EA024516B1 - Biologically active food additive - Google Patents

Abstract

The invention relates to the field of medicine, concerns a biologically active food additive (BAFA), namely vitamin-mineral-probiotic complex with wide spectrum of physiological effects, providing balanced supply of vitamins and mineral substances into organism, as well as increase of organism resistance to impact of unfavourable factors due to stimulation of growth and activity of lactobacilli of natural endogenic intestinal microflora, activation of non-fermentative component of antioxidant system and reduction of damaging effect of free radicals, activation of factors of general non-specific protection of organism and enhancement of the immune system, demonstrating adaptogenic and cytoprotective effects, increasing physical and mental workability, which can be used in pharmaceutical and food industry. BAFA is made in solid dosed form as capsules and includes vitamin-mineral and probiotic constituents as well as a carrier. In two capsules with the total weight 1000 mg vitamin-mineral constituent contains an antioxidant combination, which includes vitamins-antioxidants (water-soluble vitamin C in the form of ascorbic acid - 94.5 mg, liposoluble vitamins E in the form of α-tocopherol acetate - 13.6 mg and A in the form of retinol acetate - 0.7 mg, beta-carotene - 3.4 mg) and a selenium-containing compound in the form of sodium selenite NaSeO- 35.1 mcg, a combination of vitamins of group B (thiamine hydrochloride (B) - 1.7 mg, riboflavin (B) - 2.0 mg, pantothenic acid (B) - 6.2 mg, pyridoxine hydrochloride (B) - 2.2 mg, folic acid (B) - 500.1 mcg, cyanocobalamin (B) - 3.9 mcg), as well as vitamins phylloquinone (K) - 174.0 mcg, cholecalciferol (D) - 14.5 mcg, nicotinamide (PP) - 21.0 mg, biotin (H) - 54.9 mg and minerals (magnesium in the form of magnesium oxide - 100.0 mg, iron in the form of iron fumarate - 10.0 mg, zinc in the form of zinc oxide - 7.0 mg, manganese in the form of manganese sulphate - 1.4 mg, copper in the form of copper sulphate - 0.9 mg, iodine in the form of potassium iodide - 129.9 mcg, chromium in form of chromium picolinate - 24.9 mcg). Sterilised dried culture liquid with metabolites of bacteria Bacillus subtilis VKPM (ARCIM) No B-2335 - 5.0 mg is used as a probiotic constituent and zeolite - 195.0 mg is used as a carrier.

Description

The invention relates to medicine, relates to a biologically active food supplement (BAA), namely, a vitamin-mineral-probiotic complex with a wide range of physiological effects, providing a balanced intake of vitamins and minerals, as well as increasing the body's resistance to adverse factors by stimulating the growth and activity of lactobacilli of the natural endogenous intestinal microflora, activating the non-enzymatic link of the antioxidant system and reducing the damaging effects of free radicals, the activation of factors of general nonspecific defense of the body and the strengthening of the immune system, exhibiting adaptogenic and cytoprotective effects, increasing physical and mental performance, which can be used in the pharmaceutical and food industries.

As you know, a special role in maintaining a high level of life of people who care about their health is played by vitamins and minerals, which contribute to energy metabolism in the human body, increase its vital energy and strengthen the immune system. Constant support of the body’s defenses with the help of vitamins, mineral salts, macro- and microelements makes the body more resilient and especially in conditions of intense physical and neuro-emotional stress, when their consumption increases significantly. With a sufficient number of them, a faster and more complete recovery of the body occurs, which, as a result, leads to good health. The problem of vitamin and mineral deficiency is very urgent at the present time, since the universally observed deterioration of the ecological situation and social living conditions lead to the depletion of the body with these valuable and sometimes vital substances. At the same time, their uncontrolled intake can cause a serious imbalance in a number of elements necessary for the normal functioning of the body.

In connection with the increase in the number of oncological, somatic, infectious and other diseases of various etiologies, the severity of their course and the increase in the duration of treatment, the problem of normalizing dysfunctional disorders of the immune system by increasing the general nonspecific resistance of the body to various impacts, which will prevent and alleviate both the diseases themselves and their complications, as well as accelerate rehabilitation after critical conditions.

Of particular importance is the problem of maintaining human activity in an unfavorable environmental situation and in combination with dysbiotic disorders of the composition of the symbiotic microflora of the gastrointestinal tract (GIT) observed in almost 90% of the population [1]. The restoration and maintenance of a healthy microflora of the gastrointestinal tract and its most complex and important biotope - the intestine - is currently receiving the closest attention. The natural physiological microflora of the intestine can be significantly impaired due to inappropriate or unbalanced nutrition, climate or water changes, stress, the development of chronic diseases of the gastrointestinal tract and other organs, during complex therapy with antibiotics or radiation, and a number of other situations. The normal intestinal microflora is extremely important for the macroorganism, as it forms the basis of its microecology and has a direct and significant effect on its condition, performing a number of vital functions. The most significant of these include metabolic and synthetic functions, the function of detoxification, participation in the digestion and formation of the natural immunity of a person, as well as ensuring colonization resistance. A change in the qualitative and / or quantitative composition of the intestinal microbiocenosis leads to a disruption in the processes of assimilation of nutrients and minor components of food and a decrease in the intake of a number of biologically active substances (BAS), including vitamins and minerals, which is manifested by a deterioration in the processes of its vital activity. As a result of such violations, the evolutionarily created barrier of the colonization resistance of the macroorganism is reduced, and, therefore, its susceptibility to the occurrence of infectious diseases of a bacterial and viral nature is increased.

When regulating the functions of the body in order to ensure its normal functioning, one must take into account the factor that one of the triggering mechanisms for the development of various disorders in it is oxidative stress - a state of mismatch in the level of production of free radicals and the capacity of antioxidant systems, as a result of which prerequisites are formed for excessive oxidation of biosubstrates , modifications and irreversible changes in enzyme systems, violation of the structure and permeability of biological membranes, energy deficiency and decreased cell viability [2-7]. Oxidative stress leads to a significant increase in the content of water and fat-soluble prooxidants in tissues and blood, primarily active oxygen metabolites, products of lipid peroxidation and nitric oxide. These substances are extremely reactogenic, and therefore, an uncontrolled increase in their content in the body causes cytotoxic and genotoxic effects. In this case, both the oxidation of polyunsaturated fatty acids of the membranes and the change of proteins, some of which perform an enzymatic function, as well as DNA, occur, which is manifested in an increase in mutagenic effects.

In an intact cell, oxidative processes are under the strict control of various mechanisms of antioxidant protection, which ensure the occurrence of free radical reactions at a stationary level, which is necessary for normal cell activity. An important role in protecting the body against active oxygen radicals is played by a system of enzymatic (superoxidecismutase, catalase, glutathione-dependent peroxidase and transferase, ceruloplasmin, glutathione reductase) [8-12] and non-enzymatic antioxidants (thiol compounds - glutathione, cysteine, taoferin acid, vitamins; , beta-carotene; biogenic amines - serotonin, histamine, catecholamines; oligopeptides - carnitine, carnosine, anserin, endorphins, enkephalins; polyphenols; phospholipids; ubiquinone), with the ability to determine Under normal conditions, suppress the processes of free radical oxidation [13-17].

The impact on the human body of various extreme factors leads to changes in the state of the antioxidant system and if the resources of this protective system are exhausted, an imbalance arises between antioxidant and oxidative processes, which is crucial in the genesis of various forms of pathology [18, 19]. The onset of imbalance leads to significant violations of both the enzymatic and non-enzymatic units of antioxidant protection. The activity of catalase, superoxide dismutase, the content of beta-carotene, tocopherol and ubiquinone in the mitochondria of hepatocytes decreases.

Specialized food products — dietary supplements containing various concentrates of natural or synthetic synthetic biologically active substances [20], are used more and more widely to use to ensure normal functioning of people who care about their health. The daily long-term use of such dietary supplements, both independently and as part of food and beverages, provides the body with the supply of the necessary biologically active substances. Some of these products (dietary supplements) allow you to quickly make up for the deficiency of substances necessary for the body - vitamins, mineral salts, macro- and microelements, certain amino acids, dietary fiber and others within their physiological norm. Others (parapharmaceutical supplements) exhibit a pronounced physiological effect and have a regulatory and stimulating effect on certain functions of the body.

The need for additional intake of special products is associated, first of all, with the use of high-tech processes in the production of basic food products that cause them to be cleansed of many irreplaceable food components, as a result of which a person loses a number of biologically active substances important for his normal life. Prolonged unbalanced nutrition using such products, which often also contain toxins (nitrates, nitrites), leads to a violation of the vital functions of the body and can cause a number of serious diseases.

Thus, the above circumstances determined the need for close attention while ensuring the normal functioning of people both to restore and maintain a healthy microflora of the gastrointestinal tract, and to regulate the processes of free radical oxidation and nonspecific protection, and the prospects for finding dietary supplements with an effective regulatory effect on the human body were associated with the creation of recipes, which, along with their main purpose as additional sources of vitamins and minerals, will be able to support s normal flora of the intestine, activate the non-enzymatic link of antioxidant system and strengthen the immune system by activating factors, general non-specific protection.

Of these, the special development of German scientists BION 3 dietary supplement, including a complex of vitamins, minerals and probiotics, is of great interest [21]. In composition, mechanisms of regulatory effects on the body and achieved beneficial effects of dietary supplement BION 3 is the closest to the claimed dietary supplement and adopted as a dietary supplement prototype.

The structure of the dietary supplement prototype, manufactured by the pharmaceutical company Megck §E1K1Te1Kyop (Germany) in solid dosage form (tablets), includes live probiotic lactic acid bacteria: Lactobacillus baclobacillus 5 Dackepa RA 16/8, bifidobacterium / 3 (in 1 tablet of each bacterium 10 ⁷ CFU / g).

In addition to probiotic bacteria, each tablet of a dietary supplement prototype weighing 1050 mg contains vitamins necessary for the body:

Vitamin C (calcium ascorbate) - 60 mg;

nicotinamide - 18 mg;

vitamin E (α-tocoforol acetate) - 10 mg;

pantothenic acid - b mg;

Vitamin Be, (pyridoxine hydrochloride) - 2 mg;

Vitamin Bg - 1.6 mg;

Vitamin B ι (thiamine nitrate) - 1.4 mg;

vitamin A (retinol acetate) - 0.8 mg;

vitamin - 200 IU;

folic acid - 200 mcg;

Biotin - 150 mcg;

Vitamin Bp - 1 mcg,

- 2 024516 as well as minerals:

calcium (in the form of calcium phosphate) - 90 mg;

magnesium (in the form of magnesium oxide) - 45 mg;

iron (in the form of iron sulfate) - 5 mg;

zinc (in the form of zinc oxide) - 5 mg;

manganese (as manganese sulfate) - 1.2 mg;

iodine (in the form of potassium iodide) - 100 mcg;

selenium (as sodium selenite) - 30 mcg;

chromium (as chromium chloride) - 25 mcg;

molybdenum (as sodium molybdate) - 25 mcg.

Due to the complex composition, the BAA prototype is recommended to be used not only as an additional source of vitamins and minerals, but also as a means of regulating the intestinal flora and strengthening immunity, which should contribute to good health. However, the BAA prototype is characterized by a number of significant drawbacks that limit the possibility of its use in clinical practice in order to regulate the natural equilibrium of the intestinal microflora, to improve due to this physiological state of the intestine and to ensure its functioning as a protective barrier against pathogenic microflora. First of all, it cannot be argued that the bacteria included in the dietary supplement prototype are close to representatives of their own endogenous microflora of various parts of the intestine and are able to multiply in it. One of the main reasons for this is their lack of biocompatibility with resident intestinal bacteria. Microorganisms artificially introduced as part of a dietary supplement prototype do not, as a rule, persist in the body and, after stopping their intake, are rapidly eliminated from the intestine and replaced by random microflora. Accordingly, the probability of these bacteria entering into the macroorganism from the outside to perform their main function (substitution), and even less in a short period of time, is low. Prescribe a dietary supplement prototype, as a rule, for a sufficiently long period of time when taking 1 tablet per day (at least 4 weeks per course with repeated repetition of courses throughout the year). In the most difficult clinical situations, it is recommended to take it throughout the year.

