RU2806364C1 - Method of obtaining hemostatic agent based on chitosan - Google Patents

Abstract

FIELD: chemistry; medicine.

SUBSTANCE: invention relates to a method of producing a hemostatic agent based on chitosan, during which a chitosan solution is prepared for impregnation of a non-woven material, after which a continuous impregnation process is performed followed by gelation, in which the non-woven material first passes through a bath with a solution chitosan, then through a bath with a gelling agent, after which the resulting material is washed from unreacted compounds, the aqueous solvent is replaced with isopropyl alcohol, and the isopropyl alcohol in the resulting material is extracted with liquid carbon dioxide under excess pressure.

EFFECT: increasing the effectiveness of the hemostatic agent by increasing the amount of chitosan in the finished product, using the original nonwoven material with a wider range of characteristics in thickness, surface density and composition, modifying the structure of the hemostatic agent, as well as increasing the density of the finished product compared to the original non-woven material.

5 cl, 4 dwg, 6 tbl, 3 ex

Description

The invention relates to the field of medicine, namely to local hemostatic agents used to stop bleeding, including massive venous and arterial ones, and can be used to provide emergency and emergency medical care for wounds and injuries at the prehospital stage and in hospital settings for wounds and injuries, be included in first aid kits and packs for various purposes, and also be used during surgical interventions in medical institutions.

Today there is a wide range of local hemostatic agents (LHA) of various designs that are used to stop bleeding. In medical practice, the following basic requirements are imposed on such drugs: effective stopping of external bleeding for a period of no more than three to five minutes, prevention of re-bleeding, ease of use, biocompatibility, minimal side effects. For a long time, MGS were most often used in the form of powders, particles or granules. Among the most famous products are such products as “Gemospas”, “Gemostop M”, “Gemospas” / “Gemospas M”, “Gemospas Bio”, “Ellarga” “Gepoglos”, “QuikClot”, “Celox”. It should be noted that the accumulated practical experience in using the Gemostop MGS (RF Patent for invention No. 20091456-52 dated December 10, 2009), as well as the Gemospas MGS similar in composition, made on the basis of zeolites, has revealed their negative effects , expressed in thermal effects on human tissue, which greatly limited the scope of its use. Similar effects were observed with the local hemostatic agent “QuikClot”, produced in the USA, which is considered as the “ancestor” of such products. MGS obtained on the basis of hydro- and aluminosilicates, such as “Gemostop M” and “Gemospas M”, in a number of cases have shown their insufficient effectiveness.

Recently, MGS based on the natural biopolymer chitosan, which, due to its structural characteristics, can itself act as a coagulant, have become most widespread. In addition, chitosan is completely safe for humans and has moderate antimicrobial activity. The mechanism of hemostatic action is based on the following effects:

- sorption of the liquid part of plasma;

- coagulation of erythrocytes;

- adhesion, aggregation and activation of platelets.

Chitosan can absorb from 50 to 300% of liquid from its initial mass, and the rate of sorption depends on the molecular weight and degree of deacetylation of chitosan. Red blood cells in the presence of chitosan lose their biconcave shape, which leads to a decrease in the distance between them, and plasma sorption leads to a concentration of red blood cells and platelets in the damaged area. Coagulation in the presence of chitosan is explained by the “cross-linking” (binding) of red blood cells with polymer chains to form a “lattice” that captures blood elements, which is associated with the positive charge of the chitosan molecule, which attracts red blood cells whose membranes are negatively charged (electrostatic effect). The additional hemostatic effect of chitosan is associated with adhesion, aggregation and platelet activation, which may be associated with the surface properties of the chitosan molecule.

An important feature of chitosan-based MGS is the absence of an exothermic reaction upon dissolution or contact with blood and sorption of its components, which avoids such a strong side effect as a burn, which is characteristic of zeolite-based MGS.

A factor limiting the use of MGS in the form of powders or granules is the inconvenience of their use in field conditions (under the influence of environmental factors such as wind and precipitation), as well as during gushing bleeding. In addition, the use of such MGS complicates subsequent surgical treatment due to the need to remove powder/granules from the wound. In this regard, MGS in the form of dressings, bandages, sheets, sponges, and plasters are of greatest interest.

A hemostatic medical product is known (Patent for invention No. 2681184, published on March 4, 2019), made in the form of a hemostatic sponge, which consists of chitosan, glycerin and an antiseptic drug cross-linked with epoxy resin. In this case, the quantitative ratio of the components should be as follows, wt. %: cross-linked chitosan 75-85, glycerin 14.5-24.5, antiseptic drug - up to 100. The invention enhances the absorption capacity of the sponge due to a certain ratio of selected components in the composition.

The disadvantages of this product include the use of epoxy resin for the chitosan crosslinking process. One of the often observed harmful effects of epoxy resins is irritation of the body, which can cause the appearance of allergic contact dermatitis. Amines are most often used as hardeners for epoxy resins, which also exhibit toxicity and irritation. The use of this precursor for the manufacture of MGS may be a risk factor when used in patients, which consists in the occurrence of unwanted side effects in the form of local irritation.

A hemostatic medical product is known (Patent for PM No. 181203, publ. 07/05/2018), made in the form of a hemostatic patch consisting of a polymer substrate, an absorbent layer located on the substrate in the center, an adhesive substance applied to the substrate from the side of the absorbent layer along the edges, and a protective coating covering the surface of the patch on the side of the absorbent layer, wherein the absorbent layer contains a composition including a chitosan salt and polyhexamethylene guanidine hydrochloride, covalently cross-linked with a polyfunctional compound from a series of glycidyl ethers.

The disadvantages of this product are the relatively narrow range of its application due to low sorption capacity, as well as the limitation of the possible dimensions of the product, which makes it impossible to use it in the case of severe and extensive wounds accompanied by gushing bleeding.

A hemostatic medical product is known (Patent for PM No. 135921, published on December 27, 2013), made in the form of a one-sided or two-sided multilayer dressing and consisting of at least a substrate and an active layer of nonwoven material based on chitosan nanofibers. The active hemostatic layer is placed on one side on the surface of the substrate, which is a non-woven material with a specific gravity of not more than 15 g/ ^m2 , consisting of at least chitosan nanofibers obtained by electrospinning. The substrate consists of one or more layers and contains at least one technological and/or contact layer, with each contact layer placed in close proximity to the active layer and represented by a nonwoven material consisting of aliphatic polyamide nanofibers obtained by electrospinning. The technological layer is made of cellulose or synthetic material. A gel-forming layer, which is a non-woven material consisting of carboxymethylcellulose nanofibers obtained by electrospinning, can be placed between the contact and active layers.

