Antimicrobial agent

FIELD: antimicrobial agents.

SUBSTANCE: invention relates to application of protein component, isolated from plant chromatin as antimicrobial agent. Claimed component is produced by homogenization of plant material to isolate chromatin therefrom; decomposition of isolated chromatin with decomposition agent in hydrophobic conditions and separation of decomposed plant chromatine into individual fractions by hydrophobic interaction Protein component obtained from one fraction has apperent molecular mass of 10-20 kDa.

EFFECT: protein with increased antimicrobial activity.

32 cl, 11 ex

 

Description

The present invention relates to the use of the protein component selected from chromatin plants. The invention applies in particular to the use of the protein component selected from plant chromatin after dissociation of the latter, as an antimicrobial agent, as well as to a method of obtaining a specified antimicrobial agent.

Most organisms eukaryotes demonstrates a wide variety of protective mechanisms against infectious agents. Several such mechanisms are based on the fundamental differences that occur in the composition and structure of the membranes of microbes and cells of a complex multicellular organism, i.e. these mechanisms are directed against the outer membrane of bacteria which are sensitive to such effects. These membranes are formed by lipids with negatively charged head groups facing outwards, and microbes, probably hard to resist this kind of pressure by changing the composition and structure of its membrane. Thus, substances with antimicrobial activity, are likely candidates to replace antibiotics.

One of the examples are proteins transfer of phospholipids, which can transfer phospholipids between membranes. Described antimicrobial proteins transfer the FOSFA the lipids, selected from a wide range of samples of plants, including cereals, and these proteins differ in their activity against various pathogens. For example, in U.S. patent 5698200 shown that parts of the plant can be protected against plant pathogenic bacteria using an aqueous extract obtained from solozhenko cereals.

However, the most studied class of protective agents are antimicrobial peptides. They are found in all forms of life from plants to insects and animals, including mollusks, crustaceans, amphibians, birds, fish, mammals and humans.

These peptides directly interact with the bacteria and kill them. They are called antimicrobial because they have an unusually broad spectrum of activity, including the ability to kill or neutralize gram-negative and gram-positive fungi (including yeast), parasites (including planaria and nematodes), cancer cells and having a shell viruses such as HIV and herpes virus. In General, these agents differ in their length, starting with only 12 amino acids, and to molecules containing more than 70 amino acid residues. Discovered more than 500 of such peptides.

The nature of antimicrobial action in the majority cationogenic antimicrobial peptides studied in detail for such is aptidon, as mellitin, magainin, gramicidin, cecropin and defensin. Antimicrobial molecules usually also damage the cell membranes of organisms, which they facultya found that cationogenic antimicrobial peptides have antibacterial activity both in vitro and in vivo. They kill very fast, difficult to select resistant mutants exhibit synergism when mixed with conventional antibiotics, other peptides, as well as lysozyme and is able to kill bacteria in animal models.

As a result, the antimicrobial peptides of animal origin are currently developing new antibiotic drugs. Examples are synthetic version magainin (pexiganan) and similar protegrin, an antimicrobial peptide that was originally isolated from pig neutrophils.

However, it appeared that natural sources economically disadvantageous for the production of new alternative antibiotics. The only exception is antimikrobnyj peptide lowlands, which can be effectively obtained from a strain of Lactococcus lactis, which has high resistance to this substance.

It was also found a large number of proteins with large sizes, or parts thereof, which have antimicrobial activity. For example, the protein of rat macrophage, ubiquitin, was identical ribose the real protein S30. In addition, two antimicrobial peptides in the stomach of a bull frog (Rana catesbeina) are derived from pepsinogen at N-end. Similarly antimicrobial peptide, called Butorin I, isolated from the tissues of the stomach of the Asian toad (BBRC 218: 408, 1996). It was shown that the amino acid sequence of this peptide, containing 39 amino acids, coincides with 37 out of 39 aminoterminal residues of histone H2A from Xenopus.

Further, the antimicrobial potential can have the whole molecule peptide. Antimicrobial activity was detected in the acid extracts of liver, intestine and stomach of Atlantic salmon (BBRC 284: 549, 2001). Appropriate antimicrobial protein can be isolated from the liver of salmon using acid extraction and subsequent precipitation with ammonium sulfate, preparative gel chromatography (gel filtration), HPLC with reversed-phase HPLC according to the size of the molecules. It was found that the antimicrobial protein salmon has a molecular mass of 27.7 kDa was identified as a protein histone H1. In the patent application WO 200110901 protein histone H1 isolated from thymus cow, used in the antimicrobial compositions compositions intended for the treatment of microbial infections in a variety of organisms eukaryotes. Thus, it was found that proteins that perform many features that are already installed, show another property, awlays is antimicrobial.

However, the use of proteins cows, in particular proteins from the thymus cows, should be avoided because this material can be contaminated with harmful viruses, especially hepatitis virus, or other pathogenic agents, such as prions. The source material obtained from cows as polluted and not polluted, if you intend to use for the treatment of a person, must be subject to particularly rigorous tests.

Further, the allocation of new alternative antibiotics include the collection of certain organs or tissues of the animal, followed by a comprehensive treatment in order to obtain a product which can be used for people or animals.

The aim of the present invention is a new antimicrobial agent, which eliminates the above problems.

Another aim of the invention is an antimicrobial agent, which eliminates the risk of transmission of infectious agents pathogenic to humans and/or animals.

Another aim of the invention is an antimicrobial agent that has no taste when using it with food.

Another aim of the invention is a method for the antimicrobial agent, which use cheap raw materials.

Another aim of the invention is a method recip is of antimicrobial agent in almost unlimited quantities.

Finally, another aim of the present invention is a method for the antimicrobial agent, which does not require high cost plants to conduct bacterial fermentation.

This is accomplished by using the method according to the present invention.

In accordance with the invention offers a method that allows simple and efficient to get the protein component, which can be used as an antimicrobial drug, such as medication, full preservative agent used in the production process and during transportation, as a functional food or nutraceutical supplements as well as nutritional supplements for animals.

The protein component can be obtained surprisingly easy from the initially inert source material containing plant chromatin. In the method according to the invention the DNA is separated from the main nuclear chromatin plants. Plant chromatin mainly derived from plant seeds. Suitable plant seeds get from oats, sorghum, wheat, barley, rye, maize, rice, rapeseed, soybean, millet or buckwheat. However, to obtain the antimicrobial protein component in large scale can be used any chromatische plant material such as algae and the others who Orsk plant.

