RU2340352C2 - Method of correction of biological activity of milk and application of immunofan and amyxine for such correction - Google Patents

Abstract

FIELD: medicine; veterinary science.

SUBSTANCE: administer an immunopotentiator to females of mammals into a lactemia initial stage. The immunopotentiator immunifan is administered to lactating females in a dose of mass of a body of 0.0006-0.0008 mg/kg. The immunopotentiator amyxine is administered to lactating females in a dose of mass of a body of 0.0017-0.0018 g/kg.

EFFECT: increase of content of immunoglobulins, leucocytes and mononuclear cells in milk of lactating animals.

Description

The invention relates to the field of agriculture, to veterinary medicine.

Milk is the perfect product of nature. Among human food, it plays an important role. A person eats the milk of many mammals - cows, mares, goats, moose and less often - milk of other mammals. The composition of milk includes various ingredients, ensuring its high biological activity. However, in the process of long-term breeding of farm animals, there was a significant decrease in biological activity, for example, of cow's milk, which is associated with the selection of milk based on the quantity and fat content without taking into account its biological activity. Therefore, the task of correcting the biological activity of milk is relevant.

There is a method of correcting the biological activity of human food products by introducing biologically active ingredients, such as vitamins, trace elements (D. Thatcell, D. Wells. You and your food. M., Rosman, 1996, p.12-17) [1] .

In this known manner, the biologically active ingredients are introduced exogenously into the products. Only some components of the biological activity of food products are subject to correction. For example, vitamins, microelements, macroelements are introduced into milk, but methods for correcting the biological activity of milk by introducing important ingredients such as immunoglobulins, white blood cells, or mononuclear cells into it are not described. The introduction of such ingredients in milk will provide an opportunity to use it for the prevention and treatment of a wide range of diseases when it is eaten.

A known method for correcting the biological activity of milk by introducing to mammalian females an agent inducing the enrichment of the milk they produce with biologically active ingredients (Sarker SA at al. Successful treatment of rotavirus diarrhea in children with immunoglobulin from immunized bovine colostrum. Pediatr. Infect. Dis. J., 1998 ; 17: 1149-1154) [2].

In this known manner, cows are immunized with rotavirus, which leads to the correction of the biological activity of their milk - it increases the content of antibodies to rotavirus.

However, the correction of the biological activity of milk by this known method is insufficient.

There is a method of correcting the biological activity of milk by introducing to mammalian females an agent that induces the enrichment of the milk they produce with biologically active ingredients (Casswall T.N. at al. Treatment of Heliobacter pylori infection in infants in rural Bangladesh with oral immunoglobulins from hyperimmune bovine colostrums. Aliment. Pharmacolums. . Ther., 1998; 12: 563-568) [3].

This known method provides for the introduction to female mammalian cultures of Heliobacter pylori cells (immunization of females), as a result of which the content of antibodies to this infectious disease agent increases in the milk of the females. The enrichment of milk produced by females with important biologically active ingredients in this known method occurs endogenously, naturally, which improves the quality of the target product, eliminates the risk of introducing extraneous microflora into it, which can occur with the exogenous introduction of biologically active ingredients into milk.

However, milk obtained from females in this known manner has a low biological activity.

Known hexapeptide arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine, named immunofan, used as an immunostimulant (Leskov VP Clinical immunology for doctors. M., "Medicine", 2005, [4] p. 126 )

However, the use of immunofan as an agent inducing a change in the biological activity of milk in female mammals is not described.

The use of amixin as an immunostimulant is known ([4], pp. 126-128).

However, its use as an agent inducing a change in the biological activity of milk in female mammals is not described.

The technical result achieved by the present invention is to increase the correction efficiency of the biological activity of milk, expanding the range of products that induce the correction of the biological activity of milk.

This technical result is achieved due to the fact that immunostimulants are used as a means of inducing the enrichment of milk produced by them with biologically active ingredients in a method for correcting the biological activity of milk by introducing to mammalian females an immunostimulant.

It is advisable to use immunofan as an immunostimulant, which is administered to lactating females intramuscularly in the initial period of lactation at a dose of 0,0006-0,0008 mg / kg body weight.

Amixin can also be used as an immunostimulant, which is administered orally to lactating females in the initial period of lactation at a dose of 0.0017-0.0018 g / kg body weight.

This technical result is also achieved by recommending the use of imunofan for a new purpose - as a means of correcting the biological activity of milk in female mammals.

This technical result is also achieved by recommending the use of amixin for a new purpose - as a means of correcting the biological activity of milk in female mammals.

The following are examples illustrating the invention.

