Method for preparing water-soluble saline complexes of hyaluronic acid (variants)

FIELD: chemical technology.

SUBSTANCE: invention relates to methods for preparing water-soluble saline complexes (associates) of hyaluronic acid with d-metals of IV, V and VI periods of Mendeleyev's periodic system of elements that can be used in pharmacology and cosmetology. Invention describes a method for preparing water-soluble saline complexes of hyaluronic acid involving preparing an aqueous solution of salt of d-metal of IV, V and VI periods of periodic system and its mixing with hyaluronic acid sodium salt, holding the mixture, its stirring, dilution with water and isolation of the end product. For mixing method involves using the amount of aqueous salt of abovementioned d-metal that is equivalent to the amount of carboxy-groups of hyaluronic acid sodium salt or in the limit from 0.95 to 1.10. After dilution with water the solution mixture is subjected for ultrafiltration on separating membranes with simultaneous washing out with aqueous salt solution of abovementioned d-metal firstly and then with deionized water followed by concentrating the product. By another variant for mixing the method involves the amount of aqueous solution of d-metal salt lesser of the equivalent amount of carboxy-groups in hyaluronic acid sodium salt. After dilution with water the mixture is subjected for ultrafiltration on separating membranes with simultaneous washing out with deionized water followed by concentrating the product also. Method is characterized by the decreased time of processes and simplicity.

EFFECT: improved preparing method.

2 cl, 1 tbl

The invention relates to the field of biochemistry, and in particular to methods of obtaining water-soluble salt complexes (associates) of hyaluronic acid with d-metals of the fourth, fifth and sixth periods of the Periodic system of elements Mendeleev, which can be used in pharmacology and cosmetology.

Hyaluronic acid is a naturally occurring linear polyanionic polysaccharide, which is part of the extracellular main substance of the connective tissue of vertebrates in the form of salts and consists of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. The amino sugar in the molecule is connected with D-glucuronic acid β-(1→4)connection, and the acid with the amino sugar β-(1→3)connection. Hyaluronic acid forms salts called hyaluronates, with various inorganic and organic cations, connected with various proteins. Solutions hyaluronates have a high viscosity. Molecular weight isolated from animal tissue hyaluronate is usually in the range from 10⁵up to 10⁷daltons, depending on the type of raw materials, methods of production and purification. Acid has an extremely high hydrodynamic volume, for example, one molecule of acid is able to retain 200-500 water molecules. In humans and animals hyaluronic acid exists in the form of its nutritiously - of sodium hyaluronate. The empirical formula [(C₁₄H₂₀NO₁₁)Na]_nwhere n=250-25000.

In the tissues of the body, hyaluronic acid is involved in the complex, including understudied, physiological processes, for example in the first day of normal uncomplicated healing of wounds in the latter there is increased concentration of hyaluronic acid [Goa K.I., Benfield P. Hyaluronic acid. A review of its pharmacology and use as a surgical aid in ophthalmology and its therapeutic potential in joint disease and wound healing // Drugs. - 1994. - V.47, No. 3, - p.536-566].

A method of obtaining a composition containing cobalt complex of hyaluronic acid, according to which the solution of sodium hyaluronate, add a solution of the corresponding salt, kept under stirring and the precipitated 3 volume parts of ethanol [description of the invention to the U.S. patent No. 5472950, N. Eng. 514/54, publ. 05.12.1995].

This method can not provide after the initial deposition ethanol satisfactory purity of the product - together with hyaluronate always seacadets excess of salts contained in the solution. Thus this method is difficult to obtain a single component (or contains a valid number of impurities) salt hyaluronate complexes.

There is a method of treatment of venous ulcers and pressure sores zinc complex of hyaluronic acid. To do this, get a set of hyaluronate with ka is a Quaternary ammonium ion in the form of sediment, clean it, is dissolved in an aqueous solution of the zinc salt and the solvent partially miscible with water. Then the phases are separated, precipitated appropriate associate alkanols, the precipitate was separated and washed [description of the invention to the U.S. patent No. 5554598, N. Eng. 514/54, publ. 09.10.1996].