Plant chromatin, which is used for separation of protein component, should be a heterochromatin (hidden chromatin or "dust" (DNA). Heterochromatin is lipoarabinomannan (deacetylation) chromatin, which is due to the higher electropolishing charge takes on a more compressed form than piperazinylcarbonyl the chromatin.

Further, the choice of source material for subsequent specific protein extraction also depends on the location of the cellular tissue of plants, and status differentiation. For example, the tissue formed by small cells have a higher cell density, and therefore probably contain more nucleic acids and concomitant antibacterial protein component than the same amount of tissue formed by cells of a larger size.

By analogy, many plants due to the high content of heterochromatin have a basal genomes of very large size, which is further enhanced due to the possible polyploidie. This paper considers the amount of DNA per haploid cell, which is measured by the number of base-pairs (p-value). Variation of DNA content in the body is reflected by its p-value or size of basal genome. P-value is defined as the DNA content, and the height to weight or number of base pairs in a single copy of the complete DNA sequence, found inside the cells of a given organism. It represents the amount of DNA in it the unreplicated haploid or geneticheskom the nucleus irrespective of the ploidy level of the taxon. Thus, the p-value is equal to the size of the genome in diploid species, but always exceeds the size of the genome in polyploid species.

It is preferable also to use self-renewing undifferentiated stem cells of plants. They are found in the meristem, i.e. areas that provide new growing tips of shoots and roots. Thus, the ends of the roots of the seedling plants are unsurpassed source material for extraction of chromatin and subsequent excretion of the protein component of the present invention. Similar to the original material readily available in unlimited quantities, as it is a waste in the production of brewing malt and oils from wheat germ.

To obtain the protein component of the present invention are suitable also other source of plant materials from cells that divide mitotically in optimal growth conditions. Can be used any developing shoots, roots or shoots in the stage of germination. Mostly use the seeds of one of the four kinds of corn, which give germinate.

Cheap raw material for the sing of the present invention is the so-called green malt, which is a starting material for the production of beer. The brewing industry produces green malt from barley, which when wet allow to grow within six days (osroene). This industrial get green malt or its by-products (roots), in accordance with the present invention, can be used to produce antimicrobial protein component.

Thus, suitable for the extraction of materials are roots diploid maize and barley (p-value DNA is 5000 megabar reason), and Luke (p-value DNA 18000 megabar reason). Preferably antibacterial protein component is extracted from the source chromatin, the DNA is exceeding 3000 megabar grounds.

Most preferably, when the source material at the stage of purification is plant chromatin isolated from bystrorazvivajushchihsja of plant cells that are in S-phase. So, germinating seeds (grains) with their roots and young leaves contain a large number of cells in S-phase.

In the method of obtaining vegetable protein component of the present invention, which possesses antimicrobial activity, included the following stages:

homogenization of plant material in order to release the e is on plant chromatin;

decomposition of plant chromatin using an agent decomposition in hydrophobic conditions;

separation is subjected to decomposition of plant chromatin at the individual fractions, one of which contains vegetable protein component, using the methods of separation by hydrophobic interaction.

Thus, the plant material is first subjected to homogenization. In this sense, the term homogenization means the destruction of cell walls of plant material so that the released plant chromatin and formed the homogenate in suspension. Cell walls can be destroyed by any means known experts from the fields of technology, including, but these examples are not limited to, intensive mixing with large shear effort, sonication, mechanical disruption, rupture under the action of pressure, etc. of the Cell wall is destroyed using a suitable device, receive the homogenate.

Plant chromatin in the homogenate then decompose in a hydrophobic environment using agent decomposition in the form of its aqueous solution. These conditions are those that contribute to hydrophobic interactions.

Suitable agents of decomposition are urea, guanidinium and salt of hydrochloric acid. Preferably the salt is hydrochloric acid is sodium chloride with high ionic strength.

Certain advantages are provided if the homogenization of plant material is carried out in the presence of agent decomposition. In this case, the cleaning process is simplified, and the number of stages of treatment decreases.

Cleaning green malt is carried out by homogenizing malt in almost saturated salt solution containing 4 M sodium chloride. The high ionic strength of the solution is the decomposition of chromatin and nucleosomes, while simultaneously preventing the degradation of the protein material under the action of proteases. Homogenization is preferably carried out in the presence of a hydrophobic matrix.

The homogenate you can skip through the sieve or wire mesh or the like to remove cellular debris or other particles released from plant chromatin, which remain on the sieve. In the end you get a clear solution, which facilitates the subsequent purification subjected to decomposition of plant chromatin.

Subjected to the decomposition of plant chromatin is then divided into individual fractions, one of which contains vegetable protein component having antimicrobial activity. The separation is preferably carried out using methods of separation by hydrophobic interaction. Method of separation by hydrophobic interactions mainly I have is hydrophobic chromatography.

Other examples of suitable separation methods are the methods of distribution in polymer systems, such as distribution chromatography, the distribution in the flow and distribution in the gas afron. The separation of components subjected to decomposition chromatin otherwise spend on a column of gel-based chelates metals or with immobilized heparin.

Functional ligand matrix used for hydrophobic interaction and/or division, should be simple ether group, ISO-propyl, butilkoi or octile group. Avoid phenyl groups. Preferably the functional ligand is bucilina band on an agarose matrix, which has a 4% crosslinking. The resulting density of the ligand is 40-50 mmol/ml, which provides the ability to bind corresponding to 7 mg IgG per milliliter.

For example, after screening homogenate green malt in the form of suspension to the resulting solution was added in several portions hydrophobic matrix, while the hydrophobic matrix is a gel for holding by chromatography hydrophobic interaction containing the active butylene group. Suitable matrices are Novarose® S-Butyl 1000/40 from the company Inovata AB, Bromma, Sweden, and Butyl Sepharose® 4 from Amersham Pharmacia Biotech, Sweden. Then guide oponou matrix is washed with a salt solution with high ionic strength, when this DNA is washed.

Hydrophobic matrix then placed in a column and subjected to stepwise gradient elution with sodium chloride solution with decreasing ionic strength. Individual antimicrobial protein component eluted at a concentration of 1M NaCl.