Example 1

10 individuals of lactating mice - females F ₁ (CBA × C57BL) were divided into two groups (experimental and control), 5 mice in each group. In the initial period of lactation (one day after the birth), the females of the experimental group were injected intramuscularly with the immunofimulator immunofan at a dose of 0,0006 mg / kg body weight. Immunostimulants were not administered to mice of the control group. On the sixth day after delivery, the content of mononuclear cells producing gamma-interferon in the blood of experimental animals was 110 · 10 ⁶ cells / ml, and in the blood of control animals - 46.67 · 10 ⁶ cells / ml. On the third and sixth day after delivery, the content of immunoglobulin IgG, leukocytes and mononuclear cells producing interferons was determined in the milk of animals. On the third and sixth days after birth, the IgG content in milk increased, respectively, by 8.2% and 12.6% relative to its content in the milk of control animals. The increase in the content of leukocytes in the milk of experimental animals compared with their content in the milk of control animals amounted to 7.9% and 18.6%, respectively, on the third and sixth days after delivery. At the same time, the content of mononuclear cells increased by 9.2% and 21.8%, respectively, compared with the control.

Example 2

10 individuals of lactating mice - females F ₁ (CBA × C57BL) were divided into two groups (experimental and control), 5 mice in each group. One day after the birth, the experimental group females were injected intramuscularly with immunofan immunostimulant at a dose of 0,0008 mg / kg body weight. Immunostimulants were not administered to mice of the control group. On the sixth day after delivery, the content of mononuclear cells producing gamma-interferon in the blood of experimental animals was 122.67 · 10 cells / ml, and in the blood of control animals - 54.61 · 10 ⁶ cells / ml. On the third and sixth day after delivery, the content of immunoglobulin IgG, leukocytes and mononuclear cells producing interferons was determined in the milk of animals. On the third and sixth day after delivery, the IgG content in milk increased, respectively, by 8.4% and 12.8% relative to its content in the milk of control animals. The increase in the leukocyte content in the milk of experimental animals in comparison with their content in the milk of control animals amounted to 8.4% and 18.88%, respectively, on the third and sixth day after delivery. At the same time, the content of mononuclear cells increased, respectively, by 9.6% and 22.1% compared with the control.

Example 3

10 individuals of lactating mice - females F ₁ (CBA × C57BL) were divided into two groups (experimental and control), 5 mice in each group. A day after delivery, the amixin immunostimulant was administered to females of the experimental group at a dose of 0.0017 g / kg body weight. Immunostimulants were not administered to mice of the control group. On the sixth day after giving birth, the content of mononuclear cells producing gamma-interferon in the blood of experimental animals was 108 · 10 ⁶ cells / ml, and in the blood of control animals - 45.67 · 10 ⁶ cells / ml. On the third and sixth day after delivery, the content of immunoglobulin IgG, leukocytes and mononuclear cells producing interferons was determined in the milk of animals. On the third and sixth day after delivery, the IgG content in milk increased, respectively, by 8.3% and 12.4% relative to its content in the milk of control animals. The increase in the leukocyte content in the milk of experimental animals in comparison with their content in the milk of control animals amounted to 7.7% and 18.8%, respectively, on the third and sixth day after delivery. At the same time, the content of mononuclear cells increased by 9.5% and 21.5%, respectively, compared with the control.

Example 4

10 individuals of lactating mice - females F ₁ (CBA × C57BL) were divided into two groups (experimental and control), 5 mice in each group. A day after delivery, the amixin immunostimulant was administered to females of the experimental group at a dose of 0.0018 g / kg body weight. Immunostimulants were not administered to mice of the control group. On the sixth day after delivery, the content of mononuclear cells producing gamma-interferon in the blood of experimental animals was 122.27 · 10 ⁶ cells / ml, and in the blood of control animals - 54.65 · 10 ⁶ cells / ml. On the third and sixth day after delivery, the content of immunoglobulin IgG, leukocytes and mononuclear cells producing interferons was determined in the milk of animals. On the third and sixth days after birth, the IgG content in milk increased, respectively, by 8.2% and 12.6% relative to its content in the milk of control animals. The increase in the content of leukocytes in the milk of experimental animals in comparison with their content in the milk of control animals increased, respectively, by 8.8% and 18.5%. At the same time, the content of mononuclear cells increased, respectively, by 9.5% and 22.3% compared with the control.

Example 5

The experiments used 15 black-motley cows of the Kholmogorsk breed weighing 450-550 kg, age 4-6 years, a month after calving. Cows were divided into three groups of 5 cows per group. 1 group of cows (No. 1-5) was injected with immunofan 0.15 mg, v / m, 10 injections daily. The second group (No. 6-10) was injected with glutoxim 20 mg, IM, 10 injections daily. The third group (No. 11-15) - control. 10 days after the last injection, the content of white blood cells and IgG in milk was evaluated. To count white blood cells, 10 ml of milk was centrifuged for 10 min at 1100 rpm. Then, in the same mode, washed three times with saline. After washing, up to 1 ml of physiological saline and gentian violet were added to the precipitate. The number of stained white blood cells was counted in the Goryaev’s chamber. IgG content was determined by ELISA using monoclonal antibodies to cow IgG. The research results are presented in table 1.