The disadvantage of this product is the labor-intensive process of obtaining chitosan and aliphatic polyamide nanofibers by electrospinning. In addition, a number of specialists note a relatively low hemostatic effect in case of massive arterial bleeding.

A special place among effective means for stopping bleeding is occupied by MGS based on chitosan aerogels. These products have such properties as high specific surface area (from 200 m ² /g and above), high porosity (98-99%) and sorption capacity, low density (0.3-0.03 g/cm ³ ), and also biological compatibility with human tissues and organs. Chitosan aerogels are capable of integrating the unique physicochemical and structural characteristics of aerogels in combination with the natural properties of chitosan itself, which makes it possible to achieve an enhanced hemostatic effect that is more pronounced compared to existing products.

A wound healing and hemostatic agent is known (Patent for invention No. 2709462, published on December 18, 2019), presented in the form of spherical particles of chitosan airgel with a specific surface area from 200 to 600 m ² /g, porosity from 95 to 99% with a combination of mesopores 1 -20 nm up to 1-2 cm ³ /g and macropores larger than 50 nm up to 20-30 cm ³ /g. The group of inventions ensures the creation of a highly effective wound-healing and hemostatic agent based on chitosan, which is biocompatible with human tissue, lacks cytotoxicity, and is easy to remove from the wound surface without causing additional damage.

A distinctive feature of this invention is the first proven hemostatic effect of products based on chitosan aerogels. The main disadvantage is the direct form of the hemostatic agent, namely small spherical particles. As stated earlier, local hemostatic agents in the form of particles are low-effective and inconvenient to use, especially in military field and extreme conditions. The low density of the presented chitosan airgel particles additionally creates difficulties in its use (the possibility of acquiring an electrostatic charge, which causes “particle sticking”). When using products in which particles or granules of chitosan are applied to any base (substrate), their shedding is noted. The combination of these disadvantages indicates a low potential when used as a local hemostatic agent to stop massive bleeding.

Also known is the “Hemostatic agent based on chitosan airgel” (Patent for invention No. 2743425, published on February 18, 2021), which is a complex medical product, namely a non-woven material modified by applying chitosan airgel. Such a product can be made in the form of a bandage, napkin or bandage. The modification is carried out through the method of impregnating the source material with chitosan gel, followed by the use of supercritical drying technology. This product is distinguished by the fact that for its production a non-woven material with a surface density of 20 g/m ² to 105 g/m ² is used, impregnated with chitosan airgel, and relates to a method for producing a hemostatic agent based on chitosan, which consists of preparing a solution of chitosan with subsequent supercritical drying, characterized in that a ready-made chitosan solution with a concentration of 1 to 4 mol/l is poured into a nonwoven material carrier to form a chitosan gel for at least 6 hours, after which the product is washed to a neutral pH and the solvent is replaced with a step-by-step increase solvent concentration of 10-50-70-90-100-100-100% by weight and subsequent drying of the hemostatic agent using a supercritical drying process.

The disadvantages of existing technical solutions are:

• Limitation of the amount of chitosan that can be included in one product, which does not allow achieving optimal hemostatic activity. For example, in the MGS manufactured in accordance with the prototype invention (patent for invention No. 2743425), the amount of chitosan does not exceed 15-20 wt.%, while for the commercially available MGS "Celox", obtained using the technology of thermal bonding of chitosan with non-woven fabric (a technology different from the technology for producing the hemostatic material used in the present invention), the amount of chitosan averages 26 wt%. This disadvantage reduces the hemostatic activity of MGS, which is determined by the chitosan content in the product;

• Limitation of the surface density of the original nonwoven material used for the manufacture of a hemostatic agent, not exceeding 105 g/m ² , and the thickness of the original nonwoven material, which is directly related to the surface density and does not exceed 1 mm. These restrictions reduce the mechanical properties of the finished product (strength and density) and reduce the compression effect on the source of bleeding when placing the product in the wound;

• Low rates of adsorption of local hemostatic agents based on chitosan, which reduces hemostatic activity due to the insufficient effect of hemoconcentration associated with the absorption of the liquid part of the plasma;

• Use of chitosan sodium hydroxide solution in the gelation process, which is a hazardous substance of the 2nd hazard class (GOST 12.1.005-76). In addition, the use of sodium hydroxide as a cross-linking agent does not allow obtaining a material with a controlled internal structure, which subsequently reduces the hemostatic effect of the final product;

• A labor-intensive and low-productivity process of impregnation of the starting nonwoven material, which consists of directly pouring a chitosan solution into special containers, as a result of which, to impregnate each product, it is necessary to separately fill it with a solution of the starting substances within one container for a period of at least 6 hours, after which in these carry out the gelation process in the same containers. This process is practically unsuitable for the industrial production of products based on nonwoven material impregnated with chitosan;

• The need for supercritical drying of each product separately, the impossibility of obtaining ready-made hemostatic material in the form of rolls;

• Lack of possible ranges of change in technical parameters characterizing the process of obtaining hemostatic material;

• In addition to the low technical level of this solution, this impregnation method leads to an uneven distribution of the chitosan solution over the surface and in the thickness of the nonwoven material, which has an extremely negative effect on the quality of the final product and the appearance of defects (the appearance of areas with insufficient or, conversely, excessive amounts of hemostatic agent; fabric creases; high percentage of shedding from areas of excess hemostatic agent content, etc.).

The technical problem is the need to create an effective MGS based on nonwoven material impregnated with chitosan, modified through the use of safe cross-linking agents, which makes it possible to obtain MGS with a developed internal structure and a large specific surface area, with high hemostatic activity. An additional task is to increase the strength, density and adsorbing capacity of the finished product, as well as to develop a fundamentally more advanced method for impregnating non-woven material, by organizing a continuous process in a special installation.

The technical result is a significant increase in the effectiveness of the hemostatic agent by increasing the amount of chitosan in the finished product, using the original nonwoven material with a wider range of characteristics in thickness, surface density and composition, modifying the structure of the hemostatic agent, as well as increasing the density of the finished product compared to the original nonwoven material, combined with an increase in the productivity of the process of obtaining a hemostatic agent and a reduction in its cost due to the automation of individual stages of production, the use of industrial installations for impregnation of the original non-woven material and the extraction of isopropyl alcohol from the finished hemostatic material, reducing the share of manual labor.