The protein component can then be cleaned using conventional methods suitable for the purification of peptides/proteins. Such methods include centrifugation, precipitation at the isoelectric point, phase separation, ultrafiltration, gel chromatography (chromatography according to the size of molecules), ion exchange chromatography or HPLC, as well as combinations of these methods. Subsequent separation process is preferably carried out by gel-chromatography or ion exchange chromatography.

The stage of the preparative gel chromatography is most preferably carried out on a column filled with a gel limit exceptions, equal to 100 kDa. Before downloading a fraction of 1 M NaCl solution, exhibiting antimicrobial activity, a column lead in equilibrium with distilled water. Then the column elute with distilled water or ammonium acetate. In this case, the desalting and purification occurs at the same time and on the same stage. The protein component can be concentrated to a dry state using leofiles the tion without the use of further stages of refinement.

Allocate a fraction of the protein with an apparent molecular mass from 10 to 20 kDa, which exhibits antimicrobial properties.

An experienced specialist should be understood that the purification of the protein component can be realized by using many other methods known to experts of the art, all of which are encompassed by the present invention.

As present and degrades agent can also be used complexing agent, such as heparin, alginic acid, phytic acid or a compound of vanadium, provided that it decomposes plant chromatin at the individual components. As present and degrades agent is particularly preferred alginic acid, alginate forms complexes with protein component having antimicrobial activity. Such complexes can be used for subsequent treatment or be used by themselves for the slow release of these antimicrobial activity.

Before separating decomposed plant chromatin at the individual fractions, in accordance with the invention, using, for example, methods of separation by hydrophobic interaction, generally no antimicrobial activity is not detected in the source material or the unfolded chromatin. After vegetable protein component of the s cleared in accordance with the description of the present invention, automatically use the heterochromatin. Thus, the biological activity sequentially formed by physical separation of components of chromatin. Theoretically, the procedure division can lead to a change in the molecular structure of components.

It is believed that antimicrobial peptides exert their effects on prokaryotes through a positive charge. It is expected that the most probable mechanism of action of the protein component of the present invention is not different from that known for other cationogenic polypeptides. However, the known basic peptides allow interaction with cell membranes, which resembles the action of detergents. A similar mechanism of action of the protein component by its activity towards both gram-positive and gram-negative bacteria.

In accordance with the invention from other plant materials after elution of hydrophobic matrix when using other cleaning procedures can be obtained from other protein components with antimicrobial activity. This is due to the fact that proteins from different biological materials have different polusinteticheskie modifications that reflect the activity of cells of the plant material. So, on the partitioning scheme should have the influence, for example, the degree of acetylation, phosphorylation, methylation, ubiquitination, glycosylation, and ADP-ribosylate protein component obtained according to the present invention.

In addition, the protein component of plant chromatin, can then be chemically modified. Such modifications include changes in molecular weight and/or the level of acetylation and can lead to forms of drugs that are more specific biological activity.

The antimicrobial effect of the protein component, obtained by the method of the present invention, can be installed with the standard method Bioscreen®while as a control substance use lowlands. Compared with Nizina for the protein component was the effect corresponding to 2-4 mg/ml, while the effect is manifested in the death. Moreover, the observed effect against gram-negative bacteria, which is absent in the lowlands.

A simple method of cleaning according to the present invention allows to produce antimicrobial protein component of virtually unlimited scale. The exit process is approximately 1 g of protein from 1 kg of raw material (e.g., roots). When using germinating seeds with maximum protein synthesis can be made of the th maximum, what has been shown, for example, when solaimani within six days. If you are using fast growing plant cells in S-phase, the synthesis of natural protein chromatin is maximum and its content can be up to 80% of the total amount of protein synthesized.

The action of the protein component is selected in accordance with the invention as antibacterial agents from plant chromatin, can be synergistically enhanced when combined with one or more antimicrobial agents. An example of such a synergistic funds are lysozyme, Protamine, hepatoblastoma agents, compounds of copper (II) and bacteriocins.

The protein component of plant chromatin suitable for the preparation of pharmaceutical compositions for the treatment of microbial infections. The invention also relates to methods for treating microbial infections in mammals, including humans, when administered therapeutically effective amount of the protein component.

The protein component can be used for oral administration for the treatment of diseases of the teeth and gums, for local use for outdoor use in the treatment of dermatological diseases, skin diseases and diseases of the hair and for the treatment of OTO-ophthalmological diseases. Protein component of the plant is about chromatin can also be used in the treatment of body cavities, such as the mouth, throat, lungs, vagina and rectum, and can also be used orally in the treatment of gastrointestinal diseases after exposure to pathogenic microorganisms.

In that case, if the protein component you intend to use as an antimicrobial agent, it can be prepared in a buffered aqueous medium containing various salts and buffer additives. The salts are preferably the halides of the alkali and alkaline-earth metals, in particular sodium chloride, potassium chloride and sodium sulfate. Can be used in a variety of buffers, such as citrate, phosphate, HEPES, Tris, etc. in the amount in which these buffers are physiologically acceptable for the intended method of application.

In the case when the protein component is prepared in the form of liofilizirovannogo powder, intended for later use in the solution, it can be used in a variety of fillers or other additives. Additives may include various polyols, inert powders or other fillers.

The application of the present invention also includes compositions that contain purified protein component in an amount effective to kill bacteria or fungi, and a suitable carrier. Such compositions together with well known from the technical field of the media can nanodimension use to fight bacteria and fungus, for example, in the form of household or laboratory specimens.

Various compositions have varying degrees of activity against different organisms. The effective amount to be used for the destruction of harmful microorganisms, such as bacteria and fungi, and other harmful agents that can be easily defined.

Protein component of the present invention can also be combined with other proteins acting as preservatives to protect the protein component from proteolytic degradation. Alternatively, the protein component or composition according to the present invention can be used as preservatives or disinfectants in the form of numerous formulations, such as solutions for contact lenses, ointments, shampoos, medicines, foods, etc. the Amount of the protein component may vary depending on the type of other components, a desired level of antimicrobial protection and the intended use of the composition.

The protein component may, for example, be used together with a suitable carrier in a composition for disinfection and cold sterilization of surfaces and as an aid in the process of pasteurization of food under high pressure, as well as in the composition in the quality of the ve preservative for water, in particular, in fish farming. The protein component can also be used in amounts effective to kill bacteria in the composition of the packaging material from which the protein component can slowly be released.

