RU2779914C1 - Method for producing dna-containing preparation for oral use and preparation obtained by this method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology and can be used in medicine, food and pharmaceutical industries. The method for obtaining a DNA-containing preparation for oral administration consists in isolating the target product from a natural source - the seminal glands (milk) of fish of the salmon family or the sturgeon family, by homogenizing the raw material in a citrate-salt solution, followed by diluting the homogenate with the same solution until a homogeneous mixture is obtained. Dry sodium chloride is introduced into the resulting mixture with constant stirring until a saturated sodium chloride solution with increased viscosity is formed. Then the solution is diluted with water at least 10 times, the stirring is stopped and kept until viscous clots appear, floating to the top of the solution, where these clots are collected, redissolved in a citrate-salt solution, and sonicated to obtain DNA fragments no longer than 500 pairs nucleic bases. Purification and isolation of the target product is carried out by adding at least 2.5 volumes of ethyl or isopropyl alcohol cooled to 4°C. In the final stage, the product is dried, crushed and stored in a dark room at a temperature not exceeding 20°C.

EFFECT: method makes it possible to obtain a DNA-containing preparation that has high bioavailability, efficiency, and is also suitable as a source of DNA in dietary supplements.

13 cl, 5 tbl, 1 ex, 6 dwg

Description

The invention relates to biotechnology and can be used in medicine, food and pharmaceutical industries.

Currently, DNA and its low molecular weight derivatives are widely used both as part of biologically active food supplements (BAA) as a source of nucleotides for the synthesis of deoxyribonucleic acids in the body, and as part of medicines.

So known patent RU 2106149 (A61K 31/70, publ. 10.03.1998), which describes the subcutaneous or intramuscular injection of 1.5% sodium salt solution of DNA in 0.1% sodium chloride solution (DERINAT); SU 1804852 (A61K 35/60, publ. 03/30/1993) discloses the parenteral administration of a DNA sodium salt solution for the treatment of Hodgkin's disease.

Patent GB 808296 (IPC C07C, publ. 04.02.1959) describes a method for extracting nucleic acid from animal and plant materials, by treating the crushed material with an alkali solution, precipitating an alkali metal nucleate by adding alcohol, mixing the precipitate with an aqueous suspension of calcium sulfate, heating and filtering mixture and precipitation of essentially pure injectable nucleic acid by adding a mixture of alcohol and hydrochloric acid to the filtrate;

The description of the invention to the author's certificate SU 652187 (IPC С07Н 21/04, publ. 03/15/1979) describes a method for obtaining deoxyribonucleic acid (DNA) for injection use from fish sperm by homogenization, salt extraction of deoxyribonucleoprotein, its dissociation into DNA and protein, followed by detergent deproteosis, in which gonads of III-U stages of puberty of fish, preserved in concentrated ethyl alcohol, are used as raw materials, and the homogenized tissue is treated with a proteolytic enzyme - trypsin or pronase;

patent RU 2036652 (MPK A61K 35/60, publ. 06/09/1995) describes a method for obtaining DNA enriched with mineral additives, including heating salmon fish milk to 100-150 ° C in a 7-10% sodium chloride solution, separating the extract by filtration , precipitation of the nucleoprotein complex with ethyl alcohol, introduction of a solution of trace elements in amounts equimolar with DNA, washing and drying of the final product, which is used as a biologically active food supplement, 95% of whose DNA is decomposed in the intestine into individual nucleotides;

patent RU 2034554 (MPK A61K 35/60, publ. 05/10/1995) describes a method for extracting DNA from sturgeon milt, which consists in homogenizing sturgeon milt, DNA deproteinization is carried out directly in the homogenate, for which the homogenate is treated with sodium chloride solution at 55-60°C, then treated with diatomaceous earth, concentrated in an electrodialysis unit or in a membrane apparatus and voiced. DNA from the solution is precipitated with alcohol and dried in an oven at 40°C. The drug obtained by this method is used as an injection.

Known methods for large-scale industrial processing of fish milk, allowing you to quickly process large volumes of raw materials, so the patent JP S552634 (IPC С07Н 21/04, publ. 01/10/1980) describes a method for extracting DNA and protamine without the use of an expensive device and raw materials, by processing unrefined fish milk directly with concentrated inorganic salt solution. Nucleoprotamine is extracted from raw fish milt directly with a 1-2M aqueous solution of an inorganic salt such as alkali metal chloride, sulfate, nitrate, ammonium chloride, sulfate, etc., at room temperature - 60 degrees (Fahrenheit). The extract is diluted with water, and separate DNA and separately protamine sulfate are isolated from the same portion of the raw material;

patent JP S554308 (IPC C07H 01/08, publ. 01/12/1980) discloses a method for isolating DNA from homogenized fish milk, in which the homogenized mass is adjusted with acid to a pH of 4.5-6.5 and stirred at 80-100 deg. C for 0.5-2 hours. Then, an alkaline aqueous solution is added to adjust the pH to more than 8, and stirring is continued, the solid part is separated, and acid is added to the filtrate to isolate DNA;

patent CN 103865919 (IPC C12N 15/10, publ. 06/18/2014), describes a method for the industrial production of pure DNA from calf thymus, fish sperm or fish milt, which includes the separation of the DNA solution after the dissociation of SDS sodium dodecyl sulfate, bringing the pH to alkaline, leaving it for 1-2 hours and collect it by centrifugation. Bringing the pH of the supernatant to 7.0-7.5, then adding ethanol to precipitate, collecting the precipitate, washing and drying the product DNA;

patent JP S552635, IPC C07H 21/04, publ. 01/10/1980, describes a method for simple extraction of DNA without the use of expensive devices and material, by treating raw fish milk directly with 0.5-3 M aqueous alkali solution, for example, alkali metal hydroxide, alkali metal acetate, alkali metal carbonate, etc. Extraction is carried out at a relatively low temperature and low alkali concentration to prevent product degradation. Since fish milt is not milled, complex and inefficient operations such as centrifugal separation, filtration, etc. are eliminated.

The methods described above make it possible to obtain high-molecular-weight DNA preparations containing many impurities, but which, nevertheless, can be used in the form of biologically active food additives, the DNA of which is decomposed in the intestine into individual nucleotides, and in cosmetic preparations for external use.

There are few works devoted to the mechanism of action of exogenous DNA-Na. At the same time, the issue of the assimilation of DNA-Na and distribution in organs and tissues depending on the molecular weight has been studied in most detail. In particular, it was shown (Rykova E.Yu. The activating effect of DNA on the immune system / E.Yu. Rykova, P.P. Laktionov, V.V. Vlasov // Usp. modern biol. - 2001. - T. 121 - No. 2. - P. 160-171; Serebryanaya N.B. DNA as an immunostimulant / N.B. Serebryannaya, A.A. Novik // Medical Immunol. - 2001. - V. 3, No. 1. - pp. 27-34) that DNA-Na entering the body accumulates mainly in the bone marrow, spleen and small intestine epithelium.

Nucleic acids are such a valuable material that all cells instantly try to capture parts of DNA that appear after the decay of obsolete cells. They capture and insert into their structure even without parsing into its component parts. This mechanism has been well studied in bacteria (“How Horizontal Gene Transfer in Bacteria Occurs and What Limits.” S.V. Shestakov Ecological Genetics Volume V No. 2 2007), and is now widely studied in humans (Alexander Markov, The BIRTH OF COMPLEXITY, Evolutionary biology today: unexpected discoveries and new questions, 580 pages, 2014, Corpus (AST).

It has been shown that polymeric DNA is absorbed by the cell much more strongly than hydrolyzed (split into small fragments), and DNA remains in its original form for a long time without being destroyed (Khaitov R.M. Immunomodulators: mechanism of action and clinical application / R.M. Khaitov, B.V. Pinegin // Immunology. - 2003. - V. 24, No. 4. - P. 196-203). Cells and tissues of organs that fall into extreme stressful conditions capture DNA extremely actively (Khaitov R.M. Immunomodulators and some aspects of their clinical use / R.M. Khaitov, B.V. Pinegin // Clinical Medicine. - 1996. - No. 8. - S. 7-13).

The therapeutic efficacy of exogenous DNA is associated with the preservation of its polymeric structure. Small fragments - oligos or mononucleotides are much less effective. . - S. 37-43)

The universal effect of polymer DNA fragments when taken orally (orally - lat. Per os) is the effect of reducing the severity of antinuclear syndrome and reducing the severity of most autoimmune diseases (Khaitov R.M. Immunomodulators and some aspects of their clinical use / R.M. Khaitov, B.V. Pinegin // Clinical Medicine. - 1996. - No. 8. - P. 7-13; Yerne NK, Nordin FN Plaque formation in agar by single antibody producing / NK Yerne, FN Nordin // Science. - 1963. - Vol. 140. - P. 405-407.)

In 1959, Kanazir et al. published a paper on increasing the survival of irradiated rats by injecting them with isologous DNA-Na obtained from the spleen and liver. At the same time, the survival rate of irradiated animals increased from 2.6% in the control to 30-40% in the experimental group (Korogodin V.I. Problems of post-radiation recovery, M., Atomizdat, 1966).

