RU2797514C1 - Pharmacological substance of membranotropic homeostatic tissue-specific bioregulators isolated from various tissues and biological fluids of animals - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the pharmacological substance of membrane-tropic homeostatic tissue-specific bioregulators MHTB in the form of their solutions in a pharmaceutically acceptable carrier and/or diluent, which has the ability to maintain and restore the function of organ tissues after the development of various pathological processes in them. The pharmacological substance of membranotropic homeostatic tissue-specific bioregulators MHTB in the form of their solutions in a pharmaceutically acceptable carrier and/or diluent has the ability to maintain and restore the function of organ tissues after the development of various pathological processes in them, the substance is isolated from various tissues and biological fluids of animals selected from the group, including blood serum, tissue of the liver, lung, intestines, heart, pancreas, thyroid, prostate and mammary glands, bile, containing as an active basis protein-peptide complexes, which consist of proteins of the serum albumin multifamily, having a molecular weight in the range of 65,000–68,000 Da and biologically active peptides with molecular weights of 1,000–10,000 Da, being proteolysis products of a number of proteins, as well as calcium ions, lipids, inositol, carbohydrates, such as mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues, which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

EFFECT: abovementioned invention expands the arsenal of means for creating pharmacological preparations of natural origin based on previously insufficiently studied biologically active (protein-peptide) complexes contained in various tissues and biological fluids of animals.

1 cl, 18 dwg, 8 tbl, 38 ex

Description

The invention relates to biotechnology and concerns a pharmacological substance based on membranotropic homeostatic tissue-specific bioregulators isolated from various tissues and biological fluids of animals and can be used in the pharmaceutical industry and medicine.

The invention is aimed at identifying the composition and structure of a previously insufficiently studied biologically active substance such as membranotropic homeostatic tissue-specific bioregulators (MHTB) found in various tissues and biological fluids of animals.

The invention allows to expand the existing ideas about biologically active substances of natural origin.

The biologically active substance MHTB is extracted from animal tissues in saline at low temperatures. Biological fluids (milk, bile) and a commercial preparation of bovine blood serum can also be used as raw materials. Further, purification of MHTB and its individual components can be carried out in several ways, using the methods of preparative protein chemistry - salting out of proteins, chromatography of proteins and peptides, electrophoresis in PAAG, isoelectrofocusing (IEF), etc. The invention presents several methods for isolating and purifying the substance of MHTB, which do not affect its composition, structure and biological activity. For example, a method is presented, consisting of obtaining a tissue extract, salting out impurity proteins in a saturated solution of ammonium sulfate, obtaining a supernatant fraction and then separating it by the IEF method in a sucrose gradient in the pH range of 3.5-10.0. Fractions of acidic proteins were collected and studied, which were collected in the pH range of 1.5-3.0,

During the purification process, the presence of the pharmacological substance MHTB and its individual fractions was determined using the original adhesiometric method, which is based on the assessment of its membranotropic activity - the effect on the viscoelastic properties of the plasma membrane (PM) of mammalian cells under in vitro conditions. Given the fact that the MHTB substance exhibits a biological effect at low concentrations, a dose dependence of its activity is studied in the concentration range corresponding to 10 ^-3 - 10 ^-19 mg of protein per ml. The concentration line of solutions of the MHTB substance is obtained by successive 10-fold dilution of the initial solution with an established concentration, which is determined by standard methods.

Previously, on various experimental models in vitro and in vivo, it was shown that the MHTB substance in relation to pathologically altered tissues exhibits reparative and regenerative activity, which is characterized by the presence of organ tissue, but the absence of species specificity, and therefore can be used to prepare pharmacological agents.

For example, in published monographs [1] - [4] (Konstantinovsky et al. 2012; Yamskova et al. 2009; 2010; 2012) it was shown that fractions obtained from extracts of various eye tissues, as well as bull blood serum, in low doses activate the processes of restoration and reparation in mammals on the model of an experimental wound of the cornea of the eye in vivo. The bioregulator isolated from the cornea of the bull's eye had a pronounced protective effect on the state of this tissue when the cornea of the eye of vertebrates was cultivated in vitro due to the ability to additionally activate cellular sources of regeneration. A bioregulator isolated from blood serum stimulated corneal wound healing in a rabbit in an in vivo experiment, as well as in a person with post-traumatic recurrent erosion and corneal burns. The bioregulator isolated from the pigment epithelium of the bull's eye contributed to the stabilization of the differentiation of pigment epithelium cells and the maintenance of the viability of bipolar and Muller cells of the retina under conditions of organotypic cultivation of eye tissues in vitro.

From RU 2725491 02.07.2020, a substance for the production of drugs is known that promotes the maturation of mammalian spermatozoa and exhibits biological activity when diluted in an aqueous pharmaceutically acceptable carrier and / or diluent to a protein concentration of 10 ^-8 to 10 ^-15 mg / ml, including peptides with molecular weights of 1000-6000 Da, and bovine serum albumin with a molecular weight of 65712 Da and an amino acid sequence that has 100% homology with the N-terminal sequence of a mature molecule of bovine serum albumin, at a ratio by weight of peptides: protein as 1: 1-7, isolated from the testes of bulls IEF fractions, separated and collected by isoelectric focusing in the range of pH<3.0 using reverse phase HPLC.

In particular, from RU 2701566 09/30/2019 a pharmacological composition is known that has a protective effect on the state of the tissues of the posterior part of the eye - the sclera, retina, pigment epithelium, choroid. This substance contains a protein-peptide complex formed by mixing a protein with a molecular weight in the range from 66,000 to 68,000 Da, belonging to the blood serum albumin family, and peptides with a molecular weight from 1,000 to 6,000 Da, as well as lipids, carbohydrates and calcium ions, and is an aqueous solution of BOD at a concentration of 10 ^-7 -10 ^-17 mg/ml. BOD was obtained by extracting the scleral tissue of the eye of vertebrate animals, and then separating the resulting tissue extract by isoelectric focusing in a sucrose gradient or/and reverse-phase HPLC.

The known invention ensures the maintenance of adhesion in the scleral tissue between individual tissues of the eye, as well as adhesive intercellular interactions within individual tissues, the integrity and preservation of the spatial organization of collagen fibers, an increase in the viability of scleral fibroblasts, and the preservation of the status of their cell proliferation and differentiation.

EFFECT: invention makes it possible to obtain a highly purified preparation intended as a substance for a pharmacological (drug) agent for the prevention and treatment of eye diseases associated with the claimed disorders.

According to the above-mentioned well-known invention (RU 2701566 09/30/2019), the method for isolating and purifying the VPC consists in isolating the macular area of the retinal PE from freshly enucleated eyes of a bull or another vertebrate, washing it in saline, keeping the PE tissue in an aqueous saline solution, tissue extract is centrifuged, supernatant is collected, proteins are precipitated in 100% ammonium sulfate solution, the fraction floating at 100,000 g - 105,000 g is collected by centrifugation, dialyzed until ammonium ions are completely removed, separated by high performance liquid chromatography in a 0.1% acetonitrile gradient trifluoroacetic acid at pH 2.2-3.0, temperature 22-25°C for 30-60 minutes, fractions of hydrophilic peptides and protein - a representative of the blood serum albumin family are collected, from which acetonitrile is removed and mixed in the presence of 10 ^{- 3} M CaCl ₂ in equimolecular amounts.

Thus, in these works it was shown that extracts of various animal tissues, obtained by water-salt extraction of tissues at low temperature, contain biologically active substances localized extracellularly, which have a complex structure - they contain proteins, peptides, carbohydrates, which have a tissue-specific effect. on adhesion, migration, differentiation, proliferation of cells, cause an increase in their viability under in vitro cultivation conditions. Biologically active substances contained in these extracts of animal tissues activate the processes of restoration and reparation in pathologically altered tissues due to additional activation of cellular sources of regeneration in the body.

A significant drawback of all these well-known technical solutions is the lack of data on the composition, structure and mechanism of action of biologically active substances contained in the pharmacological substance of MHTB and exhibiting tissue-specific reparative and regenerative activity, causing the restoration of pathologically altered tissues without the formation of a connective tissue scar. The data of the conducted studies indicated a new regeneration mechanism, which was activated as a result of the ability of biologically active substances that make up the substance under study to cause the restoration of the structure and function of damaged tissues without the formation of a connective tissue scar (fibrosis mechanism). Therefore, the elucidation of issues related to the determination of the composition, structure and mechanism of action of biologically active substances seemed extremely relevant, since the results obtained in this aspect would contribute to the most effective use of the pharmacological substance MHTB as a basis for the development of new drugs.

In the claimed invention, it was possible to identify the composition, structure, determine the physicochemical properties and mechanism of the biological action of the MHTB substance isolated from various tissues and biological fluids of animals. This invention presents the results of studies that show that the basis of the MHTB substance isolated from various tissues and biological fluids of animals, make up protein-peptide complexes (BPK), which, in turn, consist of peptides with molecular masses from 1000 Da to 10000 Da, which are proteolysis products of known studied proteins (enzymes, adhesive and membrane proteins) that interact with proteins of the blood serum albumin multifamily that modulate the activity of these peptides. The presence of calcium ions (10 ^-3 M CaCl _{2 )} in the MHTB complex is indicated by the information in examples 11, 32, which shows the CD spectra of MHTB isolated from blood serum after exposure to EDTA and other physicochemical factors. In this study, it was demonstrated that the conformation of this MHTB was most disturbed after treatment with a Ca-chelating agent (EDTA): in the case of exposure to a temperature of 100°C or guanidine chloride (disaggregating agent), no such violation of the BOD conformation was found (Fig. 13-15).

In addition, it was found (18) that the method of equilibrium microdialysis showed that the MHTB substance has a high Ca ²⁺ -binding activity (Kd=10 ^-4 -10 ^-6 mg/ml).

The study of the physicochemical properties of the MHTB substance showed that its water-salt solutions contain nanosized particles that tend to further enlargement. Interactions with lipids, the presence of an inositol-containing glycosidic structure in the composition of MHTB, its nanoscale state are extremely important for the manifestation of the biological effect of the substance - it is through this mechanism that the extracellular structure of MHTB is linked to the plasma membrane of cells and maintains its stable conformation, which determines the biological effect of this substance.

Comparing these data with the results of the study of the substance MHTB - its composition, structure, physicochemical properties, biological action [2] (Yamskova et al., 2009), we can conclude that it is involved in intercellular adhesion and processes of cellular regulatory transduction due to its ability change the properties of the PMC.

In fact, the BODs that make up the substance of MHTB are the previously discovered “tissue forms” of serum albumin, the function of which has remained poorly understood until now [5] (Peters, 1996). According to the results obtained according to the invention, these "tissue forms" of serum albumin are formed as a result of the transport of certain of its isoforms through the histohematogenous barriers of the body, their penetration into the intercellular spaces of tissues and interaction by a calcium-dependent mechanism with a constant set of peptides for each tissue. Peptides are the products of the ongoing proteolysis of various proteins. The resulting BPC, which is the basis of the MHTB substance, is then capable of interacting with the PM of cells through binding to the macromolecular system of the inositol-containing glycosidic anchor.

It is possible that it is these BOD interactions that underlie the “self-assembly” of the MHTB substance in the intercellular space. This assumption is supported by the unique ability of the serum albumin molecule to bind a wide range of ligands, transporting a huge amount of various substances in the body. Such an important function of albumin is based on the structural mobility of its molecule [5].

In contrast to the above inventions, the MHTB substances of animal origin presented in the present invention are identified BPCs, the structure and structure of which have been studied: they include a constant set of biologically active peptides for each tissue with established molecular weight values, as well as proteins that modulate activity these peptides, the structure of which was identified using the PMF (peptide mass fingerprinting) method in the NCBI database.

The isolation of the substances claimed as an invention for pharmacological preparations from tissues of animal origin is carried out by extracting small fragments of freshly obtained animal tissue in saline at low temperature. This excludes tissue homogenization, enzymatic processing and other influences that cause violation of cell integrity. The main factor determining the transition to the external environment (extracting solution) of bioregulators of the MHTB group, which form the basis of the claimed substances, is the temperature of the extracting solution of +4°C-0°C. At this temperature, a phase transition of the PLA bilayer occurs, as a result of which the supramolecular structure of BOD, which is part of MHTB, associated with it (for example, through “membrane anchors”) changes its interaction with PLA and passes into the extracting solution. The presence of calcium ions in the extracting solution does not affect the isolation of MHTB.

