RU2478652C1 - Electrochemical method of purifying chondroitin sulphate - Google Patents

Abstract

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

Description

The invention relates to methods for producing, or rather purifying chondroitin sulfate, obtained from tissues of marine hydrobionts, such as cartilage of fish, muscle and muscle sac of mollusks, etc., and can be used in food, cosmetic industry, medicine.

The main properties of chondroitin sulfate, which are crucial for its successful application in various fields, are high bioavailability, biological compatibility, low toxicity, and the ability to selectively accumulate in cartilage tissue (Kofuji K., Ito T., Murata Y., Kawashima S. Effect of chondroitin sulfate on the biodegradation and drug release of chitosan gel beads in subcutaneous air pouches of mice // Biological and Pharmaceutical Bulletin. - 2002. - Vol.25, No.2. - P.268-271). The listed properties are determined by the chemical structure of chondroitin sulfate molecules, namely, the molecular weight, degree and place of sulfation. (Michelacci YM, Dietrich S.P. 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 RJ 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), comprising dissolving chondroitin sulfate in an alkaline medium, enzymatic hydrolysis of proteins, separation of the high molecular weight carbohydrate fraction by precipitation from low molecular weight protein hydrolysis products remaining in solution, washing the resulting precipitate and drying the finished product (Takai M., Kono H Salmon-origin chondroitin sulfate: European Patent EP 1270599. IPC A61K 31/737. - Declared 12/15/2000; No. EP 20000981747; Published 02.01.2003).

This generally accepted technology is implemented by various authors in different ways: the sequence, number of operations, temperature conditions, nature and concentration of the reagents used change.

A known method of separation and purification of cholesterol, which consists in the hydrolysis of the feedstock and the subsequent precipitation of cholesterol from the hydrolyzate by adding ethyl alcohol (US Pat. RF. No. 2056851, publ. 03/27/1996; US Pat. No. 2061485, publ. 06/10/1998; Pat. RF No. 2383351, published March 10, 2010). The disadvantages of the method are as follows. Requires a large consumption of ethyl alcohol, as well as the need for its regeneration. In addition, the deposition is not complete, so a fairly significant portion of cholesterol remains in solution.

A known method of purification of a polysaccharide based on electrochemical precipitation of a polysaccharide from a solution (US Pat. US No. 7883615, publ. 08.02.2011). As a result of the discharge of negatively charged polysaccharide molecules, in particular chitosan, the polysaccharide in the form of a hydrogel is deposited on the anode. The resulting product is subsequently removed from the electrode, washed in distilled water and dried. This method is closest to the proposed and adopted as a prototype. The method is intended to produce polysaccharide films for further use as boards in the production of biosensors. Electrochemical deposition in the prototype method is carried out in an acidic medium PH = 5.0-5.5, the current density is high and is 20-100 A / m ² , the duration of electrochemical deposition is 2-30 minutes The stabilization of the hydrogel of the polysaccharide is carried out in an alkaline medium (sufficiently basic solution).

The objective of the invention is to obtain cholesterol with an increased mass fraction of the main substance, i.e. increasing the degree of purification of the target product.

It is solved in the electrochemical method for the purification of chondroitin sulfate, which involves electrochemical deposition to obtain a hydrogel XC, stabilization, removal from the electrode, washing and drying, XC is dissolved in a solution of 0.01-0.1N alkali in a ratio of 1: 50-1: 200 and conduct electrochemical deposition in an alkaline medium with constant cooling and stirring the solution at a speed of 10-20 rpm, a current density of 1-10 A / m ² , the voltage required for deposition, the stabilization of the hydrogel cholesterol is carried out in a solution of 0.05-0.5 n Hcl. In this case, the voltage of electrochemical deposition is not less than 2.7 V, and the deposition time is from 2 to 60 minutes, depending on the degree of purity of the initial cholesterol, the hydrogel cholesterol is dried first in air and then in an oven at a temperature not exceeding 60 ° C. The reaction mixture is cooled with running water at a temperature of 4 ° C-15 ° C.