This method is complicated further purification of the product from the excess of salts of Quaternary ammonium cations of zinc and the corresponding anions. When dissolved associates, which were obtained by the method of deposition of Quaternary ammonium salts, formed opalescent solutions, which degrades the quality of the product and ultimately allows you to get full of salt hyaluronate.

A method of obtaining biologically active composition, in which the associates of hyaluronic acid ions With²⁺or Zn²⁺in pharmaceutically effective amount mixed with targeted supplements [description of the invention to the patent of Russian Federation №2021304, IPC 08 L 5/08 And 61 To 31/728, a 61 P 17/02, publ. 15.10.1994, bull. No. 19]. In the invention described two typical technological scheme of obtaining cobalt or zinc complexes of hyaluronic acid.

One scheme associat formed of hyaluronate acid and Quaternary ammonium salt in aqueous suspension dissolved in a mixture of solvents containing aqueous solutions ion With²⁺or Zn²⁺with n-butanol, the donkey which associat precipitated using alkanol or Alcanena, allocate the precipitate from the solution and dried final product.

This scheme is complex and requires a large number of chemical ingredients, in addition, it is not possible to obtain full salt of hyaluronic acid.

Most similar in essential characteristics to the first claimed invention is different, described in the patent of the Russian Federation No. 2021304 scheme for water-soluble associates, which includes the preparation of an aqueous solution of metal salt (chloride of zinc or cobalt chloride) and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, its mechanical mixing, dilution with water and subsequent filtration for separation of the final product.

Obtained using this scheme the final product is not sufficiently stable quality. For example, in the filtered product will meet the low molecular weight fragments of hyaluronic acid, unreacted zinc chloride or cobalt chloride, sodium chloride, sodium salt of hyaluronic acid. In this case, you may need additional cleaning. It can be assumed that obtaining declared in the invention technical result is possible with a strict observance of the quantitative ratios of ingredients and consistency technologists is a mini-stages, high purity chemical reagents, as well as the source of sodium hyaluronate.

The task of the first invention group is the development of another method of obtaining high-purity water-soluble salt hyaluronate complexes with metal cations, mainly d-metals of the fourth, fifth and sixth periods of the Periodic system of elements Mendeleev, for medical and cosmetic purposes, which is relatively simple and short process.

The technical result will be the development of a fairly simple method of obtaining a high-purity single-component water-soluble salt complexes of hyaluronic acid containing cations of d-metal, for medical and cosmetic purposes, including the minimum amount of impurities added metal salts and controlled amount released during the reaction of salts of sodium, which differ little time processes and low cost.

To solve the problem in a method of producing a water-soluble salt complexes of hyaluronic acid, comprising obtaining an aqueous solution of salt of d-metal IV, V and VI of the periods of the Periodic system and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, stirring, diluted with water and the selection of course what about the product for mixing use a quantity of an aqueous solution of salt of the above d-metal equivalent to the number of carboxyl groups of the sodium salt of hyaluronic acid or lying in the range from 0.95% up to 1.10% from the equivalent, after dilution with water the mixture solution is subjected to ultrafiltration separation membranes with simultaneous washing first with an aqueous solution of salt of the above d-metal, and then with deionized water, after which the product concentrate.

In addition:

after concentration, the product is subjected to spirooxazine or lyophilized;

after concentration the product is sterile-filtered;

after sterile filtration, the product is subjected to spirooxazine or lyophilized.

Most similar in essential characteristics to the second present invention is also described in the patent of the Russian Federation No. 2021304 scheme for water-soluble associates, which includes the preparation of an aqueous solution of metal salt (chloride of zinc or cobalt chloride) and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, its mechanical mixing, dilution with water and subsequent filtration for separation of the final product.