MHTB substance is obtained from biological fluids (for example, bile) by first subjecting it to fractionation, for example, by salting out impurity proteins by adding salts or organic solvents, followed by the use of traditional methods (biological chemistry) of protein chemistry - isoelectric focusing, chromatography, etc.

The technical objective of the present invention is to obtain a pharmacological substance of nanosized membranotropic homeostatic tissue-specific bioregulators of MHTB with increased biological activity due to a certain (identified) composition of the components included in the previously unexplored structure and organization of the pharmacological substance of MHTB, as well as the studied mechanism of its functioning in living organisms, which allows most effectively apply the results obtained for the prevention and treatment of common diseases, pathologies of tissues of various organs.

The technical result of the claimed invention in accordance with the technical task is to expand the arsenal of means for creating pharmacological preparations of natural origin, which are based on previously insufficiently studied biologically active (protein-peptide) complexes contained in various tissues and biological fluids of animals.

The set technical task and the technical result are achieved by the pharmacological substance of membranotropic homeostatic tissue-specific bioregulators MHTB claimed as an invention, which, in the form of their solutions in a pharmaceutically acceptable carrier and / or diluent, has the ability to maintain, stimulate and restore the function of organ tissues after the development of various pathological conditions in them. processes isolated from various tissues and biological fluids of animals selected from the group including blood serum, tissues of the liver, lung, intestines, pancreas, prostate, mammary gland, bile, containing protein-peptide complexes as an active base, which consist of proteins of the serum albumin multifamily and biologically active peptides with molecular weights of 1000Da-10000Da, which are products of proteolysis of a number of proteins, as well as Ca ⁺² calcium ions, lipids, carbohydrates, such as residues of mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Claimed as an invention, the pharmacological substance of membranotropic homeostatic tissue-specific bioregulators MHTB is intended for the preparation on its basis of a drug for the prevention and treatment of common diseases in humans and animals in the form of solutions in the concentration range from 10 ^-17 to 10 ^-5 mg protein / ml when diluted in a pharmaceutical acceptable carrier and/or diluent, and present in the form of nano-sized particles of 50-200 nm.

The claimed pharmacological substance of membranotropic homeostatic tissue-specific bioregulators MHTB is tissue-specific, but not species-specific, helps to maintain and restore tissue function during the development of various pathological processes.

So, the technical result is achieved by the claimed invention, which contributes to the development of the main criteria for the use of the previously insufficiently studied substance MHTB to create on its basis new pharmacological agents intended for the prevention and treatment of common diseases in animals and humans. The obtained technical result will contribute to the most effective use as a basis for the development of new drugs based on the MHTB substance isolated from tissues or biological fluids of animals containing biologically active peptides with molecular weights of 1000 Da - 10000 Da, proteins of the serum albumin multifamily, as well as ions calcium, lipids, inositol-containing glycosidic structure, which, when diluted in a pharmaceutically acceptable carrier and/or diluent at low concentrations from 10 ^-17 to 10 ^-5 mg/ml, has a biological effect, which is characterized by the presence of organ tissue (but not species) specificity.

The following is a brief description of the general method for obtaining the MHTB pharmacological substance claimed as the proposed invention, which is based on protein-peptide complexes isolated from tissues and biological fluids of various vertebrates.

Pharmacological substance MHTB, isolated from tissues or biological fluids of animals, is obtained on the basis of highly purified fractions of tissue extracts, separated and collected by the methods of isoelectric focusing, electrophoresis and reverse phase HPLC. The composition, structure and homogeneity of these fractions were studied using modern methods of biochemistry, bioorganic chemistry, and the biological effect - using the methods of biophysics, cell biology and medical biotechnology.

It has been established, for example, that for the MHTB substance isolated from tissues and biological fluids of a bull, these are the isoforms of serum albumin gi|1351907; gi|367460260; gi|74267962, in the SwissProt database, which differ from each other in the composition of amino acids only in four positions of the polypeptide chain: 116, 214, 429, and 579. , isolated from various tissues of the Wistar rat, also includes three isoforms of rat serum albumin - gi| 158138568; gi|55628; gi| 124028612, which differ from each other by three amino acid substitutions of the polypeptide chain, respectively, at positions 262, 317 and 431. The established amino acid substitutions in the primary sequence of serum albumin isolated from bovine and rat tissues are located in the second and third domains of its molecule. Such a correlation of amino acid substitutions in the primary sequence of albumins can explain their ability, as modulator proteins, to change the activity of peptides that make up protein–peptide complexes in various ways.

It was also established and shown that in the pharmacological substance of MHTB, protein-peptide complexes interact with lipids, calcium ions, which stabilize their conformation and, among other things, determine them.

The pharmacological substance MHTB, isolated from various tissues and biological fluids of animals, is used, for example, in the form of a drinking solution in a low concentration, which eliminates the development of adverse reactions from both individual tissues of the gastrointestinal tract and from the body as a whole. The solution is applied orally for 2-6 months.

To determine the carbohydrate composition of the studied IEF fraction, it is lyophilized beforehand. Then, 1 mg of the lyophilizate is hydrolyzed in 2 ml of 4 M trifluoroacetic acid in a glass ampoule with a Teflon cap for 3 hours at 110°C; after the reaction time, the trifluoroacetic acid is removed on a rotary evaporator . Modification of monosaccharides - obtaining 3-methyl-1-phenyl-2-pyrazolin-5-one-(PMP)-derivatives of carbohydrates is carried out according to the method [S. Honda, E. Akao, S. Suzuki, M. Okuda, K. Kakehi J. Nakamura Anal. Biochem. 180 (1989) 351]. The hydrolyzed sample is dissolved in 0.5 ml of 0.3 M NaOH, 0.5 ml of a 0.5 M solution of PMP in methanol is added to the solution and incubated for 2 hours at 70°C. After neutralization with 0.3 M HCl, the excess PMP is removed by extraction with chloroform, the aqueous phase is evaporated to dryness. Chromatographic analysis of PMP derivatives is carried out on a Kromasil C18 column (250 × 4.6 mm) by elution with 0.1 M CH3COONH4, pH 5.6 in 20% acetonitrile, detection is carried out at 245 nm. As a result of the analysis, it was found that the IEF fraction contains carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues), which form an inositol-containing glycosidic structure (in particular, 22.5% glucose, 22 .6% glucosamine, 20.2% mannose, 11.6% galactose, 8.5% xylose, 7.4% fucose, 6.5% N-acetylglucosamine).

The data obtained indicate the presence of a number of glycans in MHTB (containing BOD) isolated from the biological fluid and tissues of animals, which determine, on the one hand, its interaction with the plasma membrane of the cell, and, on the other hand, the tissue-specific nature of its biological action.

To determine lipids in the composition of the IEF-fraction, the method of gas chromatography-mass spectrometry is used, their derivatives are preliminarily obtained - the corresponding trimethylsilyl and methyl esters. To obtain trimethylsilyl ethers, HPLC fractions with a retention time of 53 min were treated with a silating agent, N,O-bis(trimethylsilyl)acetamide, at 60°C. To obtain methyl esters, a multicomponent sample was treated with a methylating reagent, tetramethylammonium hydroxide (25% solution in methanol). Pyrolysis of the intermediately formed tetramethylammonium salts with the formation of the corresponding methyl esters takes place directly in the column during chromatography of the analyzed fraction. The determination of the component composition of the studied fraction is carried out on an Agilent 7890 gas chromatograph (USA) on an HP-5 column (USA) (50 m × 320 μm × 1.05 μm) equipped with a 5975C mass spectrometric detector with a quadrupole mass analyzer. The analysis is carried out at the temperature program of chromatography: isotherm 2 min at 40°C; heating up to 250°С at a rate of 5°С/min; isotherm 15 min at 250°С; heating up to 320°C at a rate of 25°C/min; isotherm 5 min at 320°С. Interface temperature 280°C. Carrier gas - helium, flow rate 1 ml/min. Fraction chromatogram by total ion current. Mass spectrometric detection conditions: energy of ionizing electrons 70 eV; registration of mass spectra in positive ions in the range (m/z) from 20 to 450 at a rate of 2.5 scans/sec. Software - chemStation E02.00. Identification of the component composition (qualitative analysis) is carried out according to the library of complete mass spectra and the corresponding values of the chromatographic linear retention indices (Ilin). As a result, it was found that the IEF fraction contains components that are components of glycerides, phospholipids, glycolipids, as well as saturated fatty acids (C6:0, C8:0, C9:0, C10:0, C12:0, C14:0 , C15:0, C16:0, C17:0, C18:0) and a monoenoic fatty acid (C16.1).

The invention is illustrated by the following examples, 18 figures (18 ill.) and 8 tables, which do not limit the scope of his claims.

The following is a brief description of the attached figures (FIGS. 1-18) and tables (Tables 1-8).

Fig. 1. Isoelectric focusing of supernatants of tissue extracts isolated from:

A - rat liver; B - rat lung; B - rat small intestine; G - bile of a bull. On the ordinate 1 - pH values; along the ordinate 2- D280; abscissa - numbers of fractions.

Fig. Fig. 2. Influence on the viscoelastic properties of the plasma membrane of mouse liver cells during multiple organ cultivation in vitro, a number of subfractions (A-D), collected in the pH range less than 3.0 after isoelectric focusing of the tissue extract supernatant: A - rat liver; B - lung of rats; B - bile of a bull; D - small intestine of rats. On the ordinate - the magnitude of the effect Ea (%); on the abscissa - an indicator of the degree of tenfold dilution.

K - control; columns - exposure to the IEF-fraction at a dilution of 10 ^x , where x is the tenfold dilution of the study drug, (p<0.05).

Fig. Fig. 3. Influence on the viscoelastic properties of the plasma membrane of mouse liver cells during multiple organ cultivation in vitro, a number of subfractions (A-D), collected in the pH range of 8.5-9.5 after isoelectric focusing of the tissue extract supernatant: A - rat liver; B - lung of rats; B - bile of a bull; D - small intestine of rats. On the ordinate - the magnitude of the effect Ea (%); on the abscissa - an indicator of the degree of tenfold dilution.

K - control; columns - exposure to the IEF-fraction at a dilution of 10 ^x , where x is the tenfold dilution of the study drug, (p<0.05).

Fig. Fig. 4. Effect on the viscoelastic properties of the plasma membrane of mouse liver cells during multiple organ cultivation in vitro, a number of subfractions (A-D), collected in the range of values in the pH range of 4.5-5.1 after isoelectric focusing of the tissue extract supernatant: A - rat liver ; B - lung of rats; B - small intestine of rats; G - bile of a bull. On the ordinate - the magnitude of the effect Ea (%); on the abscissa - an indicator of the degree of tenfold dilution.

K - control; columns - exposure to the IEF-fraction at a dilution of 10 ^x , where x is the tenfold dilution of the study drug, (p<0.05).

Fig. Fig. 5. Membranotropic effect of the MHTB substance fraction isolated from rat liver, collected in the pH range of 6.7-7.2 (bioassay method, p<0.05). A - dialysis against water; B - dialysis against physical. solution.

On the ordinate - the magnitude of the effect Ea (%);

on the abscissa - an indicator of the degree of tenfold dilution.

k - control; columns - the effect of the supernatant at a dilution of 10 ^x , where x is the exponent of a tenfold dilution of the studied drug with an initial concentration of 0.02 mg / ml.

Fig. 6. Isoelectric focusing of supernatants of tissue extracts isolated from: A - bull's heart; B - bull blood serum.

On the ordinate 1 - pH values; along the ordinate 2- D280; abscissa - numbers of fractions.

Fig. Fig. 7. Isoelectric focusing of the supernatants of the tissue extract of the bovine prostate gland (A) and the tissue extract of the mammary gland of the cow (B), according to the abscissa fraction number; along the ordinate 1 - the values of the optical density of the corresponding fractions at 280 nm., along the ordinate 2 - pH values.

Fig. Fig. 8. Membranotropic activity of the fraction collected in the pH range less than 3.0 after isoelectric focusing of the supernatant of the bovine prostate tissue extract (A) and the supernatant of the bovine mammary tissue extract (B). On the ordinate - the magnitude of the effect Ea (%); on the abscissa - an indicator of the degree of tenfold dilution.