Known RU 2387456 (publ. 27.04.2010) injection and in the form of microclysters introduction to the patient of fragmented human DNA in complex with non-histone protein protamine, while the length of human DNA fragments is 200-6000 base pairs and the ratio of fragmented human DNA / non-histone protein protamine is 1 /0.8.

Thus, the creation of new double-stranded DNA preparations that provide the most convenient delivery of DNA to stimulate bone marrow cells and maintain a high level of the human immune system under adverse stressful conditions is an urgent task of today and tomorrow.

The main problem in providing a person with fragments of polymeric, double-stranded (double-stranded) DNA is that nucleic acids that come in the form of dietary supplements with food are 95-98% destroyed in the small intestine to purine and pyrimidine bases.

This problem is partially solved by the fact that for oral administration it is proposed to use immobilized oligonucleotides, which are DNA fragments with a molecular weight of 500-700 kDa, highly purified with proteolytic enzymes, obtained from raw materials - “Salmon milk extract - low molecular weight deoxyribonucleic acid”, immobilized on polyethylene oxide (patent RU 2414223, publ. 20.03.2011). In the same place (patent RU 2414223) a method for obtaining a DNA preparation for oral use is described by immobilizing DNA fragments highly purified by proteolytic enzymes with a molecular weight in the range of 500-700 kDa, obtained from raw materials - "Salmon milk extract - low molecular weight deoxyribonucleic acid", which is carried out by irradiating a 10% aqueous solution of polyethylene oxide (PEO) with a molecular weight of 1.5 kDa with a stream of accelerated electrons at a dose of 1.5 Mrad and introducing salmon milt DNA oligonucleotides into the irradiated solution to a final concentration of 10 mg per 1 ml, the mixture is stirred for 10 minutes until obtaining an opalescent solution. Pre-irradiated polyethylene oxide forms micelles containing condensed DNA, which makes it possible to protect the DNA molecule from degradation in the gastrointestinal tract and facilitates the permeability of the micelles through the intestinal wall.

However, the implementation of this method requires complex and expensive equipment - an electron accelerator, specialists who know the technique of irradiation, and highly purified DNA fragments.

The technical problem to be solved by the present invention is to eliminate the need to use complex equipment that requires special knowledge and highly purified oligonucleotides by obtaining a DNA preparation for oral use in the form of deoxyribonucleoprotein (DNP), in which the native DNA structure is most fully preserved in the form double-stranded (double-stranded) polymeric DNA with preservation of the natural packaging of the deoxyribonucleoprotein, namely, the preservation of the DNA bond with protamine proteins, which makes it possible to ensure the delivery of double-stranded polymeric DNA fragments to the small intestine upon oral intake, and then to ensure the possibility of the DNA fragments entering the blood.

The problem is solved by the fact that a method is proposed for obtaining a DNA-containing preparation for oral use from the seminal glands (milk) of fish, mainly the salmon family or the sturgeon family, which consists in the fact that the raw material is homogenized in a citrate-salt solution, followed by dilution of the homogenate with this the same solution until a homogeneous mixture is obtained, into which dry sodium chloride is introduced with constant stirring until a saturated solution of sodium chloride is formed and the viscosity of the solution increases, which indicates the beginning of the isolation of the DNA-containing preparation, which is continued to be stirred for at least another hour, then the solution is diluted with water at least 10 times, stirring is stopped and kept until viscous clots appear, floating to the top of the solution, where these clots are mechanically collected, redissolved in a citrate-salt solution, treated with ultrasound until DNA fragments no longer than 500 pairs are obtained nucleic wasps innovations, the target product is purified and isolated by adding at least 2.5 volumes of ethyl or isopropyl alcohol cooled to 4°C, dried, crushed and stored in a dark room at a temperature not exceeding 20°C.

In this case, a 0.9% sodium chloride solution prepared in a 0.01 M solution of sodium citrate (sodium citrate) pH 7.14 is taken as a citrate-salt solution.

To dilute the solution, take cold (temperature not higher than 15 ° C) drinking water GOST R 51232-98.

The mechanical collection of clots is carried out, in particular, with a sieve.

Purification and isolation of the target product is carried out by adding a sorbent to the citrate-salt solution treated with ultrasound, stirred, kept for at least 2 hours, filtered, then at least 2 5 volumes of cold ethyl or isopropyl alcohol, mixed and placed in a refrigerator at a temperature not exceeding 4 ° C to form a dense precipitate, which is collected on a sieve, washed with 80% alcohol and dried in a vacuum oven at a temperature not exceeding 40 ° C for 38 -45 hours.

As a sorbent, for example, diatomaceous earth or kromasil, or any other finely porous silica gel is used.

Predominantly, sonication is carried out to obtain DNA fragments with a length of not more than 400 nucleic base pairs.

Sonication is controlled by determining the size of the DNA fragments in the resulting preparation, for example by high performance liquid chromatography of the preparation sample or by agarose gel electrophoresis of the sample.

In a particular case of the implementation of the method for sonication, a bath with a membrane connected to a magnetostrictive emitter connected to an ultrasonic generator, for example, UZG17-2.0/22, providing ultrasound with an operating frequency of 22 kHz, is used, and the treatment is carried out for at least 20 minutes.

Filtration of a mixture of citrate-salt solution of a DNA-containing preparation treated with ultrasound with a sorbent is carried out under vacuum on a suction filter covered with a layer of belting and calico, on which the remains of insoluble fragments of the feedstock and the sorbent are retained.

The DNA-containing preparation for oral use, obtained according to the proposed method, contains at least 80.0 wt.% DNA in the form of a mixture of double-stranded fragments with a length of not more than 500 nucleic base pairs (bp), and, according to at least 10.0 wt.% protein in the form of a mixture of proteins having a molecular weight of from 6 KD to 8 KD.

Preferably, a DNA-containing preparation for oral administration contains DNA in the form of a mixture of double-stranded fragments from 200 to 400 base pairs in length, and a mixture of protamines as a protein.

DNA-containing preparation additionally contains water in the amount of 1.0 - 4.0 wt.% and mineral impurities in the form of sodium chloride up to 100 wt.%.

DNA in the preparation obtained by the proposed method has, depending on the type of raw material, a hyperchromic effect from 35 to 37%.

The DNA-containing drug is a deoxyribonucleoprotein - a complex of DNA with protamines.

The essence of the proposed method is as follows:

- at the initial stage of obtaining a DNA-containing preparation, milk cells are destroyed by mechanical homogenization, which leads to the destruction of cell membranes and the release of soluble proteins into a solution of 0.9% sodium chloride in 0.01M sodium citrate, which inhibits the action of nucleases;

- use the most gentle methods of isolation and purification of the drug;

- this leads to the fact that it is possible to preserve the natural packaging of deoxyribonucleoprotein as much as possible;

- the DNA contained in the preparation has a range of lengths of double-stranded fragments from 200 to 400 bp, which corresponds to a molecular weight from 130 to 260 kDa, that is, much lower than 500 kDa and, therefore, does not contain genes;

- hyperchromic effect of the resulting low molecular weight DNA is not less than 35%;

- DNA, which has retained its connection with protamines due to the preservation of a compact package that allows DNA to overcome the stomach, is practically not attacked by DNases in the small intestine and is more easily absorbed by the intestinal epithelium, due to arginine, the main component of protamine proteins, which well shields the negative charge of DNA, therefore, obtained the drug in the form of deoxyribonucleoprotein (DNP) does not have a negative charge and penetrates into cells more easily.

Thus, it was possible to obtain a DNA preparation in the form of a deoxyribonucleoprotein (DNP preparation) in an extremely simple way, in which the native structure of DNA in the form of a double-stranded polymeric DNA is most fully preserved with the preservation of the natural packaging of the deoxyribonucleoprotein, namely, the retention of DNA binding to protamine proteins, which makes it possible to ensure the delivery of fragments. double-stranded (double-stranded) polymeric DNA into the small intestine with oral intake of the DNP drug.

The invention is illustrated by graphic materials, where

in FIG. 1 shows a photograph of electrophoresis in the horizontal direction of a DNA-containing preparation obtained by the proposed method in agarose gel with visualization of DNA with the addition of ethidium bromide, where 1 is a blurred band of the indicated preparation at the stage of its preparation after filtration through kromasil; 2 - the specified drug after precipitation with alcohol and drying, a clear band is visible in the zone between 200-500 bp; 3 - markers of molecular weight expressed in nucleic base pairs (bp) - 1000 bp, 900 bp, 800 bp, 700 bp ., 600 b.p., 500 b.p., 200 b.p.;

in FIG. 2 shows the ultraviolet spectrum recorded on a Perkin Elmer scanning spectrophotometer when measuring in a 1 cm quartz cuvette the optical density of a solution of a DNA preparation (DNP) obtained by the proposed method in 0.9% sodium chloride solution - curve N1. The absorption maximum of the DNP preparation corresponds to 260 nm. Curve N2 is a measurement of the optical density of the sodium chloride solution, in which the DNA preparation obtained by the proposed method was dissolved;

Fig. 3 shows gel filtration of a solution obtained by the proposed method of the DNA preparation (DNP preparation) in saline on a TSK 3000 column at low elution rates (see Osterman L.A. Chromatography of proteins and nucleic acids. M., Nauka, 1985. 556 s pp. 159-161),

where the X-axis indicates the value of adsorption (absorption) at 260 nm of the resulting DNP preparation, the Y-axis indicates the time of 30 and 60 minutes passed by the pen of the recorder.