As mentioned above, obtained using this scheme, the final product of the while not to be stable quality. In the filtered product meet low molecular weight fragments of hyaluronic acid, unreacted zinc chloride or cobalt chloride, sodium chloride, sodium salt of hyaluronic acid. As impurities can be metal salts or Quaternary ammonium salts. The large number of components in the final product does not allow you to talk about predictable pharmacological and cosmetic actions received multicomponent drug on the human body.

The task of the second invention group is the development of another method of obtaining high-purity water-soluble salt hyaluronate complexes with metal cations, mainly d-metals of the fourth, fifth and sixth periods of the Periodic system of elements Mendeleev, for medical and cosmetic purposes, which is relatively simple and short process.

The technical result will be the development of a fairly simple method of obtaining high purity, typically two-component water-soluble salt complexes of hyaluronic acid containing cations of any d-metal cations are sodium, for medical and cosmetic purposes, including the minimum amount of impurities added metal salts and controlled the number is about released during the reaction of salts of sodium, which differ little time processes and low cost.

To solve the problem in a method of producing a water-soluble salt complexes of hyaluronic acid, comprising obtaining an aqueous solution of salt of d-metal IV, V and VI of the periods of the Periodic system and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, stirring, diluted with water and isolation of the final product, for use mixing the aqueous solution of salt of the above d-metal, less equivalent to the number of carboxyl groups of the sodium salt of hyaluronic acid after dilution with water the mixture solution is subjected to ultrafiltration separation membranes with simultaneous washing with deionized water, after which the product concentrate.

In addition:

after concentration, the product is subjected to spirooxazine or lyophilized;

after concentration the product is sterile-filtered;

after sterile filtration, the product is subjected to spirooxazine or lyophilized.

Some information about the effects on the human body some d-metals of the fourth, fifth and sixth periods of the Periodic system of the elements in the composition of various salts and complexes, including the composition of the salt complexes of hyaluronic acid is, you can bring to the table "Biological effects of d-metals in the body (see table).

The essence of the invention group is that of high-purity single-component or multicomponent salt complexes of d-metals hyaluronic acid for medical and cosmetic purposes, containing the minimum amount of impurities, are obtained by a rather simple ways for small periods of time and at low cost. Hereinafter, the term "high-purity single-component salt systems", you should understand almost pure single-component salt complexes of hyaluronic acid, where the percentage of substitution of carboxyl groups in any d-the metal is in the range from 95% to 98% (or higher, if it is economically justified). The remaining carboxyl groups are unsubstituted cations sodium, which in no way affects the quality of the salt of hyaluronic acid and its physico-chemical, pharmacological and cosmetic properties. The term "high-purity multi-component salt systems" should be understood as a rule, two-component salt complexes of any d-metal hyaluronate sodium, where the percentage of substitution of sodium carboxyl groups can be arbitrary, for example, 49% of the salt of d-metal hyaluronic acid and 48% sodium salt (adjusted n the processes of hydrolysis), or 90%+8%, or 8%+90%, etc. depending on the desired pharmacological and or cosmetic effect. Theoretically, two-component, for example 95%+3% salt complex of hyaluronic acid should be considered as a single component, as pharmacological and cosmetic effect will be determined by the component that is 95%. It is possible that in some cases a small amount of one of the components of the salt complex, for example cations of silver as an antibacterial agent or cations of platinum as an antitumor agent, will also affect the living organism the desired effect. However, in these cases, however, it is better to use the sodium salt of hyaluronic acid in combination with specific drugs that have been proven at a certain pharmacological or cosmetic effect, or proceed from considerations of expediency. As for the three (or more) salt complexes of d-metals with sodium hyaluronate, their expected pharmacological and cosmetic effects are difficult to predict. Typically, in such salt complex to a greater extent manifest properties of only one of the incoming d-metals.

