RU2759517C1 - Method for obtaining pharmaceutical sulfated glycoaminoglycans from biological tissues - Google Patents

Abstract

FIELD: pharmaceutical industry.SUBSTANCE: invention relates to the pharmaceutical industry and can be used to obtain sulfated glycoaminoglycans from biological tissues. Raw materials are cleaned, disinfected with sodium hypochlorite, washed with water, and crushed. Alkaline hydrolysis is carried out until the precipitate is completely dissolved. The resulting mixture is neutralized by acidification to pH 5.5-6.5. Enzymatic hydrolysis is carried out using a complex of enzymes papain, trypsin and chymotrypsin at a temperature of 30-40°C. Enzymes are deactivated by heating to a temperature of 65-70°C and the hot solution is sequentially filtered on membranes with a pore size on the final membrane of no more than 0.2 mcm. Ultrafiltration of the enzymatic hydrolyzate is carried out on a membrane with a retention limit of 2.5 kDa. The product retained on the membrane is washed with sodium chloride solution and concentrated to obtain a solution of sulfated glycosaminoglycans. A solution of sulfated glycosaminoglycans is filtered through a membrane with a pore size of 0.2 mcm and used as a finished product or sent to obtain a dry finished product.EFFECT: invention makes it possible to obtain pharmaceutical compositions with a given percentage of specific sulfated glycosaminoglycans, to use a wider range of low-value by-products from which chondroitin sulfates can be isolated, to increase the yield of the target product, to more fully isolate accompanying aGAGs (dermatan sulfate, keratan sulfate), and also to increase the purity of the final product.4 cl, 2 tbl, 4 ex

Description

The invention relates to the field of pharmaceutical industry and can be used to obtain sulfated glycoaminoglycans, such as chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate.

Glycosaminoglycans are major components of many tissues, including cartilage, skin, tendons, and ligaments. Glycosaminoglycans were found in the umbilical cord, arteries, cornea of the eye, in the valves of the heart and aorta, spleen, brain, saliva. Several types of sulfated glycosaminoglycans (sGAGs) are known. The most studied four representatives: chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate.

Functions of sGAG: substitution of proteoglycans in the structure, suppression of biosynthesis of inflammatory mediators, increase of biosynthesis of matrix components, suppression of the synergistic action of enzymes and oxygen radicals, suppression of apoptosis, masking of secondary antigenic determinants, masking of secondary antigenic determinants and suppression of chemotaxis, building collagen fibers, stimulates the synthesis of hyaluronic acid.

SGAG is used in pharmacology and cosmetology as basic substances, as well as in combination with sodium hyaluronate.

To obtain sGAG, by-products are used: cartilage, ligaments, trachea, skin, veins of cattle, cornea of eyes, tissues of marine organisms. The composition of the final product depends on the by-products used. The content of certain sGAGs varies in different tissues, which is shown in Table 1.

Table 1

SGAG name Molecular weight, Yes Where is it contained or was found Keratan sulfate from 4 × 10 ³ to 20 × 10 ³ First isolated from the cornea of the eye, later found in other tissues, including the bones of the skeleton. It was found that the proteoglycan complexes of CS in the skeleton and cornea differ structurally and have different metabolism. CS is present in the central nervous system, its function as a regulator of neuronal regeneration was found.
According to a comparative study, the cornea contains 10 times more CS than cartilage and 2–4 times more than any other tissue. Chondroitin-
4-sulfate (X4C) from 5 × 10 ³ to 100 × 10 ³ Contained in cartilaginous and bone tissues, trachea, heart septa, prevails in tissues of animals and humans (~ 70%). Chondroitin-
6-sulfate (X6C) from 5 × 10 ³ to 100 × 10 ³ Contained in cartilage and bone tissues. Х6С predominates in tissues
Aquatic organisms (up to 80%), previously it was obtained from the cartilage (fins) of a shark Dermatan sulfate (DS) from 15 × 10 ³ to 40 × 10 ³ It predominates in skin tissues, also occurs in heart valves, tendons and walls of arteries, lungs, equine aorta, umbilical cord, pig skin, pig intestinal mucosa, spleen, intervertebral disc, meniscus, etc.