The technical result is achieved by the fact that during the implementation of the method for producing a hemostatic agent, a solution with chitosan is prepared for impregnation of the non-woven material, after which a continuous impregnation process is carried out followed by gelation, in which the non-woven material first passes through a bath with a chitosan solution, equipped with a special device for removing excess chitosan, then through a bath with a gelling agent, then the resulting material is washed from unreacted compounds and the aqueous solvent is replaced with isopropyl alcohol, after which the resulting material is subjected to extraction with liquid carbon dioxide under excess pressure, while the material is supplied to the extraction stage in the form of rolls, and not individual products. Also in a preferred embodiment, sodium triphosphate (sodium polyphosphate) is used as a gelling (cross-linking) agent.

In a preferred embodiment of the invention, chitosan with a molecular weight of 60 to 600 kDa and a degree of deacetylation of 70 to 99% is used. Also, in a preferred embodiment of the invention, a non-woven material with a thickness of 0.5-2.0 mm is used, containing at least 50% cellulose fibers. Also, in a preferred embodiment of the invention, the cellulose content in the finished product is at least 25%.

In a preferred embodiment of the invention, the nonwoven material passes through a bath of chitosan solution and a bath of gelling agent at a speed of up to 0.5 m/min. Also, in a preferred embodiment of the invention, solvent extraction with carbon dioxide under excess pressure is carried out with the following parameters: pressure 120 atm, temperature 40 °C. Trisodium phosphate (sodium polyphosphate) is preferably used as a gelling agent.

The proposed invention ensures the production of a highly effective hemostatic agent by modifying its composition and structure, expanding the range of surface density and thickness of the original non-woven material in comparison with the original non-woven material, in combination with increasing the productivity of the process of obtaining the hemostatic agent and reducing its cost due to the automation of individual stages of production , the use of industrial installations for the impregnation of the original nonwoven material and the extraction of isopropyl alcohol from the finished hemostatic material, reducing the share of manual labor.

The method for producing a hemostatic agent based on chitosan is that the non-woven material sequentially and continuously passes through several units of a special installation. First, it goes into a bath, where it is impregnated with a chitosan solution. At the outlet of the bath, a device is installed to separate excess solution. The impregnated nonwoven material then enters a gelation bath, which contains the substances necessary to initiate the process directly within the bulk of the nonwoven material. These substances can also be applied by spraying. The resulting material, if necessary, goes through the stages of washing from unreacted compounds and replacing the solvent (water) with isopropyl alcohol (with a step-by-step increase in the solvent concentration of 30-60-90-100-100-100% by weight) or by carrying out the solvent replacement process in a continuous countercurrent mode. After the solvent replacement process is completed, the material in the form of rolls enters the final stage of the technological process - extraction of isopropyl alcohol from the material with liquid carbon dioxide under excess pressure. Before extraction, additional additives may be added to the material by passing the impregnated material through a solution containing the necessary substances. After extraction, the finished hemostatic material in the form of rolls is sent for storage or goes to the packaging stage in individual packaging.

Increasing the efficiency of hemostasis when using the proposed invention, improving the quality of the finished hemostatic agent, as well as optimizing the process of its production are due to the following factors:

(1) increasing the concentration of chitosan, which has a pronounced hemostatic effect, in the finished product;

(2) the use of non-woven material based on cellulose, which has an additional hemostatic effect, for the manufacture of a hemostatic agent;

(3) the use of non-woven material with a surface density of more than 105 g/ ^m2 and a thickness of more than 1 mm to obtain the finished product, which leads to an improvement in the mechanical properties of the finished product compared to the original non-woven material (increased strength and density) and an increase in the compression effect of hemostatic remedies for the source of bleeding;

(4) the use of sodium triphosphate as a cross-linking agent, which makes it possible to obtain a more developed internal surface of the finished material and ensures an increase in its specific surface area. This helps to increase the area of contact of the hemostatic agent with the blood;

(5) the use of an initial nonwoven material of greater density and thickness, which makes it possible to achieve an increase in the sorption capacity of the finished product;

(6) the use of a special technology for impregnating non-woven material with a chitosan solution, which makes it possible to achieve uniform coverage of the non-woven material with a hemostatic agent and reduce the fraying of the final product;

(7) production of hemostatic material in a special installation, including continuous successive stages of impregnation of nonwoven material, gelation, washing and replacement of the solvent, introduction of additional substances, followed by extraction of isopropyl alcohol with carbon dioxide under excess pressure, which makes it possible to increase the stability of obtaining material with specified parameters and reduce the percentage of defects, increase production productivity and reduce its cost.

The use of the proposed invention will provide a more effective stop of massive venous and arterial bleeding, including by activating one's own blood clotting processes and increasing the compressive effect of the product placed in the wound cavity on the source of bleeding. This will ensure a faster onset of hemostasis without the risk of re-bleeding, as well as a more favorable wound condition after using a hemostatic agent, which will facilitate surgical treatment and speed up recovery. The claimed hemostatic agent is convenient to use due to the uniform distribution of impregnation, which prevents its shedding.

The inventive hemostatic agent is a modified complex medical product, namely a non-woven material based on cellulose, with an applied composition consisting of nanoporous chitosan and additional additives, with a developed internal surface. This product can be made in various designs: bandage, bandage, napkin, sponge.

The modification is achieved through the use of sodium triphosphate (sodium polyphosphate) as a cross-linking agent, as well as by changing the technology of impregnation of the nonwoven material and introducing additional additives at the solvent replacement stage, followed by extraction with liquid carbon dioxide under excess pressure.

Thanks to the modification of the hemostatic agent, a more effective stop of bleeding is ensured when it enters the wound surface or into the wound cavity, in conjunction with a further favorable course of the wound healing process.

Method for producing a hemostatic agent based on nanoporous chitosan

The production of a modified hemostatic agent, which is a non-woven material impregnated with nanoporous chitosan, is carried out in several stages.