K - control; columns - the effect of the IEF-fraction at a dilution of 10 ^x , where x is the tenfold dilution of the studied drug, (p<0.05)

It was shown that these MHTBs change the viscoelastic properties of mouse liver tissue in vitro, and there is a polymodal dose dependence of the biological action of MHTBs.

Fig. Fig. 9. Separation of fractions of protein-peptide complexes that are part of MHTB, obtained after isoelectric focusing of supernatants of tissue extracts and bile, in 12% PAAG with the addition of sodium dodecyl sulfate.

Painted Coomassie G250.

a) M - markers, from 1 to 4 fractions of acidic MHTB, isolated from the liver, respectively; lung; small intestine and bile, b) M - markers, from 1 to 4 fractions of the main MHTB, isolated, respectively, from the liver; lung; small intestine and bile.

Fig. 10. Mass spectrum of the lipid fraction obtained by extraction of the acid subfraction of MHTB isolated from bovine serum.

Fig. 11. Distribution of MHTB particles in aqueous solutions along the hydrodynamic radius (method of laser dynamic light scattering). A - acidic protein-peptide complex, which is part of MHTB, isolated from the liver of rats; B - acidic protein-peptide complex, which is part of MHTB, isolated from the lung of rats; B - acidic protein-peptide complex, which is part of MHTB, isolated from the small intestine of rats; D - acidic protein-peptide complex, which is part of MHTB, isolated from bull bile.

Fig. Fig. 12. Determination of nanosized particles in HPLC fractions of MHTB obtained from (A) rat liver, (B) lung, and (C) bovine bile by AFM.

Fig. 13. CD - spectra of MHTB, isolated from the blood serum of a bull, after heating to 100°C for 30 min. On the ordinate - the value of ellipticity at 222 nm; the abscissa shows the temperature of the MHTB solution. Solid line - acid fraction of MHTB; large dotted line - MHTB subfraction collected in the pH range of 4.5-5.1; the small dotted line is the subfraction of the main MHTBs.

Fig. 14. CD - spectra of MHTB, isolated from the blood serum of a bull, after exposure to guanidine chloride for 14 hours, 21-23°C. On the ordinate - the value of ellipticity at 222 nm; on the abscissa - the values of the concentrations of guanidine chloride in mmol/l. Solid line - acid fraction of MHTB; large dotted line - MHTB subfraction collected in the pH range of 4.5-5.1; small dotted line - subfraction of the main MGTB.

Fig. 15. CD - spectra of MHTB, isolated from the blood serum of a bull, after exposure to EDTA. On the ordinate - the value of ellipticity at 222 nm; on the abscissa - the value of the decimal exponent 10 ^x mol/l EDTA. Solid line - acid fraction of MHTB; large dotted line - MHTB subfraction collected in the pH range of 4.5-5.1; small dotted line - subfraction of the main MGTB.

Fig. 16. ^1H NMR spectrum of MHTB substance isolated from bovine prostate gland (A), cow mammary gland (B), inositol signal (C).

Fig. 17. Isoelectric focusing of the supernatant of the tissue extract of the bovine pancreas, along the abscissa of the fraction number; along the ordinate 1 - the values of the optical density of the corresponding fractions at 280 nm, along the ordinate 2 - pH values.

Fig. 18. Membranotropic activity of the fraction collected in the pH range less than 3.0 after isoelectric focusing of the supernatant of the tissue extract of the pancreas (A) and thyroid (B) glands of the bull. On the ordinate - the magnitude of the effect Ea (%); on the abscissa - an indicator of the degree of tenfold dilution.

Table 1 shows the characteristics of the IEF - fractions of the liver supernatant * indicating the dilution interval of the fraction in which membranotropic activity is manifested (*data are given for a tissue extract obtained from 230 g of tissue wet weight).

Table 2 shows the membranotropic activity of acidic and basic MHTB IEF fractions isolated from extracts of various rat tissues and bovine bile. Table 3 shows data from comparative studies of the tissue specificity of MHTB fractions isolated from the liver and lung, collected in the pH range of 4.5-5.1.

Table 4 shows the data on the effect of MHTB isolated from various animal tissues and bile on the adhesion of enterocytes in vitro.

Table 5 provides information on the change in the particle size of bovine bile under the influence of MHTB isolated from bile, blood serum and liver of mammals in vitro.

Table 6 provides information on studies of the secondary structure of acidic MHTBs. It is shown that the secondary structure of BOD, which are part of MHTBs, is described mainly in terms of β-structures (approximately 70%) and a statistical coil (approximately 30%). Such a state of the secondary structure of the BOD of bioregulators of this group is their characteristic property.

Table 7 shows the effect of MHTB isolated from bovine pancreas on experimental diabetes in rats in vivo.

Table 8 provides information on the antiviral effect of MHTB preparations isolated from the thymus, blood serum, small intestine of mammals against infection caused by a highly pathogenic variant of the avian influenza virus.

A/H5N1 in porcine embryonic kidney cell cultures in vitro.

Thus, the data presented in Fig. 1-18 and tables 1-8 clearly demonstrate the effect of MHTB on the viscoelastic properties of the plasma membrane of liver cells, on the adhesion of enterocytes in vitro, etc., the tissue specificity of MHTB, the antiviral effect of MHTB preparations, which generally indicates the ability of the claimed substance MHTB and preparations based on it to maintain (stimulate) and restore the function of organ tissues after the development of various pathological processes in them.

The following are examples illustrating the invention, but not limiting it.

Example 1. Method for obtaining MHTB substance from bovine liver tissue

Obtaining tissue extract of bovine liver

Freshly isolated bovine liver was washed several times in cold saline. solution until complete removal of blood, cut into small fragments weighing 2-5 g, washed again with saline. solution and placed for 2-3 hours in physical. solution at 0°С-+4°С. Then the resulting extract was poured off, centrifuged at 2500g for 30 min, at +4°C. The resulting liver extract was further fractionated to obtain the substance MHTB or frozen and stored at -80°C--90°C.

Preparation of BOD from bovine liver tissue extract

Ammonium sulphate is gradually added to the cooled solution of liver tissue extract with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of extract). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C, dialyzed against distilled water until ammonium sulfate is removed, and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using pH range ampholines 3.5 - 10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected.

The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern (Fig. 1), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; a number of peptides with molecular weights in the range of 1000 Da - 10000 Da (2112, 2170, 2866, 2940, 3009, 3151, 5026, 5237 Yes).

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a protein of the mammalian albumin family with number gi|367460260 in the SwissProt database.

Thus, the IEF fraction collected in the pH <3.0 range contains the gi|367460260 protein, a representative of the bovine serum albumin multifamily with a pI value in the pH range of 3.90–4.00, as well as a mixture of small peptides with molecular masses from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates, in particular, residues of mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane cells.

Similarly, the substance according to the invention is isolated from other animal organs and biological fluids.

Example 2. Method for obtaining MHTB substance from rat liver tissue

Preparation of Rat Liver Tissue Extract

The experiment was carried out on Wistar rats, 220-250 g, of both sexes. Animals were decapitated under ether anesthesia, the liver was perfused through the portal vein of physical. solution for 1-3 minutes to completely remove blood. The gland was excised, cut into fragments, weighing 2-4 g, which are washed several times in fresh saline. solution and placed for 2-3 hours in physical. solution at 0°С-+4°С. Then the resulting extract is drained, centrifuged at 2500g for 30 minutes. The obtained liver extract was further fractionated to obtain BOD or frozen and stored at -90°C. Preparation of MHTB substance from rat liver tissue extract

Ammonium sulphate is gradually added to the cooled solution of liver tissue extract with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of extract). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C, dialyzed against distilled water until ammonium sulfate is removed, and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using pH range ampholines 3.5 - 10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; a number of peptides with molecular weights in the range of 1000 Da - 10000 Da (3649, 5025 Yes).

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in a gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a protein of the mammalian albumin family with number gi|55628 in the SwissProt database.

Thus, the IEF fraction collected in the pH <3.0 range contains the gi|55628 protein, a representative of the rat blood serum albumin multifamily with a pI value in the pH range of 3.90–4.00, as well as a mixture of small peptides with molecular masses from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues), which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane of the cell.

Example 3. Method for obtaining MHTB substance from fish liver

The liver is obtained from fish of the burbot species, cod-like order, or from carp from the carp-like order by cutting out the gland after decapitation of the fish, repeated washing of the tissue in the physical. solution, cutting into fragments with a mass of 2-4 g, even careful repeated washing of the tissue in physical. solution, and subsequent extraction of tissue in physical. solution at 0°С-+4°С. The resulting fish liver extract was poured off, centrifuged at 2500g for 30 min. The obtained tissue extract was further fractionated to obtain BOD or frozen and stored at -90°C.

Ammonium sulphate is gradually added to the cooled tissue extract solution with constant stirring until a saturated salt solution is formed (78 g of salt is added per 100 ml of extract). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The fish liver extract supernatant fraction is separated by isoelectrofocusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5–10.0 (Serva, Germany) (Osterman, 1983 ). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using a-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. Isoelectric focusing in a sucrose gradient reveals a protein, which is serum albumin with a pI value in the pH range of 4.4-5.0, which, according to a study by electrophoresis in PAAG, has a molecular weight of 65,000-67,000 Da.

Thus, the IEF-fraction collected in the pH range>3.0 is a BOD, consisting of a protein - a representative of the fish serum albumin multifamily, has a pI value in the pH range of 4.4-5.0, with a molecular weight of about 65000-67000 Yes, and mixtures of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues), which form an inositol-containing glycosidic structure that provides it connection with the plasma membrane of the cell.

Example 4. Method for obtaining MHTB substance from chicken liver tissue

The liver is obtained from birds of white breeds, for example, Leggorn, females 7-9 months old, by cutting out the gland, repeatedly washing the tissue in physical. solution, cutting into fragments with a mass of 2-4 g, even careful repeated washing of the tissue in physical. solution, and subsequent extraction of tissue in physical. solution at 0°С-+4°С. The resulting liver extract was poured off, centrifuged at 2500g for 30 min. The obtained tissue extract was further fractionated to obtain BOD or frozen and stored at -90°C.

Ammonium sulphate is gradually added to the cooled tissue extract solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml of extract). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The tissue extract supernatant fraction is separated using the isoelectric focusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. The sucrose gradient isoelectrofocusing method detects a protein which is serum albumin with a pI value in the pH range of 4.5-4.9, which, according to the PAAG electrophoresis study, has a molecular weight of approximately 66,000 Da-68,000 Da.

Thus, the IEF fraction collected in the pH range>3.0 is a BOD, consisting of a protein - a representative of the chicken blood serum albumin multifamily, has a pI value in the pH range of 4.5-5.1 with a molecular weight of about 66000 Da-68000 Yes, and mixtures of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues, which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane of the cell.

Example 5. Method for obtaining MHTB substance from bovine lung tissue

Obtaining tissue extract of bovine lung

The bovine lung was washed several times in cold saline. solution until complete removal of blood, cut into small fragments weighing 1-2 g, washed again with saline. solution and placed for 2-3 hours in physical. solution at 0°C-+4°C. Then the resulting extract was poured off, centrifuged at 2500g for 30 min. The resulting lung extract was further fractionated to obtain MHTB or frozen and stored at -90°C.

Preparation of MHTB from bovine lung tissue extract

Ammonium sulphate is gradually added to the cooled solution of lung tissue extract with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern (Fig. 1), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Spent electrophoresis in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a protein of the mammalian albumin family with number gi

|367460260 in the SwissProt database.

Thus, the IEF fraction collected in the pH range <3.0 contains the gi|367460260 protein, a representative of the bovine serum albumin multifamily with a pI value in the pH range of 3.90-4.00, as well as a mixture of small peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - residues of mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane of the cell.

Example 6. Method for obtaining MHTB substance from rat lung tissue

Obtaining tissue extract of the rat lung

Freshly isolated rat lung was washed several times in cold saline. solution until complete removal of blood, cut into small fragments weighing 1-2 g, washed again with saline. solution and placed for 2-3 hours in saline. solution at 0°C-+4°C. Then the resulting extract was poured off, centrifuged at 2500g for 30 min. The resulting lung tissue extract was further fractionated to obtain MHTB or frozen and stored at -90°C.