It has been shown that clinical efficacy and safety depend on the nature and quality of raw materials used for production, methods of technological processing, degree of purification (Chondroitin sodium sulfate - impurities and problems of standardization (literature review) E.L. Komarova, S.V. Chernova, K. V. Kasumova, M. S. Tabachnaya, L. V. Ovsyannikova, K. I. Eller, "Russian Biotherapeutic Journal", volume 18, No. 1 (2019), pp. 25-36).

Known methods of obtaining chondroitin sulfate from marine echinoderms, for example, sea cucumbers (Japanese application 63-10601, 1988) or cartilage of animals ("Scand. Arch. Physrol." 1989. VI, p. 210-215), however, get crude preparations containing a significant amount of impurities of other polysaccharides and proteins.

The known method described in the application FR 2491475 (1982), upon receipt of chondroitin sulfate A, crushed bovine trachea is hydrolyzed with a 1.25% aqueous solution of pepsin at pH 4.5 and a temperature of 50 ^o C for 12 hours, the enzyme is inactivated at 80 -90 ^o C, the precipitate is separated by centrifugation, washed with hot water, the washings and the supernatant are combined and sequentially treated first at pH 9-10, and then at pH 6.5, heated to boiling, cooled, the precipitate is separated, the supernatant is treated 5 % solution of cetylpyridinium chloride, the precipitate is separated, washed with methanol, dissolved in water, centrifuged and the precipitate is dried.

A known method of producing chondroitin sulfate A, including enzymatic hydrolysis of bovine trachea with pepsin, inactivation of the enzyme at 80-90%, separation of the hydrolyzate by centrifugation, precipitation with cetylpyridinium chloride, dissolving the precipitate in water and drying it, hydrolysis is carried out in 2-3 volumes of 0.5-1% solution pepsin at 38-40 ° C and pH 1.5-2.0 for 4-6 hours, cetylpyridinium chloride is used in an amount of 2-4 g / l solution, while before precipitation, hydrolyzate chromatography is carried out on a column with diethylaminoethyl cellulose using 1-2% HCl as an eluent, and after dissolving the precipitate in water, the product is salted out with sodium chloride at the rate of 20-30 g / l at 2-4 ° C and the target product is obtained by desalting dialysis against water for 12-16 hours (RU 2089557 C1, publ. 09/10/1997 )

There is a known method for producing chondroitin sulfate from salmon nasal cartilage, which involves grinding raw materials, dissolving chondroitin sulfate in an alkaline medium, enzymatic hydrolysis of proteins, separating a high molecular weight carbohydrate fraction by precipitation from low molecular weight protein hydrolysis products remaining in solution, washing the resulting precipitate, and drying the finished product A199 published 02.01.2003).

There is also known a method for the isolation of sulfated glycosaminoglycans (RU 2304441 C1, publ. 20.08.2007), which consists in mechanical cleaning of biological tissue, washing, hydrolysis of the washed tissue with activated papain, ethanol precipitation, while after mechanical cleaning, the crushed tissue is washed twice with heated phosphate buffer in a ratio of 1.5 - 2 volumes of buffer per 1 part of tissue and then kept in a new portion of heated buffer for 15 - 30 minutes, then digested in a solution of activated papain with heating for 6 - 24 hours, the digest is cooled to 4 ° C and kept at this temperature for 2-24 hours, removed by filtration fats and undigested tissue residues, then, within 10 to 24 hours, sulfated glycosimnoglycans are selectively isolated on a solid carrier obtained from bone collagen, the carrier is washed with 0.05-0, 1 N hydrochloric acid solution, defatted, eluted from the carrier with a solution of 0.6-1.5 N mineral salt in 0 , 8 n acetic acid or 0.01-0.025 n alkali solution, precipitated sulfated glycosaminoglycans in ethanol, centrifuged for 15 minutes at 1500 rpm at 4 ° C, the precipitate is washed with ethanol or acetone, dried and sterilized.

This method uses the method of selective adsorption of sGAG on a solid collagen carrier followed by elution. This method is rather slow; non-regenerable solvents (hydrochloric acid, acetic acid, alkali) are used. Also, in this method, ethanol is used without exception to obtain the final product. In this method, it is impossible to obtain the final product with a given percentage of specific sulfate-containing glycosaminoglycans.