No. Animal name Protein% C. B-VA% Pl The number of leukocytes in 100 ml IgGMg / L one Summary 3,7 9.71 27.3 8 290 2 Point 3,1 11.5 29.0 6 295 3 Tikhonya 3.2 11.53 31.3 7 300 four Darkness 3,7 11.84 26.8 6 320 5 Earring 3,1 11,4 29.8 8 300 6 Selyanka 2.82 10.00 28.0 eleven 285 7 Candle 2,8 10.68 26.9 13 290 8 Sawdust 2.83 10.28 27.9 12 270 9 Mat 2.99 9.58 29.5 9 280 10 Infantry 2.6 13.84 22.3 10 282 eleven Quiet 3.0 9.50 29.7 8 220 12 Petunia 3.02 11.10 29.9 8 280 13 Chenille 3.08 10.50 30.5 7 240 fourteen Motley 3,1 10.80 30.6 6 265 fifteen Titania 2.91 11.00 28.7 9 270

Table 2 presents the average values of the content of white blood cells and IgG in milk

TABLE 2 Group number Used immunostimulant The cell content in milk in thousand / 100 ml IgG content, mg / l one Imunofan 7 ± 0.9 300 ± 10 2 Glutoxim 11 ± 1.4 282 ± 6.6 3 No (control) 7.6 ± 1 265 ± 22 Note: ± - confidence interval.

The tables show that ten days after the cessation of the administration of immunostimulants, the leukocyte content in the milk of cows injected with glutoxim is statistically significantly increased. The IgG content is statistically significantly increased in cows that were injected with immunofan. It is assumed that the joint administration of these drugs can increase the content in the milk of both white blood cells and IgG.

Table 3 shows the results of the administration of amixin to cows in comparison with control individuals.

Table 3 No. Animal name The number of leukocytes in 100 ml IgG Mg / L Amixin one Slingshot 8 295 2 Motley 6 295 3 Eyelet 7 300 THE CONTROL four Quiet 8 220 5 Petunia 8 280 6 Chenille 7 240 7 Motley 6 265 8 Titania 9 270

Example 6

Female mice F ₁ (CBA × C57BL) (no. 5 mice in the group) were injected with diuciphon 250 μg / animal one day after delivery. After one day, the content of gamma-interferon producing cells was evaluated by the Elisspot method. It was shown that the number of gamma-interferon producing cells increases from 46.67 ± 7.94 per 10 ⁶ mononuclear cells in the control to 88 ± 10.5 in animals that were injected with diucifon.

From the above results it is seen that under the influence of immunostimulants the immunological potential of milk increases. This is evidenced by an increase in the content of IgG and white blood cells in milk. IgG is the main immunoglobulin responsible for the neutralization of pathogenic microorganisms. White blood cells - cells that secrete substances (interferons, interleukins, lactoferrin, etc.), contributing to the formation of the immune and hematopoietic systems, and reduce the allergization of the body.

Thus, the invention allows to expand the range of tools that contribute to the correction of the biological activity of milk of mammalian animals in the direction of its increase. The use of these products for cows will provide milk with improved biological properties. These funds provide an increase in the content of immunoglobulins in milk, which help to strengthen the human immunity when eating such milk. Means also increase the content of white blood cells in the milk, which absorb the cells of pathogens. The content of mononuclear cells, which are producers of interferons, also increases in milk. The use of these agents by the method according to the invention eliminates the use of techniques for exogenously replenishing milk with necessary, biologically active ingredients important for humans. The means recommended by the invention are means of changing, improving the composition of milk, increasing its biological activity, enriching it with the qualitative and quantitative composition of important biologically active ingredients.

Claims (5)

1. A method for correcting the biological activity of milk by administering to female mammals an agent that induces the enrichment of the milk they produce with biologically active ingredients, characterized in that immunostimulants are used as a means of inducing the enrichment of milk with immunoglobulin, leukocytes or mononuclear cells. 2. The method according to claim 1. characterized in that immunofan is used as an immunostimulant, which is administered to lactating females intramuscularly in the initial period of lactation at a dose of 0,0006-0,0008 mg / kg body weight. 3. The method according to claim 1, characterized in that amixin is used as an immunostimulant, which is administered orally to lactating females in the initial lactation period at a dose of 0.0017-0.0018 g / kg body weight. 4. The use of immunofan as a means of correcting the biological activity of milk in female mammals. 5. The use of amixin as a means of correcting the biological activity of milk in female mammals.