(1) dissolution of the dry substance of chitosan. In this stage, dry chitosan powder is dissolved in an aqueous acid solution. The chitosan content in the solution should be in the range from 0.5 to 4 wt%. To prepare the specified aqueous solution of the acid, weak organic acids can be used, for example, acetic, lactic, oxalic, etc. The aqueous solution of the acid is prepared so as to ensure the pH of the solution in the range from 2 to 5 units, it is within these limits that chitosan can be dissolved. It is important to note that the pH of the aqueous acid solution is selected depending on the molecular weight of chitosan. The higher the molecular weight, the lower the required pH of the aqueous acid solution. Thus, a given amount of chitosan powder is added to an aqueous solution of an acid of a given composition, which dissolves with vigorous stirring for at least 6 hours. After this, if there is a solid precipitate in the solution, it is filtered off.

(2) preparation of a gelling (cross-linking) agent.

(3) preparing a solution for washing off unreacted compounds.

(4) impregnation of the nonwoven material with a chitosan solution. At this stage, a continuous impregnation process is implemented, which is carried out in a special installation. The installation is shown in Fig. 1 includes a nonwoven material supply system 1, an impregnation bath 2, a gelation bath 3, a material winding device 4, a device for separating excess solution 5, a material feed rate control system 6. The nonwoven material is fed directly into the impregnation bath 1 with using the nonwoven material supply system 1 and is impregnated in the impregnation bath 1 with a chitosan solution. Next, the impregnated material passes through a device for separating excess solution 5 to form the required thickness of the material. The nonwoven material impregnated with a chitosan solution, after separating the excess solution, enters the gelation bath 3. It consists of a bath and a winding unit. The gelation bath 3 is filled with a solution of a gelling agent (sodium triphosphate), and the winding unit of the gelation bath 3 is also immersed in the corresponding solution. The impregnated material passes through a gelation bath 3, where the entire thickness of the material is converted into a solid gel. Next, it is rolled into a roll using a material winding device 4.

The speed of movement of the material through the installations should not exceed 0.5 m/min, which ensures the necessary uniformity of impregnation and prevents material from being pulled out.

(5a) Instead of passing through the gelation bath 3, the said composition can be applied to the surface of the nonwoven material by spraying. For these purposes, an installation is used for impregnation (production stage 4) and gelation (production stage 5a) using the spraying method, shown in Fig. 2. Such an installation includes a nonwoven material supply system 7, an impregnation bath 8, a conveyor 9, a device for winding material 10, a device for separating excess solution 11, a system for spraying a solution of a gelling (cross-linking) agent 12, a system for controlling the feed rate of material 13.

The proposed impregnation methods greatly increase the productivity of the process of obtaining a hemostatic agent compared to using the direct pouring method in molds of a given size and make it possible to obtain ready-made hemostatic material in the form of rolls, rather than individual products.

(6) washing the impregnated material and replacing the solvent. The resulting material then goes through the stage of washing from unreacted compounds and replacing the aqueous solvent with isopropyl alcohol. Two solvent replacement methods can be used:

a) in the first method, to successfully replace the solvent, the alcohol concentration is increased step by step: 10-50-70-90-100-100-100 wt%. Such a step-by-step replacement allows for satisfactory shrinkage of the gel during replacement, that is, maintaining the specified structural characteristics of the material and increasing the efficiency of the final product;

b) within the framework of the second method, an installation is used to implement stages 6 of washing and replacing the solvent using the method shown in FIG. 3. Compared to the installations described above, the gelation bath can be replaced by a conveyor with a solvent dosing system in a countercurrent mode, when the impregnated material moves towards the solvent flow. This modification will significantly increase the productivity of the installation. In general, such an installation includes a nonwoven material supply system 14, a washing conveyor 15, a solvent replacement conveyor 16, a material winding device 17, a cleaning solution spray system 18, a solvent spray system 19, and a material feed rate control system 20.

(7) introduction of additional additives. If it is necessary to introduce additional additives into the product, they are dissolved in a special bath through which the impregnated material is passed after replacing the solvent. The amount of additional additives is selected to ensure their content in the final material is not less than 0.5 and not more than 5%.

This stage may be absent if the finished product does not involve modification by introducing additional components.

(8) extraction with liquid carbon dioxide under excess pressure, the progress of which is shown in Fig. 4. The material obtained during the previous stages, impregnated with a modified chitosan gel, in the form of rolls, is then subjected to extraction with liquid carbon dioxide under excess pressure in a flow mode. Process parameters: pressure 120 atm, temperature 40 °C, carbon dioxide consumption is selected depending on the volume of the apparatus so as to ensure a process time of no more than 6 hours. To organize the operation of the installation in flow mode, it is equipped with a tank for storing carbon dioxide 21, pumping equipment 22, heat exchangers 23 to ensure temperature conditions, an apparatus 24 for extraction with liquid carbon dioxide, a throttle 25 for relieving pressure in the flow before separating the waste medium, a separator 26 for separation into gaseous carbon dioxide and liquid organic solvent. If necessary, the installation can be equipped with a carbon dioxide and solvent recovery system. The proposed method makes it possible to obtain nanoporous material in the form of rolls, rather than individual products, which increases the productivity of both the extraction process itself and the subsequent packaging of the product in individual packaging, and also simplifies its storage and transportation until packaging.

The finished hemostatic agent is stored in sterile individual packaging.

To obtain a hemostatic agent, chitosan with a molecular weight from 60 to 600 kDa and a degree of deacetylation from 70 to 99% is used. The carrier of chitosan within the framework of the invention is a non-woven material based on cellulose, with a cellulose concentration of at least 50%, the surface density of which should be in the range from 20 to 300 g/ ^m2 (preferably from 60 to 175 g/m2 and a thickness of 0.5 up to 2.0 mm).

The inventive hemostatic agent is used as follows

If bleeding is detected, apply a hemostatic tourniquet or tourniquet above the bleeding wound or, if this is not possible, stop the bleeding with finger pressure. Then clean the wound from blood clots and foreign bodies with a sterile bandage. Next, open the package with the inventive hemostatic agent and apply it, injecting it into the depth of the wound as close as possible to the source of bleeding. A medical dressing package (according to GOST 1179-93) is applied over the wound and manual or other type of wound compression is provided for 1-3 minutes. Then a pressure bandage with gauze or other bandage is applied over the wound. At the stage of providing specialized medical care, the inventive hemostatic agent is completely removed from the wound. In most cases, this occurs atraumatically without pain or trauma to the underlying tissues. Moreover, after removing the hemostatic agent, the wound remains clean, since particles of the active composition do not fall into the wound, and blood clots are adsorbed on the surface of the hemostatic agent; the color of the underlying tissues after removal of the hemostatic agent is not changed. There is no recurrence of bleeding after removal of the hemostatic agent associated with failure or detachment of a blood clot at the site of vessel damage. When removing a product adhering to a wound, it is recommended to moisturize it to reduce trauma to the wound tissue and eliminate pain effects in patients.

