Electrochemical method of purifying chondroitin sulphate

FIELD: chemistry.

SUBSTANCE: invention relates to a method of purifying chondroitin sulphate and can be used in food and cosmetic industry and in medicine. The method involves electrochemical deposition to obtain a hydrogel of chondroitin sulphate, stabilisation, removal from the electrode, washing and drying. The chondroitin sulphate is dissolved in a 0.01-0.1 n alkali solution in ratio of 1:50-1:200 and deposited in an alkaline medium with constant cooling and stirring. The solution is stirred at a rate of 10-20 rpm. Current density is equal to 1-10 A/m2. Voltage is preferably not lower than 2.7 V. The hydrogel of chondroitin sulphate is stabilised in a 0.05-0.5 n HCl solution.

EFFECT: invention enables to obtain chondroitin sulphate with high weight ratio of the basic substance and increases output of the end product.

5 cl, 1 ex

 

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

The basic properties of chondroitin sulfate that are crucial for its successful application in various fields such as 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., Amomrut C., Linhardt R.J., Structure and bioactivity of sulfated polysaccharides // Trends in Glycoscience and Glycotechnology. - 2003. - Vol.15, No.81. - P.29-46).

A known method of purification of chondroitin sulfate (CS), providing for the dissolution of chondroitin sulfate in an alkaline environment, the enzymatic hydrolysis of proteins, separation of high molecular weight carbohydrate fraction by precipitation from low molecular weight is of Reducto protein hydrolysis, remaining in the solution, washing the obtained precipitate and drying the finished product (Takai M., Kono H. Salmon-origin chondroitin sulfate: European Patent EP 1270599. IPC AC 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.

Known isolation and purification of cholesterol, which consists in the hydrolysis of the feedstock and subsequent precipitation of cholesterol from the hydrolysate by adding ethyl alcohol (U.S. Pat. Of the Russian Federation. No. 2056851, publ. 27.03.1996; Pat the Russian Federation No. 2061485, publ. 10.06.1998; U.S. Pat. Of the Russian Federation No.. 2383351, publ. 10.03.2010). The disadvantages of the method are the following. Requires a large consumption of ethyl alcohol, and the necessity of regeneration. In addition, the precipitation is not complete, so a considerable part of the cholesterol remains in solution.

A known method of purification of polysaccharide based on the electrochemical deposition of the polysaccharide from the solution (U.S. Pat. U.S. No. 7883615, publ. 08.02.2011). In the discharge of negatively charged molecules of the polysaccharide, in particular chitosan, occurs at the anode deposition of the polysaccharide in the form of a hydrogel. The resulting product is subsequently removed from the electrode, washed in distilled water and dried. This method is most similar to that proposed and adopted for the prototype. Spoopendyke to obtain films of polysaccharide for further use as cards in the production of biosensors. Electrochemical deposition method prototype is carried out in an acidic environment (PH=5.0-5.5, the current density is high and ranges from 20 to 100 a/m2the duration of the electrochemical deposition is 2-30 minutes Stabilization hydrogel of a polysaccharide is carried out in an alkaline environment (rather basic solution).

Object of the present invention to provide CHS with increased mass fraction of the primary substance, i.e. increasing the degree of purification of the target product.

It is solved in an electrochemical method of purification of chondroitin sulfate, which provides for electrochemical deposition of obtaining hydrogel XC, stabilization, removal from the electrode, rinse and drying, XC is dissolved in a solution of 0.01-0.1 n alkali in the ratio of 1:50-1:200 and conduct electrochemical deposition in an alkaline medium at a constant cooling and stirring the solution with a speed of 10-20 rpm, current density of 1-10 a/m2, the voltage required to precipitation, stabilization hydrogel XC is carried out in a solution of 0.05-0.5 n Hcl. The voltage of electrochemical deposition is not less than 2.7 V, and the deposition time from 2 to 60 min, depending on the purity of the original CS, the drying of the hydrogel XC carried out first in air and then in a drying Cabinet at a temperature not exceeding 60C. the Cooled reaction mixture is performed proto is Noah water with a temperature of 4C-15C.

Electrochemical deposition is carried out in an alkaline medium, pre-dissolved source CS in 0,01-0,10 n solution of alkali, such as NaOH or another. Dissolution of cholesterol in alkali and conducting electrochemical deposition in an alkaline environment due to the fact that it dissolves much better in alkaline than in acidic or neutral media, therefore the cleanup process will be more complete. The mass ratio chondrotin sulfate and alkali solution is from 1:50 to 1:200 (concentration of cholesterol in the solution is from 0.5 to 2.0%). The resulting solution is poured into the electrolytic cell.