Numbers 1, 2, 3 on the gel filtration curve show that there are three distinct peaks of different areas absorbing at 260 nm. Peak 3 corresponds to the DNP preparation. Peaks 1 and 2 correspond to the admixture of DNA fragments from salmon milk, which are destroyed in the gastrointestinal tract. The elution profile shows that the preparation obtained by the proposed method leaves the column and is recorded in the flow cell of the detector at a wavelength of 260 nm in the form of a wide Gaussian curve, and the content of low molecular weight (peaks 1, 2) and high molecular weight (not indicated on the profile) impurities is insignificant . This once again confirms the data obtained by electrophoresis of the resulting preparation in agarose gel, on the homogeneity of the isolated fraction of double-stranded DNA fragments in complex with protamines.

Figure 4. shows a graph of the linear dependence of the molecular weight of the DNA preparation obtained by the proposed method (DNP preparation) on the relative distance (in cm) of migration during agarose gel electrophoresis,

where the X-axis indicates the molecular weight of the markers, indicated by the numbers 1-1500 bp, 2-1000 bp, 3-800 bp, 4-700 bp. , 6-100 b.p.

The y-axis indicates the migration distance in cm, obtained by measuring the distance from the bottom edge of the pocket to the luminous strip of each marker: 1,2,3,4,6.

Wide zone 5 on the graph corresponds to a wide luminous band of the pocket into which a sample of the DNP preparation was introduced, indicated by the number 5 on the graph. On the Y axis, the migration distance in cm for pocket 5 is measured from the lower edge of the pocket to the beginning of the upper edge of the luminous zone (p.n. .o. 450) and from the lower edge of the pocket to the lower edge of the luminous zone (b.l. 200) and is outlined on the graph by an elongated oval. The straight line drawn through the points obtained at the intersection of the straight lines drawn from the x and y axes forms the indicated graph of the linear dependence of the molecular weight and the migration distance of the samples in the agarose gel.

On FIG. Figure 5 shows a photograph of electrophoresis of extracellular DNA from the blood of rabbits fed with the DNA-containing preparation obtained by the proposed method (DNP preparation), and control - without DNA and with the Derinat preparation (purified DNA), where on the first strip 1 - DNA molecular weight markers, where the stripes from top to bottom correspond to the mass: 3000 bp, 2000 bp, 1200 bp, 800 bp, 500 bp, 200 bp. but.

Lane 2 is a sample of the blood processing of the control rabbit treated with saline; Lane 3 represents a 5-fold sample of the blood treatment of a control rabbit treated with saline taken in relation to lane 2; Lane 4 - represents a sample of the processing of the blood of a rabbit treated with Derinat; Lane 5 - is taken in 5-fold size with respect to lane 4, a sample of the rabbit's blood treatment treated with Derinat; Lane 6 is a blood processing sample from a rabbit treated with DNP preparation.

On FIG. 6 is a graph of lg of the molecular weight of low molecular weight proteins - X axis, versus distance (in cm) of protein band migration in a gel with sodium dodecyl sulfate (SDS-Na) - Y axis, where 1 is chymotrypsin, mol.weight 25,000 daltons (D); 2 - cytochrome C, mol. weight 15000D; 3 - RNase A, mol.weight 13700D; 4 - protamines, mol. weight 6000 - 8000 D; 5 - insulin Ch, mol.weight 5700D; 6 - C - peptide, mol.weight 3000D. The graph was obtained by the known electrophoresis method of separating proteins by size using sodium dodecyl sulfate (SDS-Na) in PAGE gel and adding 0.1% SDS-Na to the electrode buffer (see Methods for the study of proteins and nucleic acids: Electrophoresis and ultracentrifugation (practical guide, page 54 -65), Science Publishing House 1981).

An exemplary embodiment of the invention

In order to obtain a DNA-containing preparation, in a particular case, salmon fish milk was taken (you can take milk from fish of the sturgeon family). The raw material in the amount of 10 kg was thoroughly washed and cleaned of films, fat and other foreign matter, after which the prepared raw material was subjected to thorough grinding in a knife homogenizer with the addition of 1 liter of a 0.9% sodium chloride solution cooled to 4°C at 0.01 M citrate sodium pH 7.14 until homogeneous (usually within 15-30 minutes).

20 liters of a solution of 0.9% sodium chloride in 0.01 M sodium citrate pH 7.14 was poured into a vessel (reactor) made of stainless steel with a jacket, equipped with a propeller-type submersible mechanical stirrer and with a hinged top lid, with a volume of 250 liters, and the stirrer was switched on. . In portions, with the stirrer turned on, milk homogenate was added and stirring was continued until the mixture was completely homogeneous, for about 2 hours. Thus, at the initial stage of obtaining a DNA-containing preparation, milk cells are destroyed by mechanical homogenization, which leads to the destruction of cell membranes and the release of soluble proteins into a citrate buffer pH 7.14 (solution of 0.9% sodium chloride in 0.01 M citrate sodium pH 7.14), which inhibits the action of nucleases.

Then, dry sodium chloride was added here at the rate of 56 g per liter of the mixture, until a saturated solution of sodium chloride was formed and viscosity appeared. The resulting mass continued to be stirred for about 2 more hours, then this mass was diluted tenfold with cold drinking water, to reduce the concentration of sodium chloride by ten times, the stirrer was turned off and the mass was left for several (approximately 5-6) hours to form viscous clots that float up top of the solution in the reactor.

The resulting viscous clots were collected with a fine sieve, re-dissolved in 10 l of 0.9% sodium chloride solution in 0.01 M citrate buffer pH-7.14 and sonicated on a unit consisting of an ultrasonic generator, in a particular example, USG 17-2.0 /22 (http://www.petsonic.ru/uzo-12-02-03.shtml), and a polished stainless steel bath with a diameter, in a particular example, of about 54 cm and a height of about 6 cm, to the bottom of which a connected with an ultrasound generator, a magnetostrictive emitter also connected to a stainless steel membrane fixed inside the bath at a height of 2 cm from the bottom and at a distance of 1 cm from the walls of the bath. At a height of 3 cm from the bottom of the bath and at least 1 cm from the emitter membrane, a stainless steel coil is placed, through which cold water is passed to cool the treated solution during its ultrasonic treatment.

The generator UZG 17-2.0/22 provides ultrasound with a working frequency of 22 kHz, with a rated power of 1600 watts.

In a bath cooled with cold tap water, 10 l of a solution of viscous clots in a 0.9% solution of sodium chloride in 0.01 M citrate buffer pH-7.14 was placed, and after balancing the temperature of the material being processed and the bath, the ultrasonic generator was switched on. Processing of 10 liters of material was carried out for about 25-30 minutes, then a sample of the sounded material was taken to analyze the size of DNA fragments, which, in the preferred case, should not exceed 400 nucleic base pairs (bp).

The degree of ultrasonic treatment of viscous clots was checked using electrophoretic separation of DNA fragments in agarose gel with staining with ethidium bromide in the presence of known molecular weight markers in the control pocket. At the end of electrophoresis, under a UV light source, it is clearly visible where the main amount of DNA from the sample taken for the sample is located.

At the same time, the size of DNA fragments in the preparation was visually assessed by sedimentation of a sample of this product taken from the ultrasonic bath by adding 2.5 volumes of ethyl alcohol cooled to 4°C to the sample. The DNA preparation after sonication forms a suspension in the form of small flakes.

If in the process of adding chilled ethyl alcohol to the sample, large flakes were observed, then the ultrasonic treatment of the solution of clots was continued for another 5–10 minutes and the control test for DNA precipitation in the sample was repeated using chilled ethanol.

To confirm the correct selection of the mode of treatment of the resulting preparation with ultrasound, we additionally checked the length of DNA molecules by electrophoresis in agarose gel (https://evrogen.ru/kit-user-manuals/DNA_Ladder_NL002.pdf) using a DNA length marker 100+ bp DNALadder - standard to determine the length of double-stranded DNA molecules in the range of 100 - 1000 bp. (nucleic base pairs) when analyzed by agarose gel electrophoresis.

Subsequently, these markers were also used to assess the mass of DNA fragments in the sample of the drug obtained by the proposed method by comparing the intensity of the sample band with the DNA marker band of the same size. The assessment of the mass of DNA fragments from the preparation obtained by the proposed method is presented in the form of a photograph in Fig. 1 (comparison of the intensity of the band of the sample obtained by the proposed method of the DNA preparation with the band of the marker of the same size).

As can be seen from FIG. 1, obtained by the claimed method, a DNA-containing preparation contains a mixture of DNA fragments from 200 to 400 bp in length. (pairs of nucleic bases).

