Method of producing chondroitin sulphate from sea hydrobiont tissue

FIELD: chemistry.

SUBSTANCE: method involves preparation of material for enzymatic hydrolysis. Alkaline hydrolysis is carried out with proteolytic enzyme preparations with neutralisation of the obtained solution to pH=7. A salt is added to the obtained enzymatic hydrolysate to a value of not less than 0.1 mol/l. Successive ultrafiltration is carried out, first on a membrane with maximum retention of 50 kD with separation of high-molecular weight impurities, and then on a membrane with maximum retention of 5 kD with separation of low-molecular weight substances. The chondroitin sulphate solution retained at the membrane is washed on the same membrane with distilled water until complete removal of salts. Final washing with distilled water is carried out on a membrane with maxim retention of 50 kD.

EFFECT: invention enables to obtain a chondroitin sulphate preparation with weight ratio of the basic substance.

7 ex

 

The invention relates to the fishing industry, in particular to methods of producing chondroitin sulfate from the tissues of marine organisms, such as cartilage fish, muscle-muscular bag molluscs etc, and can be used in food, cosmetic industries, in medicine.

The basic properties of chondroitin sulfate that are crucial for its successful application in various fields, high bioavailability, biocompatibility, low toxicity, ability to selective accumulation in cartilage (Kofuji K., Ito T., Murata Y., Kawashima S. Effect of chondroitin sulfate on the biodegradation and drug release of chitosan gel beads in the subcutaneous air pouches of mice // Biological and Pharmaceutical Bulletin. - 2002. - Vol.25, No. 2. - P. 268-271). These properties are determined by the chemical structure of the molecules of chondroitin sulfate, namely, molecular weight, degree and location of sulfation. (Michelacci Y. M., Dietrich S.R. Structure of chondroitin sulphate from whale cartilage: distribution of 6 - and 4-sulphated oligosaccharides in the polymer chains // International Journal of Biological Macromolecules. -1986. - Vol.8, No. 2. - P. 108-113. Toida T., Amornrut C., Linhardt, R. J. Structure and bioactivity of sulfated polysaccharides // Trends in Glycoscience and Glycotechnology.-2003.-Vol.15, No. 81.-P. 29-46).

To get chondroitin sulfate is the most widely used is known a method comprising dissolving chondroitin sulfate in an alkaline environment, the enzymatic hydrolysis of proteins, separation of high-molecular ug is evadney fractions by precipitation from low molecular weight products of protein hydrolysis, remaining in the solution, washing the obtained precipitate and drying the finished product (Takai M, Kono N. Salmon-origin chondroitin sulfate: European Patent EP 1270599, IPC A61K 31/737. Appl. 15.12.2000; No. EP 20000981747; publ. 02.01.2003).

This conventional technology is implemented by different authors in different ways: changing the sequence, the number of operations, temperature, nature and concentration of reagents used.

The closest technical solution is the method of producing chondroitin sulfate using ultrafiltration (Khare A. C., Houliston S. A., Black, T. J. Isolating chondroitin sulfate: USPTO Application 20070166798. - Appl. 14.02.2007; No. 11/674695; publ. 07/19/2007).

This method of producing chondroitin sulfate includes collection (training) of raw materials, including connective tissue, hydrolysis of the feedstock proteolytic enzyme preparations to obtain a solution of the hydrolyzate and undissolved substances, the processing liquid hydrolysate reagent comprising the hydroxide of the divalent alkaline earth metal with a pH greater than 10 to precipitate impurities of protein hydrolysate, separating at least a portion of the residue from the solution of the hydrolyzate and the processing liquid hydrolysate using membranes with the formation of the filtrate (permeate) low molecular weight substances and "detainee" concentrate, which contains high molecular weight fraction - chondroitin sulfato patent it is proposed to use membranes with a molecular mass the limit of propriety from 5 to 15 kDa (preferably from 8 to 10 kDa).

The product obtained in this way is a concentrate, which among other substances it contains chondroitin sulfate. Thus, the degree of purification of the target product is not high enough.

The inventive method is also based on ultrafiltration separation of the hydrolysate fractions with different molecular weights and uses the property of high molecular weight molecules of chondroitin sulfate separated from low molecular weight products of hydrolysis of proteins by ultrafiltration membranes.