In General, the method of obtaining water-soluble salt complexes of hyaluronic to the slots includes obtaining an aqueous solution of metal salt, primarily one of the d-metals of the fourth, fifth and sixth periods of the Periodic system of elements Mendeleev, and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, stirring, diluted with water and isolation of the final product. These methods are based on high complexing ability of d-metals and chemical equilibrium shift due to leaching of cations of sodium during ultrafiltration separation membranes.

To obtain a one-component salt complexes of hyaluronic acid for mixing use a quantity of an aqueous solution of metal salt, equivalent to the amount of carboxyl groups of the sodium salt of hyaluronic acid or lying in the range from 0.95 to 1.10 is the equivalent, after dilution with water the mixture solution is subjected to ultrafiltration separation membranes with simultaneous washing first with an aqueous solution of the salt of the obtained metal, and once with deionized water, using membranes with a certain diameter (size) long, allowing you to break free from of polymer molecules with a molecular mass below the limit of excess inorganic salts and hold in solution concentrate molecules with specific molecular weight, after what stops adding deionized water to the system, the remaining water molecules pass through the membrane, which in turn decreases the volume and increases the concentration of the solution of the finished product.

The number of carboxyl groups of sodium hyaluronate can be calculated from the known formula based on the weight of the sample of sodium hyaluronate. The number of disaccharide glycosides of units n is calculated by the formula

n=m/M

where m is the mass of a sample of sodium hyaluronate;

M - molar mass of disaccharide glycosides units of sodium hyaluronate (equal 401,22 g/mol).

The number of disaccharide glycosides units of sodium hyaluronate in the sample and is equal to the number of carboxyl groups, which gives the possibility to calculate the required number and weight of salts of d-metal to replace the sodium carboxyl groups. For example, in a 1.00 gram of sodium hyaluronate will be the following number of carboxyl groups:

n=1,00/401,22=0,00249 mol.

Depending on the valence d-metal he can attach a different number of carboxyl groups. For example, two carboxyl groups in the hyaluronate have one divalent cation metal, hence the need to take 1/2 of 0,00249 mol carboxyl groups, salts of divalent d-metal for a theoretical 100% replacement of sodium carboxyl groups. For monovalent d-metal it is necessary to take 0,00249 mol salts thereof, etc. In practice, the replacement will be in the range from 95% to 98% of the carboxyl groups.

When COI is whether the amount of aqueous solution of metal salt above is equivalent to the number of carboxyl groups of the sodium salt of hyaluronic acid, for example, 150% of the equivalent turns a full salt of hyaluronic acid, in which, for example, 97.5% of the carboxyl groups are occupied by cations of d-metal. Excess salts will be washed in the ultrafiltration process, however, adding such a quantity of salt is impractical because it leads to overuse of chemicals.

To obtain, for example, two-component salt complexes of hyaluronic acid for use mixing an aqueous solution with a quantity of metal salt is less than an equivalent amount of carboxylic groups of the sodium salt of hyaluronic acid after dilution with water the mixture is subjected to ultrafiltration separation membranes with simultaneous washing with deionized water, in this case, as in the previous case, using membranes with a certain diameter (size) long, allowing you to break free from of polymer molecules with a molecular mass below the limit of excess inorganic salts, and hold in solution concentrate molecules with specific molecular weight, and then stops adding deionized water to the system, the remaining water molecules pass through the membrane, which in turn decreases the volume and increases the concentration of the solution of the finished product.

When using the amount of salt of d-metal in aqueous solution below the equivalent of the CSOs number of carboxyl groups of the sodium salt of hyaluronic acid, for example 50% of the equivalent, the result is a two-component salt complex of hyaluronic acid, which is theoretically 50% of the carboxyl groups are occupied by cations of d-metal and 50% sodium cation.

In both these cases, the concentrated product can be subjected to sterilizing filtration for removal of a possible pathogenic microflora that is required for use of the obtained solution in the form of ready-to-use injectable form. In General obtained in the process of implementing the above-mentioned technical solutions concentrated one - or multicomponent salt complexes of hyaluronic acid acquire marketable after spirooxazine or freeze drying. In this kind of product is tightly Packed, sterilized (if necessary) and is offered for sale.