EXAMPLES

The invention is further explained and illustrated by reference to the following examples. It must, however, be noted that these examples should not be construed as fact or otherwise limiting the present invention.

Example 1. Antimicrobial analysis

Wells of microtiter tablet fill 300 ál medium for cultivation (nutrient broth+1% glucose) with a double concentration. Two sample added to the final concentration of the protein component, (2) respectively, 0.5 and 0.2 mg/ml

Use two test organism: Pseudomonas fluorescens (gram-negative aerobic bacteria) and Listeria innocua (gram-positive aerobic bacteria).

Culture of bacterial strains after incubation over night, diluted with peptone water and 50 μl of each strain is added to the wells in an amount of 104cells/ml For comparison, use holes (positive control wells), in which no test material.

Tablets incubated at 30°C for 72 h in the apparatus Bioscreen® to analyse and every 15 min govern the growth of bacteria will increase the coverage in the visible region of the spectrum.

Example 2. Extraction and fractionation

When carrying out the extraction and fractionation of plant proteins and polypeptides difference in the structure, physiology and biochemistry of plant tissues often do not allow to apply modern or conventional animals buffers and/or methods of maceration. More complex macromolecular structure and interactions within plant tissues (in particular, the presence of phenolic, carbohydrate and hydrocarbon compounds), as well as more compact cell wall preparation of plant extracts require the use of more accurate methods of maceration and selection to ensure the integrity of the polypeptides.

The barley leaves (1 kg, wet wt) is subjected to extraction and fractionation according to the method specified in Langenbuch et al., Plant Molecular Biology 2, 207-220 (1983). Nuclear chromatin barley get through a 0.4 N solution of sulfuric acid, with a typical output is 10-20 mg.

Then hold a special operation subfractionated, which allows large scale preparative isolation of the protein component. Specific protein component (molecular weight 17300) obtained by the method of differential solubility in a mixture of ethanol (80%): HCl (0,25 N).

Protein components is examined for antimicrobial activity against common strains Lsteria and Pseudomonas.

Complete inhibition of growth of both organisms is achieved using fractions, soluble in ethanol and water. The effect depends on the concentration, i.e. must exceed a certain threshold value (no effect is detected when diluted 1:1).

Antimicrobial activity is limited solely to the specific protein components with molecular weight 17300. No activity is detected for protein components having larger sizes.

Example 3. Alternative extraction and fractionation

A two-week seedlings of pea, wheat, rye and cotton are subfractionated according to methodology described Sidorova and Conevyt in the journal Biochemistry 46, 1298 (1981).

Get specific protein component with a molecular mass 17300, as well as the protein component of a larger size.

Protein components is examined for antimicrobial activity, with results identical to those obtained in Example 1.

Example 4. Improved fractionation procedure

Green malt is produced from local Breweries. Malt for 6 days germinated in the normal process of solaimani.

Green malt (100 g) is suspended in 4 M NaCl and homogenized for about 10 minutes in the mixer company Braun. The homogenate was filtered through a 20 μm sieve and the resulting solution was then added to 20 g of the guide is atomnoi matrix (Novarose® S-Butyl 1000/40, Inovata AB, Sweden), which result in equilibrium with 4 M NaCl solution. The mixture is stirred for 10 min and the matrix is filtered through a 40 µm sieve.

Next, the matrix is placed in a column (25×50 mm) and before the elution was washed with 4 M NaCl. At such high ionic strength solution of DNA is not associated with the matrix, which is set by increasing the absorption of the eluate at 254 nm, and thus, the DNA eluted in the process of filling and washing of the column.

Individual protein peaks recorded using UV absorption at 276 nm after stepwise elution, respectively, 2 M NaCl, 1 M NaCl and water. This corresponds to the desorption does not contain chromatin material 2 M NaCl solution.

Example 5. Procedure subfractionated

Fractions that desorbers in 1 M NaCl solution in Example 4 is subjected to subfractionated using preparative gel chromatography in 0.1 M solution of ammonium acetate in column (25×500 mm) with Novarose® SE-100/40 (Inovata AB, Sweden).

Receive and lyophilized three protein fractions. The middle fraction is the amount of the deduction, which corresponds to a molecular weight in the range from 10 to 20 kDa.

Thus, plant chromatin is a convenient source for selection, in this range of molecular weight, hypoallergenic snowmaggedon proteins, carrying a large electrophoretically charge.

After conducting analytical gel chromatography in 0.1 M phosphate buffer with pH 7.0 in column (8×300 mm) with Novarose® SE-100/17 (Inovata AB, Sweden), it was found that this fraction corresponds to medium molecular mass of approximately 14 kDa.

Example 6. An alternative procedure fractionation

A comparative experiment is carried out under the same conditions given in Example 4, however, as a hydrophobic matrix used Novarose® S-Phenyl (Inovata AB, Sweden). When the elution of 1 M NaCl desorption of the protein is not detected and only one fraction obtained by elution with water. Desorption of the protein material of the hydrophobic matrix does not occur when using 22%solution of ethanol in water, which suggests that the binding between the matrix and protein material is highly hydrophobic.

Example 7. Determination of antimicrobial activity

Average fraction obtained in accordance with Example 5 is subjected to further study for its antimicrobial activity. Samples receive, dissolving liofilizovannye fraction in phosphate buffer (pH 7.0) to a concentration of 4 mg/ml of the resulting solution is then diluted from 4 to 10 times the phosphate buffer.

The results obtained were not distinguishable from those that described in Example 5 for specific is belkovogo component with a molecular weight 17300, selected by the method described in Examples 4 and 5.

It should be noted that the other fraction obtained after conducting gel chromatography, generally does not show any antimicrobial activity.

Example 8. Impact p-value

Protein components with similar antimicrobial activity receive the same methodologies as described in Examples 4 and 5 of chromatin 72-hour seedlings of Arabidopsis and wheat. The average outputs of the fractions of mass in the range of 10-20 kDa obtained from these samples differ significantly from each other, while wheat is a more appropriate source for carrying out the extraction. Compared with barley desired fraction is approximately twice more.

Example 9. Analytical electrophoresis

Analytical electrophoresis, performed according to standard methods (Panyim & Chalkley, Biochem. 8: 3972, 196), shows many variations of the specific protein component with a molecular weight 17300 and the absence of any sub-fractions of protein components with higher molecular weight in the range from 28000 to 35000.