The technical result of the present method is to increase the degree of purification of the target product by using the ability of molecules of chondroitin sulfate strongly modify the hydrodynamic radius when changing the ionic strength of the solution (concentration of the electrolyte, for example, NaCl), which allows to achieve higher compared to the prototype of the purification of the target product by sequential ultrafiltration obtained hydrolysate on membranes with different threshold of propriety.

As a raw material for production of chondroitin sulfate can be used containing cartilage raw materials resulting from the processing of various marine life. When using ice cream raw materials previously conducted its devastatio.

The prepared raw material is crushed and loaded into reactionremedy, in which carry out alkaline, and then the enzymatic hydrolysis.

Hydrolyzed cartilage tissue contains various cleavage products of proteins, salts, high molecular weight polysaccharides (chondroitin sulfate).

Dissolution dilaceration substances, including proteins and chondroitin sulfate, is carried out at a temperature of from 25°to 50 ° C for 3 h with constant stirring.

After alkaline hydrolysis, the mixture is neutralized to pH 7 and separate nerastvorim precipitate by filtration or by centrifugation.

Carrying out alkaline hydrolysis provides pre-separation nerastvorim impurities and, consequently, increases the yield of the target product, increasing its purity.

In the resulting solution is added enzyme preparation (FP) or a pre-prepared solution of AF with proteolytic activity, for example an enzyme obtained from hepatopancreas crab.

Enzymatic hydrolysis of proteins is carried out at the optimum for a given AF temperature of the incubation mixture and the duration of treatment (when using AF from hepatopancreas Kamchatka temperature from 45 to 55°C and duration of hydrolysis of from 4 to 8 h), separating a solid residue.

To the resulting solution add salt, such as sodium chloride, reducing the salt concentration to 0.1 the ol.

Then spend the ultrafiltration of a solution through a membrane with a molecular weight limit of the retaining less than 50 kDa for separating remaining after hydrolysis of high molecular weight proteins and particles.

A concentrated solution containing chondroitin sulfate, washed with salt solution, for example sodium chloride or other salt concentration of 0.1 mol/L.

To do this, in the resulting solution add sodium chloride or other salt to maintain its concentration in the solution to a value not less than 0.1 mol/L. If, after neutralization of the alkali concentration of NaCl above, the additional amount of sodium chloride is added.

When the concentration of NaCl in the solution is higher than 0.1 mol/l molecules of chondroitin sulfate strongly globulinovoy that it is not possible to separate the components of the hydrolysate.

Used salt concentration reduces the hydrodynamic radius of the molecules of chondroitin sulfate and their passage through the membrane with a molecular weight limit of the retaining less than 50 kDa, membrane linger not hydrolyzed proteins, such as collagen.

The resulting solution of chondroitin sulfate, low molecular weight peptides, amino acids and salts is subjected to ultrafiltration separation on the membrane with a molecular weight limit of the retaining 5 kDa providing detention molecules of chondroitin sulfate and separating molecules of salts, amino acids and low molecular weight peptides.

It is known that lowering the concentration of salt in the solution is less than 0.001 mol/l, for example NaCl, molecules of chondroitin sulfate are deployed, which leads to the increase of the hydrodynamic radius. The maximum radius is observed in distilled water.

Retained on the membrane chondroitin sulfate is washed with distilled water, the salt concentration is reduced and the molecules of chondroitin sulfate, hydrodynamic radius, which significantly increased, can be concentrated on the membrane with a molecular weight limit of retaining 50 kDa.

On the membrane with a limit of detention 50 kDa chondroitin sulfate finally washed with distilled water from the remaining peptides average molecular weight and exercised by the concentration of its solution by ultrafiltration.

High-molecular fraction of chondroitin sulfate is concentrated on the membrane, and low molecular weight peptides and amino acids pass through it.

The obtained concentrated solution of chondroitin sulfate is further used to highlight dry preparation or as a solution in the preparation of drugs with chondroitin sulfate.

Allocation of dry chodroitin sulfate deposition is carried out by adding an excess of the precipitant (for example ethanol) or drying (sublimemovies, spray drying and other).

