Method of glycosaminoglycan release from mineralised connective tissue

FIELD: medicine.

SUBSTANCE: invention refers to medicine and describes method of glycosaminoglycan release from mineralised connective tissue, containing enzymatic hydrolysis, deproteinisation, deposition, and characterised by the fact that demineralisation is performed within 20 h using 0.5 H HC1, enzymatic hydrolysis is performed within 18 h using pepsin enzyme, deproteinisation is performed by adding of ammonium sulphate to 75% saturation, glycosaminoglycans are deposited with 4% potassium acetate in 96% ethanol, re-deposited with ethanol and lyophilised. Glycosaminoglycans are exposed to chromatographic separation using "ДЕАЕ"-sephadex A-25, and lyophilised. This method is easy-to-use, realised in laboratory environment of patient care institutions, and time-saving.

EFFECT: easy and saving method of glycosaminoglycan release from mineralised connective tissue.

1 ex, 2 dwg

 

The invention relate to the field of chemical technology and cosmetology, perhaps its use in medicine and healthcare for the production of biologically active food additives, which are aimed at improving the state of the connective tissue.

It is now known that glycosaminoglycans (GAGS) of the different types of connective tissue are used for chondroprotective action in arthrology and use of natural and synthetic glycosaminoglycans, which are a number of drugs for the regeneration of epithelial tissues. [Osteoarthritis: current status of the problem (analytical review) / Mironov S. p. Pp.96-99].

The literature describes how the selection of glycosaminoglycans of cartilage (Adams E.V., Kosygin A.I. Determination of glycosaminoglycans serum // lab. case. - 1982. No. 10. - P.15-17).

For carrying out enzymatic hydrolysis in these methods, the use of proteolytic enzymes (pronase E), and for the deposition of glycosaminoglycans - specific precipitators GAG - Quaternary ammonium base (pyridinium chloride or pyridinium bromide) (Kosygin D.V., Vasilenko pre-cast Deposition of GAG urine salts of aliphatic ammonium bases and their cleanup // lab. case. - 1988. No. 2, - P.57).

Known method of extraction of aggregates of proteoglycans, to whom or dialysis to remove salts, freed from soluble collagen and non-aggregated proteoglycans by deposition of citric acid, then dialysis to remove the acid, the resulting solution was treated with H+the form of the cation exchange resin to obtain the acid form of aggregates of proteoglycans, then salt form of a cation exchange resin to obtain the acid salt form of aggregates of proteoglycans which is then treated not less than double the volume 96,6° ethanol saturated with metal acetate, for deposition of the main form aggregates of proteoglycans, with subsequent washing and drying the precipitate in ethyl alcohol and ether, while the acid-salt and basic salt form aggregates proteoglycanes can be dried. (Patent RF №2096038, publ. 20.11.1997).

However, this method involves a long process of dialysis and the use of a large quantity of distilled water. In addition, the final product is uglevodorami connection, i.e. not sufficiently released from the protein glycosaminoglycans. The use of cation exchangers requires regeneration, and rinsing and drying the ethanol-ether mixture is toxic procedure at the final stage of purification GAG because of sorption processes of ether on the target product.

It is also known cosmetic anti-aging skin based as AK the active ingredients used enzyme Pancreatin and hyaluronic acid, included in the fatty basis hydrogenated cottonseed oil, hydroline and glycerol using preservatives, antiseptics and antioxidants (RF Patent No. 2078561, publ. 10.04.1996).

However, the known tool is expensive at cost, energy-intensive, time-consuming.

There is a method of allocation of glycosaminoglycans (GAGS) of fibrous cartilage, in which the cartilage is subjected to proteolysis with papain of deproteinization hydrolysate of trichloroacetic acid and deposition of glycosaminoglycans ethanol containing acetic acid salts of potassium or sodium (RF Patent No. 2089201 publ. 10.09.1997 year).

However, in the known method is proposed allocation of GAG from demineralizing connective bone tissue is fibrous cartilage and it is not intended to highlight the GAG from the bones of farm animals.

The present invention is to develop a consumption of time and reagents allocation method of glycosaminoglycans from different types of mineralized connective tissue, primarily from the bones of agricultural animals was launched.

The problem is solved in that in the method of extraction of glycosaminoglycans from mineralized connective tissue, including enzymatic hydrolysis, deproteinization, sedimentation, desalination carried out for 20 h using a 0.5 N. HCl, enzymatic g is droles carried out for 18 h, using the enzyme pepsin, the deproteinization carried out by adding ammonium sulfate to 75%saturation, the deposition of glycosaminoglycans spend 4%potassium acetate in 96% ethanol, periostat ethanol, conduct lyophilic drying, share glycosaminoglycans chromatography using DEAE-Sephadex A-25, conduct lyophilic drying.