The closest to the proposed method is a method for producing chondroitin sulfate using ultrafiltration (RU 2458134 C1, publ. 08/10/2012), according to which chondroitin sulfate is obtained from the tissues of marine organisms, providing for the preparation of raw materials for enzymatic hydrolysis, hydrolysis by proteolytic enzyme preparations with precipitation of protein impurities and separating the precipitate from the hydrolyzate solution, isolating the target product by ultrafiltration, while preliminarily carrying out alkaline hydrolysis with neutralizing the resulting solution to pH 7 and separating the solid precipitate, salt is added to the enzymatic hydrolyzate to a value of at least 0.1 mol / l, ultrafiltration of the enzymatic hydrolyzate on a membrane with a retention limit of 50 kDa and separation of a high-molecular impurity, the resulting solution with a salt concentration in it of at least 0.1 mol / L is subjected to ultrafiltration on a membrane with a retention limit of 5 kDa and low-molecular substances are separated, retained On the chondroitin sulfate membrane, the chondroitin sulfate is washed on the same membrane with distilled water until the salts are completely removed, then the chondroitin sulfate solution is washed with distilled water and the chondroitin sulfate solution is concentrated by ultrafiltration on a membrane with a retention limit of 50 kDa.

The disadvantages of this method is the impossibility of obtaining various sulfated glycosaminoglycans, such as keratan sulfate, dermatan sulfate, since a membrane is used during ultrafiltration with a sufficiently large pore size, which leads to the loss of keratan sulfate and dermatan sulfate. Also disadvantages are the relatively low yield and the impossibility of using a wide range of by-products, as well as the inability to control the composition of the final product.

Sulfated glycosaminoglycans (sGAGs) in tissues are always present together, only the quantitative composition differs. Industrial division into separate sGAGs is a complex, lengthy and costly process that reduces the yield and the possibility of using a variety of low-value by-products. It is advisable to obtain precisely sGAG complexes, which have a greater affinity for the tissues of the human body. It is promising to use these complexes in cosmetology practice to create biologically compatible heterogeneous matrices, as well as in ophthalmology.

This invention solves the technical problem of obtaining pharmaceutical-quality pharmaceutical compositions of a certain composition with a given percentage of specific sulfated glycosaminoglycans, using a wider range of low-value by-products, from which chondroitin sulfates can be isolated (4 and 6), increasing the yield of the target product, more complete isolation of accompanying sGAGs (dermatan sulfate , keratan sulfate), as well as increasing the purity of the final product.

The technical result is achieved by the method of obtaining sulfated glycosaminoglycans from biological tissues, which consists in the fact that the raw material is purified, crushed, alkaline hydrolysis is carried out, the resulting mixture is neutralized and enzymatic hydrolysis is carried out, the enzymatic hydrolyzate is ultrafiltered, the product retained on the membrane is washed with a sodium chloride solution and concentrated with obtaining a solution of sulfated glycosaminoglycans, while, according to the invention, after purification before grinding, the raw material is disinfected with sodium hypochlorite and washed with water, alkaline hydrolysis is carried out until the precipitate is completely dissolved, the mixture is neutralized followed by acidification to pH 5.5-6.5, enzymatic hydrolysis carried out using a complex of enzymes papain, trypsin and chymotrypsin at a temperature of 30-40 ° C, after which the enzymes are deactivated by heating to a temperature of 65-70 ° C (indicate the interval) and the hot solution is filtered sequentially on membranes with a size pores on the final membrane not more than 0.2 μm, ultrafiltration of the resulting enzymatic hydrolyzate is carried out on a membrane with a retention limit of 2.5 kDa, and the solution of sulfated glycosaminoglycans obtained after washing and concentration is filtered through a membrane with a pore size of 0.2 μm and used as a finished product or sent to obtain a dry finished product.

In addition, it is preferable to use a complex of enzymes in the form of a solution with the following concentration values: papain 0.25-0.5 wt.%, Trypsin 0.5-1 wt.%, Chymtrypsin 0.5-1 wt.%.

It is also preferable to concentrate to achieve a concentration of 0.5-1% sulfated glycosaminoglycans.

When a dry finished product is obtained, precipitation with ethanol from the solution of sulfated glycosaminoglycans obtained after filtration is carried out and the resulting precipitate is dried.

