RU2080327C1 - Method of preparing mucopolysaccharide - keratosulfate - Google Patents

Abstract

FIELD: biotechnology, polysaccharides. SUBSTANCE: method involves homogenization of animal raw, its extraction, extract separation, protein removing, precipitation with 50-60% ethanol, deposit dissolving, purification by chromatography on column with cellulose and drying. Animal raw: calf trachea. Extraction is carried out with 5-6 volumes of 0.5-1% sodium hydroxide aqueous solution at 40-60 C for 3-4 h, protein is removed by extract shaking with 3-4 volumes of chloroform for 1-2 h; precipitate after treatment of extract with ethanol is dissolved in 1-2 volumes of 0.2-0.4% hydrochloric acid solution and the purified keratosulfate is prepared by cellulose column washing out with 1-2% solution of methylpyridinium chloride. Before extraction the raw is defatted by multiple washing out with acetone. Product is used in biochemical investigations and in ophthalmology. EFFECT: increased yield of keratosulfate, improved method of preparing.

Description

 The invention relates to the field of polysaccharide chemistry, and specifically to a method for producing high molecular weight polysaccharide keratosulfate, which acts as a moving and bonding material in the body and used in biochemical studies and in ophthalmic practice.

 Known methods for producing keratosulfate from the cornea of the bull and from the intercostal cartilage of sick people with impaired skeleton functions (J. Biol. Chem. - 1953, v. 205. pp. 611-635, as well as Biochim. Biophys. Acta. 1958. v. 30 p. 184). The yield of the target product, however, is not more than 0.08 0.1% of the initial mass of raw materials.

 There is also known a method of producing keratosulfate according to the source (Acta Chem. Scand. 1957. v.11.r. R. 668-674), in accordance with which the animal raw material is homogenized in the central part of the intervertebral cartilage of a person, lyophilized, extracted with boiling water, the extract is separated, deproteinized by incubation with pancreatic extract, keratosulfate precipitated with 50-60% ethanol, the precipitate was dissolved in a 0.3% solution of barium acetate, purified by chromatography on a cellulose column using ethyl alcohol with osleduyuschey drying the eluate freeze. The disadvantage of this method is the low yield of the target product (0.12-0.32% of the initial mass of raw materials).

According to the prototype adopted, closest to the claimed technical solution according to (ed. Certificate of the USSR N 918294), the therapeutically active polyester of sulfuric acid with mucopolysaccharide from cattle trachea (keratosulfate) is isolated by fractionation with lower aliphatic alcohol (ethyl alcohol) crude mucopolysaccharide a mixture obtained by extraction of the trachea; subsequent precipitation at pH 1-2 and a temperature of 20 ^o 30-34 about. solution of ethyl alcohol, and then 70-74 vol. ethyl alcohol, hydrolysis of the precipitate with hydrochloric acid and sulfation with chlorosulfonic acid in dimethylfamamide, dialysis of a 5% solution of the product at pH 9-10 and a temperature of 4-25 ^o through hollow fiber membranes with an exclusion limit of ≅ 5000, purification on a strongly acidic cation exchange resin and discoloration of H ₂ O ₂ . 71.5 g of product are obtained from 10 kg of trachea. The disadvantage of the above method is its complexity, the need to use a dangerous and aggressive chemical compound of chlorosulfonic acid, as well as the duration of the dialysis purification process. The invention solves the problem of simplifying the process of isolating keratosulfate from cattle trachea while maintaining a high yield of the target product.

This goal is achieved by the fact that in the method for producing keratosulfate from cattle trachea, including their degreasing with acetone and homogenization, extraction with an aqueous solution of alkali, treatment of the alkaline extract with an aqueous solution of ethyl alcohol with a precipitate, hydrolysis of the obtained precipitate with hydrochloric acid, followed by chromatographic purification of the target mucopolysaccharide , fat-free and homogenized trachea cattle extraction is carried out with 5-6 parts of 0.5-1% aqueous solution of caustic soda p and 40-60 ^o for 3-4 hours, extract further deproteiniziruyut shaking-ring with 3-4 volumes of chloroform for 1-2 hours, deproteinized extract is treated with 50-60% ethyl alcohol, the hydrolysis is carried out in the resulting precipitate of 1- 2 parts of a 0.2-0.4% hydrochloric acid solution, and the chromatographic purification of the target product keratosulfate is carried out by chromatography of the obtained hydrochloric acid solution on a column filled with cellulose, with a 1-2% aqueous solution of cetylpyridinium chloride as an eluent, followed by lyophilic with shkoy.