The product in the form of a bandage is convenient to use to stop massive bleeding. The product in the form of a napkin or bandage is convenient to use when applied to a wound with light or moderate bleeding. Moreover, after removing the napkin, the wound remains clean, since particles of the active composition remain on the surface of the napkin and do not enter the wound. When using a napkin, you must apply it to the wound and hold it until the bleeding stops completely.

The claimed invention is illustrated by examples.

Example 1.

Preparation of various variants of a chitosan-based hemostatic agent, including a nonwoven material impregnated with a composition with nanoporous chitosan.

The density, thickness and cellulose content of the original nonwoven material were varied, as well as the molecular weight and degree of deacetylation of the chitosan used to impregnate the nonwoven material.

At the beginning of the method, a solution with chitosan was prepared for impregnation of the nonwoven material, after which a continuous impregnation process was carried out, followed by gelation, in which the nonwoven material sequentially passed through a bath with a chitosan solution and then through a bath with a gelling agent, after which the resulting material was washed from unreacted compounds and replacing the aqueous solvent with isopropyl alcohol. The final stage of obtaining hemostatic material was the extraction of isopropyl alcohol with liquid carbon dioxide. As a result, a hemostatic material was obtained, in which the density indicators, chitosan and cellulose content were varied, with the following parameters (Table 1).

The yield of the resulting products was 85% (based on chitosan and the original nonwoven material), the estimated productivity of the production method was about 10,000 products per month (with dimensions of 8.0 * 150 cm).

Table 1. Parameters of prepared hemostatic agents. Sample No. Parameters of the original non-woven fabric Chitosan used Finished product parameters Cellulose, wt.% Staple fiber (polyester), wt.% Thickness, mm Molecular weight, kDa Deacetylation degree, % Density, g/ ^m2 Amount of chitosan, wt.% Amount of cellulose, wt.% 1-1 50 50 0.4 50 80 105 25 37.5 1-2 70 31 2.1 300 60 201 25 52.5 1-3 50 50 0.5 300 80 105 24 38.0 1-4 50 50 1 300 80 115 15 42.5 1-5 33 67 0.5 300 80 106 25 24.5 1-6 50 50 1 300 80 125 25 37.5 1-7 70 thirty 1.5 300 80 149 25 52.5 1-8 90 10 2 300 80 200 25 67.5 1-9 50 50 1.5 300 90 185 35 32.5 1-10 50 50 1.2 300 80 165 45 27.5 1-11 50 50 1 300 80 140 50 25 1-12 50 50 0.5 300 95 125 55 22.5 1-13 50 50 1 60 90 106 25 37.5 1-14 50 50 1 600 70 106 25 37.5 1-15 50 50 1 300 80 106 25 37.5 1-16 50 50 1 300 90 106 25 37.5 1-17 50 50 1 300 99 106 25 37.5 1-18 50 50 0.5 300 65 106 25 37.5 1-19 50 50 0.4 300 70 106 25 37.5 1-20 50 50 2 605 80 175 25 37.5 1-21 50 50 2.2 600 80 200 25 37.5

The obtained samples were studied for the main parameters characterizing hemostatic activity under in vitro conditions. The sorption capacity for water (1), the sorption capacity for blood (2) and the blood coagulation time when adding a hemostatic material/agent of a standard size (3) to the sample were assessed. Commercially available local hemostatic agents based on chitosan were used as comparison samples: “Celox Z-folded hemostatic dressing” - comparison sample 1 (non-woven material on the surface of which chitosan is applied by thermal bonding) and “Hemotex hemostatic dressing agent” - comparison sample 2 (non-woven material impregnated with chitosan airgel, manufactured on the basis of patent RU 2743425, which is used as a prototype in this invention).

To evaluate the effectiveness of samples in vitro, the following methods were used:

1) Checking the sorption capacity for water.

A sample measuring 5*5 cm (area 25 cm2) is cut out from the hemostatic agent and weighed (weight of the dry sample - W1), after which it is placed in a dry Petri dish. For work, use medical tweezers. 20 ml of distilled water, preheated to a temperature of 37 °C, is added to the sample and left for 30 minutes in a thermostat at 37 °C. After 30 minutes, the sample is removed and weighed (weight of the sample with liquid - W2). The sorption capacity of the sample is calculated using the formula:

Sorption capacity for water = (W2 - W1)*4,

and is expressed in the mass of liquid in grams absorbed by a sample with S = 100 cm2.

The analysis was carried out for five samples of identical composition; the median, 1st and 3rd quartiles were calculated for each. A value of at least 12 g/100 cm2 was considered satisfactory. The obtained values for samples developed in accordance with the present invention were compared with comparison sample 1 (Celox) and comparison sample 2 (Hemotex) using the Wilcoxon method. Differences were considered significant at p<0.05.

2) Checking the sorption capacity of the blood.

Absorbency was determined according to the method [Terrill P., Sussman G., Bailey M. Absorption of blood by most wound healing dressings // Primary Intension. 2003. - Vol. 11, N 1. - P. 7-10, 12-17].

A sample measuring 5*5 cm (area 25 cm ² ) is cut out from the hemostatic agent and weighed (weight of dry sample - W1), after which it is placed in a dry Petri dish. For work, use medical tweezers. 20 ml of whole donor blood containing the anticoagulant sodium citrate in a ratio of 1:9 is added to the sample and left for 30 minutes in a thermostat at 37 °C. After 30 minutes, the sample is removed and weighed (weight of the sample with liquid - W2). The sorption capacity of the sample is calculated using the formula:

Sorption capacity for blood = (W2 - W1)*4 ,

Absorbency is expressed in the mass of liquid in grams absorbed by a sample with S = 100 cm ² .

The analysis was carried out for five samples of identical composition; the median, 1st and 3rd quartiles were calculated for each. A value of at least 12 g/100 cm2 was considered satisfactory. The obtained values for samples developed in accordance with the present invention were compared with comparison sample 1 (Celox) and comparison sample 2 (Hemotex) using the Wilcoxon method. Differences were considered significant at p<0.05.

3) Determination of coagulation time.

To determine the coagulation time, a modified method is used [Wu Sh., Huang Zh., Yue J. et al. The efficient hemostatic effect of Antarctic krill chitosan is related to its hydration property // Carbohydrate Polymers. 2015. - N 132. - P. 295-303].