For example, the resulting solution was precipitated by the addition of alcohol in the ratio of 1:2, can withstand up to full deposition of chondroitin sulfate, separating the precipitate by filtration or by centrifugation, washed with alcohol, acetone, and dried in the freeze dryer, vacuum or other dryer.

Get preparation of purified chondroitin sulfate mass fractions of the basic substance is not less than 90%.

Target product - chondroitin sulfate is a white amorphous powder, odorless, hygroscopic, mass fraction of water is not more than 10%mass fraction of chondroitin sulfate is 90-95%

Determination of the mass fraction of the basic substance was performed by the method of acid hydrolysis in hydrochloric acid (26% Hcl, 100°C, 1 h) and determine the resulting glucuronic acid by the method of the DISHA.

Identification was performed by the method of infrared spectroscopy. As the standard used drug chondroitin 6-sulfate sodium salt from shark cartilage (Catalogue Fluka, cat. No. 27043-1G-F).

The results of the comparison showed that the samples of chondroitin sulfate obtained by the present method, are almost similar numbers with those of the standard sample, which confirms the high degree of purification of the target product.

The use of ultrafiltration is possible immediately after the selection of the drug hon is routine sulfate after enzymatic hydrolysis of raw materials or when cleaning precipitated with ethanol drug after its dissolution in water.

Examples of the method

Example 1

Receiving chondroitin sulfate of cartilage salmon

1) Raw material - nasal cartilage of salmon removed from the heads, cleaned from the remnants of the surrounding tissues, to grind.

2) 400 g of crushed raw materials were loaded into a flask with 1600 g of NaOH solution (0.2 mol/DM3), heated in a boiling water bath to 37°C and at this temperature for 3 h with constant stirring spent dissolution dilaceration substances.

3) After the process is finished, the mixture was neutralized to pH 7 using 0.1 N. acetic acid.

4) Separated undissolved precipitate in the centrifuge at 5000 rpm-1.

5) In the hydrolysate was loaded enzyme preparation of hepatopancreas crab at the rate of 3 g per 1 kg of raw material. Enzymatic hydrolysis was performed at 50°C for 5 hours. The precipitate was separated in a centrifuge at 5000 rpm-1.

Used enzyme preparation was obtained from hepatopancreas red king crab Paralithodes Camtschaticus by known techniques (Sugars, YOU Way to obtain collagenase. /Youshare Avjunkie, AAC, WAV, Agestandardized, Aphthosa. Institute of immunology and Pacific Institute of Bioorganic chemistry: A.S. SU 1343591 A1, MCI UK 35/56. - Appl. 13.12.85; No. 3992368 /28-14. - 2 C.) and had a proteolytic activity on casein sodium And=280 m is the mol tyrosine*g -1*min-1. (Substrate: 1%solution of sodium Caseinate. Enzyme: 1 mg/ml solution of AF. Conditions of incubation: 37°C, 10 minutes)

6) Spent the ultrafiltration of the solution through the membrane 50 kDa. Washed concentrated solution of the sodium chloride solution with a concentration of 0.1 mol/l (To increase the ionic strength in the hydrolysate added 9.36 g of sodium chloride.)

7) Then the resulting solution of chondroitin sulfate, low molecular weight peptides, amino acids and salts was subjected to ultrafiltration separation on the membrane of 5 kDa, ensuring the retention of molecules of chondroitin sulfate.

8) Rinse with distilled water. A solution of chondroitin finally washed on the membrane 50 kDa from the remaining peptides average molecular weight.

9) Finally, the solution of chondroitin sulfate concentrated by ultrafiltration.

10) the resulting solution was besieged by the addition of alcohol in the ratio of 1:2, withstood the solution for 20 hours.

11) Separated precipitate by centrifugation at 5000 rpm-1, washed with 100 ml of alcohol.

12) the resulting substance was dried in the freeze dryer.

Got 6,15 g of dry chondroitin sulfate mass concentration of the basic substance, defined by the reaction with carbazole (method DISHA), 91%.

Example 2

Receiving chondroitin sulfate from shark cartilage

Same as in example 1, but in the quality of raw materials used in isovale shark cartilage.

