 50 mg / kg body weight in an in vivo experiment). The action of azathioprine caused a decrease in both the activity and the intensity of phagocytosis in both experiments by 45% and 40%, respectively. The polypeptides were introduced into the medium in an in vitro experiment at a concentration of 0.1 mg / ml and orally for 7 days at a dose of 100 μg / kg body weight in an in vivo experiment.

Analysis of the obtained data showed that the introduction of polypeptides in these experiments, against the background of preliminary exposure to azathioprine, significantly exceeded both indicators and restored them to the level of an intact group of animals.

According to these data, it can be concluded that the polypeptides have a stimulating effect on the phagocytic activity of macrophages both in vitro and in vivo.

Example 2

Lymph nodes are cleaned of connective tissue, homogenized in 0.15 M NaCl in a ratio of 1: 3. The resulting homogenate is autolysed at 4-6 ° C for 16 hours, centrifuged at 4000 rpm. 10 minutes. Then the supernatant is heated with stirring in a water bath to 80 ° C and maintained at this temperature for 10 minutes. After cooling, a copious amount of denatured proteins is removed by centrifugation at 5000 rpm for 30 minutes. In the supernatant, the amount of protein is determined by Lowry. Its content is 8.2 mg / ml. The product is poured into 20 ml and frozen in a special chamber (-12 ° C). The frozen product is placed in an atmospheric sublimation unit for 30 hours at a temperature of -10 ° C. The drying is carried out using infrared drying at a temperature of 40 ° C for 15 minutes. The finished product - lymph node polypeptides - a yellowish-white powder.

Recovery of the freeze-dried product: 20 ml of distilled water are added to the dried product. Determine the amount of protein according to Lowry. Its content is 8.0 mg / ml.

The biological activity of the polypeptides obtained by the proposed method was studied on male guinea pigs weighing 150-250 grams in the reaction of the "active rosette" of thymocytes with rabbit erythrocytes, which was carried out according to the standard method [Temporary pharmacopoeial article BC 42-1571-85], studied the effect of the obtained preparation for expression of receptors for these immunocompetent cells. The polypeptides were administered at a concentration similar to the first example. From the analysis of the results it follows that the number of rosetting cells (ROCK) among intact thymocytes was 42%, and after trypsin treatment it was 10%. The introduction of polypeptides into a system containing trypsin-treated thymocytes restored the amount of ROCK to 51%, which indicates an increase in the expression of receptors on the surface of guinea pig thymocytes by polypeptides and the growth-promoting activity of polypeptides in relation to these cells.

In our experiment, we studied the morphofunctional state of the inguinal lymph nodes of mice when exposed to azathioprine and AFL. The need to study the structural organization of somatic lymph nodes is associated with the identification of the reactivity of peripheral inguinal lymph nodes distant from the injection site of azathioprine immunosuppressant and a bioregulator from the lymph nodes.

The data are presented in the following tables.