A sample of MGS measuring 1 cm * 1 cm is placed at the bottom of the test tube/vial. Next, 1 ml of whole donor blood (without coagulation inhibitors) and 100 μl of 0.2 M calcium chloride solution are added to the test sample at 37°C, mixed and the stopwatch is turned on. The tubes are tilted every 30 s and the time of clot formation is recorded.

The analysis was carried out for five samples of identical composition; the median, 1st and 3rd quartiles were calculated for each. An indicator value of no more than 15 minutes was considered satisfactory. The obtained values for samples developed in accordance with the present invention were compared with comparison sample 1 (Celox) and comparison sample 2 (Hemotex) using the Wilcoxon method. Differences were considered significant at p<0.05.

Additionally, for all samples, the temperature increase during humidification, which in all cases did not exceed 5 °C, and the weight loss during drying, which did not exceed 10%, were assessed.

Samples with the shortest coagulation time were considered preferable; then samples with the highest values of sorption capacity for water and blood were identified (Table 2).

Table 2. Test results of the obtained hemostatic agents Sample No. Sorption capacity, g/100 cm ² Coagulation time, min on water by blood Comparison sample 1 (Celox) 17.5 (17.5; 17.8) 15.4 (15.3; 15.6) 14.8 (14.6; 15.4) Comparison sample 2 12.4 (12.3; 12.5) 15.7 (15.3; 15.8) 13.6 (12.7; 13.9) 1-1 11.4 (10.8; 12.1) ^* 12.3 (11.4; 13.2) ^*,# 24.4 (22.7; 26.0) ^*,# 1-2 15.4 (14.4; 16.3) ^*,# 13.0 (12.0; 13.8) ^*,# 23.8 (22.1 25.3) ^*,# 1-3 11.9 (11.2 12.6) ^* 14.1 (12.9; 15.0) ^* 20.7 (19.2; 22.0) ^*,# 1-4 19.2 (18.0; 20.4) ^*,# 14.3 (13.3; 15.4) 24.7 (23.0; 26.3) ^*,# 1-5 14.5 (13.7; 15.4) ^*,# 15.1 (14.1 16.2) 12.5 (11.6; 13.3) ^** 1-6 17.3 (15.9; 17.6) ^# 16.0 (15.8; 16.2) ^**,## 9.1 (8.5; 9.7) ^**,## 1-7 18.6 (17.4; 19.7) ^# 15.9 (14.9; 17.1) 10.3 (9.6; 11.0) ^** 1-8 20.1 (18.8; 21.1) ^**.# 15.6 (14.3; 16.6) 13.3 (12.4; 14.2) ^** 1-9 18.0 (16.8; 19.1) ^# 15.4 (14.3 16.5) 12.4 (11.5; 13.2) ^** 1-10 17.5 (16.4; 18.6) ^# 15.2 (14.2 16.3) 12.9 (12.0; 13.7) ^** 1-11 18.0 (17.4; 18.1) ^# 16.2 (15.7; 17.3) 9.4 (8.8; 10.0) ^**,## 1-12 17.0 (16.0; 17.8) ^# 15.8 (15.4; 16.9) 9.6 (9.4; 9.6) ^** 1-13 14.0 (13.1; 14.8) ^*,# 15.7 (14.6; 16.8) 14.0 (13.7; 14.9) 1-14 14.2 (13.3 15.1) ^*,# 15.4 (14.2; 16.5) 12.0 (11.2; 12.8) ^** 1-15 15.0 (14.0; 15.9) ^*,# 15.9 (14.5; 17.0) 9.7 (9.0; 10.3) ^**,## 1-16 14.7 (13.7; 15.5) ^*,# 15.8 (14.8; 17.0) 9.5 (8.8; 10.1) ^**,## 1-17 14.3 (13.4; 15.2) ^*,# 15.0 (13.9; 16.0) 12.7 (11.8; 13.5) ^** 1-18 14.0 (13.1; 14.8) ^*,# 15.2 (14.0; 16.2) 14.0 (13.0; 14.9) 1-19 14.2 (13.3; 15.0) ^*,# 15.1 (14.0; 16.2) 14.7 (13.6; 15.6) 1-20 17.9 (16.7; 19.0) ^# 15.6 (14.5; 16.8) 13.6 (12.6; 14.5) ^** 1-21 19.0 (17.8; 20.2) ^# 15.0 (13.7; 15.7) 13.8 (12.9; 14.7) Notes:
* - differences with the group “comparison sample 1” are significant at p<0.05 (deterioration of the indicator relative to comparison sample 1);
** - differences with the group “comparison sample 1” are significant at p<0.05 (improvement of the indicator relative to comparison sample 1);
# - differences with the “comparison sample 2” group are significant at p<0.05 (deterioration of the indicator relative to comparison sample 2);
## - differences with the group “comparison sample 2” are significant at p<0.05 (improvement of the indicator relative to comparison sample 2)

The results obtained allowed us to draw the following conclusions:

- the best in vitro efficiency indicators were obtained for samples 1-6, 1-11, 1-15, 1-16. For these samples, the coagulation time value was statistically significantly less, for comparison samples 1 and 2, the sorption capacity for blood was comparable to the comparison samples, and the sorption capacity for water was higher than for comparison sample 2;

- for samples 1-5, 1-7 - 1-10, 1-12, 1-14, 1-17, 1-20, the coagulation time value was statistically significantly less than for comparison sample 1, and did not differ from the comparison sample 2. For these samples, the sorption capacity for blood was comparable to the corresponding values for comparison samples 1 and 2, and the sorption capacity for water was no worse than for comparison sample 2;

- for samples 1-13, 1-18, 1-19, 1-21, the values of coagulation time and sorption capacity for blood were comparable with comparison samples 1 and 2, and the sorption capacity for water was no worse than for comparison sample 2;

- for samples 1-1, 1-2, 1-3, 1-4, the coagulation time was statistically significantly longer, and the sorption capacity for blood was lower than for comparison samples 1 and 2.