It is obvious that accurate determination of the size of DNA fragments in the resulting preparation, for example, by high-performance liquid chromatography of a preparation sample or by electrophoresis of a sample in agarose gel, is necessary during the initial use of an ultrasound generator that provides certain sonication modes characteristic of it. After that, it is sufficient to control the time of ultrasonic treatment visually by deposition of a sample of this product taken from the ultrasonic bath, adding to the sample 2.5 volumes of ethyl alcohol cooled to 4°C. The DNA preparation after sonication forms a suspension in the form of small flakes.

Kieselguhr sorbent was added to the sounded DNA-containing preparation, mixed, kept for at least 2 hours and filtered under vacuum on a suction filter, which was covered with a layer of belting and calico, and on which the remains of insoluble fragments of milk and the sorbent were retained. .

2.5 volumes of cold (4°C) ethyl (can be isopropyl) alcohol was added to the clear, colorless filtrate with the DNA preparation, mixed, and placed in a refrigerator overnight to form a dense precipitate. Later it was found that this technique removes from the resulting product short DNA fragments having a length of less than 200 nucleic base pairs.

In the morning, most of the alcohol solution was decanted from the sediment and, after mixing the precipitate with the rest of the alcohol solution, it was collected on a sieve with a fine nylon mesh (with a pore size of 900 μm).

The resulting precipitate of the DNA preparation was washed with 80% alcohol and dried in a vacuum oven at 40°C for at least 38 hours. The precipitate was ground in a ball mill, its DNA content was determined according to Diche with diphenylamine and the content of protamine proteins was determined by the color reaction for arginine according to Sakaguchi, and stored at room temperature in a well-closed dark glass jar in a well-ventilated room. Yield of the target product from 10 kg of raw materials was about 500 g (approximately 5% of the milk weight).

The resulting preparation was subjected to a biochemical study.

The molecular weight of DNA in the preparation obtained by the proposed method was determined by HPLC (high performance liquid chromatography) on a TSK 3000 column (Osterman L.A. Chromatography of proteins and nucleic acids. M: Nauka, 1985. 536 pages. Chapter 4 Gel filtration page 154 ), where the column was calibrated using a standard set of plasmid DNA, as well as by electrophoresis in agarose gel, staining with ethidium bromide (Osterman L.A. Methods for studying proteins and nucleic acids: Electrophoresis and ultracentrifugation (practical guide), M., Nauka 1981 288 pages, Chapter 4 Nucleic acid electrophoresis page 120). When the agarose gel is illuminated with an ultraviolet lamp, DNA-containing bands are visible. The resulting chromatogram is shown in Fig. one

As can be seen from FIG. 1 and the study obtained by the proposed method of the drug by HPLC (Fig. 3) found that obtained by the claimed method, the DNA preparation has a range of lengths of DNA fragments from 200 to 400 bp. (pairs of nucleic bases), which corresponds to a molecular weight of 130 to 260 kDa. that is, much lower than 500 kDa and, therefore, does not contain genes in its composition.

DNA yield was calculated from absorbance at 260 nm using a conversion factor of 1 pu. (optical unit) = 40 μg. The content of DNA in the target product was determined spectrophotometrically, analyzing the ratio of the absorption of solutions at wavelengths of 260 and 280 nm (Maniatis T. Molecular cloning. - M.: "Mir", 1984. - 480 S.). It was found that the target product contains DNA in the amount of 80.0 - 85.0 wt.%.

The hyperchromic effect of DNA was determined by comparing the optical density at 260 nm of a solution of a DNA preparation obtained by the proposed method from salmon milk, before and after hydrolysis with 5% perchloric acid at 100°C for 20 min (Lazurkin Yu.S. Physical methods for studying proteins and nucleic acids - M.: Nauka, 1967. - 342 p.). The analysis results were processed by obtaining the arithmetic mean of five parallel determinations. The increase in optical density was 36.2%, which corresponds to an increase (35-37%) for a standard sample of salmon DNA, and indicates that the initial solution of the DNA preparation obtained by the proposed method is indeed a double-stranded (double-stranded) DNA polymer. (A standard sample of highly purified polymeric DNA was obtained by phenol extraction and deproteonization according to Kirby-Georgiev (see Filippovich Yu.B. et al. Workshop on General Biochemistry, M., Enlightenment, 1975, p. 159.)

An example of determining the authenticity of the presence of DNA in the preparation obtained by the proposed method

0.375 g obtained from salmon milk by the proposed method of a DNA-containing preparation (composition: 80.0 wt.% DNA in the form of a mixture of double-stranded fragments 200-400 bp long, 10.0 wt.% protein in the form of a mixture of protamines with molecular weight from 6 to 8 kD, water in the amount of 3.0 wt.% and sodium chloride - the rest) were placed in a flask with a capacity of 30 ml and dissolved in 25 ml of 0.1% sodium chloride solution, obtaining solution No. 1. 0.1 ml of solution No. 1 was transferred into test tube A with a capacity of 10 ml with a ground stopper, 2 ml of water and 2 ml of Dische's reagent were added. Tube A was placed in a boiling water bath for 10 minutes, and a blue color appeared.

0.3 ml of solution No. 1 was placed in a volumetric flask with a capacity of 200 ml, the volume of the solution was brought up to the mark with 0.1% sodium chloride solution and mixed. The UV spectrum of the resulting solution was recorded in the wavelength range from 220 to 300 nm relative to 0.1% sodium chloride solution. The resulting spectrum (Fig. 2) has an absorption maximum at (260 ± 2) nm and a minimum at (230 ± 2) nm.

The drug gives a characteristic reaction B to sodium (GF XI, issue 1, p. 159), since the implementation of the proposed method uses a large amount of sodium chloride and DNA is an acid, it binds cations. The presence of a yellow qualitative reaction for the sodium cation also confirms the presence of a certain amount of sodium chloride in the resulting DNA-containing preparation.

UV spectrum (Fig. 2) confirms the presence of DNA in the preparation obtained by the proposed method.

Note:For the preparation of Dische's reagent 1 g of diphenylamine was dissolved in 100 ml of glacial acetic acid and 2.75 ml of concentrated sulfuric acid was added, the reagent was used until the expiration date of the solution was 3 months. To prepare a sodium chloride solution, 0.1 g of sodium chloride was placed in a volumetric flask with a capacity of 100 ml and dissolved in water, then the volume of the solution was brought up to the mark with water and stirred, the reagent was used until the expiration date of the solution - 6 months.

An example of determining the hyperchromic effect

0.03 g obtained from pink salmon milk by the proposed method of a DNA-containing preparation (composition: 80.0 wt.% DNA in the form of a mixture of double-stranded fragments 200-400 bp long, 10.0 wt.% protein in the form of a mixture of protamines with a molecular weight of 6 to 8 kDa, water in an amount of 3.0 wt.% and sodium chloride - the rest) (accurately weighed) were placed in a volumetric flask with a capacity of 100 ml, dissolved in 50 ml of 0.1% sodium chloride solution and the volume was adjusted a solution of 0.1% sodium chloride solution to the mark, obtaining solution A. 1 ml of solution A was placed in a test tube with a ground stopper with a capacity of 20 ml, 4 ml of 0.1% sodium chloride solution and 5 ml of 1.0 M perchloric acid solution were added, obtaining solution B. As a control, 5 ml of 0.1% sodium chloride solution and 5 ml of 1.0 M perchloric acid solution were added to another similar tube. Both tubes were placed in a boiling water bath and heated for 20 min, then the tubes were cooled and the optical density of solution B was measured at a wavelength of 260 nm in a cuvette with a layer thickness of 10 mm against the control. At the same time, 9.0 ml of 0.1% sodium chloride solution was added to 1 ml of solution A in another test tube with a ground stopper and mixed to obtain solution C. The optical density of solution C was measured at a wavelength of 260 nm. A 0.1% sodium chloride solution was used as a control solution.

Hyperchromic effect in percent (G) was calculated by the formula:

G \u003d [(D' _B - D _C ) / D _C ] × 100%

where: D _C - optical density of solution C, in this particular example, obtained equal to 0.600;

D' _B - optical density of solution B, in this particular example, obtained equal to 0.810.

The hyperchromic effect of the DNA-containing preparation from pink salmon milk was 35%.

Note: To prepare a 1.0 M solution of perchloric acid, 117 ml of perchloric acid were placed in a 1-liter flask, the volume of the solution was brought to the mark with water and stirred, the reagent was used until the solution expired - 1 year.

Thus, it has been proved that the native structure of DNA in the form of a double-stranded (double-stranded) polymeric DNA is preserved in the preparation obtained by the proposed method with the preservation of the natural packaging of the deoxyribonucleoprotein (CHARACTERISTICS OF THE SECONDARY STRUCTURE OF NUCLEIC ACIDS BY THE HYPERCHROMIC EFFECT, Big workshop "Biochemistry". Laboratory work: study guide, compiled by M. G. Kusakina, V. I. Suvorov, L. A. Chudinova (Perm State National Research University, Perm, 2012, 148 pp.)