Preparation of MHTB from rat lung tissue extract

Ammonium sulphate is gradually added to the cooled solution of lung tissue extract with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected.

The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the separation pattern obtained, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 s/five changes of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Spent electrophoresis in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 65,000 Da - 67,000 Da. The tryptic hydrolysis of the protein in a gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a protein of the mammalian albumin family under number gi |367460260 in the SwissProt database.

Thus, the IEF-fraction is a BOD, consisting of a protein - a member of the rat blood serum albumin multifamily and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates, in particular, the residues of mannose, galactose, glucose , N-acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane of the cell.

Example 7. Method for obtaining MHTB from rat small intestine tissue

Obtaining tissue extract of the small intestine

The small intestine is excised, placed in the physical. solution, cut into fragments about 2 cm long, washed repeatedly with the inner surfaces of the intestine, using a syringe, quickly placed in a cold physical. solution (+4°C - 0°C) for 2 hours, then the resulting extract is drained, centrifuged at 2500g for 30 minutes. The resulting tissue extract is immediately fractionated to obtain MHTB or frozen and stored at -90°C.

Preparation of MHTB from Rat Small Intestine Tissue Extract

Ammonium sulphate is gradually added to the cooled tissue extract solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent.

The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern (Fig. 1), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Spent electrophoresis in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 65,000 Da - 67,000 Da. The N-terminal fragment of the protein was determined on a sequinator by the Edman method; it corresponded to the N-terminal fragment of rat blood serum albumin.

Thus, the IEF fraction is a BOD, consisting of a protein - a member of the rat blood serum albumin multifamily and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - residues of mannose, galactose, glucose, N- acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 8 Obtaining MHTB substance from bovine bile

In the work, we used the drug "Medical canned bile", isolated from the gallbladder of a bull (1 l), purchased in pharmacies in Moscow.

Dry ammonium sulphate is added to the chilled bile with stirring until a saturated salt solution is formed, a few crystals of sodium azide are added and left for 7 days at a temperature of +4°C. Next, the protein suspension is centrifuged at 15000g for 30 minutes. The supernatant fraction, which is a transparent white liquid, is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with a fivefold change of water. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction is separated using the isoelectric focusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to a volume of 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern (Fig. 1), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Electrophoresis was performed on a 15% PAAG gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein with a molecular weight of 66366 Da is a protein of the mammalian albumin family under the number gi| 1351907 in the SwissProt database. Edman's method determined the partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE), which has 100% homology with the N-terminal sequence of the mature Bos taurus serum albumin molecule.

Thus, the IEF fraction is a BOD consisting of the gi| 1351907 representative of the Bos taurus blood serum albumin multifamily and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues, which form inositol- containing a glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 9. Preparation of solutions of various concentrations of protein-peptide complexes that are part of the MHTB substance isolated from various tissues and biological fluids of animals

Quantitative determination of protein in the studied fractions was carried out using a colorimetric method using copper bicinchoninate at 562 nm [Smith et al., 1985].

For this, two solutions were prepared:

Solution A - 1 g of bicinchoninic acid, 2 g of Na ₂ CO ₃ , 0.16 g of sodium tartrate, 1 g of NaOH, 0.95 g of NaHCO ₃ in 100 ml of distilled water (pH=11.25);

Solution B - 0.4 g CuSO ₄ *5H ₂ O in 10 ml of distilled water.

Immediately before the experiment, the third solution C was prepared by mixing 50 ml of solution A with 1 ml of solution B. To determine the protein concentration, 1 ml of solution C was added to 50 μl of the test solution, the samples were incubated for 30 minutes at 60°C, then cooled to room temperature. Absorbance was measured at 562 nm. To construct a calibration curve, solutions of bovine serum albumin (BSA) were first prepared at concentrations: 5, 10, 25, 50, 75, 100, 125, 250, 500, 750, 1000 µg/ml. 1 ml of solution B was added to 50 μl of a BSA solution of a known concentration, the resulting samples were incubated, the absorbance was measured according to the method described above, and a calibration curve was built according to the measurement results. According to the constructed calibration curve, the concentration of proteins in the studied solutions was determined.

As the initial solution of MHTB, the IEF-fraction collected in the pH <3.0 region, isolated from the corresponding bovine tissue with a protein concentration of 100±10 μg/ml, is used. In a vial containing 0.1 ml of an aqueous solution of MHTB, add 0.9 ml of distilled water, shake vigorously 25 times. Take 0.1 ml of the resulting solution into the next vial and add 0.9 ml of distilled water, shake 25 times. This procedure is repeated 17 times, thus obtaining MHTB solutions with 10-fold serial dilutions from 1 to 19 degrees. Thus, solutions of MHTB were obtained from 10 ^-1 to 10 ^-19 mg/ml. In experiments to study the safety of MHTB solution isolated from cattle blood serum, solutions were studied at concentrations of 10 ^-5 , 10 ^-12 , 10 ^-19 mg/ml. Similarly, and in the same range of concentrations, MHTB solutions were prepared in 0.9% NaCl solution. All solutions are sterilized by passing through sterile GV membrane filters with a pore diameter of 0.22 μm (MPIroge, USA). As a therapeutic dose, a solution of MHTB is examined at a concentration corresponding to 10 ^-10 -10 ^-12 mg/ml.

Example 10. Effect of MHTB substance isolated from extracts of various tissues and biological fluids of animals on the viscoelastic properties of mouse liver and lung cell membranes during multiple short-term in vitro organ cultivation (membranotropic activity)

Biotesting of fractions obtained at different stages of purification of tissue extracts and biological fluids was carried out using an adhesiometric method specially developed to identify the MHTB substance. The method makes it possible to evaluate the viscoelastic properties of a tissue under a standard deformation effect.

The experiment used CBA/C57BI mice, male, weighing 15-18 g.

After decapitation of the animal, fragments weighing 1.0–2.5 mg were cut out from the liver or lung. 1 ml of nutrient medium 199 and 0.1 ml of the test protein solution at the appropriate concentration were added to each penicillin vial of the experimental series, five fragments of liver tissue were placed or a lung mouse. The interval of active concentrations for each of the studied protein preparations was determined by studying the biological effect of its solutions obtained by sequential dilution by 10, 100, 1000 or more times. In vials of the control series, instead of a solution of the studied protein, 0.1 ml of saline was added. solution. Organ cultures of the experimental and control series were incubated for 2 h at 37°C. Next, each tissue fragment was taken out, dried for 10-15 sec on filter paper, weighed and subjected to dispersion in 0.1 ml of a 0.1% solution of trypan blue, prepared on 199 medium, in a tissue disintegrator with a gap of 50 μm under conditions standard deformation effect. The resulting suspension of cells and cell nuclei was examined microscopically in a Goryaev chamber, counting cell nuclei in a certain volume of the chamber (0.02 mm).

The parameter reflecting the effect of MHTB on the viscoelastic properties of the liver or lung tissue was determined according to the formula:

где

Where

M _a and N _k - the number of cell nuclei released from 1 mg of liver or lung tissue after dispersion, respectively, in the experimental and control series. Statistical processing of the obtained data was carried out by the method of variation statistics using Student's t-test.

In each experiment, at least 5 tissue fragments were counted per point (solution of the test drug at a certain concentration and control). Each experiment was repeated at least 3 times.

Example 11. Influence of subfractions of MHTB substance obtained by isofocusing, isolated, respectively, from liver tissue extract and lung tissue extract, on the viscoelastic properties of liver cell membranes during multiple organ tissue cultivation in vitro

Using isoelectric focusing in a sucrose gradient in the pH range of 3.0-10.0, it was shown that MHTB contained in the tissue extract of the corresponding organ can form several subfractions, the proteins of which differ in charge (Table 1). The most quantified and always present in the supernatants of tissue extracts is the subfraction of acidic proteins, which was collected after IEF in the pH region less than 3.0. Other subfractions were also identified, for example, in the supernatant of a rat liver extract, 4 subfractions were found that differ in the charge of the MHTB proteins: in the pH<3.0 region (acidic proteins); in the pH range of 4.5-5.1 (slightly acidic proteins); in the pH range of 6.7-7.2; in the pH>8.0 region (basic proteins). The main characteristics of these IEF fractions are given in Table. 1 in FIG. Figures 2-5 show dose dependence diagrams of the membranotropic action of these fractions, determined on a multiple organotypic mouse liver culture in vitro. Similarly, subfractions were obtained from a tissue extract of the lung, tissue of the small intestine and bile of rats and bulls (Fig. 2-5).

The fraction in the pH range of 6.7-7.2 is a minor fraction of MHTB, which differs from the acidic BOD fraction, firstly, by the variability of the presence in tissue extracts and biological fluids, and secondly, by the lack of resistance to the effects of various physicochemical factors (temperature, exposure to deionized medium or Ca ²⁺ - chelating agents). For example, it has been shown that the protein fraction collected in the pH range of 6.7-7.2 loses membranotropic activity after dialysis against distilled water. If the dialysis of this fraction is carried out against physical. solution containing calcium ions, then its membranotropic activity is preserved (Fig. 5). This subfraction lost its biological activity after multiple freeze-thaw procedures, as well as after being at room temperature, i.e. showed properties that significantly distinguished it from other MHTB fractions from the liver. It should be noted that this subfraction exhibited the property of tissue specificity: the fraction isolated from rat liver was active only against liver tissue, the subfraction isolated from rat lung was active only against lung tissue.

The fraction collected in the pH range of 4.5–5.1 after isoelectric focusing of the liver supernatant is a BOD consisting of serum albumin and low molecular weight peptides, the ratio of which during the formation of the complex differed from that in the BOD of the acidic subfractions: in the subfractions of the pH range 4.5–5.1 the amount of serum albumin was much higher than in the acidic BOD fraction.

In table. Figure 2 shows the characteristics of acidic and basic subfractions of BOD, which are part of MHTB, isolated by isoelectric focusing from the supernatants of several tissue extracts and biological fluids. As an example, in FIG. Figure 3 shows the results of a study of the membranotropic activity of these subfractions. It should be noted that despite the differences in the charge of MHTB proteins, in these subfractions, the main components are low molecular weight peptides and serum albumin, which form BOD. It is assumed that the differences in charges in BOD can determine the tertiary and quaternary structures of serum albumin, the composition of peptides, and lipids.

The IEF-fraction collected in the range of pH values <3.0 is most quantitatively represented in the MHTB substance. For MHTB isolated from bovine serum, it contains a protein - a representative of the bovine serum albumin multifamily gi| 1359007, pI in the pH range of 3.90-4.00, as well as a mixture of small peptides with molecular weights from 1000 to 10000 Da, among which were found, and for MHTB isolated 367460260 - a representative of the multifamily of bovine blood serum albumins with the value protein gi | 158138568 - a representative of the rat blood serum albumin multifamily with a pI value in the pH range of 3.90-4.00, as well as a mixture of small peptides with molecular weights from 1000 to 10000 Da.

In table 3. presents data on the study of membranotropic activity of subfractions isolated from the liver and lung of rats, collected in the pH range of 4.5-5.1.

It was found that the fractions isolated from the liver tissue extract and its supernatant exhibited a membranotropic effect on both liver and lung cells in vitro, i.e. were characterized by the absence of tissue specificity of biological action. Only Fractions collected in the pH range of 4.5-5.1, after isoelectric focusing, of both the lung tissue extract and its supernatant, were biologically inactive.

conclusions

1. The supernatants of tissue extracts of the liver, lung, small intestine, and bile of mammals contain MHTB in the form of several subfractions that differ in charge.

2. All of these subfractions are BOD formed by serum albumin and small peptides with molecular weights of 1000 Da - 10000 Da. The fraction that is quantitatively represented and always present in the composition of the MHTB substance is BOD, collected in the pH range less than 3.0. All other subfractions were less quantitatively represented, in addition, they were not found in a number of tissue extracts and biological fluids. Therefore, the method for obtaining MHTB from the corresponding tissue of vertebrates corresponds to the isolation and purification of the acidic BOD fraction.