In addition, in order to protect probiotic microorganisms from the effects of hydrochloric acid, bile acids and enzymes of the upper gastrointestinal tract and to ensure that they can be absorbed in large quantities by the living in the intestine, where they are expected to have a beneficial effect, in the production of a dietary supplement prototype in solid dosage form in the form The tablets use a special sophisticated technology and apply a wide range of excipients. This determines the relatively high cost of a dietary supplement prototype (700-800 rubles per 30 tablets), which, if necessary, prolonged administration also significantly limits its availability for the general population.

As can be seen, the noted shortcomings significantly limit the possibilities of using dietary supplements to maintain a high level of vital processes of people who care about their health.

The aim of the invention was to expand the range of biologically active food supplements by creating a combined vitamin-mineral-probiotic complex, which will become an additional source of balanced intake of vitamins and minerals and will have an effective regulatory effect on the body of healthy people, increasing their physical and mental performance, and also restoring the vital protective functions of their body by stimulating the growth and activity of natural substituents of the symbiotic intestinal microflora, regulation of free radical oxidation, strengthening the immune system, which does not contain substances hazardous to health, does not have a sensitizing effect, the ability to cumulate, toxic effects on reproductive function, mutagenic effect and long-term negative effects, well tolerated and accessible to wide layers population.

This goal is achieved by creating a dietary supplement, which represents a combined vitamin-mineral-probiotic complex, which provides direct balanced intake of biologically active substances necessary for maintaining optimal functioning of the body.

For this, the composition of the claimed dietary supplement includes: vitamin-mineral and probiotic components, as well as a carrier.

When forming the qualitative and quantitative composition of the claimed dietary supplement, we proceeded from the fact that its main components should have a favorable regulatory effect on the body:

make up for the deficiency of vitamins (A, E, C, Dz, K ₁ , B ₁ , B ₂ , B ₆ , B ₁₂ , nicotinamide, folic acid, pantothenic acid, biotin, beta-carotene) and minerals (magnesium, iron, zinc , manganese, copper, iodine, chromium, selenium);

stimulate the growth and activity of natural representatives of the symbiotic intestinal microflora;

to regulate the processes of free radical oxidation due to the activation of non-enzymatic

- 3,024,516 links of the antioxidant system;

influence factors of nonspecific resistance and stimulate the functional activity of immunocompetent cells to eliminate dysfunctional changes in the immune system.

A significant difference from the dietary supplement prototype is that for the effective normalizing effect on the symbiotic intestinal microflora, the inventive dietary supplement includes a probiotic strain of bacteria VasShik kiyshk VKPM No. B-2335, which is represented by its metabolites produced during the cultivation of bacilli. Use this strain of bacteria in the form of a sterilized culture fluid, since it is in it that a unique set of biologically active substances produced by the bacilli is concentrated in the process of their cultivation. The presence of the indicated lactogenic factor in the claimed dietary supplement manifests itself in selective stimulation of the growth and activity of the natural component of the symbiotic intestinal microflora - lactobacilli, which determines one of the beneficial effects of the claimed dietary supplement, which consists in restoring the natural endogenous intestinal microflora.

The value of the claimed dietary supplement consists in the fact that the action of biologically active substances produced by the bacilli VasSh 1ik kishShk leads to the synthesis in the body and the secretion of the enzymes lysozyme and myeloperoxidase into the blood. This indicates the influence of the claimed dietary supplement on factors of nonspecific resistance of the organism and determines its ability to stimulate the functional activity of immunocompetent cells.

The ability of VasShik kishShk bacilli to increase the phagocytic activity of macrophages and thereby exhibit an immunomodulating effect is also associated with the synthesis of nitrogenous bases and their derivatives (adenine, guanine, thymine, uracil, cytosine), which represent a separate group of immunomodulatory substances, are contained in and are contained in significant quantities (table. 1). For the claimed dietary supplement, this is of great importance, since these biologically active substances contribute to the restoration of the functional activity of a single macrophage system of the body, inhibited due to various adverse factors, and a significant increase in the effectiveness of non-specific protection. Due to the stimulation of the immune response, the claimed dietary supplement enhances the protective functions of the body.

The vitamin-mineral component of the complex, including vitamins, macro-and microelements in doses not exceeding the daily requirement, is introduced into the body, primarily to make up for their deficiency in the body. In conditions of increased physical, mental, as well as emotional stress, the body's need for many vitamins and minerals increases significantly. In particular, this applies to B vitamins necessary for the normal functioning of the central nervous system, as well as substances involved in the non-enzymatic link of the antioxidant defense system of the body. To do this, combinations of B vitamins and antioxidants are introduced into the vitamin-mineral component of the complex.

Vitamins of group B (B ₁ , B ₂ , B ₆ , B ₁₂ , pantothenic acid, folic acid) in the composition of the claimed dietary supplements are contained in doses that do not exceed acceptable limits, therefore, even prolonged use of the claimed dietary supplements does not increase the risk of adverse effects, possible with an overdose.

The balance of the composition of the claimed dietary supplements to ensure the antioxidant effect is achieved due to the fact that they use a complex of water- and fat-soluble antioxidant vitamins (vitamin C and vitamins E, A) to suppress free radical reactions in the aqueous medium and lipid bilayer of cell membranes. Moreover, vitamin C is used in the form of ascorbic acid;

in addition, beta-carotene is used to compensate for its deficiency in the mitochondria of hepatocytes and to prevent disorders in the non-enzymatic link of antioxidant protection;

use vitamin A and beta-carotene in combination with vitamin E to enhance their antioxidant effect (to provide more active quenching of free radicals in the lipid bilayer of biomembranes and to prevent the development of free radical processes) and to prevent destruction;

use a synergistic pair (vitamin A with zinc) to ensure the transport of the vitamin antioxidant through the intestinal mucosa through zinc-containing protein and more complete absorption of both vitamin A and zinc. The rate of zinc release is comparable with the rate of the biochemical reaction of its binding to the protein with the formation of a zinc-containing protein complex, without which the body cannot absorb vitamin A. A large intestinal area is included in the absorption process, thereby increasing the bioavailability of these biologically active components of the claimed dietary supplement;

To maintain and stimulate the activity of 8e-containing antioxidant enzymes (glutathione pyroxidases) and accelerate the recycling of their oxidized forms, a compound of 4-valence selenium (IA ₂ 8eO ₃ ) is used in a dose that allows to eliminate its toxic effect and maximize antioxidant effect. To ensure the optimal antioxidant effect, sodium selenite is used in combination with vitamin E.

- 4,024,516

The introduction of copper as an additional active participant in hematopoiesis into the composition of the claimed dietary supplement leads to the fact that, together with folic acid, vitamins B ₁₂ , B ₂ and C, the ability to induce the synthesis of amino acids (methionine and serine) is provided, and in combination with iron, it stimulates erythropoiesis.

An additional introduction to the claimed dietary supplement of vitamin K! contributes to the normalization of blood coagulation processes, as well as the initiation of synthesis in the body of a special protein (osteocalcin), which together with vitamin D ₃ provides optimal conditions for bone metabolism.

In the inventive dietary supplement, in contrast to the prototype dietary supplement, iron is used not in the form of iron sulfate (11), but in the form of iron fumarate (FeC ^ OD, in which the content of elemental iron is 33%. Iron fumarate, in contrast to iron sulfate (11), is less than soluble in water, but soluble in dilute solutions of acids such as gastric juice.This causes sufficient stability of the claimed dietary supplement, since iron in the form of fumarate does not bind to proteins in the upper gastrointestinal tract, but at the same time it is well soluble directly in the stomach and by this is not inferior in bioavailability to a water-soluble salt - iron sulfate (11).

It should also be noted that iron sulfate (11) in a humid environment is gradually oxidized to iron sulfate (111), which imposes certain restrictions on the production process, storage and use of BAA prototype.

Chrome, like zinc, is considered one of the trace elements that contribute to a long and active life. As part of the inventive dietary supplement, the trace element chromium is used in the form of chromium picolinate, a product of exposure to picolinic acid, which is a natural derivative of the amino acid tryptophan. Compared to other forms of trivalent chromium, chromium picolinate provides higher absorption of chromium in the digestive tract. The biological role of chromium is that this element slows down the synthesis of glycosized proteins that cause aging, participates in the utilization of glucose by insulin, is necessary to maintain a stable level of glucose in the blood and proper carbohydrate metabolism, prevents depression, and helps to reduce appetite for atypical depression by reducing the need for fatty and carbohydrate foods, which is required in extremely small quantities. The introduction into the body of this microelement in the composition of the claimed dietary supplement completely fills the deficit of its receipt from food.

It should be noted that the formation of the qualitative and quantitative composition of the claimed dietary supplement, namely, its vitamin and mineral component, in order to ensure a rational combination of its constituents, was performed taking into account their daily requirements, given in the methodological recommendations MP 2.3.1.2432-08 Physiological Norms energy and nutrient requirements for various groups of the population of the Russian Federation.

As a carrier in the composition of the claimed dietary supplement, a natural microporous silicate mineral zeolite is used, the main function of which is related to the immobilization of metabolites produced by the bacilli Bacillus biloba in the process of their cultivation and concentrated in the SCR. The feasibility of using zeolite as a component of the claimed dietary supplement is determined by the unique chemical structure of its molecules, when optimal conditions arise for fulfilling the functions of a sorbent and a carrier of metabolites of Vasic acid. Zeolite, not absorbed in the digestive tract and passing in transit, carries out the transport of metabolites immobilized on it throughout the intestine. The gradual release of metabolites occurs over a sufficiently long period of time (up to 1 day).

It is also important that due to the unique chemical structure of the molecules, the zeolite has a pronounced selective sorption property and provides selective binding and excretion of low molecular weight toxins, superoxide radicals (H, OH, H ₂ O ₂ , etc.), heavy metals and radionuclides from the body, exhibiting antioxidant properties and contributing to the protection of body tissues and cells from oxidative stress. In this case, there is no direct interaction with beneficial nutrients (vitamins, amino acids, proteins, etc.) and their bioavailability is even improved.

In addition, the zeolite has ion-exchange properties, since the main skeleton of its crystal lattice consists primarily of a tetrahedron and has cavities in which ions (sodium, potassium, calcium) are located, which easily exchange with each other and with the surrounding substrate. Zeolite, passing through the entire gastrointestinal tract, participates in selective ion exchange and serves as an additional source of a number of trace elements necessary for the body. The particles of the zeolite used have a size of not more than 500 microns and an oval crystal shape, which eliminates the formation of microtraumas in the intestine and is completely safe.

The ingredients are selected in such a way that the claimed dietary supplement is a balanced vitamin-mineral-probiotic complex, and their number in two capsules with a total weight of 1000 mg is:

- 5,024,516

Retinol Acetate (Vitamin A), mg - 0.7; α-tocoforol acetate (vitamin E), mg - 13.6; Ascorbic acid (vitamin C), mg - 94.5; Beta-carotene, mg - 3.4; Cholecaliciferol (Vitamin Oz), mcg - 14.5; Phylloquinone (Vitamin K |), mcg - 174.0; Thiamine hydrochloride (vitamin B O, mg 1.7; Riboflavin (Vitamin Vg), mg - 2.0; Pantothenic acid (vitamin Bz), mg - 6.2; Pyridoxine hydrochloride (vitamin B ^), mg - 2.2; Folic Acid (Vitamin B9), mcg  500.1; Cyanocobalamin (Vitamin Bp), mcg  3.9; Nicotinamide (Vitamin PP), mg - 21.0; Biotin (Vitamin H), mcg - 54.9; Magnesium (as magnesium oxide), mg - 100.0; Iron (in the form of iron fumarate), mg - ιο, ο; Zinc (as zinc oxide), mg - 7.0; Manganese (as manganese sulfate), mg - 1.4; Copper (as copper sulfate), mg - 0.9; Iodine (as potassium iodide), mcg - 129.9; Chromium (as chromium picolinate), mcg - 24.9; Selenium (as sodium selenite), mcg - 35.1; Zeolite, mg - 195.0; Sterilized Dried Culture bacterial metabolite fluid You, Peak ZiBKZ VKPM No. B-2335, mg - 5.0.