The technical result of the proposed method in comparison with the prototype is an increase in the product yield due to the use of a complex of enzymes and by-products are hydrolyzed almost completely, which increases the efficiency of separation of the target product from the hydrolyzate mixture, as well as the use of a membrane with a smaller pore size during ultrafiltration. Also, the technical result of the proposed method in comparison with the prototype is an increase in the purity of the final product as a result of the fact that the hydrolyzate solution is successively filtered before pouring it into the tangential filtration system on membranes with a pore size on the final membrane of no more than 0.2 μm, ultrafiltration of the resulting enzymatic hydrolyzate carried out on a membrane with a retention limit of 2.5 kDa, and the solution of sulfated glycosaminoglycans obtained after washing and concentration is filtered through a membrane with a pore size of 0.2 μm.

It is possible to establish in advance what the% content of components is when using various raw materials within certain limits and to obtain a composition with a component content that is obtained from a certain type of raw material. In addition, when using simultaneously different types of raw materials, the percentage in the final product of a specific sGAG will depend on the percentage of a particular by-product at the input, since different by-products contain different amounts of a certain sGAG. So, for example, the cornea contains the maximum amount of keratan sulfate, and the more of this by-product in the feedstock, the more keratan sulfate in the final product.

The proposed method is carried out as follows.

Material preparation. A feature of this method is that raw materials are used as by-products to obtain pharmaceutical compositions of various composition, which may have a different percentage of by-products, as shown in Table 2.

table 2

Offal Content in raw materials,% Cattle trachea (cattle) 0-100 Cartilage and bursae 0 - 100 Cattle nasal septum cartilage 0 - 100 Heart valves 0 - 100 Tendons and ligaments 0 - 100 Fleshed pork skin 0 - 100 Cattle cornea 0 - 100

First, the material is washed in running water to remove blood and debris. The material is carefully trimmed from the pieces of fat and meat on it. A feature of this method is that the material is disinfected with substances containing active chlorine, preferably with a sodium hypochlorite solution with a concentration of 0.5 - 0.55%, soaking it for 24 - 36 hours in a ratio by weight of 1 part of the material to 3 - 4 parts solution. After that, the material is removed from the sodium hypochlorite solution and washed with distilled water 2 - 3 times at a rate of 1: 1 by weight.

Shredding. The material is passed through a meat grinder and homogenized in a disk grinder to a particle size of 0.005-0.01 mm. The thoroughness of homogenization has a significant effect on the yield of the final product.

Alkaline hydrolysis. A feature of this method is that alkaline hydrolysis is carried out with stirring at a temperature of 20-30 ° C in a sodium hydroxide solution with a concentration of 6.0-6.5% for 24-36 hours until the precipitate is completely dissolved. For alkaline hydrolysis, take 1 part of the raw material for 1 - 2 parts of sodium hydroxide solution by weight. Then the solution is neutralized and acidified to pH = 5.5 - 6.5 with a solution of hydrochloric acid with a concentration of 0.5 - 1 mol / l.

Enzymatic hydrolysis. A feature of this method is that after alkaline hydrolysis, the mixture is acted upon with an enzymatic papain-trypsin-chymotrypsin complex with a ratio of enzymes by weight: papain from 0.25% to 0.5%; trypsin from 0.5% to 1%; chymotrypsin from 0.5% to 1%. For 1 liter of the mixture after neutralization and acidification, take 100 ml of a solution of an enzyme complex with a total concentration of enzymes of 2 - 3%. Enzymatic hydrolysis is carried out at a temperature of 30 - 40 ° C with stirring for 12 - 24 hours while maintaining a pH of 5.5 - 6.5. After a predetermined period of time, the mixture is heated to 70 ° C to deactivate the enzymes.

Filtration. A feature of this method is that the mixture after enzymatic hydrolysis of heating is not cooled, but is immediately filtered on membranes 1.2 μm, then 0.65 μm, then 0.45 mmq and then 0.2 μm. The sterile filtrate enters the filtration system using the reverse osmosis principle.

Ultrafiltration. A feature of this method is that the solution is subjected to ultrafiltration separation on a membrane with a molecular weight retention limit of 2.5 kDa for the most complete yield of sGAGs of various molecular weights and a more complete removal of the amino acid-protein fraction and other impurities. The solution after the previous normal filtration is diluted 2-3 times with a sterile solution of 0.9% sodium chloride and poured into the tangential installation. The solution is washed with 2-3 times a solution of 0.9% sodium chloride by volume of the diluted solution.