These results also reflect the difference in genome size between species. Arabidopsis has a small diploid genome with a size of 200 megabar grounds and, accordingly, the smaller the outputs are compared to wheat, which has a large hexapodal GE the om size 17000 megabar grounds.

Thus, the yield of extraction is closely related to genome size, expressed its p-value.

Comparative example 1. Comparison with histone animals

Because analytical chromatography in the separation buffer medium as described in Example 5 leads to a molecular mass of approximately 14 kDa, which corresponds to the molecular mass of histones H2A and H2B calf, then the comparison is made between the protein component of the present invention and animal histones.

Parallel control experiments with available on the market histones H2A and H2B calf (Sigma, USA) and the average fraction according to Example 4, having a molecular weight in the range from 10 to 20 kDa, is carried out using an analytical gel chromatography in 0.1 M phosphate buffer with pH 7.0 in column (8×300 mm) with Novarose® SE-100/17 (Inovata AB, Sweden).

All of the obtained fractions examined for antibacterial activity, with positive results repeated for the protein component selected from plant chromatin.

Another comparative experiment does not confirm any antimicrobial activity of the first fraction, close to the free volume of the column, where it is expected the plant histone H1. Moreover, to re-establish that the pattern of decomposition of nucleosomes in saline solution varies in animals and plants and that the standard IU is the odik, used for animals, could not be applied to plants.

Comparative example 2. Extraction of the aqueous solvent

Know a few methods that allows for the extraction of all of polypeptides from plant tissues. The methods used are favorable for obtaining fractions of different tissues that are enriched subcellular components. In contrast, for animals of systems described by a number of more or less "universal" preparative methods.

Such methods have been studied in U.S. patent 5698200, issued in the name Karen Barret, it was investigated a few simple aqueous extracts of solozhenko cereal grains.

The same subsequent methods as described in example 1 and 2 of U.S. patent 5698200, were used for the leaves of barley (see example 2) as the starting material.

In the range of 10-20 kDa were identified only spare proteins. These proteins do not show any antimicrobial activity.

The conclusion is made that the chromatin cannot be allocated on any of the methods described in U.S. patent 5698200, if the source material is the substance of the cytosole. Most likely, were obtained thionine, which are well-known defensively representing cytotoxic serosoderjaschei polypeptides of low molecular weight, namely 2-3 kDa.

1. Application b is kovago component, isolated from the plant chromatin after decomposition of the latter, as an antimicrobial agent, with the specified protein component has an apparent molecular weight in the range from 10 to 20 kDa.

2. The use according to claim 1, where the specified chromatin is heterochromatin.

3. The use according to claim 1, where the specified plant chromatin is DNA, the value of which exceeds 3000 megabar grounds.

4. The use according to any one of claims 1 to 3, where the specified plant chromatin extracted from the seeds of plants.

5. The use according to claim 4, where these plant seeds get from oats, wheat, barley, rye, maize, rice, rapeseed, soybean, millet or buckwheat.

6. The use according to claim 4, where the specified plant chromatin obtained from these seeds in seedling stage.

7. The use according to claim 6, where the specified antimicrobial agent against both gram-positive bacteria and gram-negative bacteria.

8. The use according to claim 7, where the specified gram-positive bacterium is a Listeria innocua.

9. The use according to claim 7, where the specified gram-negative bacterium is Pseudomonas fluorescens.

10. Application of the protein component according to claim 1 together with at least one synergistic antimicrobial agent.

11. The use of claim 10, where the specified at least one synergistic antimicrobial agent is lysozyme, protus is in, chelate forming agent, a compound of copper (II) or bacteriocins.

12. Application of the protein component according to claim 1 in the form of a complex with a complexing agent, from which the protein component will slowly be released.

13. The application indicated in paragraph 12, where the specified complexing agent is alginic acid.

14. Application of the protein component according to claim 1 in an amount effective to kill bacteria, and a suitable carrier in the form of a composition as a dietary Supplement.

15. Application of the protein component according to claim 1 and a suitable carrier in the form of a composition as stimulating the growth of food additives for the animal.

16. Application of the protein component according to claim 1 in an amount effective to kill bacteria, and a suitable carrier in the form of a composition for disinfection and cold sterilization of surfaces, and also as an aid in the pasteurization of food under high pressure.

17. Application of the protein component according to claim 1 in an amount effective to kill bacteria, comprising packaging material, from which it can slowly be released.

18. Application of the protein component according to claim 1 in an amount effective to kill bacteria, and a suitable carrier in the form of a composition as a preservative for water.

19. Use p in fish farming.

20. SP is a way to obtain vegetable protein component, possessing antimicrobial activity, which has an apparent molecular weight in the range from 10 to 20 kDa, comprising the following stages:

homogenization of plant material, the purpose of separation from it of plant chromatin;

the decomposition of the specified plant chromatin using an agent decomposition in hydrophobic conditions; and

the division specified decomposed plant chromatin at the individual fractions, one of which contains the specified vegetable protein component, method of separation by hydrophobic interaction.

21. The method according to claim 20, in which the specified homogenization is carried out in the specified agent of decomposition.

22. The method according to claim 20, in which the specified agent decomposition is urea, guanidine chloride or salt of hydrochloric acid.

23. The method according to item 22, which specified salt of hydrochloric acid is sodium chloride.

24. The method according to claim 20, wherein said agent decomposition is the complexing agent, the complex formed with the specified protein component.

25. The method according to paragraph 24, in which the specified complexing agent is heparin, alginic acid, phytic acid or a compound of vanadium.

26. The method according to claim 20, in which carry out the specified division.

27. The method according to p in which the specified method under the Oia by hydrophobic interaction is hydrophobic chromatography, chromatography on chelate complexes of metals, distribution chromatography, the distribution in the flow and distribution in the gas afron.

28. The method according to p, in which the functional ligand in said method of separation by hydrophobic interaction is simple ester group, ISO-propyl, bucilina or anjilina group.

29. The method according to p in which the specified method of separation by hydrophobic interaction follows the procedure division.

30. The method according to clause 29, which further specified the procedure division is gel chromatography or ion exchange chromatography.

31. The use of a protein component selected from plant chromatin after decomposition of the latter, for the preparation of pharmaceutical compositions intended for the treatment of microbial infections, with the specified protein component has an apparent molecular weight in the range from 10 to 20 kDa.