The present invention explaining a detailed description of the sample lab execution and illustrations, in which:

figure 1 - illustrates the allocation of glycosaminoglycans from mineralized connective tissue;

figure 2 shows the chromatographic profile of glycosaminoglycans bone on aminoalkenes.

The method is as follows.

After pre-processing, which consists in removing soft tissue, mineralized connective tissue (e.g. bones of cattle) were crushed to pieces about 5×5 mm Then the fabric was subjected to demineralization of 0.5 N. HCl for 20 h, after this procedure, demineralized tissue was liberated from a solution of bone mineral Nikolayevich proteins and spent basenowy proteolysis in the presence of enzyme in 0.1 M HCl with a pH=2. Pepsin was taken from a rate of 1 mg per 100 mg tissue. For 18 h incubation at 37 ° °With magnetic stirrer fabric was destroyed completely with education is the use of colloidal solution, which was subjected to centrifugation at 40.000gx. Proteins, RNA, glycoproteins besieged from the supernatant by adding ammonium sulfate to 75%saturation. The precipitate was formed within 1 h, after which it was separated by centrifugation (10 min, 40.000gx). From the obtained supernatant was performed deposition of GAG 4%potassium acetate in 96% ethanol. Sediment GAG, collected by centrifugation, was perioadele ethanol was again collected and freeze-dried. To obtain vysokochistykh fractions of GAG separated chromatographically using DEAE-Sephadex A-25, then freeze-dried and for the manufacture of cosmetic products homogenized with ointment base in a mass ratio of 1:10, respectively.

Example laboratory run

Cattle bone cleaned of soft tissue, fat-free, alcohol-ether mixture of 1:1 is crushed to pieces size 5×5 mm

100 g of degreased crushed bone tissue is placed in a round bottom flask, into which pour 200 ml of 0.5 N. HCl and left for 20 h at room temperature. Obtained after removal nadeshiko nerastvorim bone matrix filled with 0.1 M HCl and make 1 mg of pepsin. The reaction mass is placed on the magnetic stirrer and carry out the enzymatic hydrolysis of bone tissue for 18 h to dissolve bones and education colloides the solution.

Then from the solution of proteolysed by centrifugation at 40.000gx 10 min to separate the precipitate, which contains proteins, RNA, and glycoproteins. Choose glasses for centrifugation supernatant and spend the deproteinization by adding ammonium sulfate to 75% saturation. After repeated centrifugation in the same mode precipitated GAG 4%potassium acetate in 96% ethanol in the calculation of the volume of 1:4. The precipitate formed within 30 min in the cold at 4°C. After centrifugation at 1.300gx 15 min, remove supernatant, collecting exhaust 4%potassium acetate in 96% ethanol in a separate container for subsequent distillation of ethanol. Sediment GAG, collected by centrifugation, periostat ethanol, centrifuged again at 1.300gx 15 min, harvested and freeze dried. To obtain vysokochistykh fractions of GAG separated chromatographically using DEAE-Sephadex A-25 (ion-exchange capacity of 3.5 to+0.5 meg/g, grain size 40-120 μm). Linear concentration gradient eluting solution create using 0,00-2.5 M NaCl in 0.02 M Tris-HCl buffer (pH 7.0). Faction densitometric at 226 nm and selected 3 ml, identifying them by definition GUK, hexoses, total nitrogen and sulphates. After that, the obtained solutions of various fractions of GAG freeze-dried.

Selected purified GAG injected into the ointment base in soo is wearing 1:10, respectively.

The proposed method is easy to use, allows to reproduce it in the laboratory of the medical institution, for its implementation does not require the use of expensive reagents - specific precipitating GAG and a large number of proteolytic enzymes.

In addition, this method is time saving, allows re-use of ethanol during the collection it is used, after the subsequent distillation, and allows to obtain highly purified target product.

The proposed method is used in biochemical laboratories RISC "RTO" them. academician Gailizarov, ibid developed and cosmetic.

Method of extraction of glycosaminoglycans from mineralized connective tissue, including enzymatic hydrolysis, deproteinization, deposition, characterized in that the demineralization carried out for 20 h using a 0.5 N. HCl, the enzymatic hydrolysis is carried out for 18 h using the enzyme pepsin, the deproteinization carried out by adding ammonium sulfate to 75%saturation, the deposition of glycosaminoglycans spend 4%potassium acetate in 96%ethanol, periostat ethanol, conduct lyophilic drying, share glycosaminoglycans chromatography using DEAE-Sephadex A-25, conduct lyophilic drying.



 

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