Concentration. At the end of the washing process, the concentration process begins, for which the addition of the washing solution to the ultrafiltration system is stopped, as a result of which the volume of the solution containing sGAG that has not passed through the membrane decreases, and its concentration increases. The process is carried out upon reaching a concentration of 0.5 - 2% of the target product in solution.

Normal filtration on a 0.2 µm membrane. After concentration, the solution is filtered through a 0.2 μm membrane. The resulting sterile solution of the complex of target products in physiological sodium chloride solution is used directly, or precipitated with 96% ethanol at a volume ratio of solution: ethanol = 1: 3, then the precipitate is dried, or freeze-dried.

Target product. The yield of sGAG is from 1 to 10% by weight of dry matter to raw by-products. The content of basic substances is 98.3 - 98.5 wt.%. Protein content 0.02 - 0.025 wt%. The content of sodium hyaluronate is 0.5-1 wt%. The percentage of chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate and keratan sulfate ranges from 10% to 98%, depending on the ratio of various by-products in the initial substance. All percentages are by weight.

The content of the components in the target product was determined using HPLC and IR spectrometry. The content of basic substances can be 98.3 - 98.5 wt.%. Protein content - 0.02 - 0.025 wt.%. The content of sodium hyaluronate is 0.5-1 wt%. It has been shown that the content of chondroitin sulfates (4 and 6) can range from 30% to 98.5%; dermatan sulfates from 0.5% to 20.5%; keratan sulfates 0.15% to 80.5%, depending on the ratio of by-products in the raw material. The molecular weight of the components in the target product ranges from 2.5⋅10 ³ Da to 100⋅10 ³ Da. The viscosity of a 1% solution is from 50 mPa⋅s to 230 mPa⋅s.

Example 1

Obtaining sGAG from cattle trachea (100%)

Took 1 kg of cleaned and washed bovine trachea. Filled with a solution of sodium hypochlorite in the amount of 3.5 liters, containing 0.5% sodium hypochlorite and left for bleaching and disinfection of the material for 24 hours. Then the liquid was drained, the trachea was washed three times with 1 liter of distilled water.

The washed material was passed through a meat grinder. The crushed material was placed in a disc grinder. Crushed for 20 minutes.

The porridge-like mass was poured into 1 L of sodium hydroxide solution containing 65 g of base (6.5%), put on a mixer. The suspension was hydrolyzed for 36 hours at 23 ° C with stirring.

The mixture was neutralized and acidified with 1 molar hydrochloric acid solution in a volume of 1630 ml.

The mixture was poured into a solution of papain-trypsin-chymotrypsin complex in a volume of 363 ml, containing 1.452 g of papain (0.4%), 2.904 g of trypsin (0.8%) and 2.904 g of chymotrypsin (0.8%). Fermentolysis was carried out for 24 hours at a temperature of 35 ° C at pH = 5.5. After a predetermined period of time, the mixture was heated to 70 ° C to deactivate the enzymes.

The mixture after enzymatic hydrolysis and heating was not cooled, but immediately filtered on membranes 1.2 μm, then 0.65 μm, then 0.45 μm, and then 0.2 μm.

The sterile filtrate was diluted with 12 liters of 0.9% sodium chloride solution and poured into a tangential filtration system with a 2.5 kDa membrane.

During the ultrafiltration process, another 30 L of 0.9% sodium chloride solution was poured into the system for flushing.

The solution was concentrated to a volume of 8.2 liters.

Then the solution was filtered through a 0.2 μm membrane. Received a clear sterile solution containing 1% sGAG in solution in 0.9% sodium chloride solution. The target product yield was 7.7% by weight. The content of basic substances is 98.7 wt.%. The content of chondroitin sulfates 4, 6 was 97.71 wt.%; dermatan sulfates - 0.43 wt%; keratan sulfates - 0.015 wt%; sodium hyaluronate - 0.55 wt.%. The protein content in dry matter was 0.023 wt%. The viscosity of a 1% solution was 230 mPas.

Example 2.

Obtaining sGAG from bovine nasal septum cartilage (50%) and fleshed pork skin (50%).

Took 500 g of cleaned and washed cartilage of the nasal septa of cattle and 500 g of cleaned, washed and fleshed pork skin.

The process was carried out as described in example 1.

The solution was concentrated to a volume of 3 liters.

Then the solution was filtered through a 0.2 μm membrane. Received a clear sterile solution.

Then the solution was precipitated with 9 liters of 96% ethanol, cooled to a temperature of 4 ° C.