The protein content in the DNA-containing preparation obtained by the proposed method (DNP preparation) was determined by the Sakaguchi method (reaction) (Methodological guide for a small workshop on biochemistry. Teaching aid for students of the Faculty of Biology, studying in the areas of training 06.03.01 "Biology" and 44.03.01 "Pedagogical education - Biology" SARATOV 2017, p. 9, Sakaguchi reaction to arginine), using a solution of the amino acid arginine as a standard. Quantification of arginine-containing peptides by the Sakaguchi reaction: Color tests for individual amino acids are used to detect peptides containing the corresponding amino acid residues. To identify the guanidine group of arginine, the Sakaguchi reaction is used - when interacting with a-naphthol and sodium hypochlorite, guanidines in an alkaline medium give a red color. (Amino acids, peptides and proteins T. Daveny. Ya. Gergey Translation into Russian, "Mir", 1976 p. 193. Chemist's Handbook 21, chemistry and chemical technology). The measurement is carried out at a wavelength of 550 nm.

The measurement of the amount of protamines (protamine) in the DNA-containing preparation obtained by the proposed method (DNP preparation) was carried out by the Sakaguchi reaction with a sample of DNP and determining the amount of arginine in solution using a calibration curve constructed for a pure arginine preparation. The calculation of the amount of protamines was carried out based on the data that arginine is 80% of protamine. Thus, the results of quantitative determination of protamines in the resulting DNP preparation show that the content of protamines is about 10.0% by weight.

Molecular weight electrophoretic analysis indicates that the weight of the protamine molecules in the DNP preparation is between 6000 and 8000 D. This is confirmed by FIG. 6 plot of the lg molecular weight of low molecular weight proteins - X axis, from the distance (in cm) of migration of the protein band in a gel with sodium dodecyl sulfate (SDS-Na) - Y axis, obtained by a known method of protein sizing electrophoresis using sodium dodecyl sulfate (SDS- Na) (see Methods for the study of proteins and nucleic acids: Electrophoresis and ultracentrifugation (practical guide), pp. 56-60, Science Publishing House 1981), where 1 is chymotrypsin, mol.weight 25,000 daltons (D); 2 - cytochrome C, mol. weight 15000D; 3 - RNase A, mol.weight 13700D; 4 - wide band, protamines, mol. weight 6000 - 8000 D; 5 - insulin H, mol.weight 5700D; 6 - C - peptide, mol.weight 3000D.

In turn, the presence of protamines in the DNA preparation obtained by the proposed method confirms that in the process of isolating the preparation, protamines were not lost at the stages of isolation and washing, and the preparation obtained by the proposed method does indeed contain DNA and proteins - protamines and is a complex of double-stranded (double-stranded) DNA and protamines , i.e. deoxyribonucleoprotein (DNP).

The effect of the deoxyribonuclease enzyme on the delivery of the resulting drug to the patient's small intestine was assessed by identifying differences in the stability of double-stranded (double-stranded) DNA molecules, when it is free or bound into a deoxyribonucleoprotein complex with protamine proteins, using pancreatic deoxyribonuclease 1 (DNase I ), which "meets » drug exiting the stomach.

As a source of DNase I , a preparation from the company DiaM, isolated from pancreatic juice, was taken in the form of a solution with an activity of 1 unit/μl, not containing RNases (https://www.dia-m.ru/catalog/reactive/genomica/vydelenie-i- ochistka-nukleinovykh-kislot/dnkaza-i-rastvor-ne-soderzhit-rnkaz-1-ed-mkl-13235280/ ).

Performing a check

For the study, a preparation obtained by the proposed method from salmon milk was taken, containing 85.0 wt.% DNA in the form of a mixture of double-stranded fragments 200-400 bp long, 10.0 wt.% protein in the form of a mixture of protamines with a molecular weight of 6 to 8 KD, water in the amount of 3.0 wt.% and sodium chloride - the rest (DNP preparation).

In tube 1, containing 1 ml of 1% solution of the drug DNP in 0.06 M K-phosphate buffer pH 7.0, was added 0.8 ml of 0.01 M MgCl ₂ solution, and 50 μl of DNase I solution;

Tube 2 was prepared containing 1 ml of a 1% solution of pure injectable DNA from salmon milt (DNA content of at least 95% and protein impurity of less than 1%) in 0.06 M K-phosphate buffer pH 7.0, to which the same as and in tube 1, 0.8 ml of 0.01 M MgCl ₂ solution, and 50 μl of DNase I solution were added.

Instead of a DNA solution, 1 ml of distilled water was added to tube 3, and the same components were added as in tubes 1 and 2.

The volume of the mixtures in all 3 test tubes was adjusted to a volume of 4 ml with distilled water, thoroughly mixed, and incubated for 4 hours at 37°C in a thermostat to ensure that DNase I works to cleave DNA.

Then, 1 ml of a sample was taken from each tube, and 1 ml of a 1M perchloric acid solution was added to this sample as a precipitant to inactivate DNase 1.

Received three samples No. 1, 2, 3, placed in the appropriate test tubes.

In addition, another 1 ml of sample was taken from each tube heated in a thermostat at 37°C, to each of which 2.5 volumes of cold 96% ethanol were added. Thus, we received three more samples No. 4, 5, 6 placed in test tubes with a 96% ethanol precipitant. All six tubes were centrifuged on a microcentrifuge and visually analyzed for the presence of sediment and checked in the supernatant on a spectrophotometer for the absorption of nucleic acids at a wavelength of 260 nm. The results obtained are summarized in Table 1.

Table 1

Number of Tubes (Samples) Precipitation absorption at 260 nm one ++-- - - - - 2 - - - - ++++ 3 - - - - - - - - four ++++ - - - - 5 +- - - ++-- 6 - - - - - - - -

Note:

Precipitation: ++++ profuse sediment; ++- - slight sediment;

- - - no sediment.

Absorption at 260 nm: - - - - absent, ++++ - absorption greater than 2 x optical units. against the control tubes with water.

From the results presented in Table 1, it can be seen that the maximum precipitate was found in samples 4 and 1, which indicates that the DNA preparation in complex with protamines remained intact and precipitated under the action of perchloric acid (sample 1), and to a greater extent under the action of ethanol (sample 4). There is no precipitate in sample 2 with perchloric acid, and very little precipitate in sample 5. This indicates that pure DNA has been degraded, while a weak precipitate in sample 5 indicates that ethanol precipitates smaller oligonucleotides than perchloric acid. In the control sample 3 and 6 (water), there is no sediment when treated with any precipitant.

Evaluation of DNA absorption by the spectrophotometric method on a spectrophotometer at 260 nm in the sample supernatant after sedimentation on a microcentrifuge showed that the absorption is maximum in sample 2 with perchloric acid precipitant and less intense in sample 5 with ethanol precipitator. This is in good agreement with the data on the presence of a precipitate, that ethanol more completely precipitates even small DNA fragments.

The absence of absorption in the supernatant of samples 1 and 4 indicates that perchloric acid and ethanol completely precipitate the DNA preparation obtained by the proposed method and soluble low molecular weight fragments are not observed in the supernatant.

Thus, this experiment proves that the preparation obtained by the proposed method is practically not cleaved by DNase I, while pure DNA is cleaved into fragments, mainly of small molecular weight, remaining in solution and absorbing strongly at 260 nm.

In the experiment presented below on mice, it is shown that when animals are fed with a DNA-containing preparation obtained by the proposed method (DNP preparation), DNA fragments enter the blood of animals already 2 hours after feeding.

Determining the content of fragments DNA in the blood of mice after feeding them obtained by the proposed method of DNA-containing drug

The ability of DNA fragments to enter the blood after feeding them a DNA-containing preparation obtained by the proposed method was studied in an experiment on 20 non-inbrand mice weighing 38-40 g, obtained from the Stolbovaya nursery of the Russian Academy of Medical Sciences, following all the rules stipulated by the order of the Ministry of Health N755 dated 12.08 .1977.

For the study, a preparation was taken obtained by the proposed method from salmon milk and containing 85.0 wt.% of a mixture of double-stranded DNA fragments 200-400 bp long, with a hyperchromic effect of 36.5%, a mixture of proteins with a molecular weight of 6 to 8.0 KD in the amount of 10.0 wt.%, water in the amount of 3.0 wt.% and sodium chloride - the rest. This preparation (DNP, DNP preparation) was diluted with distilled water so that 1 ml of the solution contained 1.0, 10.0 and 100.0 mg of the DNP preparation, and 100 µl of the prepared DNP solution with the concentration indicated in TABLE 2. One control group received only the appropriate amount of distilled water. As a positive control, Derinat was used with a concentration of pure injectable DNA of 15 mg/ml.

After giving the animals the DNP preparation, blood sampling was carried out after 0, 1, 2, 3, 4, 10, and 24 hours, and then the amount of DNA in the blood serum was determined.

The mice were bled from the tail vessels by vacuum. Why the tip of the tail was smeared with xylene and cut off. A drop of 5% sodium citrate solution was applied to the wound, after which the tail was inserted into one of the holes of the can, the walls of which were waxed. Air was sucked out through the second hole with a vacuum pump, and blood was sucked into the test tube at a pressure of 2.6-5.3 kPa. This made it possible to obtain 0.2-0.5 ml of blood, saving the life of the animal.