Got 6,15 g of dry chondroitin sulfate mass concentration of the basic substance 93%.

Example 3

Receiving chondroitin sulfate of cartilage North slope

Same as in example 1, but in the quality of raw material used in the cartilage of the North slope. Conducted preliminary degreasing of raw materials acetone. In paragraph 5 of the enzymatic hydrolysis was carried out in two stages for 3 hours with the addition of enzyme preparation respective concentration.

Got 5,43 g of dry chondroitin sulfate mass concentration of the basic substance 92%.

Example 4

Purification of chondroitin sulfate of cartilage salmon

Chondroitin sulfate obtained by the method of example 1, except paragraphs 6-9, re-dissolved in 500 ml of distilled water, added with 2.93 g of sodium chloride, the solution was mixed for 60 minutes Then spent the ultrafiltration processing solution as described in example 1, p.6-9 and next 10-12.

Got to 5.85 g of dry chondroitin sulfate mass fraction of the main substance of 94%.

Example 5

Receiving chondroitin sulfate of cartilage salmon

Same as in example 1, but as the alkali for surgery according to claim 2) instead of NaOH used potassium hydroxide KOH (0.2 mol/DM3). When ultrafiltration 6) instead of NaCl used 0.1 mol/DM3a solution of potassium chloride KCl.

Got 6,10 g dry chondro the ina sulfate, mass fraction of the main substances 92%.

Example 6

Receiving chondroitin sulfate of cartilage salmon

Same as in example 1, but instead of NaCl at UF 6) used 0.05 mol/DM3the solution of calcium chloride.

Got 6,15 g of dry chondroitin sulfate mass fraction of the main substance of 91%.

Example 7

Receiving chondroitin sulfate of cartilage salmon

Same as in example 1, but instead of the enzyme preparation from hepatopancreas crab used it G3X based 4.5 g per 1 kg of raw material (activity protocolin G3X in relation to fish protein was 1.5 times lower than the activity of AF from hepatopancreas crab).

Got 6,35 g of dry chondroitin sulfate mass concentration of the basic substance 89%.

The invention allows to obtain the product chondroitin sulfate mass fractions of the basic substance is not less than 90% (90-95%), and to increase the yield of the target product.

The method of producing chondroitin sulfate from the tissues of marine organisms, providing training materials to enzymatic hydrolysis, the hydrolysis of proteolytic enzyme preparations with the deposition of protein impurities and separating the precipitate from the solution of the hydrolyzate, the selection of the target product by ultrafiltration, wherein the pre-spend alkaline hydrolysis of the neutralization of the resulting solution to pH 7 and the separation of a solid residue, in the enzymatic hydrolysate add salt to the value of not less than 0.1 mol/l, hold the ultrafiltration enzymatic hydrolysate on the membrane with a limit of detention of 50 kDa and separation of high molecular weight impurities, the resulting solution with a salt concentration of at least 0.1 mol/l is subjected to ultrafiltration on a membrane with a limit of detention 5 kDa and separate the low molecular weight substances retained on the membrane chondroitin sulfate is washed on the same membrane with distilled water to remove salts, followed by washing with distilled water and the concentration of the solution of chondroitin sulfate by ultrafiltration on a membrane with a limit of detention of 50 kDa.



 

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3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to traumatology and orthopedics and can be applied in complex treatment of posttraumatic, dystrophic, neurological, degenerative and scar-adhesion processes in joint area, deforming osteoarthrosis of large joints and osteochondrosis. Method of treatment includes introduction of drug mixture by ultraphonoresis for 5 minutes on a field, daily. To obtain said mixture applied are ointment "Indomethacyne", "Chondroxyde", gel "Essaven" in proportion 1:1:1, in dose, expressed in form of 3-10 cm long stripe of each ointment. These components are mixed with 50 mg of medication Karipasim, dissolved in 5 ml of physiological solution and 5 ml 2% solution of Trental. Treatment course includes 15-20 procedures.

EFFECT: method ensures fast achievement of antihydrophic and analgetic effect, reduction of total terms of patients' rehabilitation, increase of disease remission periods.

3 ex, 1 tbl, 4 dwg

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