Example 12 Effect of MHTB Isolated from Rat Intestinal Tissue on Adhesion of Rat Enterocytes in Vitro

The experiment was carried out on Wistar rats, both sexes, weighing 240-250 g. Under ether anesthesia, the animal was decapitated, and the small intestine was removed from the abdominal cavity. Fragments approximately 80-100 mm long were cut out from its middle, which was turned out with a glass rod, making the inner surface the outer one. Further, the fragments were repeatedly washed very carefully, without damaging the surface of the intestine, in 199 medium at room temperature (22–24°C), cut into smaller fragments 20–25 mm long. 5 small fragments of the intestine were placed in a Petri dish containing 5 ml of 199 medium and 10% blood serum of cattle (control), 5 small fragments were placed in other dishes containing 5 ml of 199 medium and 10% serum and MHTB at a concentration of 10 ^-9 mg protein/ml. We studied the biological effect of the following MHTB in the form of IEF fractions collected in the pH range less than 3.0:

1. MHTB isolated from rat small intestine tissue;

2. MHTB isolated from rat liver tissue;

3. MHTB isolated from rat lung tissue;

4. MHTB isolated from bovine serum;

5. MHTB isolated from bovine bile.

Organotypic cultures of the intestine were incubated for 15 minutes at 37°C. Next, each fragment was carefully removed, quickly dried on filter paper, weighed, put on a metal rod, 1.5 mm in diameter, and subjected to rotation for 3 min, lowering the rod into a cuvette with 199 medium containing 10% blood serum, 1 ml in volume. . Rotation speed 100 rpm, 22-24°C. Next, the secreted enterocytes were counted in a nutrient medium using a Goryaev camera. 5 fragments of each experimental series were counted.

As the results in Table 4 show, only when exposed to MHTB isolated from the small intestine, enterocytes were released approximately 2.0 times less than in the control or when exposed to MHTB isolated from other sources.

conclusions

In this study, it was shown that MHTB exhibit the properties of adhesive proteins, which is characterized by tissue specificity.

Example 13 Effect of MHTB substance isolated from bovine bile on particle size in human bile in vitro

A person daily secretes 250-1000 ml of bile with a complex component composition. The bile found in the ducts of the liver (hepatic bile) and the bile present in the bladder (cystic bile) differ significantly in composition, despite the fact that both are unstable colloidal systems.

An integral component of bile is bile acids (salts), which largely determine its physicochemical properties. The physiological significance of bile acids for the human body is determined by a number of provisions: the formation of micelles for the transport of water-insoluble substances (cholesterol, vitamins A, D, E, K); formation of liquid crystals with cholesterol; stabilization of hepatocyte membranes; activation of pancreatic lipase; stimulation of intestinal motility; increased secretion of sodium and water ions in the colon.

It was shown that only when MHTB, isolated from bile, was added to gallbladder bile of a bull under in vitro conditions, a statistically significant decrease in the average particle diameter was observed by about half compared to the control (Table 5). In the same experiment, it was also shown that MHTB isolated from the blood serum and liver of mammals had no effect on the particle size of bile in vitro. Thus, in this experimental series, it was possible to demonstrate the specific biological activity of MHTB isolated from bile.

Example 14 Preparation of MHTB substance from bovine heart tissue

The heart was obtained from young bulls of meat processing enterprises in Moscow and the Moscow region.

Obtaining a tissue extract of the heart of a bull

The heart of the bull was washed several times in cold saline. solution until complete removal of blood, cut into small fragments weighing 1-2 g, washed again with saline. solution and placed for 2-3 hours in physical. solution at 0°С-+4°С. Then the resulting extract was poured off, centrifuged at 2500g for 30 min. The resulting heart extract was further fractionated to obtain MHTB or frozen and stored at -90°C.

Preparation of MHTB from bovine heart tissue extract

Ammonium sulphate is gradually added to the cooled solution of the tissue extract of the heart with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000 g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from a gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the separation pattern obtained (Fig. 6), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Spent electrophoresis in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a protein of the mammalian albumin family with the number gi| 1351907 in the SwissProt database.

Thus, the IEF fraction is a BOD consisting of the gi| 1351907 representative of the Bos taurus blood serum albumin multifamily and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues, which form inositol- containing a glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 15. Preparation of MHTB substance from rat heart tissue

Obtaining tissue extract of rat heart

Freshly isolated rat heart was washed several times in cold saline. solution until complete removal of blood, cut into small fragments weighing 2-3 g, washed again with saline. solution and placed for 2-3 hours in physical. solution at 0°C-+4°C. Then the resulting extract was poured off, centrifuged at 2500g for 30 min. The resulting heart extract was further fractionated to obtain MHTB or frozen and stored at -90°C.

Preparation of MHTB from Rat Heart Tissue Extract

Ammonium sulphate is gradually added to the cooled solution of the tissue extract of the heart with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Spent electrophoresis in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 65,000 Da - 67,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a protein of the mammalian albumin family under the number gi|124028612 in the SwissProt database.

Thus, the IEF fraction is a BOD, consisting of a protein - a member of the rat blood serum albumin multifamily and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - residues of mannose, galactose, glucose, N- acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 16. Method for obtaining the MHTB substance from the blood serum of cattle

We used a commercial preparation of bovine blood serum, sterile, inactivated, used as a nutritional supplement for cell and tissue cultures.

Ammonium sulphate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of serum) and left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The resulting supernatant fractions are concentrated using a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5-10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution -15 g sucrose, up to 200 ml distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. Protein detection is carried out on any spectrophotometer at 280 nm, for each fraction, the pH value is determined using a pH meter. According to the separation pattern obtained (Fig. 6), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; a number of peptides with molecular weights in the range of 1000 Da -10000 Da are detected, including including peptides with molecular weights 1151, 1338, 1448, 1666, 1812, 1915, 2016 Da. Determination of the primary sequence using C-carboxypeptidase shows that they are products of proteolysis, respectively, for a peptide with a molecular weight of 1448 Da - MGLRGGPLASLLLR - there is complete homology with the N-terminal sequence of P-cadherin, identified in bovine aortic endothelium. For a peptide with a molecular weight of 1151 Da - (dkhgc) _n-5 -PAYVSP - homology with the amino acid sequence of a fragment of the kinase domain of the regulatory protein tribble-2 (TRB-2), obtained from granulocytes (granulosa cells) Bos taurus. For a peptide with a molecular weight of 1338 Da, homology was found with adrenomodulins, proteins produced by the vascular endothelium of Bos taurus.

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products shows that the isolated protein with a molecular weight of 66367 Da is a protein of the mammalian albumin family with the number gi| 1351907 in the SwissProt database. Edman's method determined the partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE), which has 100% homology with the N-terminal sequence of the mature bovine BSA molecule.

Thus, the IEF fraction collected in the range of pH<3.0 contains the protein gi| 1351907 - a representative of the multifamily of bovine serum albumins, with a pI value in the pH range of 3.90-4.00, and a mixture of small peptides with a molecular weight of 1000-6000 Da, as well as calcium ions, lipids, carbohydrates, in particular, mannose residues , galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane of the cell.

Identification of three peptides that make up the BPC showed that they are products of proteolysis of the adhesive protein P-cadherin and the regulatory protein tribble-2 (TRB-2) isolated from granulocytes and adrenomolulins.

Example 17. Method for obtaining the MHTB substance from the blood serum of Wistar rats

The blood serum of Wistar rats is obtained after decapitation of animals of both sexes, weighing 180-240 g, by taking blood from the heart of animals, sedimentation, centrifugation and cold sterilization through filters with a pore size of 0.45 μm (B.I. Antonov, V.A. Borisova , P.M.Volkova, etc. // Laboratory research in veterinary medicine. Handbook edited by B.I.Antonov. - M.: Agropromizdat.1986. 352 p.).

Ammonium sulphate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. The method of isoelectric focusing in a sucrose gradient reveals a protein, which is a serum albumin with a pI value in the pH range of 4.60-4.63.

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products shows that the isolated protein is a member of the mammalian albumin family with protein number gi| 124028612.

Thus, the IEF fraction is a BOD consisting of the gi| 124028612 - rat blood serum albumin with a pI value in the pH range of 4.60-4.63 and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates - residues of mannose, galactose, glucose, N-acetylglucosamine , glucosamine, N-acetylgalactosamine, which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 18. Obtaining the MHTB substance from horse serum

Horse blood serum is obtained according to the method (Antonov B.P., Borisova B.C., Volkova P.M. et al. // Laboratory research in veterinary medicine. Handbook edited by B.I. Antonov. - M .: Agropromizdat. 1986. 352 c.) from animals of both sexes by taking blood from the jugular vein of animals, sedimentation, centrifugation and cold sterilization of serum through filters.

Ammonium sulphate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. Protein detection is carried out on a spectrophotometer at 280 nm, and the pH value is determined for each fraction. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water.

The IEF fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. Polyacrylamide gel electrophoresis, as well as isoelectric focusing in a sucrose gradient, reveal a protein, which is a serum albumin with a pI value in the pH range of 4.6-4.7 and a molecular weight in the range of 66,000-67,000 Da.

Thus, the IEF fraction collected in the pH<3.0 region is a BOD consisting of serum albumin with a pI value in the pH range of 4.6-4.7 and a mixture of peptides with molecular weights from 1000 to 10000 Da, and also calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues), which form an inositol-containing glycosidic structure that ensures its connection with the plasma membrane of the cell.

Example 19. Method for obtaining MHTB substance from dog blood serum

Dog blood serum is obtained according to the method (Antonov B.I., Borisova B.C., Volkova P.M. et al. // Laboratory research in veterinary medicine. Handbook edited by B.I. Antonov. - M .: Agropromizdat. 1986. 352 c.) from animals of both sexes by taking blood from the jugular vein, sedimentation, centrifugation and cold sterilization of serum through filters

Ammonium sulphate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using a-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. The method of isoelectric focusing in a gradient of sucrose in the IEF fraction reveals a protein, which is a serum albumin with a pI value in the pH range of 4.6-4.7. By electrophoresis in PAAG, a protein with a molecular weight of about 67,000 Da is found in the IEF fraction.

Thus, this fraction is BOD, consisting of serum albumin with a pI value in the pH range of 4.6-4.7 with a molecular weight of about 67,000 Da and several peptides with molecular weights up to 10,000 Da, as well as calcium ions, lipids, carbohydrates (in particular, the residues of mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine), which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 20. Method for obtaining MHTB substance from fish blood serum

Blood serum is obtained from fish of the burbot species, cod-like order or from carp from the carp-like order (Antonov B.I., Borisova V.C., Volkova P.M. et al. // Laboratory research in veterinary medicine. Handbook edited by B.I. Antonov - M.: Agropromizdat.1986. 352 p.) by taking blood after fish decapitation, serum sedimentation and centrifugation and sterilization through filters.

Ammonium sulphate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. The method of isoelectric focusing in a sucrose gradient reveals a protein, which is a serum albumin with a pI value in the pH range of 4.5-4.6, which, according to the study by electrophoresis in PAAG, has a molecular weight of 65000-66000 Da.

Thus, the IEF-fraction collected in the pH range>3.0 is a BOD, consisting of a protein - a representative of the fish serum albumin multifamily, has a pI value in the pH range of 4.5-4.6, with a molecular weight of about 65000-66000 Yes, and mixtures of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues), which form an inositol-containing glycosidic structure that provides it connection with the plasma membrane of the cell.

Example 21. Method for obtaining substance MHTB from blood serum of chickens

The blood serum of chickens is obtained from birds of white breeds, for example, Leggorn, females 7-9 months old, according to the method (Antonov B.I., Borisova B.C., Volkova P.M. et al. // Laboratory research in veterinary medicine. Handbook ed. BI Antonova - M.: Agropromizdat 1986. 352 pp.) by taking blood from the heart of animals, sludge and cold sterilization of the serum through filters.

Ammonium sulphate is gradually added to the cooled blood serum solution with constant stirring until a saturated salt solution is formed (78 g of salt was added per 100 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution -0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. Isoelectric focusing in a sucrose gradient reveals a serum albumin protein with a pI value in the pH range of 4.60-4.75, which, according to the study by electrophoresis in PAAG, has a molecular weight of approximately 66,000 Da.

Thus, the IEF-fraction collected in the pH range>3.0 is a BOD, consisting of a protein - a representative of the chicken blood serum albumin multifamily, has a pI value in the pH range of 4.60-4.75 with a molecular weight of about 66,000 Da and a mixture peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (in particular, the residues of mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine), which form an inositol-containing glycosidic structure that provides it connection with the plasma membrane of the cell.