There is a special relationship between the ratio of sodium cations and cations of d-metals of the fourth, fifth and sixth periods of the Periodic system of the elements in the molecule, hyaluronate. Obviously, the greater the number of cations of sodium carboxyl groups of the molecule hyaluronate substituted with a biologically active metal of the d-group, the greater the biological activity of a molecule of hyaluronate. Getting hyaluronate molecules with different content of biologically active metal of the d-group, you can receive the th different biological activity of drugs hyaluronate. For example, one-component salt hyaluronate complexes of zinc, copper, cobalt, silver, etc. have a more pronounced demand, as in pharmacology and cosmetology, antibacterial properties compared to two-component systems, for example, theoretical 50%+50% (or 80%+20% and others) on the number of carboxyl groups of hyaluronate.

Two-component salt complexes of hyaluronic acid have advantages compared to single-component salt systems in cases, for example when used inorganic cation such as cobalt, shows an acute toxic effect on the body. Receiving a two-component salt complex of hyaluronic acid containing sodium cations and the minimum number, for example 5% or 10% of the number of carboxyl groups of toxic cations of d-metal, for example chromium, reduce or neutralize it to be toxic. For example, using a two-component salt complex of hyaluronic acid containing sodium cations and, for example, 10% of the number of carboxyl groups of the cations of zinc, in addition to restore skin elasticity contributes to the formation of cosmetic scar as a result, for example, various lesions of the skin, preferably in comparison with the use of cosmetic products, sod is rasih mainly zinc component.

Especially important is the reduction in the toxic effect due to minimal introduction of d-metal, when drugs are used, for example, as injection. For example, it can be assumed that the platinum drugs as anticancer agents are introduced into the body, it is better to use a two-part salt complexes containing sodium cations and the minimum content of the platinum cations, for example 1-5%, or 5-10% of the total number of carboxyl groups of sodium hyaluronate is absolutely compatible with the body (with rare exception - depending on your natural reaction) of the polysaccharide.

Water-soluble associates, hyaluronate and cations, such as zinc or copper is obtained by adding salts (preferably chlorides or other) of copper and zinc in a solution of sodium hyaluronate with subsequent ultrafiltration separation membranes, excluding the output hyaluronate, depending on its molecular weight. Zinc and copper are selected from other d-metals due to popular applications in pharmacology and cosmetology various inorganic drugs with their content and the existing consumer demand.

After washing with deionized water on the UF plant solution precipitated with ethyl alcohol to obtain a precipitate of associate hyaluronate honey is or zinc. Dry matter can also get freeze drying or the solution is used immediately after sterilization by filtration through membranes with pores of 0.20 to 0.45 μm. The method of obtaining single-component salt complex of zinc hyaluronate is the following. The sodium hyaluronate in aqueous medium was added an equivalent amount of carboxylic groups of zinc chloride. The solution is incubated with stirring, a day to complete the complexation, then diluted and start to wash out a 0.07% solution of zinc chloride on the UF plant with pores of the membrane, preventing the output of hyaluronate, to remove excess cations sodium and shift of the chemical equilibrium towards the formation of zinc hyaluronate. Then the solution is washed with deionized water to remove excess cations of zinc, chloride ions and cations of sodium. The solution is concentrated and filtered over a membrane with pores of 0.20 to 0.45 μm in order to separate possible adscititious components, such as pathogens, resulting in a ready to use solution or precipitated with ethanol, followed by drying or freeze-dried. This method allows to obtain pure samples of hyaluronate, a carboxyl group which almost completely by 95% and above - saturated cat the areas of zinc and containing less than 5% of the sodium cations.

The chemical composition of the salts: [(C₁₄H₂₀NO₁₁)Zn]_n.