The yield of keratosulfate according to the claimed method is 0.58-0.62% of the initial mass of raw materials, which is almost not inferior to the yield according to the known method (0.715%), however, the claimed method is simpler to perform, safer and not so long (due to the exclusion of ClSO ₃ H and stage dialysis).

 The invention is illustrated by examples 1-3.

Example 1: 1 kg of calf trachea is minced in a meat grinder, homogenized in a tissue micro-grinder, minced meat is treated with acetone (4 x 4 L) for 6 hours, dried to remove the smell of acetone and the powder is extracted with 5 L (5 volumes) of 0.5% aqueous NaCH at 40 ^o C for 3 h. The mixture is cooled to 20 ^o , the extract is separated by filtration on a suction filter, 13.3 l of chloroform (3 volumes) are added to 4.6 L of the solution and shaken on a shuttle machine for 1 h. The aqueous phase is separated from the chloroform containing proteins on a separatory funnel and 4 volumes of 50% are added. ethyl alcohol. After 1 h, the precipitate formed is filtered off and dissolved in 1 L of a 0.2% HCl solution (1 volume by weight of the feedstock), the solution is passed through a column (5x35 cm) with cellulose powder at a rate of 15 ml / min. the column is washed with a 1% aqueous solution of cetylpyridinium chloride, the eluate is freeze-dried and 5.3 g (0.38% yield) of purified keratosulfate are obtained.

Example 2: 1 kg of crushed and homogenized trachea of calves is degreased with acetone (4 x 4 L), dried and the powder is extracted with 5.5 L (5.5 volumes) of a 0.75% aqueous solution of NaCH at 50 ^o C for 3, 5 hours. The mixture is cooled to 20 ^° C., the extract is filtered off, 17.5 liters of chloroform are added to 5 liters of solution (3.5 volumes and shaken for 1.5 hours. The aqueous phase is separated, 4 volumes of 55% ethyl are added). alcohol, after 1 h, the precipitate is filtered off, dissolved in 1.5 l of a 0.3% HCl solution (1.5 volumes by weight of the starting material), the solution is passed through a column (35 x 5 cm) s cellulose powder at a speed of 15 ml / min, the column is washed with a 1.5% aqueous solution of cetylpyridinium chloride, the eluate is freeze-dried and 6 g (yield 0.6%) of purified keratosulfate are obtained.

Example 3:
1 kg of crushed and homogenized trachea of calves is degreased with acetone, the powder is dried, extracted with 6 l (6 volumes) of 1% aqueous NaOH solution at 60 ^° C for 4 hours. The mixture is cooled to 20 ^° C, the extract is filtered off to 5.6 L of the solution add 22.4 L of chloroform (4 volumes) and shake for 2 hours. The aqueous phase is separated, 4 volumes of 60% ethanol are added, after 1 h, the precipitate is filtered off, dissolved in 2 L of a 0.4% HCl solution. (2 volumes by weight of the feedstock), the solution is passed through a column (5 x 35 cm) with cellulose powder at a speed 15 ml / min. the column is washed with a 2% aqueous solution of cetylpyridinium chloride, the eluate is freeze-dried and 6.2 g (0.62% yield) of purified keratosulfate are obtained.

The product does not contain impurities of other polysaccharides, as evidenced by the presence of a single spot with R _F 0.73 during the chromatography of a sample on paper (upward chromatography on paper in a solvent system n-butyl alcohol concentrated ammonia water, 4: 1: 5).

Claims (1)

A method for producing keratosulfate mucopolysaccharide from cattle trachea, including their degreasing with acetone and homogenization, extraction with an aqueous solution of alkali, treatment of the alkaline extract with aqueous solutions of ethyl alcohol to isolate the precipitate, hydrolysis of the obtained precipitate with hydrochloric acid, followed by chromatographic purification of the target mucopolysaccharide, which differs, fat-free and homogenized cattle trachea spend 5 6 hours. 0.5 - 1% aqueous solution of caustic soda at 40 60 ^o C for 3-4 hours, the extract is additionally deproteinized by shaking with 3 4 volumes of chloroform for 1 2 hours, the deproteinized extract is treated with 50-60% ethanol, the hydrolysis of the precipitate is carried out in 1 2 hours. 0.2 0.4 % hydrochloric acid solution, and chromatographic purification of the target keratosulfate product is carried out by chromatography of the obtained hydrochloric acid solution on a column filled with cellulose, with 1 2% aqueous solution of cetylpyridinium chloride as an eluent, followed by freeze drying.