Example 22 Effect of MHTB substance isolated from bovine blood serum and heart tissue extract, respectively, on the viscoelastic properties of liver cell membranes during multiple short-term in vitro organ cultivation (Fig. 3).

Biotesting of fractions obtained at different stages of purification of tissue extracts and biological fluids was carried out using an adhesiometric method specially developed to identify the MHTB substance. The method makes it possible to evaluate the viscoelastic properties of a tissue under a standard deformation effect.

The experiment used CBA/C57BI mice, male, weighing 15-18 g.

After decapitation of the animal, fragments weighing 1.0–2.5 mg were excised from the liver or lung. In each penicillin vial of the experimental series, 1 ml of nutrient medium 199 and 0.1 ml of the studied protein solution at the appropriate concentration were added, five fragments of mouse liver or lung tissue were placed. The interval of active concentrations for each of the studied protein preparations was determined by studying the biological effect of its solutions obtained by sequential dilution by 10, 100, 1000 or more times. In vials of the control series, instead of a solution of the studied protein, 0.1 ml of saline was added. solution. Organ cultures of the experimental and control series were incubated for 2 h at 37°C. Next, each tissue fragment was taken out, dried for 10-15 sec on filter paper, weighed and subjected to dispersion in 0.1 ml of a 0.1% solution of trypan blue, prepared on 199 medium, in a tissue disintegrator with a gap of 50 μm under conditions standard deformation effect. The resulting suspension of cells and cell nuclei was examined microscopically in a Goryaev chamber, counting cell nuclei in a certain volume of the chamber (0.02 mm).

The parameter reflecting the effect of MHTB on the viscoelastic properties of the liver or lung tissue was determined according to the formula:

где

Where

M _a and N _k - the number of cell nuclei released from 1 mg of liver or lung tissue after dispersion, respectively, in the experimental and control series. Statistical processing of the obtained data was carried out by the method of variation statistics using Student's t-test.

In each experiment, at least 5 tissue fragments were counted per point (solution of the test drug at a certain concentration and control). Each experiment was repeated at least 3 times.

Example 23. Study of the antiarrhythmic activity of the MHTB substance isolated from blood serum and from bovine heart tissue

The study was carried out on experimental models of arrhythmias caused by the administration of aconitine or calcium chloride, in accordance with the guidelines for the experimental (pharmacological) study of drugs proposed for clinical trials as agents for the prevention and treatment of cardiac arrhythmias.

The following preparations of the MHTB substance were studied.

1. MHTB 1 - obtained from the blood serum of cattle;

2. MHTB 2 - obtained from the heart of a bull.

The experiments were carried out on 30 outbred white rats (males), weighing 200-220 g. ECG was recorded on a Neuart Mirror-1 electrocardiograph (Innomed Medical Inc, Budapest).

Statistical processing of the obtained results was carried out by the methods of variation statistics using the Statistica software for Windows, version 6.0. The Mann-Whitney test was used to reject the null hypothesis. Differences between groups were considered statistically significant at p<0.05.

In the study, MHTB preparations were given to animals of both experimental groups for 7 days in the form of drinking solutions prepared with distilled water at a concentration corresponding to 10 ^-10 mg protein / ml; in the control group, the animals were given distilled water. Animals were not allowed to drink for 1 hour before the start of the experiment. Arrhythmogen was injected into the tail vein under light ether anesthesia.

During the experiment, the following parameters were determined:

1. animal survival;

2. absolute antiarrhythmic effect (AAE) ~ complete restoration of sinus rhythm after exposure to arrhythmogen;

3. partial antiarrhythmic effect (PAE) - suppression of the frequency of ectopic contractions by 50%;

4. periods of time of occurrence and duration of arrhythmia.

1. Aconitin model of cardiac arrhythmia

After weighing before the experiment, all animals were intravenously injected with aconitine at the rate of 40 µg/kg.

When using this experimental model, polytopic extrasystole occurs. At the last stage of development, it can develop into ventricular tachycardia with a fatal outcome. In this model, a low survival rate of experimental animals is observed.

Animals of the control group (15 pcs) developed mixed forms of cardiac arrhythmias of the atrioventricular type, lasting 97±5.3 minutes; 8 animals died (the data of one specific experiment are given, a total of 3 experiments were carried out). In the experimental groups (15 pcs in each group), all animals were alive, but AAE was not observed in any group, while PAE was observed in all animals of both groups. In both experimental groups, a significant increase in the time period for the onset of arrhythmia was observed compared with the control group (1.7 ± 0.06), respectively, in group No. 1 - in 2.3 ± 0.05, in group No. 2 - in 3, 3±0.05. In animals of both experimental groups, a more rapid recovery of heart rate was observed - 40.6±1.4 min and 23.2±0.7 min, respectively, in groups No. 1 and No. 2. It is important to note that both MHTB drugs had an effect on the course of the pathological process: there was a decrease in the number of cases of ventricular tachycardia and the frequency of extrasystoles.

2. Calcium chloride model of arrhythmia

After weighing, the animals were intravenously injected with a 10% solution of calcium chloride at a rate of 65 µg/kg.

When using this experimental model, bradycardia develops with ventricular extrasystoles, which can later turn into ventricular tachycardia, ending in the case of large doses of arrhythmogen with ventricular fibrillation and death. When using calcium chloride as an arrhythmogen, there is also a dissociation between electrical impulses and mechanical contractions of the heart muscle. This is due to the ability of calcium chloride to have a direct effect on the membrane of cardiomyocytes and myocardial fibers.

In the control group of animals (15 pcs), after intravenous administration of a solution of calcium chloride, severe cardiac arrhythmia was observed, which led to the death of 8 animals caused by the development of ventricular fibrillation; in surviving animals, restoration of sinus rhythm was observed 134.2 ± 3.35 min hours after the administration of arrhythmogen (data from one specific experiment are given, a total of 3 experiments were carried out).

In both experimental groups (15 pcs in each) the animals were alive. AAE was not observed in any animal, PAE - in all animals of both groups. In both experimental groups, a significant significant increase in the time period for the onset of arrhythmia was observed compared with the control group (1.4±0.09), respectively, in group No. 1 - in 2.5±0.07, in group No. 2 - in 3 .4±0.05. In animals of the experimental groups, a much faster recovery of the heart rate was observed - 45±1.7 minutes and 35±1.9 minutes, respectively, in groups No. 1 and No. 2.

conclusions

Prophylactic administration for 7 days of two MHTB preparations isolated from the blood serum and heart of a bull, on experimental models of arrhythmia, demonstrated a pronounced antiarrhythmic effect, which consisted in the abolition of the death of animals, in a decrease in the duration of arrhythmia, as well as the severity of developing cardiopathology.

Both preparations of the MHTB substance, isolated from the blood serum and heart of a bull, respectively, are promising for the development of new antiarrhythmic drugs on their basis.

Example 24. Method for obtaining MHTB from bovine mammary tissue

The mammary glands of sexually mature heifers were obtained at the Ramensky or Sergiev-Posad meat-packing plant.

Obtaining a tissue extract of the mammary glands of cows

Freshly isolated cow glands were washed several times in cold saline. solution until complete removal of blood, cut into small fragments weighing 1-2 g, washed again with saline. solution and placed for 2-3 hours in physical. solution at 0°С -+4°С.

Then the resulting extract was poured off, centrifuged at 2500g for 10 min. The obtained tissue extract was further fractionated to obtain BOD or frozen and stored at -90°C.

Obtaining MHTB from the extract of the mammary gland tissue of cows

Ammonium sulphate is gradually added to the cooled tissue extract solution with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of extract). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern (Fig. 6), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 66,000 Da. Edman's method determined the partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE), which has 100% homology with the N-terminal sequence of the mature Bos taurus BSA molecule.

Thus, the IEF-fraction is a BOD consisting of a protein representative of the Bos taurus blood serum albumin multifamily and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine), which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Analysis of the spectra of separation by isoelectric focusing of supernatant fractions obtained after salting out of proteins contained in extracts of various tissues or biological fluids of animals showed that they are similar to each other and contain MHTB subfractions that differ in charge: acidic (pH less than 3.00), basic ( pH over 8.00), and protein-peptide complexes (BOD) with pI values in the pH range of 4.00 - 7.00.

Example 25 Obtaining MHTB from bovine prostate tissue

The prostate glands of sexually mature bulls were obtained at the Ramensky or Sergievsky meat-packing plant.

Obtaining tissue extract of the bovine prostate gland.

The freshly obtained glands of the bull were washed several times in cold saline until the blood was completely removed, cut into small fragments weighing 1-2 g, washed with saline again. solution and placed for 2-3 hours in physical. solution at 0°C-+4°C. Then the resulting extract was poured off, centrifuged at 2500g for 30 min. The resulting prostate extract was further fractionated to obtain MHTB or frozen and stored at -90°C. Obtaining MHTB from an extract of bovine prostate tissue

Ammonium sulphate is gradually added to the cooled tissue extract solution with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern (Fig. 6), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected.

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0.

A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein with a molecular weight of 66369 Da is a protein of the mammalian albumin family with the number gi| 1351907 in the SwissProt database.

Thus, the IEF fraction is a BOD consisting of the gi| 1351907 a representative of the multifamily of bovine serum albumins and a mixture of peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine residues), which form inositol- containing a glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 26. Effect of IEF fractions collected in the pH range less than 3.0 after isoelectric focusing of supernatants of tissue extracts of the bovine prostate and mammary gland of the cow on the viscoelastic properties of liver cells during multiple organotypic cultivation of mouse tissue in vitro (diagrams, Fig. 8).

The effect of MHTB isolated from the supernatants of tissue extracts of bovine prostate and bovine mammary gland on the viscoelastic properties of mouse hepatocytes in vitro was studied, as described in example 10 of the present invention. In FIG. 8 presents the results of this study. It was shown that these MHTBs change the viscoelastic properties of mouse liver tissue in vitro, and there is a polymodal dose dependence of the biological action of MHTBs.

Example 27. Polyacrylamide gel electrophoresis of protein-peptide complexes that are part of the MHTB substance isolated from various tissues and bile of animals

By electrophoresis Electrophoresis in 15% polyacrylamide gel was carried out under denaturing conditions according to the Lamley method (gel thickness - 0.75 mm, size 8×10 cm). Gels were stained with Coomassie G-250.

Composition of a 15% separating polyacrylamide gel: monomer solution (mass fractions of acrylamide 30.8%, bis-acrylamide 2.7%) - 7.5 ml, Tris buffer (1.5M Tris-HCl, pH 8.8) - 3.8 ml, 10% sodium dodecyl sulfate solution - 0.15 ml, bidistilled water - 3.6 ml, 10% ammonium persulfate solution - 75 μl, TEMED (tetramethylethylenediamine) - 5 μl. The composition of the concentrating 4% polyacrylamide gel: monomer solution - 0.44 ml, buffer solution (0.5 M Tris-HCl, pH 6.8) - 0.83 ml, 10% sodium dodecyl sulfate solution - 33 μl, bidistilled water - 2.03 ml, 10% ammonium persulfate solution - 16.7 μl, TEMED - 1.7 μl. The electrode buffer contained: 0.025 M Tris, 0.192 M glycine and 0.1% sodium dodecyl sulfate (pH 8.3).

To obtain an electrophoretically pure protein, electrophoresis was performed under native conditions, without the use of sodium dodecyl sulfate in the preparation of gels and buffers. As molecular weight markers, a reagent from Pharmacia (USA) was used (α-lactalbumin - 14.4 kDa, soybean trypsin inhibitor - 20.1 kDa, carbonic anhydrase - 30 kDa, ovalbumin - 43 kDa, bovine serum albumin -67 kDa and phosphorylase b - 94 kDa). We also used molecular weight markers LKB (horse cytochrome C - 12.3 kDa; horse myoglobin 17.2 kDa; bovine carbon anhydrase - 30 kDa; chicken egg ovalbumin - 45 kDa; bull blood serum albumin - 66.25 kDa; chicken egg ovotransferrin - 76- 78 kDa).

When staining the gels with Coomassie G-250 dye, they were first placed for 10-15 minutes in a fixing solution (25% isopropanol, 10% acetic acid), and then in a Coomassie solution (10% acetic acid, 0.06% Coomassie G-250 ) until color appears. A fixing solution (5% methanol, 7% acetic acid) was used to remove background staining.