Table 1
The distribution density of cells in the structural components of the inguinal lymph nodes of CBA mice in the experiment per unit area of the histological section (880 μm. Sq.) ANIMAL GROUP STRUCTURAL COMPONENTS Paracortical zone Mantle of lymphoid nodules Nodule Reproduction Centers Pulp Brain sinuses Control 41.6 ± 2.08 60.6 ± 3.03 43.8 ± 2.19 33.0 ± 1.65 18.8 ± 0.94 Azathioprine 47.6 ± 2.38 - - 31.8 ± 1.59 20.4 ± 1.02 Az. + AFL-2 48.6 ± 2.43 62.2 ± 3.11 41.2 ± 2.06 51.8 ± 2.59 33.8 ± 1.69 table 2
The cellular composition of the structural components of the inguinal lymph nodes of mice of various experimental groups (%) CELLS Groups of animals STRUCTURAL COMPONENTS Paracortical zone Mantle of lymphoid nodules Breeding centers Pulp Brain sinuses Reticular 1 9.2 ± 0.46 8.9 ± 0.44 11.8 ± 0.45 4.8 ± 0.24 6.4 ± 0.32 2 12.6 ± 0.63 0 0 15.1 ± 0.75 15.7 ± 0.78 3 14.8 ± 0.74 8.4 ± 0.42 13.6 ± 0.68 18.5 ± 0.93 20.2 ± 1.01 Blasts 1 4.8 ± 0.24 1.0 ± 0.05 4.1 ± 0.20 1.20 ± 0.06 2.2 ± 0.11 2 0.40.02 0 0 0 0 3 2.1 ± 0.1 1.9 ± 0.09 3.4 ± 0.17 1.2 ± 0.06 1.2 ± 0.06 Lymphocytes (total count) 1 60.1 ± 3.00 76.5 ± 3.82 50.2 ± 2.51 35.2 ± 1.76 35.2 ± 1.76 2 52.5 ± 2.62 0 0 52.9 ± 2.642 29.4 ± 1.47 3 50.6 ± 2.53 76.3 ± 3.81 58.2 ± 2.91 39.8 ± 1.99 34.0 ± 1.7 Large lymphocytes 1 11.5 ± 0.55 8.9 ± 0.37 15.5 ± 1.34 5.5 ± 0.98 2.2 ± 0.16 2 5.9 ± 0.99 0 0 5.1 ± 0.63 2.1 ± 0.12 3 12.4 ± 0.61 13.2 ± 0.66 14.1 ± 0.71 8.5 ± 0.42 13.0 ± 0.65 Plasma cells 1 0.9 ± 0.05 0 0.5 ± 0.03 19.4 ± 0.97 0 2 1.7 ± 0.08 0 0 1.9 ± 0.08 0 3 1.3 ± 0.06 1.6 ± 0.08 1.5 ± 0.07 4.3 ± 0.2 8.3 ± 0.42 Plasma blasts 1 2.8 ± 0.21 0 3.6 ± 0.21 11.0 ± 1.07 6.4 ± 0.48 2 3.4 ± 0.28 0 0 5.9 ± 0.43 5.9 ± 0.34 3 2.5 ± 0.12 0.9 ± 0.06 1.9 ± 0.08 10.1 ± 0.51 7.7 ± 0.32 Macrophages 1 2.8 ± 0.14 2.7 ± 0.14 8.7 ± 0.44 6.7 ± 0.34 19.2 ± 0.96 2 6.7 ± 0.33 0 0 1.3 ± 0.07 3.9 ± 0.19 3 10.3 ± 0.52 3.5 ± 0.17 5.4 ± 0.227 5.1 ± 0.25 10.6 ± 0.53 Destruction 1 18.7 ± 0.94 11.2 ± 0.56 21.0 ± 1.05 18.8 ± 0.94 28.7 ± 1.44 2 22.7 ± 1.14 0 0 22.0 ± 1.10 42.2 ± 2.11 3 18.5 ± 0.93 7.4 ± 0.37 16.0 ± 0.8 17.7 ± 0.88 18.4 ± 0.92 Note: 1 - intact group; 2 - azathioprine; 3 - AFL

As can be seen from tables 1 and 2, the administration of azathioprine to mice led to a decrease in the distribution density of cells in various structural components of the node compared to the intact group of animals: - in the mantle of lymphoid nodules and in the reproduction centers by 100%. The use of AFL in a dose of 0.1 mg / kg and within 8 days after the administration of azathioprine restored the levels to the level of intact animals. Along with morphological changes in the organ, the administration of azathioprine leads to marked shifts in the cellular composition - there is a violation of the processes of blast transformation and maturation of lymphoid cells, as a result of which the blast forms of cells disappear. In the paracortical zone, individual blasts remain, but their number, relative to the control, decreases by 10 times. The action of azathioprine also leads to a decrease in the number of antibody-producing cells. In the experiment, plasmocytes were not detected in the cortical substance of the inguinal lymph nodes, as well as in lymphoid nodules without reproduction centers and in the sinuses of the lymph nodes. Destructive processes are intensifying. So, in the lumens of the sinuses, the number of destructively altered and destroyed cells increases from 28% in the control to 42.25% after the administration of azathioprine. At the same time, the macrophage reaction was sharply reduced; the number of macrophages in them was 5 times less than in the control. With the introduction of AFL, equilibrium in the population of lymphoid cells is restored to the level of control values or approaches them. In the body, the reproduction centers in the lymphoid nodules are restored, the number of young forms of cells and plasma cells is increasing. Cell destruction is reduced.

As you know, biologically active peptides of the thymus, bone marrow, spleen have immunomodulating properties. We found that AFL also restores the immune response.