The ability to achieve the objectives of the invention is proved by the following examples.

Example 1. Obtaining the inventive dietary supplement in encapsulated solid dosage form for oral administration.

The inventive dietary supplement is made in an encapsulated solid dosage form, which not only greatly simplifies its use when taken orally, but also provides reliable protection of its constituent components from the effects of factors causing their degradation. The compatibility of the components in 1 capsule is provided by special production technology of the inventive dietary supplement.

Receive the claimed dietary supplement in the beginning in the form of a powder, for which the following algorithm of actions is performed. Microorganisms are grown by Vaschiz ziBiNK VKPM No. V-2335 by the method of deep cultivation in a biological reactor (fermenter). At the end of the cultivation process, QOL with microorganisms is centrifuged to separate and subsequently remove living cells of microorganisms, and then sterilized. Before sterilization, QOL is mixed with zeolite, previously crushed to particles no larger than 500 microns in size. The resulting mixture is subjected to lyophilization, in which the biologically active metabolites of the producer are immobilized on zeolite particles. Then, vitamin and mineral premixes are successively added to the mass and placed in the tank of the granulation unit for mixing and drying. After unloading, the mass is sieved on a vibrating screen and transferred to the encapsulation stage. Filling of hard capsules is carried out on a capsule filling machine, and dust removal is carried out on a capsule polishing machine.

To introduce the components of the vitamin-mineral component, vitamin and mineral premixes are used (OOO KRAFT, St. Petersburg).

A natural zeolite — a natural microporous silicate mineral of the Kholinsky deposit (Buryatia) (KRAFT LLC, St. Petersburg) [22–25] is used as a carrier.

As you can see, the process of obtaining the inventive dietary supplement in encapsulated solid dosage form for oral administration is simple, and the ingredients used are available under industrial conditions, and affordable.

Example 2. Safety assessment of the claimed dietary supplement.

The safety of the claimed dietary supplement was evaluated by the results of sanitary-chemical and radiological analysis, sanitary-microbiological studies, as well as by determining a number of toxicological and other characteristics: acute toxicity, chronic toxicity and the ability to cumulate, the possible local irritating effect on the mucous membrane of the esophagus, irritating effect on skin and eyes, inhalation exposure to dust, sensitizing effects, possible long-term effects.

Sanitary-chemical and radiological analysis.

Sample preparation was carried out according to GOST 26929-94 Raw materials and food products. Sample preparation. Mineralization to identify toxic elements. Interstate standard. Lead and cadmium were determined according to GOST 30178-96. Raw materials and food products. Atomic absorption method for the determination of toxic elements. Interstate standard. Arsenic was determined according to GOST 26930-86 Raw materials and food products. Method for determination of arsenic. Mercury was determined according to Method 6.0 Guidelines for the detection and determination of total mercury in foods by flameless atomic absorption (No. 5178-90).

The content of organochlorine pesticides (toxicants) was determined according to the Methodological guidelines for the determination of residues of organochlorine pesticides (No. 1766-77) and official methods of analysis of AOAC (O £ ic1a1 teOyuyk o £ apauyuk o £ 1ye AOAC, 1984, 141H her., Syar1et 29, pp. . 537-538).

The studies were performed using laboratory VLR-500 scales, a Carlo Erba gas-liquid chromatograph (ICOS model 5700), a Perkin-Elmer atomic absorption spectrometer (model 303), a USK-Gamma-plus gamma spectrometer (Russia), and exchange cells from the Italian company Techpor1ak1.

The results of the measurements are presented in table. 2 and 3 and indicate that in the claimed dietary supplement the content of toxicants (organochlorine pesticides HCTs and DDT), hazardous substances (lead, cadmium, mercury, arsenic), radionuclides (δτ, Sk) does not exceed the permissible levels established by the Unified Sanitary and Epidemiological and hygienic requirements for goods subject to sanitary and epidemiological surveillance (control) of the EurAsEC Customs Union (chapter II, section I, index 10.6) (ET standard (chapter II, section I, index 10.6)) [26]. The same pesticides as aldrin and heptachlor in the claimed composition were not found.

Sanitary and microbiological studies

Microbiological studies were carried out in accordance with the requirements of the guidelines MUK 4.2.577-96 Methods of microbiological control of baby food, medical nutrition and their components and MUK 2.3.2.721-98 Determining the safety and effectiveness of biologically active food additives, GOST R 52814-2007 Food . A method for detecting bacteria of the genus §-1tope11a, GOST R 52815-2007 Food products. Methods for the identification and determination of the number of coagulase-positive staphylococci and §1aryu-1ossossik aitoik, GOST R 52816-2007 Food products. Method for identifying and determining the number of bacteria of the group of Escherichia coli (coliform bacteria), GOST 10444.8-88 Food products. Method for determination of VasHik segeik, GOST 10444.12-88 Food products. Method for determination of yeast and mold, GOST 30726-2001 Food products. Methods for the identification and determination of the number of bacteria of the species Exexusa soya.

The results of the assessment of sanitary-microbiological safety indicators are presented in table. 4 and indicate that, according to sanitary-microbiological safety indicators, the declared dietary supplement complies with the Unified Sanitary-Epidemiological and Hygienic Requirements for Products Subject to Sanitary-Epidemiological Surveillance (Control) of the EurAsEC Customs Union (Chapter II, Section I, Index 10.6, 10.10) (ET standard (chapter II, section I, index 10.6, 10.10)).

Acute Toxicity Study

The inventive dietary supplement was administered to healthy outbred white rats of both sexes weighing 130-150 g and 180-200 g orally through a metal atraumatic probe in the form of intragastric water-starch suspensions, which were prepared exotrot. The studies were carried out using Litchfield-Wilcoxon probital analysis in modification 3. Rota [27]. During the experiment, 2 groups of 10 animals each were formed: experimental (introduced the inventive dietary supplement twice a day at the maximum tolerated dose of 15 g / kg) and control (introduced equivalent volumes of 1% starch suspension).

Experimental animals were observed for 14 days. with registration of indicators: mortality, time of death of animals, symptoms of poisoning, results of daily monitoring of general condition and behavior, weighing, results of monitoring feed and water consumption, results of autopsy and macroscopic description of dead and surviving animals at the end of the study (euthanasia was carried out with an overdose of ether), results determination of mass coefficients of internal organs.

The introduction of the claimed dietary supplement in the maximum tolerated doses did not cause the death of experimental animals. Not noted any other signs of negative impact of the claimed dietary supplement. In particular, no dyspeptic symptoms were noted. On all days of observation, the animals of the experimental and control groups did not differ in general condition and behavior.

The results of studying the body mass dynamics of experimental animals receiving the claimed dietary supplements during 14 days of observation made it possible to establish that the body mass dynamics of the experimental and control groups practically did not differ (Table 5).

The autopsy of the animals of the experimental group, carried out a day after the acute administration of the claimed dietary supplement, made it possible to establish that the pleura and chest organs were not changed. Light pale pink, airy, without seals to the touch. The size of the heart is within normal limits. In the cavities of the heart contains a small amount of liquid blood. The heart muscle is dense, brown in color. The stomach is filled with a small amount of solid food. The mucous membrane is shiny, folded, slightly pinkish in color.

The mucosa of the small intestine is shiny, smooth, pinkish in color. The size and shape of the liver do not differ in animals of the experimental and control groups. The surface of the liver is smooth. The capsule is thin, transparent. The liver pattern in the section is not changed. Liver tissue is moderately full-blooded. Kidneys ordinary

- 7,024,516 sizes and forms, brownish in color, dense, with a distinct cortical and medulla substance in the section. The thyroid gland, adrenal glands and pancreas do not differ in appearance. When examining the histological preparations of the stomach and parenchymal organs, differences in the structure between the experimental and control groups of animals were also not found.

The vessels of the lungs were moderately full-blooded. The epithelium of the alveoli and intrapulmonary bronchi did not represent any changes. The alveoli were filled with air. Edema or pneumonia was not observed. The myobifrils of the left ventricle of the heart and interventricular septum had a distinct transverse striation, the nuclei of cardiomyocytes were light. Myocardial vessels are moderately full-blooded. The lobular structure of the liver persisted. The boundaries of hepatocytes were clear, the cytoplasm of hepatocytes was weakly basophilic, granular, a nucleus with sufficient chromatin content and a thin nuclear membrane.

The epithelium of the convoluted tubules of the kidneys did not represent any changes. Its cytoplasm was granular, oxyphilic, and its nuclei were bright. Glomerular vessels are moderately full-blooded.

The structure of the mucous membrane of the fundus of the stomach of animals of the experimental and control groups did not differ. The nuclei of the epithelium of the mucosa and glands were light. In the basal parts of the glands, granularity in the cytoplasm was preserved. The epithelium of the villi and crypts of the small intestine mucosa was preserved; mitosis was present in the crypts.

When staining frozen sections of the liver with Sudan IV, signs of fatty degeneration were not observed.

Analysis of the mass ratios (MK) of the organs of white rats revealed that the acute administration of the claimed dietary supplement in the maximum tolerated dose does not cause any significant differences compared with the control group receiving 1% starch suspension (Table 6).

Thus, the results of toxicometry, observation of experimental animals within 14 days after acute administration, as well as necropsy data allow us to attribute the claimed dietary supplement to non-toxic (relatively harmless according to S. D. Zagolnikov) and low hazard (IV hazard class according to GOST 12.1.007 ) substances.

Study of chronic toxicity and cumulative ability

Studies of chronic toxicity were carried out on white non-linear male rats weighing 180-200 g with the introduction of the claimed dietary supplement intragastrically through an atraumatic metal probe for 30 days at a daily dose of 1.5 g / kg. Animals of the control group received 1% starch suspension in similar volumes.

The measurement of basic physiological parameters was carried out after 30 days of chronic daily administration of the inventive dietary supplement.

No lethal effects were observed throughout the observation period. The general condition and behavior of the experimental animals remained normal and did not differ from the control group throughout the experiment.

The results of the study of integral indicators of total toxicity are presented in table. 7 and 8, indicate that no statistically significant differences were found in any of the analyzed parameters with the control group of animals receiving 1% starch suspension (p> 0.05 at a 95% probability level) or pathological deviations beyond the limits of physiological norm variation.

The autopsy of rats euthanized the day after the last administration of the inventive dietary supplement showed that the size, shape and color of the internal organs had no macroscopic changes compared with the control group. The mucous membranes of the stomach and small intestine were shiny, pale pink, with no signs of irritation or inflammation.

A histological examination of preparations of the lungs, myocardium, liver, kidneys and gastric mucosa of experimental animals receiving the claimed dietary supplement did not reveal any dystrophic, inflammatory, or necrobiotic changes in organs.

The epithelium of the alveoli and intrapulmonary bronchi had no changes, the alveoli were air. Atelectasis or pulmonary edema was not observed. The transverse striation of the myocardial myofibrils was distinct. The structure of the liver was not disturbed. Borders of hepatocytes were distinct, granular cytoplasm, weakly basophilic, nuclei bright with a thin membrane and distinct nucleoli. Nephroepithelium with oxyphilic granular cytoplasm and clear, clear nuclei. The epithelium of the gastric mucosa is preserved, the main and parietal cells of the glands of the stomach are not changed.