32. A method of treating microbial infections in mammals, including humans, which is administered a therapeutically effective amount of the protein component selected from plant chromatin after decomposition of the latter and having an apparent molecular weight in the range from 10 to 20 kDa.



 

Same patents:

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to azaindoleoxoacetic derivatives of piperazine of the general formula (I): wherein Q is chosen from the group consisting of the following compounds: ; -W- represents compound of the formula: . Proposed compounds possess antiviral activity both in separate using and in combination with other antiviral, anti-infectious agents, immunomodulating agents or inhibitors HIV entering. Also, invention describes a pharmaceutical composition based on compounds of the formula (I).

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

55 cl, 10 tbl, 169 ex

FIELD: organic chemistry, chemical technology, microbiology.

SUBSTANCE: invention relates to novel 2,2,5,5-tetrabromo-1,6-di-(4'-methylphenyl)-1,3,4,6-hexanetetraone of the formula (I): that possesses antibacterial activity. Compound of the formula (I) is synthesized by interaction of 3,4-dihydroxy-1.6-di-(4'-methylphenyl)-2,4-hexadiene-1,6-dione with bromine in chloroform medium. Compound of the formula (I) possesses antibacterial activity with respect to Staphylococcus aureus with MAC (maximum allowable concentration) value = 0.0075-0.06 mcg/ml and to colon bacillus with MAC value = 0.12-0.32 mcg/ml and with the acute toxicity value LD50 = 2960 mg/ml.

EFFECT: valuable activity of compound.

1 tbl, 1 ex

FIELD: medicine, chemical-pharmaceutical industry.

SUBSTANCE: invention relates to an antiviral agent as gel. Agent comprises human leukocyte interferon and 2-% solution of styrene copolymer and maleic anhydride as gel-forming base in the following ratio of the agent components, in 1 g of the agent: concentrated interferon, 5000-10000 IU; nipagin, 0.002-0.004 g and a base, the balance. Proposed agent possesses high activity, nontoxic and possesses antiviral, immunomodulating, anti-inflammatory and regenerative effects and can be used in ophthalmology, dermatology, gynecology and surgery for topical using and in treatment of viral diseases.

EFFECT: valuable medicinal properties of agent.

2 tbl, 2 ex

FIELD: medicine, pediatrics, obstetrics.

SUBSTANCE: method involves administration of preparations balans-narine-f and calcemin. Carrying out this therapy provides decreasing the content of staphylococci in breast milk, increasing the sterility period of milk based on reducing cellular membranes penetrability and to diminish probability in development complications in treatment of bacteriolactia in nursing women. Invention can be used in treatment of staphylococcus bacteriolactia in women.

EFFECT: improved method of treatment.

1 tbl, 2 ex

FIELD: medicine, pharmacology.

SUBSTANCE: invention relates to a medicinal agent used in treatment of warts. Proposed agent contains an active compound chosen from isopropyl laurate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, ethyl myristate, propyl myristate, butyl myristate and/or ethyl oleate and at least a mixture containing (-)-epicatechol, (-)-epicatechol gallate, (-)-epigallocatechol, (-)-epigallocatechol gallate, (+)-gallocatechol and (-)-gallocatechol gallate possessing the enhanced effectiveness.

EFFECT: valuable medicinal properties of drug.

7 cl, 2 ex

FIELD: veterinary science.

SUBSTANCE: invention proposes a preparation containing myramistin, chitosan and distilled water taken in the following ratio of components, wt.-%: myramistin : chitosan : water = (0.015-0.1):0.2:100, respectively. The preparation shows high effectiveness, strongly expressed wound-healing effect and bactericidal activity and simplicity in using. Invention can be used in treatment of animals with wounds of different etiology and anatomical localization, weeping eczema, burns and others.

EFFECT: valuable medicinal properties of preparation.

1 tbl, 3 ex

FIELD: synthesis of biologically active compounds.

SUBSTANCE: invention provides novel urea-substituted imidazoquinoline ethers depicted by general formula I: (1), in which X represents -CHR5- or -CHR5-alkyl group; R1 is selected from radicals: -R4-NR8-CR3-NR5-Z-R6-Alk, -R4-NR8-CR3-NR5-Z-R6-Ph, -R4-NR8-CR3-NR5-Z-R6-furanyl, -R4-NR8-CR3-NR5R-7, phenyl being optionally substituted by one or more substituents selected from methyl, methoxy, methylthio, cyano, hydrogen, dimethylamino, and acetyl; R2 is selected from hydrogen, alkyl, and alkyl-Y-alkyl; R3 represents =O or =S; R4 represents alkyl optionally substituted by one or several O-groups; each of R5 represents C1-C10-alkyl; R6 represents ordinary bond or alkyl; R7 forms cycle together with R5; R4 represents hydrogen, C1-C10-alkyl, or forms morpholine ring together with R8; Y represents -O-; Z ordinary bond, -CO-, or -SO2-; n=0; each of R is independently selected from C1-C10-alkyl, C1-C10-alkoxy, hydroxy, halogen, and trifluoromethyl; or pharmaceutically acceptable salt of forgoing compounds. Described are further compounds of general formula II, intermediates of compounds of general formulae III and IV, pharmaceutical compositions based on compounds I and II, which are immunomodulators for synthesis of cytokines based on compounds I and II, methods of treating viral diseases utilizing compounds I and II, and methods of treating tumor diseases utilizing compounds I and II.

EFFECT: expanded synthetic possibilities in quinoline series and increased choice of therapeutically useful compounds.

