RU2139715C1 - Method of preparing glycosamonoglycans - Google Patents

Abstract

FIELD: pharmaceutical chemistry. SUBSTANCE: connective tissue is disintegrated, successively hydrolyzed with collagenase solution and chemotripsin-papain mixture. After boiling, hydrolyzed material is centrifuged at 3000-5000 rpm, dialyzed for 5-6 days, centrifuged, and treated with ion-exchange resins, after which glycosamonoglycans are precipitated by ethanol and saturated metal acetate solution, washed with 80 and 96.6% ethanol, and dried with ether. EFFECT: increased yield of solid dry preparation.

Description

 The invention relates to medicine, and more particularly to the medical industry, and can be used to obtain salts of glycosaminoglycans from connective tissue, used as medicines.

 The closest analogue to the production of glycosaminoglycans is a method for producing sulfated glycosaminoglycans from corneas of farm animals by hydrolysis with a papain enzyme solution followed by boiling, addition of trichloroacetic acid and dialysis (RF patent N 2056851).

 The disadvantage of this method is that in the tissue treated with one enzyme, part of the glycosaminoglycans remains in the undamaged tissue, especially if the undamaged tissue is dense, which leads to a decrease in the yield of the target product. Using this method, only dissolved glycosaminoglycans can be obtained.

 The objective of the invention is to develop a universal method for producing salts of glycosaminoglycans contained in connective tissues.

 The technical result of the invention is to obtain the target product with a high content of glycosaminoglycan salts in the form of a solid dry preparation.

 The technical result is achieved by the fact that according to the invention, in the method for producing glycosaminoglycan salts, the connective tissue is crushed to a homogeneous state and sequential hydrolysis is carried out with a collagenase solution and a mixture of proteolytic enzymes chymotrypsin and papain, boiled, centrifuged at 3-5 thousand rpm, dialyzed for 5 -6 days, then centrifuged again, treated with ion exchange resins, glycosaminoglycans precipitated with ethanol, saturated metal acetates, washed with 80% and 96.6% ethanol and dried ether for anesthesia.

 The method is as follows.

 Grinding of connective tissue (cornea, cartilage, shark fin, vitreous body) is carried out mechanically using an ultra-chopper until a homogeneous mass is obtained.

The hydrolysis of the tissue is carried out by the enzymatic method, and in the beginning, the enzyme collagenase is used, since this enzyme specifically breaks down collagen molecules - a protein with which glycosaminoglycans are closely connected (located between fibrils of collagen fibers). The optimal concentration of collagenase is 0.33%. The enzyme effectively breaks down collagen at a temperature of 37 ^o C. Hydrolysis with collagenase is carried out for no more than 6 hours, since with a longer treatment the yield of the final product does not increase. Then the temperature of the reaction mixture is increased to 60-65 ^o C and a mixture of proteolytic enzymes chymotrypsin and papain is added to the solution in a ratio of 1: 1, since this mixture most effectively cleaves all other components of the connective tissue. The hydrolysis is carried out at a temperature of 65 ^o C, pH 6.5, for 4 hours, as these are the optimal parameters of the enzyme-substrate interaction. After hydrolysis, the solution is boiled for 15 minutes, after adding trichloroacetic acid to it (to a final concentration of 5%). After hydrolysis, the resulting homogenate is centrifuged at 3-5 thousand rpm. When centrifuging less than 3 thousand rpm, less than 50% of the hydrolyzate precipitates, and at a speed of more than 5 thousand rpm, part of the main product precipitates.

 Dialysis supernatant hydrolyzate is carried out for 5-6 days. During dialysis for less than 5 days, impurities in the form of salts and protein are detected in the solution, and during dialysis for more than 6 days the composition of glycosaminoglycans remains constant, which makes continued dialysis impractical.

 Dialysate centrifugation removes impurities from the solution.

The next significant point in the method is the treatment of the dialysate solution with ion-exchange resins. This treatment allows you to translate glycosaminaglycan in acidic, acid-salt and basic salt forms. For this, after centrifugation, depending on the need, the dialysate is treated with the acid form (H ⁺ form of cation exchange resin with a pH of 2.0-3.0) of an ion-exchange resin to convert glycosaminoglycans into an acid form, then with a salt form to obtain the acid-salt form of the target product ( Na ⁺ form of an ion-exchange resin), which is precipitated with ethanol saturated with metal acetate and the basic salt form of glycosaminoglycans is obtained. As ion exchange resins, amberlites of the IR 40-120 grades with a pH of 2.0-7.0 are most suitable.

 After processing the solution with an ion-exchange resin, glycosaminoglycans are precipitated with ethanol saturated with metal acetate, which makes it possible to obtain acidic, acid-salt, and basic-salt forms of the final product.

 After precipitation, glycosaminoglycans are washed once with 80% ethanol and at least 3 times with 96.6% ethanol, then dried with ether for anesthesia. If necessary, the final drying is carried out in a desiccator with silica gel in vacuum.

 Quantitative and qualitative analysis of the obtained glycosaminoglycan powder is done according to the Provisional Pharmacopoeia Article N 42-2678-96 for the drug "Sulfated glycosaminoglycans."

 The implementation of the method of producing glycosaminoglycans.

 Example 1. From the eyes of pigs, corneas are cut with a razor and placed in a glass vessel to a total mass of 1 kg, after which they are crushed to obtain a homogeneous mass.

Corneal hydrolysis begins at a temperature of 37 ^o C in 0.33% collagenase, which is added to the homogenate. After 6 hours, the homogenate is centrifuged and the supernatant is drained. A mixture of enzymes is added to the homogenate - 50 ml of a 0.01% trypsin solution and 50 ml of 0.125% papain in a 1: 1 ratio. The temperature is brought to 65 ^o C for 4-5 hours. After that, trichloroacetic acid (up to 5%) is added to the hydrolyzate and boiled for 5-30 minutes. After cooling, the mixture is placed in centrifuge glasses and centrifuged at 3 thousand rpm.

The supernatant is drained and the glycosaminoglycan solution is treated with an IR-120 ion exchange resin (Amberlite H ⁺ form) to convert the glycosaminoglycans to an acidic form and then with a Na ⁺ form to obtain the acid-salt form of the final product.

 Next, the target product is precipitated with chilled 96.6% ethanol, saturated sodium acetate and get the main form of glycosaminoglycans.

The mixture is left for 24-48 hours to concentrate the precipitate at a temperature of 2-4 ^o C, washed once with 80% and three times with 96.6% ethanol and dried with ether for anesthesia. Finally, the product is dried in a desiccator with silica gel in vacuum.

 This method allows to obtain salts of glycosaminoglycans in solid dry form for use as medicines.

Claims (1)

 A method of producing glycosaminoglycan salts, including enzymatic hydrolysis of tissue, boiling with trichloroacetic acid and dialysis, characterized in that the tissue is crushed, hydrolyzed sequentially with a collagenase solution and a mixture of chymotrypsin and papain, after boiling, centrifuged at 3-5 thousand rpm, dialyzed for 5-6 days, centrifuged, treated with ion exchange resins, glycosaminoglycans precipitated with ethanol saturated with metal acetate, washed with 80 and 96.6% ethanol and dried with ether for anesthesia.