Therefore, the chronic intragastric intake of the claimed dietary supplement into the body of experimental animals does not cause dystrophic or destructive changes in the parenchymal organs and is not accompanied by irritation of the gastrointestinal mucous membranes. Thus, according to the integral indicator of cumulation and indicators of total non-lethal toxicity, the claimed dietary supplement does not have the ability to cumulate and is non-toxic.

Investigation of a possible local irritant effect

The local irritating effect of the claimed dietary supplement was evaluated on a model of the ciliated epithelium of the frog's esophagus. The cytotoxic effect was studied on marsh frogs of Capa 1 metrogap weighing 25

- 8,024,516

After immobilization by destroying the spinal cord and fixing the animals on a cork table, the chest was opened, the esophagus was secreted, and dissected in the caudal direction. Two rods were installed over the pharyngeal part of the esophagus. During the experiment, the transit time of the cork crumb of a portion of the esophagus 10 mm long, limited by rods, before and after applying distilled water or the claimed dietary supplement in the form of a powder in the amount of 50 mg to the mucous membrane of the esophagus was recorded. The exposure duration was 1 min.

It was experimentally established that the application of the inventive dietary supplement in the form of a powder to the mucous membrane of the esophagus does not affect the motor function of the ciliated epithelium of the frog’s esophagus (Table 9). Thus, the claimed dietary supplement does not have a local irritant effect.

Research on skin and eyes

The effect on the skin and skin-resorptive effect was studied on white rats weighing 130 150 g. After placing the animals in special houses, their tails were placed for 2/3 for 4 hours in a pulp, which was obtained by mixing the inventive dietary supplement in the form of a powder with cold water in amounts, respectively, 90% and 10%. 1 hour and 16 hours after the end of a single application, erythema or edema of the skin of the tails was not observed (the edema was estimated by measuring the thickness of the tail in the middle part using a thickness gauge of the type TR-1-10). It was found that a 20-fold application did not cause the death of experimental animals and did not reveal an irritating effect on the skin during the subsequent 5-day observation period.

Morphologically, the skin of the tails at the place of application of the powder had no changes. The epidermis and appendages of the skin were also unchanged. The layers of the epidermis were distinct, the basement membrane was preserved. Epithelial cells of the external and internal root vaginas of the hair follicles and connective tissue bag are well defined.

Macroscopic and microscopic changes in internal organs in experimental animals were absent.

A study of the morphological, biochemical, hematological and physiological parameters of white rats did not reveal significant differences from the control group of animals whose tails were placed in water.

Therefore, the claimed dietary supplement does not have an irritating effect on the skin and skin-resorptive effect, that is, it is non-toxic when it comes into contact with skin.

Also investigated the irritating effect of the claimed dietary supplement on the mucous membrane of the eye of a rabbit. During the experiment, it was found that 1 min after a single application of the inventive dietary supplement in the form of a powder in an amount of 50 mg to the conjunctival sac of the rabbit’s eye, moderate hyperemia and lacrimation appeared, accompanied by short blepharospasm, which can be regarded as a reaction to an indifferent mechanical foreign body. Vascular congestion lasted no more than 20 minutes, and after 40-45 minutes all the phenomena of irritation that occurred completely disappeared. Therefore, the claimed dietary supplement has a weak irritant effect on the mucous membrane of the rabbit eye, like any mechanical dust.

Investigation of inhalation exposure to dust

A single dynamic inhalation effect of the inventive dietary supplement in the form of a powder on white female rats weighing 130-150 g was performed for 2 hours in standard B.A. chambers. Courland 200 liters The air supply rate was 60 l / min at a temperature of 20 ° C. The maximum possible dust concentrations, which were determined by the gravimetric method during the experiment, were achieved using dust sprayers. Air samples were taken every 30 minutes. During the experiment, the average dust concentration was 55.5 ± 3.5 g / m ³ .

Both during and after exposure, experimental animals did not have any lethal outcomes, but only signs of dust (mechanical) irritation of the upper respiratory tract (sneezing, grooming) and eyes (lacrimation), as well as anxiety.

The autopsy of the sacrificed animals revealed no changes, except for moderate plethora of the internal organs. In the respiratory tract were determined particles of the powder of the claimed dietary supplement.

Consequently, acute inhalation exposure to dust of the claimed dietary supplement is not dangerous, since it does not cause signs of poisoning, but is only able to slightly irritate the upper respiratory tract and eyes, like any mechanical dust.

Sensitizing study

The study of the sensitizing effect of the claimed dietary supplement was carried out on guinea pigs. To do this, 0.02 ml of water dilution of the inventive dietary supplement at a dose of 10 mg / kg was injected once into the skin of the outer surface of the animal’s ear with a tuberculin syringe, and after 10 days, the inventive dietary supplement in powder form was applied and fixed on pre-trimmed skin of the lateral back surface of 2x2 cm at the rate of 20 mg / cm ² Blood sampling in animals was carried out 3 hours after the staging of skin samples.

The results of evaluating the impact of the claimed dietary supplement on indicators of sensitizing action are presented in table. 10 and indicate the absence of signs of sensitization in peripheral blood - 9,04516 vi - eosinophilia or an increase in the level of lysis in the reaction of specific leukocyte lysis (RSLL) compared with the control. Examination of the back skin of experimental animals showed no signs of irritation. Therefore, the claimed dietary supplement when epicutane contact does not have a sensitizing effect, that is, does not provoke the development of allergies.

Investigation of possible long-term effects

The study of a possible gonadotoxic effect was carried out in accordance with the guidelines for indicators required in the initial assessment of chemicals [28, 29]. Mutagenic activity was studied by the frequency of micronuclei formation in polychromatophilic bone marrow erythrocytes (from the femur) of white mice in accordance with the methodological recommendations [30, 31].

The results of a study of the possible gonadotoxic and mutagenic effects of the inventive dietary supplement when administered for 10 days at a dose of 1 g / kg, carried out 15 days after the end of its administration, indicate the absence of significantly significant changes in gonadotoxicity and mutagenicity indicators (Table 11). Inhibition of spermatogenesis or an increase in the frequency of mutations was also not observed.

Therefore, the claimed dietary supplement does not have a toxic effect on reproductive function and does not have a mutagenic effect. This indicates its safety in terms of the development of long-term negative consequences in humans.

Example 3. Evaluation of the stability of the claimed dietary supplement during storage.

We investigated the possibility of maintaining the activity and safety of the claimed dietary supplement in the form of a powder and established a guaranteed shelf life in case of storage at various temperatures and without the use of special equipment.

During the experiment, the powder was placed in 100 consumer packages in the form of double plastic bags and stored for 2.5 years at temperatures 5 ± 2 ° С, 10 ± 3 ° С, 25 ± 5 ° С, 35 ± 6 ° С 25 packs per temperature mode. After every 6 months of storage, 5 packs were taken from the appropriate temperature regime and the powder characteristics were determined: moisture, amylolytic and proteolytic activity, microbiological purity and content of vitamins (C, B ₁ , B ₂ , E) and minerals (zinc, chromium, selenium, magnesium). To assess possible changes in these characteristics during storage, their values were determined at the beginning of the study.

The data presented in table. 12, indicate that at the selected storage temperature conditions for testing (from 5 to 40 ° C), the evaluated indicators practically did not change during storage for 2.5 years. The average values of the indicators were: mass fraction of moisture - 5.9%, amylolytic activity - 2.8 ± 0.4 units. AA / g, proteolytic activity - 0.4 ± 0.1 units. PA / g The microbiological purity of the claimed dietary supplement, assessed by the absence of pathogenic microorganisms in it (Rkheyotopak aegidshoka, Etcobasipaspas. §1arcoussossica aigheik and Vaschik segeik), was maintained for 2.5 years, that is, the entire period of research. The content of vitamins and minerals was also maintained throughout the entire storage period.

Thus, the guaranteed shelf life of the claimed dietary supplement, made in solid form in the form of a powder, is at least 2.5 years at a temperature of no higher than 40 ° C, subject to the safety of packaging. Special temperature storage conditions or the use of special equipment is not required.

Example 4. The study of the effect of metabolites of VasHik kiyshk VKPM No. B-2335 on the growth and activity of probiotic lactobacilli.

In the ίη νίΙΐΌ experiment, the antagonistic activity of Vaschik kiPPK VKPM metabolites No. B-2335 against live lactobacilli Lactobacillus 8K-A3, as well as their effect on the growth and activity of these lactobacilli, close to the natural component of the endogenous intestinal microflora, was investigated. At the same time, the activity of Lactobacilli Lactobacillus palliagite 8K-A3 was evaluated by the level of their specific antibacterial activity against a number of opportunistic microorganisms (staphylococci, Escherichia, pseudomonas).

The studies were carried out using the direct antagonism method by direct application of sterile bacterial metabolites of Vaschik kiyshk VKPM No. В-2335 or live lactobacilli Lactobacillus 8K-A3 into wells or onto the surface of a solid nutrient medium (agar MPC-4) immediately after plating of Lactobacillus bacillus bacillus. p1a Chagit 8K-A3 or indicator test cultures, followed by incubation for 24 hours at a temperature of 37 ° C;

the method of two-layer agar [32], in which a suspension of Vaschik kiyshk VKPM No. B-2335 metabolites or live lactobacilli Lactobacillus 8K-A3 was added to the lower layer of solid nutrient medium (agar MPC-4), and lactobacillas bacillus Bacillus bacillus 8 was sown in the upper layer A3 or indicator test cultures.

Metabolites of Vasichik kiyshk VKPM No. B-2335 were administered at concentrations of 1 g or 0.1 g in 10 ml of agar.

Lactobacilli were seeded in various amounts of 7 1§ CFU / ml - 2 1§ CFU / ml.

As test cultures of opportunistic microorganisms, §1arcu-1ososik aigeyik was used

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209, Excepsoi M15 and Rkeibotopac aegidshock.

Using the methods of direct antagonism and bilayer agar, it was found that the metabolites of Vasilik siSiq VKPM No. B-2335 do not have an inhibitory effect on the growth of lactobacilli Lactobacillus p1ap1agit 8K-A3.

It was also found that the metabolites of YOUR SHIK KiISHK VKPM No. B-2335 as a lactogenic factor stimulate the growth of probiotic lactobacilli. In the presence of Vaschik kiyshk VKPM No. B2335 metabolites, a larger number (four or more times) of lactobacilli Lactobacillus p1aagit 8K-L3 grew (Table 13). Lactobacilli colonies, when 50 colonies were sown on a Petri dish and growth in the presence of VasShik kiyshk VKPM No. B-2335 metabolites, was 2 times larger, their diameter increased from 1 to 2 mm compared to the control (without the addition of VasShik kiyshk VKPM No. V-2335 metabolites .

In the presence of Vaschik kiyshk VKPM metabolites No. B-2335, the antibacterial effects of lactobacilli Lactobacillus p1ap1agit 8K-L3, that is, their specific antagonistic activity, against staphylococci, Escherichia and pseudomonas increased significantly (Table 14-16).

Thus, it has been experimentally established that the metabolites of YOUR SHICK KISK VKPM No. B-2335 as part of the inventive dietary supplement represent the lactogenic factor and stimulate the growth of lactobacilli and their specific antagonistic activity against opportunistic microorganisms, which is due to the beneficial effect of the inventive dietary supplement on the natural endogenous intestinal microflora .

Example 5. The study of the immunotropic effects of the claimed dietary supplement.

Studies aimed at assessing the ability of the claimed dietary supplement to influence factors of nonspecific resistance of the body were performed on white outbred laboratory mice weighing 18-23 g. 80 animals were used, which were divided into two groups of 40 individuals in each: control (received isotonic sodium chloride solution ) and experimental (received the claimed dietary supplement).