25 cl, 4 tbl, 44 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to phenylpyridazine derivative of general formula I , wherein R1 represents C1-C12-alkyl optionally comprising cyclic C3-C6-alkyl structures and optionally substituted by phenyl, which may be substituted by 1-2 halogen atoms; or C1-C12-alkenyl substituted by optionally halogen-substituted phenyl; R2 and R3, independently form each other, represent hydrogen, C1-C12-alkyl, C1-C12-hydroxyalkyl, C1-C12-dihydroxyalkyl, or C1-C12-alkynyl; or R2 and R3, together with adjacent nitrogen atom form 5-6-membered saturated heterocyclic group containing 1-2 nitrogen atoms and optionally oxygen atom, indicated heterocyclic group being optionally substituted by C1-C12-alkyl group, C1-C12-alkoxydicarboxylic group or phenyl-C1-C7-alkyl group; X, Y, and Z, independently form each other, represent hydrogen, halogen, optionally halogen(s)-substituted C1-C12-alkyl, C1-C12-alkoxy, C1-C12-alkylthio, C1-C12-alkylsulfinyl, C1-C12-alkylsulfonyl, or phenyl; and n is a number from 0 to 5; provided that R2 and R3 cannot be simultaneously hydrogen atoms or identical C1-C3-alkyl groups when R1 is benzyl or C1-C3-alkyl group; and salts of compounds I. Foregoing compounds manifest inhibitory activity against production of interleukin IL-1β being well dissoluble in water and characterized by good oral absorption. Invention also relates to therapeutical agent inhibiting production of interleukin 1β, pharmaceutical composition, employment of above-defined compounds, a method for treating disease caused by interleukin 1β production stimulation as well as methods for treating immune system disturbances, inflammatory conditions, ischemia, osteoporosis, or septicemia using above compounds.

EFFECT: expanded therapeutical possibilities.

22 cl, 4 dwg, 2 tbl, 217 ex

FIELD: medicine, gynecology, urology.

SUBSTANCE: on isolating Chlamydia out of infection foci and setting a preliminary diagnosis it is necessary to determine their sensitivity to different etiotropic preparations. Then it is important to sample a patient's blood followed by centrifuging and dividing it into plasma, erythrocytic mass and leukocytic suspension. Removed blood plasma should be substituted with physiological solution, and autoerythrocytes should be returned for a patient in physiological solution intravenously by drops. Autoleukocytic suspension should be activated with laser radiation of He-Ne laser at λ=0.63 mcm. Then it should be supplemented with etiotropic preparation moxyfloxacin to be intravenously injected for a patient. The procedure should be repeated daily for about 10-12 d. The innovation suggested enables to shorten the terms of interrupting the main clinical manifestations of the disease due to a purposeful transport of etiotropic preparation being of higher bacteriological efficiency in leukocytes activated with laser radiation, into primary (urogenital tract) and secondary (extraurogenital) foci of inflammation and, also, decrease side action of chemopreparations due to decreasing their course dosage.

EFFECT: higher efficiency of therapy.

2 ex

FIELD: medicine, immunology.

SUBSTANCE: the present innovation deals with specific prophylaxis of smallpox and viral hepatitis B. The kit contains two tablets each contains stabilizing additives, a filler and lyophilized alive viral material worked out based upon recombinant VOV strain at typical VOV properties expressing proteins preS2-S and HBs virus of hepatitis B virus, the first immunizing dosage corresponds to minimal quantity of viral material being sufficient to obtain weak immune response in the body in case of insignificant at insignificant reactogenicity, and immunizing dosage of the second - maximal quantity of viral material that causes pronounced and prolong immune response in the body at no negative side action. The technique of applying the kit of bivaccine tablets, first, one should use the 1st tablet at minimal dosage of bivaccine, as for the 2nd tablet - with maximal dosage of bivaccine it should be taken till the moment of developing humoral answer (in 7-14 d) after injecting the 1st tablet at minimal immunizing dosage of bivaccine. The innovation enables to create stable immunity.

EFFECT: higher efficiency.

4 cl, 5 ex, 6 tbl

FIELD: medicine, phytotherapy, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a composition prepared from the following raw medicinal plants: Tripterygium hypoglaucum (Levl.) Hutch, barrenwort (Epimedium), box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) taken in the following mass ratio: 1-4; 1-4; 1-4 and 1-4, respectively. Method involves the following procedures: weighing and milling of Tripterygium hypoglaucum (Levl.) Hutch and barrenwort (Epimedium), their separate boiling in water 3 times, wetting box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) in warm water at temperature 80-95°C 1-3 times, separate decantation of decocts and liquid, their separate passing through adsorption column, washing out with water up to clearing eluate, elution with 30-99.3% ethyl alcohol and mixing eluate with alcoholic liquid. The total weight of eluate is 1-8-fold more of weight of parent raw medicinal agents. Then method involves recycling each eluent of the raw medicinal agent, concentrating to specific gravity 1.1, drying extracts and their mixing. Pharmaceutical composition is used in treatment of rheumatism, rheumatic arthritis and chronic nephritis. Invention provides realization of indicated designation.

EFFECT: improved preparing method, valuable medicinal properties of pharmaceutical composition.

8 cl, 21 tbl, 13 ex

FIELD: medicine, phytotherapy, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a composition prepared from the following raw medicinal plants: Tripterygium hypoglaucum (Levl.) Hutch, barrenwort (Epimedium), box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) taken in the following mass ratio: 1-4; 1-4; 1-4 and 1-4, respectively. Method involves the following procedures: weighing and milling of Tripterygium hypoglaucum (Levl.) Hutch and barrenwort (Epimedium), their separate boiling in water 3 times, wetting box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) in warm water at temperature 80-95°C 1-3 times, separate decantation of decocts and liquid, their separate passing through adsorption column, washing out with water up to clearing eluate, elution with 30-99.3% ethyl alcohol and mixing eluate with alcoholic liquid. The total weight of eluate is 1-8-fold more of weight of parent raw medicinal agents. Then method involves recycling each eluent of the raw medicinal agent, concentrating to specific gravity 1.1, drying extracts and their mixing. Pharmaceutical composition is used in treatment of rheumatism, rheumatic arthritis and chronic nephritis. Invention provides realization of indicated designation.

EFFECT: improved preparing method, valuable medicinal properties of pharmaceutical composition.

8 cl, 21 tbl, 13 ex

FIELD: medicine, phytotherapy, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a composition prepared from the following raw medicinal plants: Tripterygium hypoglaucum (Levl.) Hutch, barrenwort (Epimedium), box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) taken in the following mass ratio: 1-4; 1-4; 1-4 and 1-4, respectively. Method involves the following procedures: weighing and milling of Tripterygium hypoglaucum (Levl.) Hutch and barrenwort (Epimedium), their separate boiling in water 3 times, wetting box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) in warm water at temperature 80-95°C 1-3 times, separate decantation of decocts and liquid, their separate passing through adsorption column, washing out with water up to clearing eluate, elution with 30-99.3% ethyl alcohol and mixing eluate with alcoholic liquid. The total weight of eluate is 1-8-fold more of weight of parent raw medicinal agents. Then method involves recycling each eluent of the raw medicinal agent, concentrating to specific gravity 1.1, drying extracts and their mixing. Pharmaceutical composition is used in treatment of rheumatism, rheumatic arthritis and chronic nephritis. Invention provides realization of indicated designation.