Thus, the best results, superior to comparison samples in terms of in vitro efficiency, were obtained for samples 1-6, 1 11, 1-15, 1-16. The efficiency assessment results for samples 1-5, 1-7 - 1-10, 1-12 - 1-14, 1-17 - 1-21 were satisfactory, which confirms the possibility of their use for the production of MGS. The results of the effectiveness assessment for samples 1-1 - 1-4 were unsatisfactory, which does not allow us to recommend them for the production of local hemostatic agents

Based on the results obtained, it can be concluded that the optimal effectiveness of the hemostatic agent is achieved in the following cases: (1) the non-woven material used for the manufacture of the hemostatic agent should have a thickness of 0.5 to 2.0 mm, (2) the cellulose content in the original non-woven the material must be at least 50%, (3) the cellulose content in the finished product must be at least 25%, (4) for the manufacture of a hemostatic agent, chitosan with a molecular weight of 60 kDa to 600 kDa and a degree of deacetylation from 70% to 99 must be used %.

Example 2.

A method for producing a hemostatic agent based on chitosan, including a nonwoven material impregnated with a composition with nanoporous chitosan.

First, a solution with chitosan was prepared to impregnate the nonwoven material, after which a continuous impregnation process was carried out, followed by gelation, in which the nonwoven material first passed through a bath with a solution with chitosan, and then through a bath with a gelling agent, then the resulting material was washed from unreacted compounds and replacing the aqueous solvent with isopropyl alcohol, after which the resulting material was subjected to extraction of isopropyl alcohol with liquid carbon dioxide under excess pressure. When implementing the method, the speed of the web, the extraction parameters, and the gelling agent used were varied. For the manufacture of a hemostatic agent, non-woven material was used with the following parameters: surface density 100 g/m ² , the ratio of cellulose and staple fiber was 70:30; Chitosan with a molecular weight of 300 kDa and a degree of deacetylation of 85% was used for impregnation.

As a result, hemostatic material with different density, chitosan and cellulose contents was obtained (Table 3).

Table 3. Parameters of prepared hemostatic agents. Sample No. Web speed, m/min Extraction parameters Gelling agent Finished product parameters Temperature, °C Pressure, atm. Density, g/ ^m2 Amount of chitosan, wt.% Amount of cellulose, wt.% 2-1 0.2 40 120 NaOH unsatisfactory properties of the sample: settling of chitosan in the form of uneven layers, peeling of chitosan after extraction; high consumption of chitosan, low process speed 2-2 0.3 40 120 NaOH 130 29 50.0 2-3 0.5 40 120 NaOH 128 28 50.4 2-4 1.0 40 120 NaOH 125 25 52.5 2-5 1.1 40 120 NaOH unsatisfactory properties of the sample: uneven impregnation, insufficient amount of chitosan 2-6 0.5 80 120 NaOH 138 28 50.3 2-7 0.5 35 120 NaOH extraction failed 2-8 0.5 85 120 NaOH unsatisfactory mechanical properties of the material after extraction (increased fragility, low tensile strength) 2-9 0.5 40 85 NaOH extraction failed 2-10 0.5 40 90 NaOH 134 28 50.4 2-11 0.5 40 200 NaOH 132 28 50.5 2-12 0.5 40 205 NaOH excess consumption of liquid CO2, increasing the cost of the material production process 2-13 0.5 40 120 Sodium polyphosphate 128 28 50.4

The effectiveness of the obtained samples of hemostatic agents was tested in vitro (Table 4). Sample No. Sorption capacity, g/100 cm ² Coagulation time, min on water by blood Comparison sample 1
("Celox") 17.5 (17.5; 17.8) 15.4 (15.3; 15.6) 14.8 (14.6; 15.4) Comparison sample 2 12.4 (12.3; 12.5) 15.7 (15.3; 15.8) 13.6 (12.7; 13.9) 2-2 17.0 (16.5; 17.2) ^# 15.8 (15.5; 16.9) 10.1 (10.0; 10.8) ^**,# 2-3 17.3 (15.9; 17.6) ^# 16.0 (15.8; 16.2) ^**,# 9.4 (8.9; 9.7) ^**,# 2-4 17.0 (15.9; 17.4) ^*,# 15.9 (14.9; 17.1) 10.4 (9.8; 10.8) ^**,# 2-6 17.2 (16.8; 18.0) ^# 15.6 (14.3; 16.6) 11.5 (11.3; 11.9) ^**,# 2-10 16.7 (16.7; 17.0) ^* 15.4 (14.3; 16.5) 12.3 (11.8; 12.6) ^** 2-11 17.0 (16.4; 17.3) ^* 15.2 (14.2; 16.3) 12.4 (12.4; 13.0) ^** 2-13 18.2 (18.1; 18.4) ^**,# 16.2 (16.1; 16.4) ^**,# 9.0 (8.7; 9.0) ^**,# Notes:
* - differences with the group “comparison sample 1” are significant at p<0.05 (deterioration of the indicator relative to comparison sample 1);
** - differences with the group “comparison sample 1” are significant at p<0.05 (improvement of the indicator relative to comparison sample 1);
# - differences with the group “comparison sample 2” are significant at p<0.05 (improvement of the indicator relative to comparison sample 2)

Hemostatic agents of satisfactory quality were obtained using the following process parameters:

- web speed: 0.3 m/min to 1.0 m/min. At a lower speed, an excess amount of chitosan was deposited on the nonwoven material, which after extraction was distributed unevenly and peeled off, which was accompanied by high shedding. At a higher speed, the amount of chitosan in the finished material decreased, and the material itself was unevenly impregnated. In addition, the material was stretched, which negatively affected its final consumer properties;

- pressure when carrying out extraction with liquid carbon dioxide under excess pressure from 90 to 200 atm. At a lower pressure value, it is technically impossible to carry out successful extraction; at a higher pressure, there was a significant increase in the consumption of the drying agent and an unreasonable increase in the cost of the process of obtaining hemostatic material;

- temperature during extraction with liquid carbon dioxide under excess pressure from 40 °C to 80 °C. At lower temperatures, it is technically impossible to carry out successful extraction, and at higher temperatures, the resulting hemostatic material was characterized by unsatisfactory mechanical properties (fragility and low tensile strength).

The best in vitro efficiency indicators are achieved with the following characteristics of the process for obtaining hemostatic material: material movement speed 0.5 m/min; extraction parameters: pressure 120 atm, temperature 40 °C. Improved performance characteristics can be achieved through use as a gelling agent.

Example 3.