A method of obtaining a two-component zinc-sodium salt hyaluronate complex is the following. The sodium hyaluronate in the aquatic environment add the required quantity, for example not more than 95% of the equivalent carboxyl groups, of zinc chloride in solution. The solution is incubated with stirring, a day to complete the complexation, then diluted and begin to wash with deionized water at UF plant with pores, preventing the output of hyaluronate. Due to the natural processes of leaching of metals is obtained, for example, a salt with the number of carboxyl groups containing cations of zinc, approximately 90%. Then, the solution concentrated and filtered over a membrane with pores of 0.2-0.45 μm, or precipitated with ethanol, followed by drying, or freeze-dried. This method allows to obtain pure samples of the two-component salt hyaluronate, a carboxyl group which is partially saturated with cations of zinc and containing unsubstituted sodium cations.

The chemical composition of the salts: [(C₁₄H₂₀NO₁₁)Zn_xNa_n-x]_n. The method of obtaining single-component salt hyaluronate complex of copper is as follows. The sodium hyaluronate in the same medium was added an equivalent amount, on carboxyl groups, chloride of copper. The solution is incubated with stirring, a day to complete the complexation, then diluted and start to wash out a 0.07% solution of copper chloride on the UF plant with pores of the membrane, preventing the output of hyaluronate, to remove excess cations sodium and shift of the chemical equilibrium towards the formation of hyaluronate copper. Then the solution is washed with deionized water to remove excess copper cations, chloride ions and cations of sodium. The solution is concentrated and filtered over a membrane with pores of 0.20 to 0.45 μm, or precipitated with ethanol, followed by drying, or freeze-dried. This method allows to obtain pure samples of hyaluronate, a carboxyl group which is almost completely saturated with copper cations and does not contain cations of sodium carboxyl groups.

The chemical composition of the salts: [(C₁₄H₂₀NO₁₁)Cu]_n.

A method of obtaining a two-component copper-sodium salt hyaluronate complex is the following. The sodium hyaluronate in the aquatic environment add the required quantity, for example not more than 95% of the equivalent carboxyl groups, chloride of copper in solution. The solution is incubated with stirring, a day to complete the reaction of complexones the project, then dilute and begin to wash with deionized water at UF plant with pores of the membrane, preventing the output of hyaluronate. Due to the natural processes of leaching of metals is obtained, for example, a salt with the number of carboxyl groups containing cations of copper, approximately 89%. The solution is concentrated and filtered over a membrane with pores of 0.20 to 0.45 μm, or precipitated with ethanol, followed by drying, or freeze-dried. This method allows to obtain pure samples of hyaluronate, a carboxyl group which is partially saturated with copper cations and containing unsubstituted sodium cations.

The chemical composition of the salts: [(C₁₄H₂₀NO₁₁)Cu_xNa_n-x]_n.

In the same way, get salt, for example, gold, cobalt, silver and other d-metals of the fourth, fifth and sixth periods of the Periodic system.

Characteristics of sodium hyaluronate used in the examples to obtain the salt of the associates of hyaluronate, zinc and copper:

- average molecular weight 1500000;

the protein content is not more than 0.05 wt.% from solids;

the viscosity of 2% solution of sodium hyaluronate 1450 MPa·C;

the content of sodium hyaluronate 98,53%;

the pH value of an aqueous solution of 6.5.

The invention is illustrated in more detail by the following examples.

Example 1. Getting onecomponent the aqueous salt complex of zinc hyaluronate.

10.0 g of sodium hyaluronate dissolved in 900 ml of deionized water to obtain a 1.1% solution. Prepare to 90.0 ml of 1.9% solution of zinc chloride. Then slowly with stirring, pour in a solution of zinc chloride in a solution of sodium hyaluronate. The solution at room temperature, stirred overnight, after which the mixture is diluted with deionized water to 10 liters of the Solution is subjected to ultrafiltration separation membranes with pores of 0.03 μm, slowly adding, as the solution through the membrane, a 0.07% solution of zinc chloride by volume of 5.0 L. Then the solution is washed with deionized water to a volume of 10 L. Then, the solution concentrated to a volume of 3.5 liters of This solution was precipitated with 3 volumes of high-purity 95% ethanol. The precipitate is collected on a glass filter and dried at a temperature of 60°With under reduced pressure. The result is a complex of zinc hyaluronate mass of 10.0, the product Yield is 93%.