Isolation of DNA from blood was carried out by phenol-chloroform extraction (PCE), based on the use of organic compounds (Kayumov A.R. Workshop on molecular genetics. Educational manual / A.R. Kayumov, O.A. Gimadutdinov - Kazan: Kazan , KFU, 2016. - 36 p., P. 10, Isolation of DNA from blood).

To do this, 150 µl of blood serum was added to a 1.5 ml microcentrifuge tube containing 0.5 ml of buffer solution. Added 15 μl of a solution containing 10 mg/ml proteinase K (to a final concentration of 0.3 mg/ml) and mixed. Then, incubation was carried out at a temperature of 56°C for at least 1 h. After purification of the DNA solution from protein impurities, an equal volume of a mixture of phenol-chloroform-isoamyl alcohol (25:24:1) was added to the test tube with lysis products. The tube was closed with a lid and thoroughly mixed, then centrifugation was carried out for 3–5 min at 10,000–15,000 rpm until the two phases separated. Transfer the aqueous phase (upper) to a new 1.5 ml microcentrifuge tube, being careful not to trap the lower phase. The DNA solution purification procedure was repeated several times, after which an equal volume of water-saturated n-butanol was added to the tube containing the aqueous phase. After mixing and centrifuging, the aqueous phase (lower) was transferred to a new 1.5 ml microcentrifuge tube, to which an equal volume of water-saturated diethyl ether was added. Centrifugation was again carried out and the upper phase containing diethyl ether was carefully removed. To remove traces of ether, the aqueous phase was incubated for 5 min at 70°C. A 5M NaCl solution and 96% ethanol were added to the DNA solution, thoroughly mixed, and left at a temperature of -20°C overnight. After centrifugation at 10,000-15,000 rpm, the supernatant was removed and 1 ml of 80% ethanol was added to the sediment, centrifuged again in the same mode, and the supernatant was removed again. The precipitate was dissolved in 50 µl of TE buffer. The isolated DNA was stored in a freezer at -20°C. The concentration of extracellular DNA (ecDNA) was determined on an EnspireTM 2300 fluorometer (Perkin Elmer) by PicoGreen fluorescence (Invitrogen, USA) at 480 nm excitation and 520 nm emission wavelengths.

Table 2. The content of extracellular DNA (ecDNA) in the blood of mice after feeding them the DNA preparation (DNP) obtained by the proposed method

No. P/P Injection drug Sampling time and determination of cnc DNA on a spectrofluorimeter in ng/ml one Remote control H ₂ O 0 1 hour 2 hours 3 hours 4 hours 10 hours 24 hours 27
25
24
28 26
27
26
thirty 27
26
thirty
29 27
25
24
28 26
2
24
27 24
25
23
24 26
27
24
23 2 DNP - 1 mg/ml 24
28
29
27 29
31
28
32 41
43
41
44 48
40
41
42 40
39
43
41 28
27
thirty
29 thirty
29
32
34 3 DNP - 10 mg/ml 23
25
29
28 60
65
58
63 thirty
95
97
91 420
410
440
430 415
405
417
411 151
148
162
144 33
35
thirty
29 four DNP - 100 mg/ml 23
thirty
29
24 150
167
160
150 310
305
315
311 4250
4100
3890
4200 2990
3120
3315
3115 250
232
215
248 32
34
29
31 5 Derinat - 15 mg/ml 26
28
25
24 28
29
24
27 thirty
29
28
29 31
29
thirty
32 42
40
39
43 27
28
26
25 26
24
27
28

As can be seen from Table 2, the concentration of extracellular DNA in the serum of animals in the norm was 26 ± 7 ng/ml, respectively. In vivo experiments have shown that when mice are injected with a DNA-containing preparation obtained by the proposed method from salmon milk, an increase in the concentration of extracellular (extracellular) DNA is observed. The concentration of cfcDNA in the blood reaches a maximum 2-3 hours after feeding, and after 24 hours it drops to the content of cfcDNA in the control group. In experiments in vivo, with the introduction of per os of the study drug, a significant increase in the concentration of cfcDNA is observed, which increases in proportion to the amount of drug administered. Thus, this experiment proves that when the DNA-containing preparation obtained by the proposed method is administered orally, it is DNA (DNA fragments) that enters the bloodstream, and not individual nucleotides and nucleic bases. See Table 2. The content of cfDNA in the blood of mice treated with DNP in samples 3 and is especially noticeable in samples 4. And there is practically no increase in cfDNA in samples 5 (control Derinat).

Study of spontaneous and mitogen-induced proliferation of mouse splenocytes (splenocyte - a monocyte formed in the reticular tissue of the spleen)

The immunotropic, mitogenic, and allergenic properties of the DNA-containing preparation obtained by the proposed method (DNP preparation) were studied using the lymphocyte blast transformation reaction (RBTL).

Using this approach with the use of T- and B-cell mitogens also allows us to evaluate the predominant effect of the studied DNP preparation on T- or B-cell populations of lymphocytes. (Khaitov R.M., Ataullakhanov R.I., Ivanova A.S. et al. Guidelines for assessing the immunotoxic effect of pharmacological substances. - M., 2000)

For the study, a preparation was taken obtained by the proposed method from salmon milk and containing 80.0 wt.% DNA in the form of a mixture of fragments 200-400 bp long. (nucleic base pairs) with a hyperchromic effect of 36.2%, 12.0 wt.% of a mixture of protamines with a molecular weight of 6 to 8 kDa, water in an amount of 3.5 wt.% and sodium chloride - the rest. This preparation (hereinafter DNP) was diluted with distilled water on the basis that 1 ml of the solution contained 1.0, 10.0 and 100.0 mg of DNP.

The immunomodulating effect of the DNP preparation was studied in experiments on 160 non-inbred mice weighing 14–18 g, obtained from the Stolbovaya nursery of the Russian Academy of Medical Sciences, following all the rules stipulated by the order of the Ministry of Health No. 755 of 12.08.1977.

Reaction setting . Animals of the experimental and control groups were sacrificed using cervical dislocation, the spleens were removed, and a cell suspension was prepared using a glass homogenizer.

The obtained splenocytes were washed with medium 199 with 5% bovine serum and 200 μl of cell suspension (with a concentration of 2×10 ⁶ /ml) in RPMI-1640 medium (Flow, England) supplemented with 10% fetal calf serum (Gibco, USA) , 0.01 M HEPES buffer (Gibco, USA), 200 mM L-glutamine (Sigma, USA), 10 μM 2-mercaptoethanol (Merck, Germany), 100 mg/ml streptomycin, and 100 U/ml penicillin were added to wells 96 well plates.

Mitogen - phytohemagglutinin was added to 48 wells at concentrations of 10 μl (10 μg/ml). No mitogen was added to the other 48 wells to assess spontaneous lymphocyte proliferation. Physiological saline (PS) was added to the top row of control wells. In the second row of wells, 50 μl of a solution of the DNP preparation with a concentration of 1 mg/ml was added. In the third row of wells, 50 μl of a solution of the DNP preparation with a concentration of 10 mg/ml was added. In the fourth row of wells, 50 µl of a solution of the DNP preparation with a concentration of 100 mg/ml was added. Then the splenic cells were incubated for 96 hours in an atmosphere of 5% CO ₂ at a temperature of 37.5° C. 24 hours before the end of cultivation, 1 μCi of a solution of ³ H-thymidine in a volume of 10 μl was added to all wells. The incorporation rate ^{of 3} H-thymidine (number of pulses/min) was determined from cells harvested with a Cell Harvester (Flow) on a scintillation counter (Marck-III).

The RBTL level of splenocytes under the influence of the mitogen and the DNP preparation introduced into the cultivation medium was evaluated in relation to the intensity of RBTL of splenocytes in the medium not containing the mitogen, but containing the DNP preparation at various concentrations. To assess the effect of drugs on RBTL, we compared the incorporation ^{of 3} H-thymidine in the spleen cells of mice that received DNP preparations with that of splenocytes of control mice that received saline (PS). The results are expressed in pulses per minute.

The average number of impulses of the radioactive label of lymphocytes in the experimental and control tubes was calculated, followed by the determination of the stimulation index according to the formula:

Stimulation Index = Average Pulses/Minute/Experience divided by Average Pulses/Minute/Control.

Determining the index of stimulation of lymphocytes allows you to evaluate the effect of the dose of the drug DNP on the level of RBTL lymphocytes. The stimulation index of the action of the drug DNP was insignificant at DNP 1 mg/ml and amounted to 1.2, with DNP 10 mg/kg and 100 mg/kg - 1.8 and 1.9 the stimulation index was significantly higher. The obtained experimental data are presented in Table 3.

Thus, when studying the effect of the DNP drug on the reaction of lymphocyte blast transformation, it was found that DNP, dose-dependently, mainly - 10 and 100 mg/ml, moderately stimulates both spontaneous and mitogen-induced RBTL. It is likely that the increase in splenocyte proliferation caused by the introduction of the DNP preparation into the cell culture is also accompanied by an increase in the proliferative response of cells to the T cell mitogen.