The study of the effectiveness of local MGS obtained in accordance with the invention in in vivo experiments was carried out on an adapted model of femoral artery damage described by B. Kheirabadi et al. [Kheirabadi B.S., Scherer M.R., Estep J.S. [et al.]. Safety evaluation of new hemostatic dressing in a model of extremity arterial hemorrhage in swine // J. trauma injury, infection and critical care. 2009. Vol. 67, N 3. P. 450-459], modified by Samokhvalov I.M. et al. [Samokhvalov I.M., Reva V.A., Denisov A.V. and others. Improvement of the experimental model for studying the effectiveness of local hemostatic agents // Military Medical Journal. - 2015. - No. 3. - P. 19-25], on adult sexually mature male rams. Application of MGS samples was performed after free arterial bleeding lasting 30 s. After placing the MGS sample in the wound, compression was applied to the wound area with both hands using three modes: for 1 minute, for 2 minutes and for 3 minutes. After compression was completed, a tight tamponade of the wound was performed. After 5 minutes, primary hemostasis was assessed, after which a pressure bandage was applied to the wound area. 180 minutes after assessing primary hemostasis, the tampons covering the wound were removed and secondary hemostasis was assessed. After this, the bandage was removed from the wound and a “march test” was performed (5 flexion-extension movements of the hind limb). After the “march test” was completed, a final assessment of hemostasis was performed.

The effectiveness of MGS was assessed by the rate of bleeding control and the presence of recurrent bleeding. During the experiments the following was recorded:

- presence of primary hemostasis;

- presence of secondary hemostasis;

- presence of final hemostasis (absence of bleeding after performing the “march test”);

- approximate volume of blood loss (by weighing the dressing material before and after the experiment).

For in vivo testing, two samples of hemostatic material were used, which showed the best effectiveness in vitro. Samples of hemostatic material were cut into bandages measuring 8.0*150 cm and folded in a Z-shape. For comparison, we used the commercially available Celox MGS. (Table 5).

Table 5. Parameters of hemostatic material samples used to evaluate in vivo effectiveness Sample No. Series of bandages Chitosan used Finished product parameters Molecular weight, kDa Deacetylation degree, % Density, g/ ^m2 Amount of chitosan, wt.% Amount of cellulose, wt.% MGS "Celox" - ^* - ^* 288±83.5 ^# 26.0±6.30 ^# - ^* 1-6 Episode 1 300 80 125 25 37.5 1-16 Episode 2 300 90 106 25 37.5 Notes:
* - parameter unknown (cannot be determined on the finished product)
# - calculated indicator obtained for 4 MGS "Celox"

The effectiveness of bandages made in accordance with the claimed invention was studied under three compression modes. For the Celox MGS, the instructions for use provide for compression lasting at least 3 minutes, and therefore one compression mode was chosen for the comparison sample - 3 minutes (Table 6).

Table 6. Evaluation of the effectiveness of MGS in in vivo experiments Sample Compression time and efficiency indicators 3 min 2 minutes 1 min primary hemostasis, % secondary hemostasis, % graduated hemostasis, % volume of blood loss, ml primary hemostasis, % secondary hemostasis, % graduated hemostasis, % volume of blood loss, ml primary hemostasis, % secondary hemostasis, % graduated hemostasis, % volume of blood loss, ml Bandage series 1 84.6 (n=13) 100 (n=13) 92.3 (n=13) 75±88.3 83.3 (n=6) 45±70.3 66.7 (n=3) 110±128.4 Bandage series 2 100 (n=9) 31±32.6 100 (n=6) 22±24.3 100 (n=3) 29±36.0 MGS "Celox" 66.7 (n=6) 100 (n=6) 83.3 (n=6) 85±92.5 not assessed not assessed

Based on the experiments conducted, it was established:

- series 2 bandages were effective in the in vivo model with all studied compression modes in 100% of cases, and for these bandages the compression time when used to stop bleeding can be determined at the level of 1 minute;

- series 1 bandages were less effective and provided 100% secondary hemostasis only with a compression time of 3 minutes. With a compression time of at least 2 minutes, they provided primary hemostasis, secondary and final hemostasis - in 83% of cases, which allows us to determine the sufficient compression time for these bandages at the level of 2 minutes;

- in terms of efficiency indicators, series 2 bandages are superior to the Celox MGS at any compression time, although the identified differences are not statistically significant, and series 1 bandages are comparable to the comparison sample.

In parallel with assessing the effectiveness of hemostasis, the subjective state of the wound was recorded after using bandages of series 1 and 2, as well as Celox MGS. The subjectively good condition of the wound (“dry” wound) was understood as the absence of discharge, clots, and stagnant blood; absence of small hemorrhages in the muscles surrounding the vascular bundle; unchanged color of adjacent tissues.

When using bandages of series 2, subjectively good wound condition was noted in 100% of cases (n=9), when using bandages of series 1 - in 76.9% of cases (n=13), when using MGS "Celox" - in 83.3% cases. In the absence of a “dry” wound, blood clots and blood clots, as well as liquid discharge, were detected in the wound pockets.

In addition to the effectiveness and study of the wound condition, the ease of use of MGS was assessed. The use of series 1 and 2 bandages was more convenient compared to Celox MGS for the following reasons:

- the bandages were softer compared to the Celox MGS, and did not have a hard “scratching” (irritating) coating, and when bent they did not form sharp and hard edges;

- bandages easily took the shape of the wound, which made it possible to better “fill” all wound cavities and pockets;

- bandages passed through the wound opening more easily, which subjectively ensured their faster use;

- when using bandages, there was practically no shedding of the composition with which the bandage was impregnated.

Claims (5)

1. A method for producing a chitosan-based hemostatic agent, during which a chitosan solution is prepared for impregnation of the nonwoven material, after which a continuous impregnation process is carried out followed by gelation, in which the nonwoven material first passes through a bath with a chitosan solution, then through a bath with a gelling agent, after which the resulting material is washed from unreacted compounds, the aqueous solvent is replaced with isopropyl alcohol, and the isopropyl alcohol in the resulting material is extracted with liquid carbon dioxide under excess pressure. 2. The method according to claim 1, characterized in that the nonwoven material passes through a bath with a chitosan solution and a bath with a gelling agent at a speed of up to 0.5 m/min. 3. The method according to claim 1, characterized in that sodium triphosphate is used as a gelling agent. 4. The method according to claim 1, characterized in that the extraction of isopropyl alcohol with liquid carbon dioxide under excess pressure is carried out with the following parameters: pressure 120 atm, temperature 40°C. 5. The method according to claim 1, characterized in that the hemostatic material after extraction of isopropyl alcohol is presented in the form of rolls.