Example 2. Getting one-component salt hyaluronate complex of copper.

10.0 g of sodium hyaluronate dissolved in 900 ml of deionized water to obtain a 1.1% solution. Prepare to 90.0 ml of a 1.8% solution of chloride of copper. Then slowly with stirring, pour in a solution of chloride of copper in a solution of sodium hyaluronate. The solution at room temperature, stirred overnight, after which the mixture is diluted with de is oneiromancy water up to 10 L. The solution is subjected to ultrafiltration separation membranes with pores of 0.03 μm, slowly adding, as the solution through the membrane, a 0.07% solution of chloride of copper by volume of 5.0 L. Then the solution is washed with deionized water to a volume of 10 L. Then, the solution concentrated to a volume of 3.5 liters of This solution was precipitated with 3 volumes of high-purity 95% ethanol. The precipitate is collected on a glass filter and dried at a temperature of 60°With under reduced pressure. The result is a complex hyaluronate copper with a mass of 10.0, the product Yield is 93%.

Example 3. Bicomponent salt complex, hyaluronate, a carboxyl group in which 50% of substituted cations of zinc.

10.0 g of sodium hyaluronate dissolved in 900 ml of deionized water to obtain a 1.1% solution. Prepare to 90.0 ml of 1.0% solution of zinc chloride. Then slowly with stirring, pour in a solution of zinc chloride in a solution of sodium hyaluronate. The solution at room temperature, stirred overnight, after which the mixture is diluted with deionized water to 10 liters of the Solution is subjected to ultrafiltration separation membranes with pores of 0.03 μm, slowly adding, as the solution through the membrane, deionized water with a volume of 10 L. Then, the solution concentrated to a volume of 3.5 liters of This solution was precipitated with 3 volumes of high-purity 95% etano the and. The precipitate is collected on a glass filter and dried at a temperature of 60°With under reduced pressure. The result is 9.7 g of a two-component complex of zinc hyaluronate, approximately 58% of the total mass, and sodium hyaluronate, which is around 35%. Thus, the product yield is 93%.

Example 4. Bicomponent salt complex, hyaluronate, a carboxyl group which 80% of substituted cations of copper.

10.0 g of sodium hyaluronate dissolved in 900 ml of deionized water to obtain a 1.1% solution. Prepare to 90.0 ml of a 1.5% solution of chloride of copper. Then slowly with stirring, pour in a solution of chloride of copper in a solution of sodium hyaluronate. The solution at room temperature, stirred overnight, after which the mixture is diluted with deionized water to 10 liters of the Solution is subjected to ultrafiltration separation membranes with pores of 0.03 μm, slowly adding, as the solution through the membrane, deionized water with a volume of 10 L. Then, the solution concentrated to a volume of 3.5 liters of This solution was precipitated with 3 volumes of high-purity 95% ethanol. The precipitate is collected on a glass filter and dried at a temperature of 60°With under reduced pressure. The result of 9.9 g of a two-component complex hyaluronate copper, approximately 80% of the total mass, and hyaluronan is sodium, approximately 13%. As can be seen, the yield is 93%.

Example 5. Getting 1,0% sterile solution of one-component salt complex of zinc hyaluronate.