Determining the content of fragments DNA in the blood of rabbits after feeding them a DNA-containing preparation obtained by the proposed method (DNP preparation)

For the study, a preparation was taken obtained by the proposed method from salmon milk and containing 85 wt.% DNA in the form of a mixture of fragments 200-400 bp long. (nucleic base pairs) with a hyperchromic effect of 36.0%, 10 wt.% of a mixture of proteins - protamines having a molecular weight of 6 to 8kD, water in an amount of 3.5 wt.% and sodium chloride - the rest (DNP preparation).

The research material was blood samples of rabbits taken at 90 days of age. For the experiment, 3 groups of rabbits were formed, 3 heads each, with an average live weight of 4.5-4.6 kg. The first (control) group of rabbits received the basic diet (RR). Composition of OR: grain of corn, wheat, barley, sunflower cake, alfalfa hay and macronutrients; the second (experimental) group was fed the basic ration (RR), with the difference that the supplement of the DNP preparation was added to the feed at the rate of 500 mg/kg of feed. The third (control) group received Derinat, an injectable DNA preparation, in the same amount as the DNP preparation. The rabbit feeds 30-35 times a day and eats about 3 g of food at a time, a total of 900 - 1050 g. That is, the animal ate up to 500 mg of the DNP preparation per day. Taking into account the metabolism in rabbits, blood sampling was carried out after two days, when a certain homeostasis of extracellular DNA was established in the blood of rabbits that received food of various compositions.

Before the start of the experiment and two days after feeding the animals, 2 ml of blood was taken from the ear vein of each group of animals (3 animals per group) into a test tube and blood serum was obtained. After sedimentation of blood cells, 1 ml of serum was taken from the upper layer and extracellular (extracellular) DNA was isolated by phenol-chloroform extraction (PCE) in the same way as described above in the study of mouse blood.

The concentration of extracellular (exc) DNA was determined on an EnspireTM 2300 fluorometer (Perkin Elmer) by PicoGreen fluorescence (Invitrogen, USA) at 480 nm excitation and 520 nm emission wavelengths. The data obtained are presented in Table 4.

Table 4. Spectrofluorometric determination of the content of extracellular DNA in the blood of rabbits after feeding the DNP preparation

No. P / P Injection drug Sampling time and determination of ec DNA on the spectrofluorimeter in ng/ml one Remote control H ₂ O 0 Two days later 26
24
26 21
22
23 2 DNP - 500 mg/kg feed 24
38
39 429
431
428 3 Derinat -500 mg DNA/kg feed 23
25
29 60
65
58

As can be seen from Table 4, the concentration of extracellular DNA in the serum of animals was normally 24-39 ng/ml, and two days after the start of feeding rabbits with DNP, the concentration of extracellular DNA in the serum of animals increased by more than 10 times and reached 430 ng/ml . In sample 3, in the blood of rabbits treated with an injection of pure DNA (Derinat) on the 2nd day, the content of cfcDNA increased very slightly.

An in vivo experiment showed that when rabbits are orally administered a DNA-containing preparation from salmon milk (DNP), obtained by the proposed method, a significant increase in blood levels of cfDNA in sample 2 and insignificant in sample 3. A slight increase in sample 3 may be associated with the stimulation of metabolic processes in the body by nucleotides and nucleosides from Derinat.

Thus, this experiment, as well as a similar experiment on mice, proves that when the DNA-containing preparation obtained by the proposed method is administered orally, it is the fragments of double-stranded DNA that enter the bloodstream, and not individual nucleotides and nucleic bases formed during the breakdown of DNA in the gastrointestinal tract, which confirmed by comparing the data on ecDNA samples 2 and 3 of Table 4.

The extracellular DNA obtained as described above in the blood serum of rabbits of the control group, the group treated with Derinat and the group treated with the DNP preparation, was subjected to agarose gel electrophoresis with visualization by the addition of ethidium bromide in the horizontal direction, since 1) the gel with a low concentration of agarose holds better , 2) less distortion (collapse) occurs during electrophoresis, and 3) DNA bands are less distorted.

This method is known and common in DNA work, as described in McDonell MW, Simon MN, Studier F. W. , 1977. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels, J Mol. Biol., 110, 119; Peacock AC, Dingman C. W. , 1968. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels, Biochemistry, 7, 668.

The molten agarose gel was poured onto a glass plate leveled horizontally. Then, at the edge of the gel melt, a comb with teeth was installed, which, after solidification of the agarose, remain immersed in the agarose gel, and when the comb was carefully removed from the solidified gel, recesses remained in place of the teeth - “pockets”, where the required volume of the sample of the material under study was introduced with a micropipette .

At the end of electrophoresis, the gel was cut off exactly along the border below the “pockets” and then photographed in UV light.

On FIG. Figure 5 shows a photograph of electrophoresis of extracellular DNA from the blood of rabbits fed with the DNA-containing preparation obtained by the proposed method (DNP preparation) and control ones, where on the first 1 lane there are DNA molecular weight markers, where the bands from top to bottom correspond to the mass: 3000 bp .o., 2000 bp, 1200 bp, 800 bp, 500 bp, 200 bp; Lane 2 is a sample of the blood processing of the control rabbit treated with saline; Lane 3 is a 5-fold sample of the blood treatment of a control rabbit treated with saline taken in relation to lane 2; Lane 4 - represents a sample of the processing of the blood of a rabbit treated with Derinat; Lane 5 - is taken in 5-fold size with respect to lane 4, a sample of the treatment of the blood of the control rabbit treated with Derinat; Lane 6 is a blood processing sample from a rabbit treated with DNP preparation.

As can be seen from the presented photograph (Fig. 5), no DNA luminescence was recorded on lanes 2, 3 and 4, 5, despite a 5-fold increase in the volume of the test sample applied to lanes 3 and 5, while at the same time on lane 6 it is good a clear DNA band is visible below 500 bp. and above 200 bp, and the DNA “tail” is not observed. Since bands 4 and 5 are not visible at all on samples from “pockets”, this confirms that in the gastrointestinal tract of a rabbit, DNA from Derinat is practically cleaved to low molecular weight oligonucleotides or nucleosides. At the same time, DNA from the DNP preparation penetrates into the blood and accumulates in the blood, which reflects the luminescence of DNA fragments no longer than 400 bp. and at least 200 b.p. in lane 6 (Fig. 5), hydrogen-bonded into complementary pairs of AT and GC, forming double-stranded DNA fragments with the help of so-called weak bonds.

Immunological and chemical parameters of the blood of rabbits when adding the DNA-containing preparation obtained by the proposed method from the milk of salmon fish (DNP preparation) to the feed

For the study, the same DNP preparation was taken as for the study of the content of extracellular DNA in the blood serum of rabbits. The research material was blood samples of rabbits taken at 90 days of age. For the experiment, 3 groups of rabbits were formed, 3 heads each, with an average live weight of 4.5-4.6 kg. The first (control) group of rabbits received the basic diet (RR). Composition of OR: grain of corn, wheat, barley, sunflower meal, alfalfa hay and macronutrients; the second (control) group was fed the basic ration (RR), with the difference that Derinat was introduced into the feed, an injectable DNA preparation at the rate of 500 mg/kg of feed; the third group was fed with an additive of the DNP preparation at the rate of 500 mg/kg of feed. Evaluation experiments immunological and chemical parameters of the blood of rabbits when using an additive obtained by the proposed method of a DNA-containing preparation from salmon fish milk (DNP preparation) to feed, was carried out in the vivarium of the institute, which had an immunochemical laboratory and specialists to carry out such work.

A rabbit feeds 30-35 times a day and eats about 3 g of food at a time, a total of 900 - 1050 g. That is, the animal ate up to 500 mg of DNP per day. Blood samples were taken on the third day of keeping groups on such feeding. Blood for the study was taken into serological and hematological tubes by puncturing the marginal ear vein of rabbits with an injection needle. The blood was delivered to the laboratory on the day of its collection. The number of samples - 3 from each group. In the study of blood, the activity of immunity was studied. The clinical and physiological state of the rabbits was determined by their daily examination. Attention was paid to their behavior, food intake, condition of the coat. Live weight was determined by individual weighing. The digital material obtained in the experiments was processed according to the methods described in the literature ( Lakin G.F. Biometrics. Textbook for biol. .- 352 p.), using a personal computer and an application package (Microsoft Excel). The difference in values was considered significant at: * - p >0.05; ** p < 0.01 and *** p < 0.001. The main results of the experiment are presented in Table 5.