To obtain a phoretically pure fraction of the test sample, colored bands were excised from the gel, eluted with MilliQ deionized water with the addition of sodium azide, and then dialyzed against deionized water. Next, the resulting protein solution was concentrated to small volumes. To study the biological activity and conduct biological experiments, only the protein fraction obtained under non-denaturing (native) conditions was used.

In this experiment, it was shown that the main component of all studied IEF fractions is the Coomassie-stained fraction, which is characterized by high electrophoretic mobility (Rf 0.90±0.05) (Fig. 9). To study its biological activity, electrophoresis in PAAG was performed in the absence of sodium dodecyl sulfate. Further, this fraction was eluted from the gel and examined by biotesting. It was found that these low molecular weight fractions exhibited membranotropic activity characteristic of MHTB.

Example 28. The study of lipids that are part of MHTB

To determine the fatty acids in the MHTB substance isolated from various animal tissues, they were extracted with sulfuric ether from the IEF fraction collected in the pH range less than 3.0. To do this, to an aqueous solution of MHTB cooled to 4°C - 8°C in a concentration corresponding to 10 ^-3 -10 ^-4 mg of protein per ml, an equal volume of also cooled sulfuric ether is added, the vessel is tightly closed and exposed to an ultrasonic bath for 10-15 min, bleed off excess sulfuric ether vapor and repeat the process several times. Then separated from the aqueous solution of MHTB. The sulfuric ether fractions are combined, the ether is distilled off using a vacuum rotary evaporator, an oily residue is obtained, which is further examined by mass spectrometric method.

MHTB substances isolated from bovine blood serum, bovine liver, bovine lung, bovine bile, bovine mammary gland, and bovine prostate were studied. In FIG. 10 as an example shows the spectrum of fatty acids obtained from the substance MHTB, isolated from the blood serum of a bull. The presence of such fatty acids as decanoic, linoleic, cholic, deoxycholic and palmitic has been shown. Similar results were obtained when studying the composition of lipids isolated from other tissues.

These fatty acids are permanent components not only of the blood serum of mammals, but are also part of almost all tissues of vertebrates. They are involved in such important biological processes as gene expression, cell migration, proliferation, adhesion, and differentiation [7] (Lapillonne et al., 2004).

Example 29. Determination of the secondary structure of protein-peptide complexes that make up MHTB using the circular dichroism method

The method of circular dichroism is based on the analysis of deviations in the plane of polarized light caused by various conformations of the polypeptide chain. A measure of such a deviation is the angle of ellipticity, the definition of which makes it possible to estimate the proportion of α-helices, β-structures, and a statistical coil in the molecule of the studied polypeptide. Circular dichroism spectra in the UV region (195–260 nm) were recorded on a Jasco 720 CD spectrometer (Japan) at 20°С in quartz cuvettes with an optical path length of 1 mm. The scanning speed was 50 nm/min, the step was 1 nm, the accumulation of each step was 2 sec. The concentration of the studied protein in the aqueous solution was approximately 100 μg/ml. The final spectrum was obtained by averaging the data of three scans and subtracting the spectrum of the baseline (control). The content of secondary structure elements was estimated using the CDNN program (http://bioinformatik.biochemtech.uni-halle.de/cdnn) and the k2d program [9] (Venyaminov and Yang, 1996).

The secondary structure of BOD of acidic MHTB isolated from mammalian tissues, as well as bile and milk, was characterized by the circular dichroism method (Table).

It was shown that the secondary structure of the BODs that make up MHTB is described mainly in terms of β-structures (approximately 70%) and a random coil (approximately 30%). The data of these studies show that such a state of the secondary structure of the BOD of bioregulators of this group is their characteristic property.

Literature data show that the secondary structure of proteins that tend to form macromolecular associates is often characterized by a high content of β-structures and disordered regions of the polypeptide chain [8],][13] (Ramis, 2005).

Example 30

The study of macromolecular associates present in aqueous solutions of MHTB was carried out using laser dynamic light scattering and atomic force microscopy. In this experimental series, solutions of acidic MHTB fractions obtained after IEF were studied.

и

где η - вязкость растворителя, k - константа Больцмана, D - коэффициент диффузии, q- волновой вектор.Experiments to determine the hydrodynamic radius of particles in fraction solutions were performed by dynamic light scattering on a PhotoCor Complex device (PhotoCor, Russia) equipped with an automatic goniometer, a PhotoCor-FC multi-time real-time correlator, and a helium-neon laser. Uniphase 1135R" with a power of 20 mW (λ=633 nm) as a light source. The measurements were carried out at a scattering angle of 90°C and a temperature of 25°C. Dust was removed from the solutions by filtration through Durapore membranes with a pore diameter of 0.45 μm (Millipore). The homodyne intensity correlation function G ⁽²⁾ (t) was determined in the delay time interval from 2×10 ^-8 to 10 ³ s/ The correlation time distribution was obtained by the inverse Laplace transform method using the CONTIN program. hydrodynamic radius (R _h ) used the following relationships

And

where η is the viscosity of the solvent, k is the Boltzmann constant, D is the diffusion coefficient, q is the wave vector.

The results of a study carried out by the dynamic scattering method showed that the solutions of acidic MHTB fractions contain particles with an average hydrodynamic radius of 40–60 nm. The distribution of these particles in all MHTB fractions was unimodal. In FIG. Figures 11 and 12 show particle size distribution curves present in MHTB fractions isolated from rat liver, lung, small intestine, and bovine bile.

The results of the study of aqueous solutions of MHTB, carried out by dynamic laser light scattering, are consistent with the data obtained by atomic force microscopy (AFM).

Example 31

The sizes of the particles present in the aqueous solution of MHTB were determined using atomic force microscopy. The particle radius was determined using a FemtoScan scanning probe microscope (Russia). Solutions containing BOD were preliminarily dedusted using Durapore filters with a pore diameter of 0.45 μm (Millipore). For data, single-lever cantilevers of the Standard FemtoScan category (Russia) with a stiffness of 0.03 N/m were used. Scanning was carried out by the contact method.

AFM showed that after removal of the solvent from the BOD fraction, which is part of the MHTB, particles with a size of 30-80 nm are found on the substrate (Fig. 15).

The results obtained in the study of individual subfractions that make up the MHTB substance isolated from the liver, lung, small intestine, and bile show that nanosized particles are present in their aqueous solutions. Literature data indicate the ability of substances in the form of nanosized particles (10–1000 nm) to exhibit unique physicochemical properties and biological effects [13] (Ramis, 2005). Note the similarity between nanosized particles present in water–protein systems both in vitro and in vivo [13] (Ramis, 2005). It was suggested that the formation of nanoparticles in biological objects may be the result of the ability of matter to self-organize in nonequilibrium systems [8] (Ramis, 2006). In connection with these data, it can be assumed that the biological activity of the MHTB substance is due to the ability to form stable nanoparticles.

Example 32. Study of the resistance of MHTB to the effects of physico-chemical factors by the method of circular dichroism

The studied substance MHTB exhibits very high resistance to such extreme physical and chemical factors as heating its solution to 100°C, as well as to the action of guanidine chloride, used as a deaggregating agent, and Ca ^{+ 2} -chelating agent - EDTA. As an example, in FIG. 13-15 shows the values of ellipticity, reflecting the state of the secondary structure of BOD, which is part of the MHTB substance isolated from the blood serum of a bull, under the influence of physicochemical factors. The data obtained indicate that there are no changes in the secondary structure of BOD with an increase in the temperature of the solution to 100°C and the action of guanidine chloride compared to the initial solution of MHTB.

The impact of EDTA leads to a change in the ellipticity of BOD solutions (all subfractions) isolated from the blood serum of a bull, and with an increase in the concentration of EDTA, the value of ellipticity tended to decrease. These data indicate the participation of Ca ⁺² ions in the organization of the secondary structure of BOD, which are part of MHTB.

Example 33. Study of the MHTB substance by NMR spectroscopy

произведенный в Германии. Измерение спин-решеточных времен релаксации осуществляли в ампулах с диаметром равным 5 мм. Внешним стандартом являлся раствор дейтерированного ацетона, который в ампуле погружали в капилляр и центрировали специальными тефлоновыми вставками. Использовали стандартную программу восстановления. Точность измерения времени релаксации равна ±5%.The acidic fractions of the MHTB substance isolated by isoelectric focusing from the supernatants of the extract of various tissues, including blood serum, bovine prostate gland, and cow mammary gland, were studied. The study was carried out on an Avance 600 NMR spectrometer

produced in Germany. Spin-lattice relaxation times were measured in ampoules with a diameter of 5 mm. The external standard was a solution of deuterated acetone, which was immersed in an ampoule into a capillary and centered with special Teflon inserts. Used the standard recovery program. The relaxation time measurement accuracy is ±5%.

In all MHTB substances studied by the NMR method, similar spectra were found, which indicates that the composition of the MHTB substance isolated from various sources is practically the same.

As an example, in FIG. 16 shows the ¹ H NMR spectrum of the MHTB substance isolated from the bovine prostate gland (A), FIG. 16 (B) - MHTB substance isolated from the mammary gland of a cow, in Fig. 16 (B) - spectrum of inositol. The ¹ H NMR spectrum was recorded in the range 25÷-8 ppm. Wide unresolved signal in the area 1÷2.5 ppm refers, apparently, to the protons of a part of the protein located in the associated microparticles, interconnected by interaction through water molecules by hydrogen bonds, which is confirmed by their destruction and the disappearance of this line upon additional action (pumping) on the water signal or slight heating (up to 40 °C). In the area where aromatic protons are located, the signals of protons of phenylalanine (δ=8.7; 8.5; 8.0 ppm) and histidine (δ=7.67; 7.36 ppm) shifted to more weak field than usual due to acidic solution (pH<3.0). In the same region there are signals of protons of NN ₃ ⁺ -groups (δ=7.16; 7.07; 7.00 ppm). In the high field (0.4÷1.5 ppm) there are lipid signals (very low density δ=1.3÷1.18 ppm; low density δ=1.10÷1.04 m .d.; broad high-density signal with δ=0.75 ppm). Doublet with δ=1.28 ppm refers to lactate protons. Proton signals in the region of 2÷4 ppm can be attributed to the protons of the carbohydrate components: NAc - glucosamine (δ=2.13 ppm), glucose (δ=3.30 and 3.38 ppm .), choline (δ=3.28÷3.30 ppm), etc. Singlet at 2.6 ppm appears, possibly due to some impurity with an acetyl group. Thus, according to ^1H NMR data, it can be concluded that the composition of the MHTB fractions under study includes lipid and carbohydrate, as well as protein components. A distinctive feature of the spectrum of proteins of this fraction is a large number of well-resolved lines, both in the region of manifestation of aromatic protons and in the region above 5 ppm, which indicates the possibility of the presence of rather short peptide chains. These data are in good agreement and support the assumption about the composition and structure of MHTB.

Example 34 Obtaining MHTB substance from bovine pancreas and thyroid tissue

Obtaining a tissue extract of the bovine pancreas

The obtained glands of the bull - pancreas or thyroid, are washed several times in cold saline. solution until the blood is completely removed, cut into small fragments weighing 2-4 g, washed again several times with saline. solution and placed for 2-3 hours in physical. solution at 0°C-+4°C. Then the resulting extract is drained, centrifuged at 2500g for 30 minutes. The resulting gland extract is further fractionated to obtain MHTB or frozen and stored at -90°C.

Preparation of MHTB from bovine pancreatic tissue extract

Ammonium sulphate is gradually added to the cooled tissue extract solution with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the resulting separation pattern (Fig. 20), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; a number of peptides with molecular weights in the range of 1,000 Da - 10,000 Da are detected. Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a representative of the bovine albumin multifamily. Edman's method determined the partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE), which has 100% homology with the N-terminal sequence of the mature Bos taurus serum albumin molecule. Thus, the IEF fraction collected in the pH range <3.0 contained bovine serum albumin and a mixture of small peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine), which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 34 Obtaining MHTB substance from bovine pancreas and thyroid tissue

Obtaining a tissue extract of the pancreas of a bull The obtained glands of a bull - pancreas or thyroid, are washed several times in cold saline. solution until the blood is completely removed, cut into small fragments weighing 2-4 g, washed again several times with saline. solution and placed for 2-3 hours in physical. solution at 0°C-+4°C. Then the resulting extract is drained, centrifuged at 2500g for 30 minutes. The obtained tissue extract of the gland is further fractionated to obtain MHTB or frozen and stored at -90°C.