Electrochemical deposition is carried out in an alkaline medium, after dissolving the initial cholesterol in a 0.01-0.10 N alkali solution, such as NaOH or another. The dissolution of cholesterol in alkali and electrochemical deposition in an alkaline medium is due to the fact that it dissolves much better in alkaline than in acidic or neutral environments, therefore, the cleaning process will be more complete. The mass ratio of chondrotin sulfate and alkali solution is from 1:50 to 1: 200 (the concentration of cholesterol in the solution is from 0.5 to 2.0%). The resulting solution is poured into an electrolytic cell.

A cell is a container (a glass or polymer cup) in which electrodes and a mixing device are located. The anode is made in the form of a cylinder located at the side walls of the vessel, and the cathode is in the form of a central rod. Between the cathode and the anode there is a rotating mixer, which is made either in the form of a frame or a blade, the axis of which is inclined at an angle to the horizontal plane of the container.

To cool the reaction mixture, the tank is equipped with a cooling device in the form of an external jacket into which cooling water is supplied. For cooling, the tank can be immersed in a vessel with running cooling water or in a liquid thermostat. The temperature of the cooling water is from 4 to 15 ° C. Cooling is necessary for a more complete deposition of hydrochloride hydrochloride on the electrode.

A constant voltage of at least 2.7 V is applied to the electrodes; at a voltage lower than 2.7 V, no deposition will occur on the electrode. The current density is from 1 to 10 A / m ² , this density is quite enough for the process. High current densities cause significant heat generation, which will not contribute to a more complete deposition of cholesterol on the electrode.

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

At the end of electrolysis, the voltage from the electrodes is removed, a cylindrical electrode with precipitated hydrogel XC is transferred to a vessel with distilled water and washed for 1-5 minutes.

Then, the electrode with the precipitated hydrogel XC is transferred to a vessel with 0.05-0.5 n HCl for the final formation of a precipitate, i.e. carry out stabilization of the resulting hydrogel, then washed with distilled water.

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

The resulting product is ground in a device of any design.

Example

A portion of the initial cholesterol 5.0 g was dissolved in 500 ml of a 0.05 N NaOH solution (or other alkali) and poured into an electrolytic cell.

The electrolytic cell was a glass in which a cylindrical anode and cathode are located in the form of a central rod. The electrodes are made of stainless steel. Between the cathode and the anode is placed a rotating frame mixer.

To cool the reaction mixture, the cell was placed in a container with running tap water. The temperature of the cooling water was 8 ° C.

A constant voltage of 2.7 V was applied to the electrodes. The current in the electrolyte was 2.5 A / m ² .

The electrolysis is carried out with continuous slow 20 rpm stirring the solution between the electrodes for 10 minutes

At the end of electrolysis, the voltage is removed from the electrodes. A cylindrical electrode with a precipitated chondroitin gel is transferred to a vessel with distilled water and washed for 1-5 minutes.

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

The wet gel is cut off from the surface of the electrode and dried in air, and then in an oven at a temperature not exceeding 60 ° C.

The resulting product is ground in a mortar.

The yield of cholesterol was 4.3 g, i.e. 86%

The invention allows to obtain a preparation of chondroitin sulfate with an increased mass fraction of the main substance, as well as to increase the yield of the target product.

Claims (5)

1. The electrochemical method of purification of chondroitin sulfate, including electrochemical deposition to obtain a hydrogel of chondroitin sulfate (CS), stabilization, removal from the electrode, washing, drying, characterized in that CS is dissolved in a solution of 0.01-0.1 n alkali in a ratio of 1 : 50-1: 200 and carry out electrochemical deposition in an alkaline medium with constant cooling and stirring the solution at a speed of 10-20 rpm, current density 1-10 A / m ² , voltage required for precipitation of cholesterol, stabilization of hydrogel cholesterol 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 the electrochemical deposition is at least 2.7 V. 3. The method according to claim 1, characterized in that the hydrochloride hydrochloride is dried first in air and then in an oven at a temperature not exceeding 60 ° C. 4. The method according to claim 1, characterized in that the time of electrochemical deposition is from 2 to 60 minutes, depending on the initial degree of purity of the cholesterol. 5. The method according to claim 1, characterized in that the cooling is carried out with running water with a temperature of from 4 ° C to 15 ° C.