Table 5 . The results of immunological parameters in the control (group 1) and with the addition of the Derinat injection preparation containing fragments of high-purity double-stranded DNA to the feed (group 2) and with the addition of the DNP preparation obtained by the proposed method containing DNA in the form of a complex with protamines (group 3)

Blood index Control group 1 Group 2 (Derinat) Group 3 (DNP drug) Leukocytes (WBC), 10 ⁹ /l 5.17±0.857 7.47±0.733* 7.8±0.723 Hematocrit (HCT), % 23.17±2.371 24.47±1.848 33.2±0.418*** ESR, mm/hour 2.33±0.333 1.33±0.333 1.67±0.333 FA (30 min), % 41.33±0.667 44.3±0.882** 47±2.517* FC (30 min), units 0.65±0.024 0.65±0.018 0.8±0.018*** ZF, units 1.1±0.059 1.27±0.033* 1.36±0.024* KM, unit 2.0±0.033 2.7±0.344* 2.78±0.331* T, % 58.778±1.176 60.333±1.764 62±1* AT, % 27.267±0.504 28.667±1.333 30.8±1.665 T, 10 ⁹ /l 1.033±0.176 1.31±0.456 1.567±0.145* B, 10 ⁹ /l 0.733±0.12 0.757±0.171 0.81±0.08 T-l % / W-l % 2.156±0.043 1.976±0.159 2.176±0.143

FA - phagocytic activity of leukocytes, PF - phagocytic number, CF - completeness of phagocytosis, KM - mobilization coefficient, T-lymphocytes, B-lymphocytes. (. Immunology. Under the editorship of Voronin E.S. M.: Kolos-Press, 2002. - 408 p. - S. 34.) T and B are given as a percentage or based on the number. (10 to 9 degrees) in Goryaev's cell.

In the blood of adult rabbits, the variation of leukocytes is considered normal in the range of 5.5-10 thousand per 1 mm³ (Berhof P.K. Small pets. Diseases and treatment / Translated from German. I. Kravets. Ed. 2, corrected and added. - M .: OOO "AQUARIUM PRINT". - 2004.- P. 60; Georgievsky V.I. A practical guide to the physiology of farm animals. Proc. the allowance for page - x. universities. - M., "Higher School", - 1976. - S. 184-185).

As can be seen from Table 5, the average values of leukocytes in the blood of the 2nd and 3rd experimental groups were within the physiological norm and were higher than in the control 1st group by 43.2% and 52.3% (p > 0 .05). The phagocytic activity of leukocytes is the ability of phagocytes to capture, kill and digest pathogenic and conditionally pathogenic microorganisms. The phagocytic activity of neutrophils usually increases at the beginning of the development of the inflammatory process. A decrease in the phagocytic activity of blood cells contributes to the development of a chronic inflammatory process and the emergence of aggression against the body's own tissues (the appearance of autoimmune processes).

Phagocytic activity in rabbits of the second experimental group was normal and higher than the control group 1 by 7.3% and in the third group higher than the control 13.8% (p > 0.05).

The most informative for assessing phagocytic activity should be considered the absorption index, that is, the phagocytic number, the phagocytic number coefficient, which reflect the completion of phagocytosis (CF) and the bactericidal index - the ability of the phagocyte to digest the captured microbe (Nagoev B.S. Handbook of Immunology. - Nalchik: Elbrus, 2002 - 192 p. - P.53).

Completion of phagocytosis was noted in animals of the 2nd and 3rd groups, but higher rates were observed in group 3. 3rd group. This indicates an increase in the activity of the microbicidal system of phagocytes in animals of these groups.

To characterize cellular and humoral immunity, quantitative determination of T- and B-lymphocytes was carried out. The parameters of the cellular immunity of the 3rd experimental group of rabbits were higher than in the control group. The absolute number of T-lymphocytes was higher by 27 and 52% (p > 0.05) in the 2nd and 3rd groups. The absolute number of B-lymphocytes showed an increase of 3% and 5% (p > 0.05), respectively, in the 2nd and 3rd groups relative to the control - the 1st group. At the same time, the percentage of T- and B-lymphocytes in both groups did not significantly change compared to the background values.

Thus, based on the general analysis of studies of the immunological composition of blood, the advantage of the experimental group 3 was noted. The use of DNP supplements in feed has a stimulating effect on the body of rabbits, which is manifested by the activation of protective blood systems and an increase in the immune properties of animals. This is consistent with the known fact that fragmented double-stranded (double-stranded) DNA supports the immune response by activating macrophages and T-lymphocytes. These effects have been found to be related to cell activation via the receptor apparatus (Nat Rev Immunol. 2004 Apr; 4(4):249-58).

The above experiments unequivocally prove that the DNA-containing preparation obtained by the proposed method overcomes the stomach due to its protein component, and DNA is actively absorbed by the intestinal mucosa, which is fixed by an increase in extracellular DNA in the blood of mice and rabbits, in proportion to the amount introduced by them through the mouth (lat. per os,) of the drug.

Therefore, the DNA-containing preparation obtained by the proposed method is suitable both for oral use without additional processing, both as a source of DNA with high bioavailability for the body for the manufacture of pharmaceutical preparations and in biologically active food supplements, and as a reagent for various areas of biochemistry, biotechnology and pharmaceuticals, containing DNA fragments of a certain length of a sufficiently high degree of purity.

The claimed method for obtaining a DNA-containing preparation for oral use is a successful solution to the problem of delivering such a valuable drug as fragments of polymeric DNA through the intestines and blood to where it is most needed.

The non-obviousness of the invention lies in the fact that in an extremely simple way it was possible to obtain a drug that is not inferior in its properties to injectable DNA preparations, as well as DNA immobilized on polyethylene oxide.

Oral preparations of polymeric DNA fragments in the form of capsules or tablets will be especially convenient for use by personnel involved in the maintenance and elimination of accidents at nuclear reactors at nuclear power plants or reactors on icebreakers or nuclear submarines, since the implementation of the universal effects of exposure to the body of nucleic acid fragments makes it possible to achieve positive results of health correction in a wide range of pathological conditions, acute and chronic diseases (Karaskov A.M., Weinberg Yu.P., Volkov A.M., Kazanskaya G.M. // Military Medical Journal. - 1995, No. 2. - P. 64 -65).

Claims (13)

1. A method for obtaining a DNA-containing preparation for oral use from the seminal glands (milk) of fish, which consists in that the raw material is homogenized in a citrate-salt solution, followed by dilution of the homogenate with the same solution until a homogeneous mixture is obtained, into which dry sodium chloride is introduced with constant stirring until a saturated sodium chloride solution is formed and the viscosity of the solution increases, which continues to be stirred for at least for an hour, then the solution is diluted with water at least 10 times, the stirring is stopped and kept until viscous clots appear, floating to the top of the solution, where these clots are collected, redissolved in a citrate-salt solution, treated with ultrasound before obtaining DNA fragments with a length of not more than 500 pairs of nucleic bases,purified and isolated by adding at least 2.5 volumes of ethyl or isopropyl alcohol cooled to 4°C, dried, crushed and stored in a dark room at a temperature not exceeding 20°C. 2. The method according to claim 1, characterized in that a 0.9% sodium chloride solution prepared in a 0.01 M solution of sodium citrate pH 7.14 is taken as a citrate-salt solution. 3. The method according to claim 1, characterized in that the purification and isolation of the target product is carried out by adding a sorbent to its citrate-salt solution treated with ultrasound, mixed, incubated for at least 2 hours, filtered, then to a transparent , at least 2.5 volumes of cold ethyl or isopropyl alcohol are poured into the colorless filtrate with DNA containing the preparation, mixed and placed in a refrigerator at a temperature not exceeding 4 ° C to form a dense precipitate, which is collected on a sieve, washed with 80% alcohol and dried in a vacuum oven at a temperature not exceeding 40°C. 4. The method according to claim 3, characterized in that finely porous silica gel, for example, kieselguhr or kromasil, is used as a sorbent. 5. The method according to claim 3, characterized in that the filtration of a mixture of a citrate-salt solution of a DNA-containing preparation treated with ultrasound with a sorbent is carried out under vacuum on a suction filter covered with a layer of belting and coarse calico, on which the remains of insoluble fragments of the feedstock and the sorbent are retained . 6. The method according to claim 1, characterized in that sonication is carried out until DNA fragments are obtained, preferably not longer than 400 base pairs. 7. The method according to claim 1, characterized in that, in particular, for sonication, a bath with a membrane is used, connected to a magnetostrictive emitter connected to an ultrasonic generator, for example, UZG17-2.0/22, which provides ultrasound with an operating frequency of 22 kHz. 8. The method according to claim 1, characterized in that the raw material is taken mainly from the milk of fish of the salmon family or the sturgeon family. 9. Immunomodulatory DNA-containing preparation for oral use, obtained according to the method according to any one of claims 1-8. 10. The drug according to claim 9, characterized in that it contains at least 80.0 wt.% DNA in the form of a mixture of double-stranded fragments with a length of not more than 500 nucleic base pairs and at least 10.0 wt.% of the mixture proteins with a molecular weight of 6 to 8 kD. 11. The drug according to claim 10, characterized in that it contains DNA in the form of a mixture of double-stranded fragments with a length of 200 to 400 nucleic base pairs. 12. The drug according to claim 10, characterized in that it contains DNA with a hyperchromic effect from 35 to 37%. 13. The drug according to claim 10, characterized in that it contains predominantly protamines as a protein.

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