10.0 g of sodium hyaluronate dissolved in 900 ml of deionized water to obtain a 1.1% solution. Prepare to 90.0 ml of 1.9% solution of zinc chloride. Then slowly with stirring, pour in a solution of zinc chloride in a solution of sodium hyaluronate. The solution at room temperature, stirred overnight, after which the mixture is diluted with deionized water to 10 liters of the Solution is subjected to ultrafiltration separation membranes with pores of 0.03 μm, slowly adding, as the solution through the membrane, a 0.07% solution of zinc chloride by volume of 5.0 L. Then the solution is washed with deionized water to a volume of 10 L. Then, the solution concentrated to a volume of 990 ml Then the solution is filtered under aseptic conditions through a membrane with pores of 0.45 μm at a pressure of 2-4 bar. Receive 1,0% transparent sterile solution full of salt hyaluronate, zinc.

Example 6. Receive a 0.5% sterile solution of one-component salt hyaluronate complex of copper.

10.0 g of sodium hyaluronate dissolved in 900 ml of deionized water to obtain a 1.1% solution. Prepare to 90.0 ml of a 1.8% solution of chloride of copper. Then slowly with stirring, pour in a solution of chloride of copper in solution Gia is uronate sodium. The solution at room temperature, stirred overnight, after which the mixture is diluted with deionized water to 10 liters of the Solution is subjected to ultrafiltration separation membranes with pores of 0.03 μm, slowly adding, as the solution through the membrane, a 0.07% solution of chloride of copper by volume of 5.0 L. Then the solution is washed with deionized water to a volume of 10 L. Then, the solution to condense the volume 1980 ml. Then the solution is filtered under aseptic conditions through a membrane with pores of 0.45 μm at a pressure of 2-4 bar. Get 0.5% transparent sterile solution full of salt hyaluronate copper.

Examples 7-10. Getting a variety of one-component and two-component salt hyaluronate complexes of copper or zinc corresponds to the methods indicated in examples 1-4, except that after ultrafiltration of the solution on the separation membranes he immediately goes on the lyophilic drying. The product yield accounts for 93.4%.

Thus, the use of the invention may fairly simple ways to get one-component high-purity salt complexes of hyaluronic acid containing cations of any d-metal, or, as a rule, two-component salt complexes of hyaluronic acid, in addition to containing cations of any d-metal and sodium cations, for medical and cosmetic purposes, include the minimum number of impurities added metal salts and controlled amount released during the reaction of salts of sodium, which differ little time processes and low cost.

1. The method of obtaining water-soluble salt complexes of hyaluronic acid, comprising obtaining an aqueous solution of salt of d-metal IV, V and VI of the periods of the Periodic system and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, stirring, diluted with water and isolation of the final product, characterized in that mixing is used, the amount of aqueous solution of salt of the above d-metal equivalent to the number of carboxyl groups of the sodium salt of hyaluronic acid or lying in the range from 0.95 to 1.10 is, after dilution with water the mixture solution is subjected to ultrafiltration separation membranes with simultaneous washing first with an aqueous solution of salt of the above d-metal and after deionized water, after which the product concentrate.

2. The method according to claim 1, characterized in that after concentration, the product is subjected to spirooxazine or lyophilized.

3. The method according to claim 1, characterized in that after koncentrirane the Oia product is subjected to sterile filtration.

4. The method according to claim 3, characterized in that after sterile filtration, the product is subjected to spirooxazine or lyophilized.

5. The method of obtaining water-soluble salt complexes of hyaluronic acid, comprising obtaining an aqueous solution of salt of d-metal IV, V and VI of the periods of the Periodic system and its mixing with the sodium salt of hyaluronic acid, keeping the mixture, stirring, diluted with water and isolation of the final product, characterized in that mixing is used, the amount of aqueous solution of salt of the above d-metal, less equivalent to the number of carboxyl groups of the sodium salt of hyaluronic acid after dilution with water the mixture solution is subjected to ultrafiltration separation membranes with simultaneous washing with deionized water, after which the product concentrate.

6. The method according to claim 1, characterized in that after concentration, the product is subjected to spirooxazine or lyophilized.

7. The method according to claim 1, characterized in that after concentration, the product is subjected to sterile filtration.

8. The method according to claim 7, characterized in that, after sterile filtration, the product is subjected to spirooxazine or lyophilized.