The inventive dietary supplement was administered orally 1 time per day at a dose of 150 μg / mouse for 7 days. Evaluation of the effect of exposure was carried out after 1, 3 and 7 days. For this, blood was collected for the study from animals of the control and experimental groups, and blood was taken before the start of the study and then after 1, 3, and 7 days. At each stage of the study, material was collected from 10 animals from the group, individually from each mouse after its decapitation.

The dynamics of changes in non-specific resistance factors was evaluated by determining the functional state of the cells of the phagocytic system (the digesting activity of granular leukocytes), as well as the concentration in the blood serum of lysozyme and myeloperoxidase enzymes, which are mediators of cell-cell interaction during the development of the immune response to any antigenic effect.

The digesting activity of granular leukocytes of peripheral blood of experimental animals was evaluated by the method of Zelenova E.G. and Nikiforova V.A. (1972). Serum was obtained by standard methods, separating the blood clot from the liquid phase. To assess the functional state of phagocytic cells, blood was collected in tubes pretreated with silicone (§egua, Germany) and containing a heparin solution at the rate of 20 PIECES per 1 ml of blood.

The phagocytic index (Phi), which represents the ratio of the severity of the absorbing and digesting functions of phagocytic cells, was determined after 15 min and 30 min of incubation with a phagocytosis test object, which was used as a test microbe (M. 1ukobeyuysik) grown on meat and peptone agar in a thermostat at a temperature of 37 ° C for 24 hours. The index of digesting activity of granular leukocytes (PI), representing the ratio of the number of phagocytic cells in a smear after 30 min of incubation of blood with t there is an object of phagocytosis to the number of phagocytic cells in the smear after 15 min of incubation of blood with a test object of phagocytosis.

The IP values were determined by the formula:

PI = B: A, (1) where A is the number of phagocytic cells per 100, counted in a smear of granular leukocytes, which absorbed the test microbe during 15 min of incubation;

B is the number of phagocytic cells per 100, counted in a smear of granular leukocytes, which absorbed the test microbe within 30 minutes of incubation.

The value of PI was expressed in arbitrary units. Experimental data on the assessment of the functional state of granular leukocytes of peripheral blood of mice treated with the inventive dietary supplement (Fig. 1) indicate that the use of the inventive dietary supplement contributes to a significant increase in the functional activity of granular leukocytes in peripheral blood, since the IP values significantly exceed the level of the control group (differences with the control group was significant at p <0.05).

The concentration of lysozyme in serum was determined by the method of Bukharin O.V. et al. (1974). For this, the blood of the experimental animal was collected in a centrifuge tube and serum was obtained. Then 0.1 ml of blood serum was transferred to a bacteriological tube and 0.9 ml of physiological saline solution was added. The resulting mixture was mixed and 1.5 ml of a suspension of the test microbe was added to it, which was previously standardized on an FEK-56M photoelectrocolorimeter to an extinction of 0.66.

The prepared suspension containing the test microbe and the test serum was mixed and placed in a thermostat for 30 min at a temperature of 37 ° C. After the incubation time, the tubes were removed from the thermostat and the extinction of the material was determined. The obtained extinction values using special tables were translated into values characterizing the concentration of lysozyme in the blood serum, and expressed in μg / ml.

The level of myeloperoxidase (MPO) in the blood serum was determined by the method developed on the basis of generally accepted approaches to the detection of this substrate in blood serum and immunocompetent cells (Oe11 R.S.N. et al. (1968); Rade K.S. et al. (1978 )). To determine the concentration of MPO in serum in 96-well plates for immunological reactions with a flat bottom, starting from the second well of row A, 0.1 ml of sera from experimental animals were poured. Then, 0.1 ml of the reagent, which is a mixture of benzidine and 3% hydrogen peroxide, was added to them. Instead of serum, 0.1 ml of physiological saline was poured into the first well of row A of the panel (control). After adding the reagent, the panels were shaken, placed in a thermostat and kept at a temperature of 37 ° C for 60 minutes. After the incubation time, the panels were removed from the thermostat and viewed on a vertical OupaUSn spectrophotometer (Germany) at a wavelength of 495 nm. The concentration of the enzyme in blood serum was obtained in units of extinction and expressed in arbitrary units.

As shown in FIG. 2-3 data on the assessment of the concentrations of lysozyme and myeloperoxidase in the blood serum of mice receiving the claimed dietary supplement, the immunotropic effects of the claimed dietary supplement are manifested at the level of the phagocytic blood system cells and processes associated with the synthesis and secretion of these enzymes into the blood. The effects of a significant increase in the functional activity of factors of nonspecific immunity (differences with the control group were significant at p <0.05) were recorded 1 day after the administration of the claimed dietary supplement and they lasted at least 7 days from the time of administration. The stimulating effect of the claimed dietary supplement on the mechanisms of nonspecific resistance took place after its single administration. The concentration levels of lysozyme and myeloperoxidase in the blood serum of animals treated with the claimed dietary supplement, after 1 day increased, respectively, 2.2 times and 1.5 times in comparison with the control group of animals that were injected with isotonic sodium chloride solution.

Example 6. Assessment of the biological activity of the claimed dietary supplement.

The biological activity of the claimed dietary supplement was evaluated on the basis of a study of its adaptogenic activity, neurotrophic and cytoprotective effects, antioxidant properties.

The experiments to assess these characteristics were performed on outbred white rats-males weighing 180-200 g and white outbred laboratory mice weighing 18-23 g after intragastric (through a probe) administration of the inventive dietary supplement. The required dose of administration of the inventive dietary supplement was 100 mg / kg (a prophylactic dose adapted for rats, taking into account an interspecific transfer coefficient of 6, based on the need to administer the contents of 2 capsules of 1000 mg per day for humans with an average weight of 60-70 kg) or 150 mg / kg (a prophylactic dose adapted for mice, taking into account the interspecific transfer coefficient of 10, based on the need to introduce the contents of 2 capsules, amounting to 1000 mg per day for humans with an average weight of 60-70 kg).

The effect of the claimed dietary supplement on the resistance of rats to high temperatures.

The adaptogenic properties of the claimed dietary supplements were evaluated by the effect of the temperature factor on experimental animals, which allows a comprehensive assessment of the effect of the claimed dietary supplements on metabolic systems, the stability of which in conditions of hyperthermia determines the survival of individuals.

To assess the stability of rats under hyperthermia, the experimental and control groups of 6 animals in a cage were placed in a ventilated thermostat and kept at a temperature of 60 ° C. The device of the heat chamber made it possible to provide adequate gas exchange, sufficient to prevent oxygen deficiency, and made it possible to observe the animals through a glazed inspection hole. During the observation, the development of symptoms of the lesion was recorded and the time of manifestation of the convulsive syndrome and death of individuals was noted.

Experimental data indicate that the claimed dietary supplement with a four-week administration has a positive effect on the resistance of rats to hyperthermia (table. 17). The rate of development of the convulsive syndrome in rats receiving the claimed dietary supplement has a distinct tendency to decrease, and their lifespan in conditions of hyperthermia is significantly higher. Thus, the claimed dietary supplement has an adaptogenic effect and with the course of application can increase resistance to extreme factors.

The impact of the claimed dietary supplement on the processes of short-term memory and mental performance of rats.

The neurotrophic effects of the claimed dietary supplement were evaluated by the effect on the short-term memory of rats and in comparison with the dietary supplement prototype [33]. The basic model for assessing the impact on the formation and reproduction of a memorial footprint in the norm and in the conditions of its experimental violation is

- 12 024516 technique of the conditioned reflex of passive avoidance (passive avoidance reaction) [34]. The main advantages of this method are the speed of reflex production (learning from one sample) and the possibility of differentiated effects on different phases of memory.

The experimental animals were divided into 4 groups: the control and three experimental animals, 10 animals each. Before developing passive avoidance reaction, experimental animals of the first and second experimental groups were administered intragastrically for 10 days, respectively, the claimed dietary supplement and dietary supplement prototype at a dose of 100 mg / kg each. The animals of the third experimental group were also injected intragastrically with the Noopept® comparison drug 10 mg tablets (ZAO Masterclone, Moscow) in the form of an aqueous suspension at a dose of 5 mg / kg (the maximum therapeutic dose adapted for rodents for humans based on the active component ) Animals of the control group were injected with 1% starch suspension in a volume of 0.2 ml.

The installation for generating the passive avoidance reflex was a plywood box - a compartment, which consisted of an illuminated part with a 40 W incandescent lamp mounted and darkened with 2 mm diameter copper rods on the floor. Current was supplied from the mains to the rods through a lather and a closing key. Between themselves, the parts were separated by a partition with an opening that was blocked by a shutter.

On the 11th day of the study, the passive avoidance reaction was performed in rats in an installation, the triggering stimulus of which was the animal in a brightly lit space.

The rat was placed in the bright part of the compartment. After the rat transitioned to the dark part, fatal pain was immediately applied with 5 consecutive stimuli of alternating current (1 mA, each lasting 1 s with an interval of 2 s).

During testing (repeated placement in the illuminated part of the compartment after 24 h in order to determine the preservation of passive avoidance reaction), each animal was observed for 180 s. In this case, the latent period of the rat’s transition to the dark part of the compartment, the time spent in the light and dark parts of the compartment, and the number of animals that did not enter the dark part of the compartment (number of trained animals) were recorded.

The results of the assessment of passive avoidance reaction indicators testify to the achievement of the maximum effect in the case of using the claimed dietary supplement (table. 18). The use of the claimed dietary supplement allows you to activate cognitive processes and to a greater extent to consolidate the formed defensive reflex. Compared with the control group, the latent period of the transition reaction of animals and their time in the light part of the compartment increased, respectively, by 89.3 and 71.4%, and the time spent in the dark part of the compartment decreased by 4.4 times. Moreover, skills were developed in all 10 animals of the first experimental group, which is 2.0 times higher than the control group.

When using a dietary supplement prototype, in comparison with the control group, the latent period of the transition reaction of animals and their time in the light part of the compartment increased, respectively, by 73.3 and 54.2%, and the time spent in the dark part of the compartment decreased by 2.8 times . At the same time, skills were developed in 8 animals, which was 1.6 times higher than the control group.

Under similar conditions, when using the well-known drug noopept in comparison with the control group, the latent period of the animals transition reaction and their residence time in the light part of the compartment increased, respectively, by 53.3 and 35.7%, and the residence time in the dark part of the compartment decreased 2.9 times. In this case, skills were developed only in 7 animals out of 10, which is 1.4 times higher than the control group.

Thus, it was found that the claimed dietary supplement, if used prophylactically, improves the mnestic functions of the central nervous system, has a positive effect on cognitive processes and helps improve memory and learning ability. Moreover, it is more effective than the dietary supplement prototype and is not inferior to the drug noopept.

The influence of the claimed dietary supplement on the resistance of rats to the effects of hypercapnic hypoxia

The adaptogenic and cytoprotective effects of the claimed dietary supplement were evaluated under hypoxic loads due to the universality of this condition under various extreme influences and pathological conditions. Hypoxia was modeled using a desiccator with a water lock, which made it possible to provide a hypoxic load when air was discharged equivalent to the atmosphere at an altitude of 11.5 km and with a constant increase in the amount of carbon dioxide due to the intake of exhaled air from animals. Rats were placed in a chamber in pairs (according to one of the experimental and control groups) and kept in it until they died, recording the frequency and rate of development of the convulsive syndrome, as well as the life span of the animals. Before the experiment, the animals of the experimental group for four weeks were intragastrically administered the claimed dietary supplement in a dose of 100 mg / kg Animals of the control group were injected with 1% starch suspension in a volume of 0.2 ml.

It was found that the claimed dietary supplement contributes to a significant increase in the resistance of animals to the effects of adverse factors modeling the conditions of enclosed spaces, a smoky environment and underground transport, that is, it exhibits a cytoprotective effect (Table 19).