The effect of AFL on the indicators of the cellular (graft versus host reaction - GVHD, delayed-type hypersensitivity reaction - HRT) and humoral (determining the number of antibody-forming cells - AOK in the spleen) of the immune response was studied in mice of both sexes with CBA and F ₁ lines (CBA × C57B1 / 6) weighing 18-20 g. The immunosuppression state was modeled by oral administration of the classic immunosuppressant, azathioprine, at a dose of 50 mg / kg of animal weight for 5 days.

Table 3
The degree of development of the delayed-type hypersensitivity reaction (HRT) when exposed to AFL in mice with azathioprine (M ± m), n = 10 No. p / p Groups Dose mg / kg The reaction index (%), P 1. Intact animals (control) 0 46.7 ± 3.4 2. Azathioprine (Az.) fifty 32.2 ± 1.4 p ₁ <0.01 3. AFL 0.1 48.5 ± 2.8 4. Az + AFL 0.01 44.7 ± 2.7 p2 <0.001 5. Az + AFL 0.1 46.4 ± 2.9 p2 <0.001 6. Az + AFL 1 37.3 ± 1.5

As can be seen from table 2, the inhibition of the delayed-type hypersensitivity reaction with azathioprine was 31.05%. The use of AFL in a dose of 0.1 mg / kg of weight led to a complete restoration of indicators to the control level.

Table 4
The effect of AFL on the number of antibody-forming cells (AOK) of the spleen of CBA mice with azathioprine immunosuppression (M ± m), n = 10 No. p / p Groups Dose mg / kg The absolute amount of AOK in the spleen parenchyma Relative amount of AOK (per 10 ⁶ splenocytes) 1. Intact animals 263161 ± 19135 1688 ± 101 2. Azathioprine fifty 104011 ± 7625 * ¹ 1046 ± 74 * ¹ 3. AFL 0.1 332485 ± 16935 2704 ± 105 4. Az + AFL 0.1 245999 ± 21445 * ² 1668 ± 107 * ² 5. Az + AFL 0.01 201136 ± 16734 * ² 1447 ± 125 ** ² 6. Az + AFL 1 173012 ± 13496 * ² 1031 ± 59 * ^{1 (2)} - significance p <0.001 relative to group No. 1 (2)
** ² - reliability p <0.02 relative to group No. 2 Table 5
The degree of development of the reaction "graft versus host" (GVHD) with azathioprine immunosuppression and exposure to AFL (M ± m), n = 10 No. p / p Groups Dose mg / kg Lymph node enlargement index, P 1. Intact animals 0 2.7 ± 0.2 2. Azathioprine fifty 1.67 ± 0.04 p ₁ <0.001 3. AFL 0.1 2.64 ± 0.16 4. Az + AFL 0.1 2.22 ± 0.12 5. Az + AFL 0.01 2.04 ± 10.07 p ₂ <0.05 6. Az + AFL 1 1.84 ± 0.05 p ₂ <0.05 p ₁ - significantly relative to control;
p ₂ - significantly relative to the level of suppression (Az.)

The results presented in tables 3, 4 and 5 indicate that AFL restores indicators of both the cellular component of the immune response and the humoral response in immunosuppression caused by azathioprine.

According to the results of studies of the activity of the substance obtained by the proposed method, it is clear that it is a stimulator of the body's immune system. This, in turn, allows us to recommend the resulting immunomodulator as an integral component of food products in conditions accompanied by suppression of the immune system.

Thus, the above information indicates that when using the claimed invention, the implementation of the "method of obtaining an immunomodulator", having the following set of advantages compared to the prototype: the inventive method allows to obtain a polypeptide preparation, simplifying the process, and expanding the raw material base for obtaining a food immunomodulator using cheap local raw materials.

Therefore, the claimed invention meets the condition of "industrial applicability".

Claims (1)

A method for producing an immunomodulator, including the isolation of polypeptides from animal feedstock, homogenization in sodium chloride solution, autolysis, centrifugation, heating of the supernatant, cooling, sediment removal, drying of the supernatant, characterized in that the lymph nodes of the animals are used as raw materials, while the drying of the supernatant carried out by freezing it and placing in an atmospheric freeze-drying unit, followed by drying using infrared drying.