The material was dried to obtain 14.3 g of dry target product.

The target product yield was 1.43%. The content of basic substances - wt. 98.3%. The protein content in dry matter was 0.025 wt%. The viscosity of a 1% solution was 152 mPa · s. The content of components in the target product was determined using HPLC and IR spectrometry. The content of chondroitin sulfates 4, 6 was 89.4%; dermatan sulfates - 9.3 wt%; keratan sulfates - 0.17 wt%; sodium hyaluronate - 0.95 wt.%.

Example 3.

Obtaining sGAG from the cornea of the eye (100%).

Took 1000 g of cleaned and washed corneas of cattle.

The process was carried out as described in example 1.

The solution was concentrated to a volume of 2.6 liters.

Then the solution was filtered through a 0.2 μm membrane. Received a clear sterile solution.

Then the solution was precipitated with 7.8 liters of 96% ethanol, cooled to a temperature of 4 ° C.

The material was dried to obtain 10.9 g of dry target product.

The yield of the target product was 1.09%. The content of basic substances is 98.45 wt%. The protein content in dry matter was 0.02 wt%. The viscosity of a 1% solution was 50 mPa · s. The content of components in the target product was determined using HPLC and IR spectrometry. The keratan sulfate content was 77.8% by weight; chondroitin sulfates (4 and 6) - 18.2 wt.%; dermatan sulfates - 2.6 wt%; sodium hyaluronate - 1.05 wt.%.

Example 4.

Obtaining sGAG from articular cartilage (50%), bursae (25%) and the cornea of the eyes (25%).

Took 500 g of articular cartilage of cattle, 250 g of synovial bags of cattle and 250 g of the cornea of the eyes of cattle.

The process was carried out as described in example 1.

The solution was concentrated to a volume of 4.5 liters.

Then the solution was filtered through a 0.2 μm membrane. Received a clear sterile solution.

Then the solution was precipitated with 13.5 liters of 96% ethanol cooled to a temperature of 4 ° C.

The material was dried to obtain 55.7 g of dry target product.

The yield of the target product was 5.57%. The content of basic substances is 98.35 wt%. The protein content in dry matter was 0.022 wt%. The viscosity of a 1% solution was 97 mPa · s. The content of components in the target product was determined using HPLC and IR spectrometry. The keratan sulfate content was 19.7% by weight; chondroitin sulfates (4 and 6) - 78.1 wt.%; dermatan sulfate - 1.8 wt%; sodium hyaluronate - 0.22 wt.%.

Claims (4)

1. A method of obtaining sulfated glycosaminoglycans from biological tissues, which consists in the fact that the raw material is purified, crushed, alkaline hydrolysis is carried out, the resulting mixture is neutralized and enzymatic hydrolysis is carried out, ultrafiltration of the enzymatic hydrolyzate is carried out, the product retained on the membrane is washed with sodium chloride solution and concentrated to obtain solution of sulfated glycosaminoglycans, characterized in that after purification before grinding, the raw material is disinfected with sodium hypochlorite and washed with water, alkaline hydrolysis is carried out until the precipitate is completely dissolved, the mixture is neutralized followed by acidification to pH 5.5-6.5, enzymatic hydrolysis is carried out using complex of enzymes papain, trypsin and chymotrypsin at a temperature of 30-40 ° C, after which the enzymes are deactivated by heating to a temperature of 65-70 ° C and the hot solution is subsequently filtered on membranes with a pore size on the final membrane of no more than 0.2 μm, ultrafiltration according to The obtained enzymatic hydrolyzate is passed through a membrane with a retention limit of 2.5 kDa, and the solution of sulfated glycosaminoglycans obtained after washing and concentration is filtered through a membrane with a pore size of 0.2 μm and used as a finished product or sent to obtain a dry finished product. 2. The method according to claim 1, characterized in that a complex of enzymes is used in the form of a solution with the following concentration values: papain 0.25-0.5 wt.%, Trypsin 0.5-1 wt.%, Chymtrypsin 0.5- 1 wt%. 3. A method according to claim 1, characterized in that the concentration is carried out until a concentration of 0.5-1% of sulfated glycosaminoglycans is reached. 4. The method according to claim 1, characterized in that the production of a dry finished product is carried out by precipitation with ethanol from the solution of sulfated glycosaminoglycans obtained after filtration and drying the resulting precipitate.