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The effect of the claimed dietary supplement on the degree of ischemic brain damage in stenosis of two carotid arteries

We studied the effectiveness of the claimed dietary supplement on models of cerebral ischemia (ischemic stroke) in terms of the development of oxidative stress with registration of both its severity and outcomes in the form of accumulation of peroxidation products. Of the four vessels involved in the blood supply to the rat brain, 2 carotid arteries were ligated simultaneously. The operation was performed under ketamine anesthesia. Observation of anesthetized animals was carried out for 2.5 hours after stenosis, after which they were decapitated and the brain was removed for biochemical studies. The erythrocyte catalase activity was evaluated by the decay rate of hydrogen peroxide introduced into the medium in a known amount. The antiradical capacity of blood plasma and brain homogenates was evaluated by restoration of the free radical (diphenylpicrylhydrazyl). Moreover, lower optical density of this colored product indicated the restoration of the radical and the effect of the antioxidant effects of the claimed dietary supplement. Diene conjugates of fatty acid hydroperoxides were extracted from blood and brain homogenates with isopropanol and heptanol for separate determination in the phospholipid fraction of the membranes. It is known that peroxidation products are capable of absorbing transmitted light with a wavelength of 232 nm, while unoxidized double bonds at a wavelength of 220 nm. The ratio of extinction indices indicates the proportion of peroxidation products in the total lipid fraction. The optical density indices were determined using an SF-26 spectrophotometer.

Before the experiment, the animals of the experimental group were injected intragastrically for four weeks with the claimed dietary supplement at a dose of 100 mg / kg, and the animals of the control group received 1% starch suspension in a volume of 0.2 ml. Significant differences were found between the blood indices of the animals of the control and experimental groups (Table 20). The activity of catalase in the blood in animals treated with the claimed dietary supplement remains low, which reflects the lack of substrate for this enzyme, which is formed during oxidative stress reactions. In comparison with animals of the control group, diene conjugates of fatty acid hydroperoxides were detected in rat blood in much smaller amounts.

Along with the study of the characteristics of the content of lipid peroxidation products in the blood, we studied the processes of peroxidation directly in the lesion. The results of spectrophotometry of samples of brain homogenates (Table 21) showed that when the inventive dietary supplement is used, the content of diene conjugates in the phospholipid membrane fraction extracted by isopropanol is reduced, which ensures a protective effect at the level of cells and tissues. The inventive dietary supplement activates the non-enzymatic link of antioxidant protection.

Thus, the introduction of the claimed dietary supplement for four weeks allows you to achieve an adequate cytoprotective effect, coupled with the restriction of lipid peroxidation and activation of the non-enzymatic link of the antioxidant system.

The influence of the claimed dietary supplement on the duration of swimming mice

Swimming is a heavy physical dynamic load and allows you to evaluate the effectiveness of adaptogens and the activity of redox processes in the muscles of mice. During the experiment, swimming was carried out at a water temperature of 38-39 ° C and was carried out with a load, which is a lead tube on a rubber ring attached to the root of the tail of the mouse and comprising 5% of body weight. At the same time, 5 animals from the control and two experimental groups swam in a large bath. The animals of the first and second experimental groups were administered intragastrically daily, respectively, the claimed dietary supplement and dietary supplement prototype at a dose of 150 mg / kg each. Mice in the control group received daily intragastric 1% starch suspension in a volume of 0.2 ml.

The criterion of fatigue and cessation of swimming was considered the first diving of the mouse with the immersion of the nasal passages in water. The duration of swimming of the mice was recorded from the moment they were placed in the bath until the moment of the first diving. The results of testing conducted 14 days after the daily administration of the inventive dietary supplement showed that the inventive dietary supplement increases the swimming time of mice by 33.9%, that is, it has a protective effect and reduces fatigue (Table 22).

In similar conditions, the BAA prototype increases the swimming time of mice only by 14.4%, that is, it is 2.4 times less effective than the claimed BAA

The effect of the claimed dietary supplement on the static-power endurance of mice.

The effect of the claimed dietary supplements on the static force endurance was studied by recording the time of hanging of mice of the experimental and control groups on a vertical grid. The inventive dietary supplement, as in the swimming test with the load, was administered to mice of the experimental group at a dose of 150 mg / kg. The animals of the control group received daily intragastric 1% starch suspension in a volume of 0.2 ml.

The animals were tested after a 14-day administration of the inventive dietary supplement. The criterion for the depletion of static strength was considered the time when the mouse could no longer support the weight of its body and fell down from the net (the dead weight of mice averaged 25 g). The experimental results demonstrate an increase in the static physical endurance of experimental animals under the influence of the claimed dietary supplement (Table 23).

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The effect of the claimed dietary supplement on the muscle strength of rats

Muscle strength was evaluated in white rats using a rat dynamometer. The results presented in table. 24, indicate that daily intake of the claimed dietary supplement for 14 days contributed to an increase in the muscle strength of experimental animals by 15.3% compared with the background in the first experimental group and by 17.1% compared with the control group receiving 1% starch Suspension also for 14 days. The use of a dietary supplement prototype for 14 days contributed to an increase in the muscular strength of rats by 5.4% compared to the baseline in the second experimental group and by 5.8% compared to the control group. Thus, in terms of influence on muscle strength, the claimed dietary supplement surpasses the dietary supplement prototype by 2.8 times.

Thus, the studies showed that the claimed dietary supplement, which is a combined vitamin-mineral-probiotic complex, provides a rational balanced intake of biologically active substances in the body, exhibits a wide range of physiological effects and restores the vital protective functions of the body, increasing its resistance to adverse factors:

stimulates the growth and activity of probiotic lactobacilli and normalizes due to this the natural endogenous intestinal microflora;

possesses a cytoprotective effect, associated with the restriction of lipid peroxidation and activation of the non-enzymatic link of the antioxidant system of the body, which is expressed in a decrease in fatigue and an increase in static physical endurance;

exhibits immunotropic effects and increases the functional activity of non-specific immunity factors;

possesses adaptogenic effect and increases resistance to adverse factors;

has a positive effect on cognitive processes, improves the mnestic functions of the central nervous system, helps to improve memory and learning ability, increases mental performance;

does not have a sensitizing effect and does not provoke the development of allergies;

does not have a toxic effect on reproductive function, does not have a mutagenic effect and is safe in terms of the development of long-term negative effects.

In addition, the claimed dietary supplement does not contain toxic and hazardous components in quantities exceeding the acceptable levels, is a low-hazard substance, non-toxic, does not have the ability to cumulate and is well tolerated, since it does not exhibit undesirable side effects in the form of skin-resorptive and topical irritating action.

The claimed invention meets the criterion of novelty, since the composition of a biologically active food supplement, which is a combined vitamin-mineral-probiotic complex, the components of which are selected in such a way as to ensure a balanced intake of vitamins and minerals, and also increase the body's resistance to adverse effects, is first proposed. factors by stimulating the growth and activity of lactobacilli of the natural endogenous intestinal microflora, an asset non-enzymatic link of the antioxidant system and reducing the damaging effects of free radicals, activation of factors of general nonspecific defense of the body and strengthening the immune system, exhibiting adaptogenic and cytoprotective effects and increasing physical and mental performance.

The claimed invention meets the criterion of inventive step, since from the available information it is advisable to use Vaschik kiyshk VKPM metabolites No. B-2335 as a probiotic component and the possibility of providing a lactogenic effect and a beneficial effect on the growth and activity of lactobacilli of natural endogenous intestinal microflora, an adequately justified combination of water - and fat-soluble antioxidant vitamins, as well as the optimal amount of selenium when introduced in the form of selenite on dence does not seem obvious.

The compliance of the claimed invention with the criterion of suitability for use is confirmed by the above examples, which showed a simple technology for the production of the inventive biologically active additives in encapsulated solid dosage form, the ingredients of which are manufactured under industrial conditions, are affordable and inexpensive, as well as the results of numerous studies confirming the durability of the claimed dietary supplement during storage during 2.5 years, safety and high biological activity.

List of references

1. Intestinal dysbiosis. Guidelines for diagnosis and treatment / Edited by prof. E.I. Tkachenko, prof. A.N. Suvorov. St. Petersburg: SpetsLit, 2007.238 s.

2. Abramova Zh.I., Oksengendler G.N. Man and antioxidants. L .: Science. Leningra. rest 1985.232 s.

3. Bilenko M.V. Ischemic and reperfusion damage to organs. M, 1989.368 s.

- 15,024,516

4. Vladimirov Yu.A., Azizova O.A., Deev A.I., Kozlov A.V., Osipov A.A., Roshchupkin D.I. Free radicals in living systems // Itogi Nauki i Tekhniki. Ser. Biophysics. / VINIT.-M. 1991.Vol. 29.250 s.

5. Zhukov A.A., Zhirnov G.F. The mechanism of oxygenase reactions: the main, intermediate and by-products of the oxygenase cycle // Bulletin of the USSR Academy of Medical Sciences. 1988. No. 1. S. 33-43.

6. Zhuravlev A.I. Spontaneous bio-chemiluminescence of animal tissues // Biochemiluminescence. M .: Science. 1983, p. 3-29.

7. Zenkov NK, Menitsikova E.B. Activated Oxygen Metabolites in Biological Systems // Achievements in Lying, Biology. 1993. T. 113, No. 3. S. 286-296.

8. Menitsikova E.B., Zenkov N.K. Antioxidants and inhibitors of radical oxidative processes // Successes in modern biology. 1993. V. 113, No. 4. S. 442-455.

9. Pluzhnikov N.N., Sofronov G.A. Pharmacological correction of the state of the body's antioxidant system // Pathophysiology of extreme conditions: Selected lectures and reports. / Ed. V.Yu. Shanina and V.I. Zakharova. St. Petersburg, 1993.S. 108-113.

10. Vguap S.L., Satrée Ο.Ό., ba \\ tepse K.A., copycope δ.Ο. ΌίρΙιοφΙιΟΓνΙ Ιίρίά-A го11есес1 гак га1 1ей тот тот тот 1 1ейейейейей // // // // // // // // I. L. SNP. May. 1992. Wo. 161, No. 2. R. 559-566.

11. Sayepak E., Voteuy A., Sapke V. Yo \ y-1ex'e1 siLitseksepse o оo1od1sa1 kuk1etk // Rgee Kaika1k sh Vyuodu. 1984. V1. 6. R. 211-242.

12. Oxidative stress and inflammation: pathogenetic partnership: Monograph / Ed. O.G. Khurtsilavy, N.N. Pluzhnikova, Y.A. Nakatisa. SPb .: Publishing house of SZGMU named after I.I. Mechnikova, 2012.S. 97-111.

13. Rpjuyu I. 8irehokhje yaitShakek - apayar1ayop Yu a ratatadpace dak // ί. Vu1. Eat. 1989.-Uo1. 264, No. 14.-R. 7761-7764.

14. Voskresensky O.N., Bobyrev V.N. Bioantioxidants obigatnye nutrition factors // Vopr. honey. chemistry. 1992. No. 4. P. 21.

15. Metelitsa D.N. Activation of oxygen by enzyme systems. M .: Nauka, 1982.256 s.

16. Maeda X., Akaike T. Nitric oxide and oxygen radicals in infection, inflammation and cancer // Biochemistry. 1998, T. 63, issue 7. S. 1007-1019.

17. Voskresensky O.N., Bobyrev V.N. Bioantioxidants - obligate nutritional factors // Vopr. honey. chemistry. 1992. No. 4. P. 21-26.

18. Sokolovsky V.V. Thiol antioxidants in the molecular mechanisms of the nonspecific reaction of the body to extreme effects // Vopr. honey. chemistry. 1988.V. 34, No. 6. S. 2-11.

19. RNKK N., Mepagy M. Ohyay-shyisey tol / uop o £ / mc £ goth te1a11oLiopet. L0e // Arcb. It is. Wurk. 1992. Uo1.293, No. 1. P.195-199.

20. Fedorova V.S., Budantseva E.P., Pavlyuchenko I.V. Food supplements as an object of invention // Patents and licenses. 2003. No. 6. S. 18-21.