Obtaining MHTB from an extract of tissue of the pancreas or thyroid gland of a bull Ammonium sulphate is gradually added to the cooled solution of the tissue extract with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the resulting separation pattern (Fig. 20), a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000 Da - 10000 Da are detected. Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in the gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein is a representative of the bovine albumin multifamily. Edman's method determined the partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE), which has 100% homology with the N-terminal sequence of the mature Bos taurus serum albumin molecule.

Thus, the IEF fraction collected in the pH range <3.0 contained bovine serum albumin and a mixture of small peptides with molecular weights from 1000 to 10000 Da, as well as calcium ions, lipids, carbohydrates (mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine), which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell.

Example 35 Effect of MHTB substance isolated from bovine pancreatic or thyroid tissue on the viscoelastic properties of the plasma membrane of mammalian cells in vitro. (Fig. 18)

Biotesting of fractions obtained at different stages of purification of tissue extracts and biological fluids was carried out using an adhesiometric method specially developed to identify the MHTB substance. The method makes it possible to evaluate the viscoelastic properties of a tissue under a standard deformation effect.

In the experiment, CBA/C57BI mice, males, weighing 15-18 g were used. After decapitation of the animal, fragments weighing 1.0-2.5 mg were excised from the liver or lung. In each penicillin vial of the experimental series, 1 ml of nutrient medium 199 and 0.1 ml of the studied protein solution at the appropriate concentration were added, five fragments of mouse liver or lung tissue were placed. The interval of active concentrations for each of the studied protein preparations was determined by studying the biological effect of its solutions obtained by sequential dilution by 10, 100, 1000 or more times. In vials of the control series, instead of a solution of the studied protein, 0.1 ml of saline was added. solution. Organ cultures of the experimental and control series were incubated for 2 h at 37°C. Next, each tissue fragment was taken out, dried for 10-15 sec on filter paper, weighed and subjected to dispersion in 0.1 ml of a 0.1% solution of trypan blue, prepared on 199 medium, in a tissue disintegrator with a gap of 50 μm under conditions standard deformation effect. The resulting suspension of cells and cell nuclei was examined microscopically in a Goryaev chamber, counting cell nuclei in a certain volume of the chamber (0.02 mm).

The parameter reflecting the effect of MHTB on the viscoelastic properties of the liver or lung tissue was determined according to the formula:

где

Where

M _a and N _k - the number of cell nuclei released from 1 mg of liver or lung tissue after dispersion, respectively, in the experimental and control series. Statistical processing of the obtained data was carried out by the method of variation statistics using Student's t-test.

In each experiment, at least 5 tissue fragments were counted per point (solution of the test drug at a certain concentration and control). Each experiment was repeated at least 3 times.

Example 36 Effect of MHTB substance isolated from bovine pancreas on the development of experimental type 2 diabetes in rats in vitro

The experiment was carried out on Wistar rats, males, 250 g. solution at a dose of 60 mg/kg 4 groups were formed (15 pieces each):

Group 1 - control (intact rats)

Group 2 - control (which was injected with saline)

Group 3 control (SPT + saline solution)

4 experimental (SPT+MGTB fraction pH<3.0)

As a protective substance, we studied the MHTB fraction isolated from the pancreas of a bull, with pH <3.0, which was fed to animals in the form of a drinking solution in drinkers daily at a concentration corresponding to ^10–10 mg/ml. The experiment was carried out for 2 weeks. The content of glucose and insulin was determined using a standard glucometer.

After the introduction of SPT, one animal died. All other rats that received SPT and SPT+MHTB from the pancreas remained alive during the experiment. Rats of the 4th experimental group looked more active on the third day of the experiment: they took food and drank actively, the stool was normal. The results of the study of the content of glucose and insulin in the blood (tail vein) on the 14th day after the administration of SPT are shown in Table 7. In rats of the 4th group, the results were divided into two subgroups: in one of them (9 pcs), the parameters of glucose and insulin did not differ statistically from 1 control group (intact animals). These results indicate a pronounced not only protective, but also therapeutic effect of MHTB isolated from the bovine pancreas in relation to the model pathology of type 2 diabetes in mammals. The remaining 6 animals of this group had indicators higher than those of intact animals, however, they were approximately two times lower than the results of animals in the control group taking SPT. The obtained results indicate a protective effect of the MHTB substance isolated from the bovine pancreas, taken orally in the form of a drinking solution at a protein concentration of ^10-10 mg/ml, in relation to the development of induced diabetes in rats in vivo. The data obtained allow us to consider MHTB as a potential substance for the development of a pharmacological drug for the prevention and treatment of type 2 diabetes at the initial stages of development.

Example 37. Study of the effect of the substance MHTB isolated from the extract of the thyroid gland of a bull on the state of the thyroid gland in rats in an experimental model of hypothyroidism in vivo

Mercazolil is an antithyroid drug, the mechanism of action of which is to block the peroxidase enzyme, which is responsible in the thyroid gland for the activation of iodine molecules in the form of free radicals, which carry out the iodination of thyroglobulin, which, in turn, leads to a decrease in the concentration of thyroid hormones in the blood.

The study was carried out on 45 Wistar rats of both sexes, 200-220 g.

The control group (C1) - intact animals (15 pcs) took free access to distilled water and food.

Experimental hypothyroidism was induced in 30 rats by daily administration of mercazolil at a dose of 10 mg/kg for 28 days intraperitoneally.

After that, blood was taken from all 30 rats for research from the tail vein and the level of thyroid-stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) was determined.

Then 30 rats were divided into two groups:

The control group (K2) - the animals took free access to distilled water and food.

Experimental group (OP1) - the animals of this group took a solution of the substance MHTB, isolated from the extract of the thyroid gland of a bull, at a concentration corresponding to 10 ^-10 , prepared in distilled water, with free access and food.

After 21 days, all animals were withdrawn from the experiment. Blood was taken from the animals from the aortic bifurcation and the level of TSH, T3 and T4 was determined in it by radioimmunoassay. Statistical processing of the obtained data was carried out using Student's t-test. Research results

Animals with experimental hypothyroidism (group K2) showed a significant change in the level of thyroid hormones compared with the data of the control group K1 (intact animals): the level of TSH increased by 4.6 ± 2 times, T3 decreased by 11 ± 6%, and the level of T4 decreased by 32±4%. The results obtained showed that by administering Mercazolil according to the indicated method, an adequate model of experimental hypothyroidism was obtained in rats of the K2 group and the OP1 group.

The study of the level of thyroid hormones in animals of the OP1 group on the 21st day of the experiment showed that the level of TSH decreased by 53 ± 3%, the level of T3 increased by 43 ± 5%, and the level of T4 increased by 2.8 times compared with the control group K2. The obtained data show that the MHTB substance isolated from the bovine thyroid gland caused the restoration of gland function in the model of experimental hypothyroidism in rats in vivo.

conclusions

The data obtained show that the MHTB substance isolated from bovine thyroid tissue in the form of a drinking solution helps restore thyroid function in an in vivo model of experimental hypothyroidism in rats and can be used to develop new drugs for the treatment of thyroid pathologies.

Example 38. Study of the antiviral activity of the MHTB substance

The study was carried out in collaboration with the Institute of Virology named after. DI. Ivanovsky RAMS.

We used a highly pathogenic strain of avian influenza virus A (H5N1). The virus-containing material was a culture fluid collected from H5N1 virus-infected cultures of embryonic porcine kidney epithelial cells (SPEV) at the height of development of cytopathic manifestations. We used infectious doses of virus-containing material equal to 100 TCD50/0.2 ml.

Cell cultures. The study of the antiviral activity of preparations of MHTB substances was carried out in cultures of SPEV cells grown as a one-day monolayer in 48-well panels on nutrient medium 199 with the addition of 10% fetal calf serum and antibiotics.

Preparations. We studied the effect of MHTB substances, which are fractions of the corresponding tissue extracts collected after IEF in the pH range less than 3.0:

- MGTB No. 1 - MGTB substance isolated from rat thymus extract;

- MHTB No. 2 - MHTB substance isolated from the blood serum of a bull;

- MGTB No. 3 - MGTB substance isolated from the extract of the small intestine of rats;

-No.K4 - "composition" of these MHTB substances, obtained by mixing solutions of MHTB No. 1-3 in one volume (1:1:1 by volume).

All MHTB preparations were studied at a final concentration of ^10–12 mg protein/ml.

Depending on the time of adding the studied preparations of MHTB No. 1-K4 to the nutrient medium of SPEV cell cultures, the following experimental series were formed:

1st series - MHTB preparations No. 1-K4 were added 1 hour before cell infection;

2nd series - MGTB preparations No. 1-K4 were added simultaneously with infection with the virus;

3rd series - MGTB preparations No. 1-K4 were added 1 hour after infection with the virus;

4th series - control series in which the cells were infected with the H5N1 virus;

5th series - control series, intact cultures.

The results of the study were taken into account by the effect of drugs on the viability of H5N1-infected SPEV cells in comparison with the control series, which used avian influenza A virus-infected SPEV cell cultures.

As can be seen from the results shown in Table 8, the maximum antiviral activity of MHTB preparations No. 1-K4 was registered when the cell culture was treated 1 hour before infection with the virus, i.e. The most pronounced was the preventive effect of MHTB preparations. It should be noted that under these experimental conditions, when MGTB preparations No. 1 and No. 3 were added to the culture medium, 100% of the cells survived within 48 hours after infection with the virus. Treatment with No.K4 led to the preservation of viable 90% of the cells, but only 25% of the cells survived in the case of treatment of cells with MHTB No. 2.

The introduction of MHTB No. 3 and No. K4 into the culture medium 1 hour after infection of cell cultures with the virus resulted in 100% protection of cells within 48 hours after infection with the virus in both cases, respectively.

conclusions

The study of the antiviral activity of 4 MHTB drugs against the development of an infection caused by a highly pathogenic variant of the A/H5N1 avian influenza virus revealed a high therapeutic and prophylactic activity of the MHTB substance isolated from the small intestine and the drug, which is a “composition” of the studied MHTB substances. In this study, it was shown that the MHTB substance isolated from the small intestine of rats, when introduced into the culture medium in all three variants of the experiment for 48 hours, contributed to 100% viability of target cells under cultivation conditions. It is important that all studied MHTB substances showed prophylactic activity within 24 hours. The results of this study indicate the tissue-specific nature of the antiviral action of MHTB: the MHTB substance isolated from the small intestine and the “composition” of MHTB substances in which it was present turned out to be the most active against the H5N1 avian influenza virus that infects intestinal epithelial cells.

It has been shown that the secondary structure of BOD, which is part of MHTB, is described mainly in terms of β-structures (approximately 70%) and a statistical coil (approximately 30%). Such a state of the secondary structure of the BOD of bioregulators of this group is their characteristic property.

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Claims (2)

1. Pharmacological substance of membranotropic homeostatic tissue-specific bioregulators MHTB in the form of their solutions in a pharmaceutically acceptable carrier and / or diluent, which has the ability to maintain and restore the function of organ tissues after the development of various pathological processes in them, isolated from various tissues and biological fluids of animals selected from group, including blood serum, tissue of the liver, lung, intestines, heart, pancreas, thyroid, prostate and mammary glands, bile, containing as an active basis protein-peptide complexes, which consist of proteins of the serum albumin multifamily, having a molecular weight in the range 65000-68000 Da and biologically active peptides with molecular weights of 1000-10000 Da, which are products of proteolysis of a number of proteins, as well as calcium ions, lipids, inositol, carbohydrates, such as mannose, galactose, glucose, N-acetylglucosamine, glucosamine, N- acetylgalactosamine, which form an inositol-containing glycosidic structure that provides its connection with the plasma membrane of the cell. 2. The pharmacological substance of the membranotropic homeostatic tissue-specific bioregulators MHTB according to claim 1, characterized in that it is intended for the preparation on its basis of a drug for the prevention and treatment of various diseases in humans and animals in the form of solutions in the concentration range from 10 ^-17 to 10 ^-5 mg protein/ml when diluted in a pharmaceutically acceptable carrier and/or diluent, and present as nanoparticles 50-200 nm.

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