The cell consists of a container (glass or plastic Cup), which houses the electrodes and mixing device. The anode is made in the form of a cylinder, located at the side walls of the container, the cathode is in the form of a Central rod. Between the cathode and the anode is rotating mixer, which is made either in the form of frames or blades, the axis of which is inclined at an angle to the horizontal plane of the vessel.

For cooling the reaction mixture, the tank is provided with a cooling device in the form of the external jacket in which cooling water is supplied. For cooling capacity may be immersed in a vessel with a flowing cooling water or liquid thermostat. The cooling water temperature from 4 to 15C. Cooling is necessary for more than the full deposition of hydrogel XC pole.

The electrodes serves a DC voltage of not less than 2.7 V, when the voltage is lower than 2.7 In deposition on the electrode will not occur. The current density is from 1 to 10 a/m2this density is quite enough for the process. High current density causes a significant release of heat that will not contribute to a more complete cholesterol deposition on the electrode.

The electrolysis is carried out at constant slow 10-20 rpm stirring of the solution between the electrodes during the whole process of electrochemical deposition, i.e. 2-60 minutes of Slow mixing helps to include in the electrochemical deposition of the entire volume of the solution in the cell, mixing the same with a speed of more than 20 rpm will break deposited on the electrode hydrogel XC.

At the end of the electrolysis, the voltage from the electrodes are removed, a cylindrical electrode with the deposited hydrogel XC transferred into the vessel with distilled water and wash out within 1-5 minutes

Then the electrode with the deposited hydrogel XC transferred into the vessel from 0.05-0.5 n Hcl for the final formation of the precipitate, i.e. they perform the stabilization of the formed hydrogel is then washed with distilled water.

The wet gel is cut from the surface of the electrode is dried first in air and then in a drying Cabinet at a temperature not exceeding 60C.

Obtained the product is crushed in the device of any design.

Example

A portion of the original XC 5.0 g was dissolved in 500 ml of 0.05 n NaOH solution (or other alkali) and filled in the electrolytic cell.

The electrolytic cell consisted of a glass, which is a cylindrical anode and a cathode in the form of a Central rod. The electrodes are made of stainless steel. Between the cathode and the anode is placed a rotating anchor agitator.

For cooling the reaction mixture, the cell was placed in a tank with running water. The cooling water temperature was 8C.

The electrodes were applied constant voltage of 2.7 Century, the Current in the electrolyte was 2.5 a/m2.

The electrolysis is carried out at constant slow 20 rpm stirring of the solution between the electrodes for 10 minutes

At the end of the electrolysis the voltage from the electrodes are removed. A cylindrical electrode with the deposited gel chondroitin transferred into the vessel with distilled water and wash out within 1-5 minutes

Then a cylindrical electrode with the deposited gel XC is transferred into a vessel with 0.1 n Hcl for the final formation of the precipitate (stabilization), then washed with distilled water.

The wet gel is cut from the surface of the electrode and dried in air and then in a drying Cabinet at a temperature not exceeding 60C.

The resulting product is pulverized in a mortar.

Output XC was 4.3 g, i.e. 86%.

The image is a buy allows you to get the drug chondroitin sulfate with increased mass fractions of the basic substance, and to increase the yield of the target product.

1. Electrochemical method of purification of chondroitin sulfate, including electrochemical deposition with obtaining hydrogel chondroitin sulfate (CS), stabilization, removal from the electrode, washing, drying, wherein XC is dissolved in a solution of 0.01-0.1 n alkali in the ratio of 1:50-1:200 and conduct electrochemical deposition in an alkaline medium at a constant cooling and stirring the solution with a speed of 10-20 rpm, current density of 1-10 a/m2, the voltage required for the deposition of cholesterol, stabilize the hydrogel XC is carried out in a solution of 0.05-0.5 n Hcl.

2. The method according to claim 1, characterized in that the voltage of electrochemical deposition is not less than 2.7 Century

3. The method according to claim 1, characterized in that the dried hydrogel XC first in air and then in a drying Cabinet at a temperature not exceeding 60C.

4. The method according to claim 1, characterized in that the time of electrochemical deposition ranges from 2 to 60 min, depending on the initial purity XC.

5. The method according to claim 1, characterized in that the cooling is carried out by flowing water with temperatures ranging from 4C to 15C.



 

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