21. Certificate of state registration of biologically active food additives BION 3. Ki.77.9.11.003.E.043698.10.11 of 10/14/2011

22. Avramenko V.A., Vasilevsky V.A. New sorbents based on modified zeolites and their use in ecology, agriculture and medicine // Zeolites of Primorye. Abstracts of the scientific and practical conference. Vladivostok, 1994, S. 16-19.

23. Panichev A.M., Gulkov A.N. Natural minerals and causal medicine of the future. Vladivostok: DVGTU Publishing House, 2001, - 216 p.

24. Pylev L.N., Vasilyeva L.A., Valamina I.E. Analysis of the biological aggressiveness of zeolites of various deposits of the Russian Federation // Natural minerals in the service of man. Materials of the scientific and practical conference. Novosibirsk, 1999, S. 68-70.

25. TU 9197-033-16925875-03 Natural zeolite - raw materials for the production of biologically active food additives.

26. Unified sanitary and epidemiological and hygienic requirements for goods subject to sanitary and epidemiological surveillance (control). Chapter P. Section 1. M., 2010, S. 6-451;

27. Glanz E. Biomedical statistics. M., 1999.

28. Methods of experimental research to establish thresholds for the effects of industrial poisons on the generative function. Guidelines. M., 1969, 25 p.

29. Methods of experimental studies to establish thresholds for the effects of industrial poisons on the generative function for the purpose of hygienic standardization. Guidelines 1744-77. M., 1978, 35 p.

30. Assessment of the mutagenic activity of chemicals by the micronuclear method. Guidelines 28/10. M., 1984, 21 p.

31. Evaluation criteria and methods for predicting the long-term effects of exposure to harmful substances in the air of the working area. Information mail. Research institute gig. tr and prof. the sickness. M., 1985, 15 pp.

32. Ermolenko E.I., Isakov V.A., Zhdan-Pushkina S.Kh., Tets V.V. Quantification of the antagonistic activity of lactobacilli. // Journal. microbiol. epidemic., immunobiol. 2004.-No5. S. 94-98.

33. Guidelines for the preclinical study of drugs with a nootropic type of action (in the book A Guide for Preclinical Studies of Medicines. Edited by AN Mironov. Part One. M: Grif and K, 2012, pp. 276-297 )

34. Buresh Ya. Methods and basic experiments on the study of the brain and behavior. M .: Higher. school., 1991.-399 s.

Table 1. Characteristics of the biological and physico-chemical properties of the sterilized culture fluid obtained by deep cultivation of the bacterial strain VasShik kiyshk VKPM No. V-2335

Name of the analyzed indicators The values of the analyzed indicators in the SKZh bacteria Vasyshiz ziibi VKPM No. V-2335 Residual cochlea schlämlapfobaproducer (cells), CFU / ml 0 Protein content *, μg · cm ' 541.5 Proteolytic activity (PA), units Pa-dm ' ³ 8.7 Amylolytic activity (AA), units AA-dm ' ³ 42.7 pH units pH 5,6 OP, units OP 0.31 WITH,% 0.68 The total concentration of total amino acids, mcg-cm ' ³ 568.8 The total concentration of free amino acids, mcg-cm ' ³ 60.8 The concentration of total, mcg-cm ^ - hexosamines - glucosamines - diaminopimelic acid 104.9 95.6 19.6 The concentration of free glucosamine, mcg-cm ' ³ 6.5 The concentration of free / bound nitrogen bases, μg / cm ³ -adenine / Xa ** - guanine / Xg ** - thymine / Xt ** - uracil / Xy ** - cytosine / Xc ** 12.2 / 3.2 0 / 16.3 8.2 / 0 11.4 / 8.6 0 / 12.1

Notes:

* - protein content was determined by the Lowry method;

** -Xa, Xg, Xm, Xy, Xc - unidentified derivatives, respectively, of adenine, guanine, thymine, uracil and cytosine in terms of the basic substance.

Table 2. The content in the inventive dietary supplement of toxic elements and organochlorine pesticides

Research indicators The content of toxic elements and organochlorine pesticides, mg / kg ET standard (chapter I, section I, index 10.6) In the claimed dietary supplement Toxic elements, mg / kg: - lead 6.0 0.02 - cadmium 1,0 0.01 - mercury 1,0 0.004 - arsenic 3.0 0.05 Organochlorine pesticides, mg / kg: - HCH (sum of cc, β and χ isomers) 0.1 <0.01 - DCT and its metabolites 0.1 <0.01 - Aldrin no tolerance. (<0.002) <0.001 - Heptachlor no tolerance. (<0.002) <0.001

Table 3 - the Total specific radioactivity of the claimed dietary supplement

Radionuclide Total specific radioactivity, Bq / kg Standard Y (chapter II, section I, index 10.6) In the claimed Dietary supplement ’° 5g one hundred 17 ^{, 37} Sz 200 23

- 17,024,516 my dietary supplement

Table 4. The results of studies of sanitary-microbiological safety indicators are declared

Research indicators Values of sanitary and microbiological indicators Standard ET (chapter II, section I, index 10.6) In the claimed Dietary supplement KMAFAiM, CFU / g Not more than 1-10 ’ Less than 10 BGKGT (coliforms), 0.1 g Not allowed Not detected 81 arb1lossoss5 aigeis, 0.1 g Not allowed Not detected Pathogenic microorganisms, including Salmonella, 10.0 g Not allowed Not detected VasShiz segeiz, CFU / g. in 1.0 g No more than 200 Not detected Yeast CFU / g No more than 100 Not detected Mold, cfu / g No more than 100 Not detected Spore-forming microorganisms of the type Vashiv vivbnv, 1.0 g Living cells of the bacteria Bacillus sylvidae are not allowed Living cells of the bacteria VasSi® ziN »VKPM No. B-2335 not found

Table 5. Dynamics of changes in body weight of rats treated with the claimed dietary supplement

G group ANIMALS View animals Animal body weight (g) over time background 2 days 7 days 14 days Control (receiving 1% starch suspension) male rats 200.4 ± 4.3 198.7 ± 3.8 201.7 ± 4.2 211.3 ± 4.3 female rats 197.1 ± 2.3 191.4 ± 3.3 193.9 ± 3.0 207.8 ± 3.7 Experienced (received the claimed dietary supplement) male rats 193.4 ± 3.2 191.8 ± 4.6 197.9 = 4.8 208.1 ± 3.4 female rats 187.0 ± 2.5 183.6 ± 2.6 190.5 ± 2.7 195.7 ± 2.8

Table 6. Mass ratios (MK) of organs in white rats with acute administration of the inventive dietary supplement

Analyzed organ Organ mass ratios (mg / 100 g body weight) of animal groups Control (receiving 1% starch suspension) Experienced (received the claimed dietary supplement) male rats female rats male rats female rats A heart 3.8 ± 0.2 4.1 ± 0.3 4.4 ± 0.4 4.3 ± 0.3 Lungs with trachea 7.2 ± 0.4 7.7 ± 0.7 8, GCD 7.9 ± 0.5 Thymus 1.34 ± 0.13 1.38 ± 0.06 1.18 ± 0.08 1.21 ± 0.13 Liver 37.3 ± 1, b 35.3 ± 1.8 31.9 ± 2.0 35.5 ± 1.5 Spleen 4.0 ± 0.3 4.4 ± 0.2 3.9 ± 0.3 3.4 ± 0.5 Kidney (left) 4.6 ± 0.1 4,5 ± 0,2 5.0 ± 0.2 4.4 ± 0.2 Adrenal glands 0.10 ± 0.01 0.08 ± 0.01 0.09 ± 0.02 0.10 ± 0.01 Brain 9.2 ± 0.4 8.5 ± 0.4 8, O ± O, 3 9.2 ± 0.2 Testicles or ovaries 13.6 ± 0.2 0.32 ± 0.02 11.9 ± 0.2 0.22 ± 0.08

th field after chronic administration of the claimed dietary supplement (registration within 5 min), M ± t

Table 7. Changes in the behavioral responses of white non-linear rats according to the open-method

Group animals Indicators of Behavioral Reactions Using the Open Field Method Horizontal motor activity Vertical motor activity Mink reflex Boluses Integral activity Control (received 1% starch suspension 35 ± 2 15 ± 3 13 ± 1 4 ± 1 67 ± 4 Experienced (received the claimed dietary supplement) 37 ± 5 15 ± 2 154 = 1 6 ± 2 75 ± 5

toxicity of nonlinear white rats, M ± t

Table 8. The effect of chronic administration of the claimed dietary supplement on indicators of total non-lethal

Total non-lethal toxicity The value of the indicator of total non-lethal toxicity The control After chronic administration of the claimed dietary supplement Body weight g 205 ± 10 201 ± 4 Mass indices of organs, g / 100 g body weight: -lungs b, 2 ± 0.1 6.4 ± 0.1 - liver 28.9 ± 1.0 28.0 ± 1.7 - kidneys 7.7 ± 1.1 8.0 ± 0.6 - spleen 4.7 ± 0.2 5.1 ± 0.2 - a heart 3.7 ± 0.2 3.2 ± 0.1 Urine counts: - urine protein, mg% 4.2 ± 0.6 3.4 ± 0.5 - specific gravity of urine, g / mm ³ 1,045 ± 0,035 0.985 ± 0.025 Blood biochemical parameters; - blood sugar, mg% 89 ± 3 82 ± 7 serum protein, g% 8.43 ± 0.13 7.89 ± 0.15 -lipid phosphorus, mmol / l 33.82 ± 0.51 32.11 ± 0.38 - Alt, mkkat / l 0.82 ± 0.07 0.85 ± 0.10 - AST, mkkat / l 0.71 ± 0.11 0.80 ± 0.14 - alkaline phosphatase, mkkat / l 1.01 ± 0.09 0.85 ± 0.20 thym test, unit muddle. 1.79 ± 0.15 1.81 ± 0.10 Blood morphological parameters: the content of red blood cells, million Umm ³ 7.26 ± 0.05 6.94 ± 0.10 - hemoglobin content, g% 15.9 ± 1.2 15.6 ± 0.5 - the content of leukocytes, thousand, / mm ³ 10.28 ± 0.11 8.43 ± 0.49 Physiological indicators: - nbsp; in 12.8 ± 0.5 14.9 ± 1.3 -hexenal sleep duration, min. 25.4 ± 2.4 23.6 ± 1.3

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Table 9. The effect of the claimed dietary supplement on the motor function of the ciliated epithelium

Group of animals number EXPERIENCE The passage time of cork crumb section of the esophagus with a length of 10 mm, s Source After a period of time after application 1-3 min 7-10 mick Control (distilled water was applied to the mucous membrane of the esophagus) 10 23.5 ± 1.4 25,842,6 27.9 ± 1.7 Experienced (the claimed dietary supplement was applied to the mucous membrane of the thread guide) 10 21.1 ± 1.4 24,341,2 28,441,1

the introduction of the claimed dietary supplement, M ± t

Table 10. Sensitization of peripheral blood of guinea pigs after intradermal

Index sensitizing actions Sensitizing effect value The control After the introduction of the claimed dietary supplement White blood cells, thousand / mm '' 10.5 ± 0.6 10.4 ± 0.1 Eosinophils,% 7 ± 2 6 ± 1 Lymphocytes,% 33 ± 4 3645 Monocytes,% 4 ± 1 6 ± 1 Neutrophils,% 56 ± 2 52 ± 5 RSLL,% lysis 8 ± 1 942

Table 11. Indicators of gonadotoxic and mutagenic effects of the claimed dietary supplement, M ± t

The indicator of gonadotoxic and mutagenic effects Indicator value gonadotoxic and mutagenic effects The control After the introduction of the claimed dietary supplement Gonad weight coefficient of male rats, g / 100 g body weight 6.9640.35 7.0840.47 The number of sperm, 10 ⁶ / ml 152 ± 24 191439 Sperm movement duration, min 96412 104 ± 3 Osmotic resistance MG \ aC1 2.5140.09 2.40 ± 0.12 The number of analyzed bone marrow cells of mice 40514104 42664114 The frequency of cells with micronuclei,% 0,03640,003 0,03340,012