EFFECT: improved preparing method, valuable medicinal properties of pharmaceutical composition.

8 cl, 21 tbl, 13 ex

FIELD: medicine, phytotherapy, pharmaceutical technology, pharmacy.

SUBSTANCE: invention relates to a composition prepared from the following raw medicinal plants: Tripterygium hypoglaucum (Levl.) Hutch, barrenwort (Epimedium), box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) taken in the following mass ratio: 1-4; 1-4; 1-4 and 1-4, respectively. Method involves the following procedures: weighing and milling of Tripterygium hypoglaucum (Levl.) Hutch and barrenwort (Epimedium), their separate boiling in water 3 times, wetting box-thorn matrimony-vine berries (Lycium) and dodder seeds (Cuscuta) in warm water at temperature 80-95°C 1-3 times, separate decantation of decocts and liquid, their separate passing through adsorption column, washing out with water up to clearing eluate, elution with 30-99.3% ethyl alcohol and mixing eluate with alcoholic liquid. The total weight of eluate is 1-8-fold more of weight of parent raw medicinal agents. Then method involves recycling each eluent of the raw medicinal agent, concentrating to specific gravity 1.1, drying extracts and their mixing. Pharmaceutical composition is used in treatment of rheumatism, rheumatic arthritis and chronic nephritis. Invention provides realization of indicated designation.

EFFECT: improved preparing method, valuable medicinal properties of pharmaceutical composition.

8 cl, 21 tbl, 13 ex

FIELD: medicine, pharmacology.

SUBSTANCE: invention relates to a medicinal agent used in treatment of warts. Proposed agent contains an active compound chosen from isopropyl laurate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, ethyl myristate, propyl myristate, butyl myristate and/or ethyl oleate and at least a mixture containing (-)-epicatechol, (-)-epicatechol gallate, (-)-epigallocatechol, (-)-epigallocatechol gallate, (+)-gallocatechol and (-)-gallocatechol gallate possessing the enhanced effectiveness.

EFFECT: valuable medicinal properties of drug.

7 cl, 2 ex

FIELD: method and composition for oral cavity care.

SUBSTANCE: claimed form contains a) 1-40 % of retaining agent, Selected from group containing water soluble hydrophilic resins, water soluble hydrophilic polymers or mixtures thereof, wherein retaining agent is capable of hydration under water or saliva action to produce retention coefficient of 1-4; and b) 60-90 % of local carrier. Moreover composition contains 65 wt.% of water-insoluble particles. Further disclosed is dental paste composition containing a) 30-65 % of water-insoluble particles as retaining agent, wherein solubility of retaining agent in water is less then 1 g/30 g at 25°C; b) 0.01-40 % of active agent for oral cavity care; c) 0.1-25 % of buffer agent. Claimed composition has retention coefficient of 1-4.

EFFECT: improved compositions for oral cavity care.

9 cl, 3 dwg, 5 ex

FIELD: method and composition for oral cavity care.

SUBSTANCE: claimed form contains a) 1-40 % of retaining agent, Selected from group containing water soluble hydrophilic resins, water soluble hydrophilic polymers or mixtures thereof, wherein retaining agent is capable of hydration under water or saliva action to produce retention coefficient of 1-4; and b) 60-90 % of local carrier. Moreover composition contains 65 wt.% of water-insoluble particles. Further disclosed is dental paste composition containing a) 30-65 % of water-insoluble particles as retaining agent, wherein solubility of retaining agent in water is less then 1 g/30 g at 25°C; b) 0.01-40 % of active agent for oral cavity care; c) 0.1-25 % of buffer agent. Claimed composition has retention coefficient of 1-4.

EFFECT: improved compositions for oral cavity care.

9 cl, 3 dwg, 5 ex

FIELD: method and composition for oral cavity care.

SUBSTANCE: claimed form contains a) 1-40 % of retaining agent, Selected from group containing water soluble hydrophilic resins, water soluble hydrophilic polymers or mixtures thereof, wherein retaining agent is capable of hydration under water or saliva action to produce retention coefficient of 1-4; and b) 60-90 % of local carrier. Moreover composition contains 65 wt.% of water-insoluble particles. Further disclosed is dental paste composition containing a) 30-65 % of water-insoluble particles as retaining agent, wherein solubility of retaining agent in water is less then 1 g/30 g at 25°C; b) 0.01-40 % of active agent for oral cavity care; c) 0.1-25 % of buffer agent. Claimed composition has retention coefficient of 1-4.

EFFECT: improved compositions for oral cavity care.

9 cl, 3 dwg, 5 ex

Massage agent // 2302854

FIELD: medicine cosmetic, in particular agent for treatment of cripple infant suffering from infantile cerebral paralysis.

SUBSTANCE: claimed agent contains cameline oil, dogrose oil, and mint oil. Said agent is uniformly distributed on skin, has good absorption, provides good sliding effect, increases blood circulation, relives muscle stress.

EFFECT: agent with increased feeding, anti-inflammation and immunostimulating action.

1 ex

FIELD: bioengineering.

SUBSTANCE: method involves producing acids polysaccharide fucoidan from laminaria by crushing raw material by treating it with 0.5-1.0% food acid solution during 4-5 h and subjecting acid extract to ultrafiltration on membrane of 100-300 kDa with following end product drying by applying lipophilic or spraying or vacuum drying method.

EFFECT: high purity of end product.

FIELD: biotechnology, medicals.

SUBSTANCE: invention relates to method for extraction, purification, and enzyme modification of β-conglycinin α'-subunit. Claimed method includes isolation of β-conglycinin by selective extraction from milled defatted soybeans and following deposition by extract treatment with ethanol aqueous solution. Enriched fraction is then subjected to affinity chromatography with metal (MAC) under denaturant condition to produce α'-subunit which further is treated with chymotrypsin and subjected to additional treatment with MAC to isolate amino-terminated region of said polypeptide having molecular weight of 28000 Da.

EFFECT: isolated fraction of increased purity.

10 cl, 2 tbl, 2 dwg, 1 ex

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