Table 12. The study of changes in the characteristics of the claimed dietary supplement when stored for 2.5 years

Estimated performance The value of the indicator in the claimed dietary supplement Before storage After storage Appearance Granular powder Granular powder Colour Light brown Light brown Smell Specific, Specific, inherent in this inherent in this product product Moisture content, % 5.8 5.9 Ampolytic activity, units AA / g 3.2 2,840,4 Proteoletic activity, units PA / g 0.6 0.4 ± 0.1 Microbiological purity (availability in 1 g of the drug): - Rzey4otopaz aegi§tosa Are absent Are absent - Ep1egoacapaseae Are absent Are absent - 31ар1 | у1ососси8 аигеиз Are absent Are absent - VasShiz segeiz Are absent Are absent The content of vitamins, mg / g: -FROM 90.5 90.2 -Βι 1,53 1,52 - at ₂ 1.81 1.80 -E 13.6 13.5 The content of minerals, mg / g: - zinc 7.1 7.0 -chromium 0,0243 0,0242 - selenium 0,0362 0,0360 - magnesium 102.5 101.9

Table 13. The effect of VasShik kiMShk VKPM metabolites No. B-2335 on the growth of probiotic lactobacilli Lactobacillus p1iagit 8K-A3

Concentration of Metabolites of Vasiliconium zabpc VKPM No. B-2335 Ten-fold dilutions of Lactobacillus p1ap1agish 8K-AZ, seeded in 10 μl *** one 2 3 4 5 6 Initial concentration * lawn THOUSANDS thousands HUNDREDS 10 Dilution 1: 10 ** lawn thousands thousands hundreds fifty 5 The control lawn thousands thousands 124 12 0

Notes:

* - 1 g per 10 ml of culture medium;

** - 0.1 g per 10 ml of culture medium;

*** -dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc .;

**** - hereinafter - the number of CFU (colony forming units)

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Table 14. Antibacterial activity of lactobacilli LacFasac Schiz p1ap1agit 8K-L3 against staphylococci

The investigated composition (number) Tenfold dilutions of 31ar11u1ososi8 aigeis from 209, seeded in 10 μl * Without breeding one 2 3 4 5 Lactobacilli bacCocassia p1ap1agit 8K-AZ (0.1 g) hundreds 150 ** fifteen 2 0 0 Lactobacilli bacillus bacillus papantum 8K-AZ (0.1 g) + metabolites Bacillus bacillus VKPM No. B-2335 (1 g) hundreds 117 10 one 0 0 The control lawn lawn THOUSANDS hundreds 14 2

Notes:

* - dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc .;

** - hereinafter - the number of CFU (colony forming units).

Table 15. Antibacterial activity of lactobacilli LacFasac Schiz p1ap1agish 8K-L3 against Escherichia

The investigated composition (number) Ten-fold dilutions of Euxepilon Coni S15, seeded in 10 μl * Without breeding one 2 3 4 5 Lactobacilli Bashobaschis p1ap1agit 8K-AZ (0.1 g) hundreds 120 ** 12 one 0 0 Lactobacillus bacillus bacillus p1ap (agish 8B.-AZ (0.1 g) + metabolites Bacillus bacillus VKPM Xy B-2335 0 g) hundreds 80 7 0 0 0 The control lawn lawn thousands hundreds 24 2

Notes:

* -dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc .;

** - hereinafter - the number of CFU (colony forming units).

Table 16. Antibacterial activity of lactobacilli LacFacac Schiz p1ap1agish 8K-L3 against pseudomonads

The investigated composition (number) Ten-fold dilutions of Rzeyskopopaz aegi ^ toza, seeded 10 μl * Without breeding one 2 3 4 5 Lactobacilli Lactobacillus p1ap1aP1t 8K-AZ (0.1 g) hundreds twenty** 3 0 0 0 Lactobacillus bac (bacillus 5 p1ap1agitp 8K-AZ (0.1 g) + metabolites of Vasilyz sulophytes VKPM No. B-2335 (1 g) hundreds eleven one 0 0 0 The control lawn lawn THOUSANDS hundreds 32 3

Notes:

* -dilutions: 1 - 1:10; 2-1: 100; 3 - 1: 1000, etc .;

** - hereinafter - the number of CFU (colony forming units)

Table 17. The effect of the inventive dietary supplement when administered for four weeks on the resistance of rats to high temperatures (M ± t, n = 6)

Group of animals Controlled indicator, min Start of cramps Death time Control. . (received distilled water) 31.8 ± 1.2 41.4 ± 1.9 ....... Experienced (received the claimed dietary supplement) 41.7 ± 1.5 45.3 ± 1.8 *

Note. * - the difference with the control group is significant.

Table 18. The effect of the claimed dietary supplement on indicators of passive avoidance reaction of rats

Group of animals Research indicators Latent period of transition reaction, s Time stay in the bright part compartment, with Time stay in the dark compartment, with The number of trained animals (% Co. All group) Control (received!% starch suspension) 75 ± 5 70 ± 5 22 ± 3 5 (50) 1 experienced (received the claimed dietary supplement) 140 ± 8 * 120 ± 10 * 5.0 ± 0.5 * 10 (100) 2 experienced (received dietary supplement prototype) 130 ± 6 * 108 ± 6 * No. 0.5 * 8 (80) 3 experienced (receiving Noopept) 115 ± 10 * 95 ± 5 7.5 ± 0.5 * 7 (70)

Note. * - significant differences from the control group of animals at p <0.05

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Table 19. A study of the effect of the claimed dietary supplement when administered for four weeks on the resistance of rats with hypercapnic hypoxia (M ± t, n = 6)

Group of animals Controlled indicator, min Beginning of cramps Death time Control (receiving 1% starch suspension) 24.3 + 1.1 27.3 + 0.6 Experienced (received the claimed dietary supplement) 43.8 + 2.1 * 46.7 + 1.9 *

Note. * - the difference with the control group is significant

Table 20 - The effect of the claimed dietary supplement with a four-week introduction on the indicators of lipid peroxidation in rat blood after bilateral ligation of the carotid arteries

Blood count Indicator level for a group of animals Intact (no dressing) Control (receiving 1% starch suspension) Experienced (received the claimed dietary supplement) Catalase activity 0.247 + 0.007 0.243 + 0.018 0.225 + 0.019 * Recovery diphenylpicrylhydrazyl 0.568 + 0.023 0.566 + 0.028 0.476 + 0.017 * Diene conjugates isopropanol fraction 0.203 + 0.024 0.233 + 0.021 0.160 + 0.032 Diene conjugates heptanol fraction 0.842 + 0.031 0.851 + 0.057 0.724 + 0.023 *

Note. * - the difference with the control group is significant

Table 21. The effect of the claimed dietary supplement when administered for four weeks on the spectrophotometry of samples of rat brain homogenates after bilateral ligation of the carotid arteries

Test indicator Indicator level for a group of animals Intact (no dressing) Control (receiving 1% starch suspension) Experienced (received the claimed dietary supplement) Recovery diphenylpicrylhydrazyl 0.088 + 0.019 0.101 + 0.031 0.049 + 0.008 * Diene conjugates isopropanol fraction 0.181 + 0.025 0.168 + 0.020 0.145 + 0.011 * Diene conjugates heptanol fraction 0.549 + 0.025 0.536 + 0.011 0.547 + 0.012

Note. * - the difference with the control group is significant

Table 22. The effect of the claimed dietary supplement on the endurance of white mice in a swimming test with a load (M ± t)

R group of animals The duration of swimming mice (min) with the introduction of the inventive dietary supplement during background 14 days Control (receiving 1% starch suspension) 565 + 42 560 + 36 1 experienced (received the claimed dietary supplement) 515 + 35 690 + 25 * 2 experienced (received dietary supplement prototype) 520 + 20 595 + 32 *

Note. * - the difference with the control group and the background indicator in the group is significant. Table 23 - Study of the influence of the claimed dietary supplement on the static-power endurance of mice in the test of power load own weight (M ± t)

Group of animals The duration of hanging mice, min Control (receiving 1% starch suspension) 20.7 + 0.5 Experienced (received the claimed dietary supplement) 28.3 + 0.3 *

Note. * - significant differences with the control group at p <0.05

Table 24. The effect of the claimed dietary supplement on the muscle strength of rats (M ± t)

Group of animals Muscle strength of rats (g) after administration of the inventive dietary supplement during background 14 days Control (receiving 1% starch suspension) 459 + 16 463 + 15 1 experienced (received the claimed dietary supplement) 470 + 15 542 + 12 * 2 experienced (received dietary supplement prototype) 465 + 12 490 + 10

Note. * - the difference with control and background in the group is significant

Claims (1)

CLAIM A biologically active food supplement, which is a balanced vitamin-mineral-probiotic complex and includes the vitamin-mineral and probiotic components, in which the vitamin-mineral component contains an antioxidant combination, including antioxidant vitamins, including water-soluble vitamin C, fat-soluble vitamins E in the form of α a tocopherol acetate and retinol as an acetate and selenium-containing compound in the form of sodium selenite Na ₂ 8eO _{3, the} combination of B vitamins, including tiami as hydrochloride (vitamin B!), riboflavin (vitamin _B2), pantothenic acid (vitamin B _5), pyridoxine hydrochloride (vitamin _B6), folic acid (vitamin B _9), cyanocobalamin (vitamin B ₁₂₎ and holekalitsiferol ( vitamin Ό ₃ ), nicotinamide (vitamin PP), biotin (vitamin H) and minerals, including magnesium in the form of magnesium oxide, zinc in the form of zinc oxide, manganese in the form of manganese sulfate, iodine in the form of potassium iodide, iron and chromium, characterized in that it further comprises a carrier, which is used as a zeolite, as a probiotic component The sterilized dried culture fluid containing bacterial metabolites of Vaschiz 5u1b3 VKPM No. V-2335 was used, the vitamin-mineral component additionally included the vitamin antioxidant beta-carotene, phylloquinone (vitamin K!), and the trace element copper in the form of copper sulfate, moreover beta-carotene is included in an antioxidant combination in which vitamin C is used in the form of ascorbic acid, iron in the form of iron fumarate, chromium in the form of chromium picolinate, and the biologically active additive Nena in a solid dosage form in the form of capsules and the amount of ingredients in two capsules a total weight of 1000 mg is: Retinol acetate (A), mg - 0.7; α-tocoforol acetate (E), mg - 13.6; Ascorbic acid (C), mg - 94.5; Beta-carotene, mg - 3.4; Cholecaliciferol (ϋ ₃ ), mcg - 14.5; Phylloquinone (K]), mcg - 174.0; Thiamine hydrochloride (B ι), mg -1.7; Riboflavin (Vg), mg - 2.0; Pantothenic acid (B5), mg 6.2; Pyridoxine hydrochloride (Wb), mg - 2.2; Folic acid (B $), mcg - 500.1; Cyanocobalamin (B12), mcg - 3.9; Nicotinamide (PP), mg - 21.0; Biotin (H), mcg - 54.9; Magnesium (in the form of magnesium oxide), mg - 100.0; Iron (in the form of iron fumarate), mg - 10.0; Zinc (in the form of zinc oxide), mg - 7.0; Manganese (in the form of manganese sulfate), mg - 1.4; Copper (in the form of copper sulfate), mg - 0.9; Iodine (as potassium iodide), mcg - 129.9; Chromium (as chromium picolinate), mcg - 24.9; Selenium (as sodium selenite), mcg - 35.1; Zeolite, mg - 195.0; Sterilized dried culture fluid containing the metabolites of the bacteria Bacillus viridae VKPM No. B-2335 mg - 5.0.