KR101858733B1 - Method for preparing super low molecular weight of hyaluronic acid - Google Patents

Abstract

The present invention relates to a method for preparing a hyaluronic acid with a low molecular weight. More specifically, the method, which is for preparing a hyaluronic acid with a molecular weight of 10,000 or less or the salt thereof, includes the steps of: (a) dispersing a hyaluronic acid or a salt thereof in a basic aqueous medium; (b) centrifuging the solution prepared by the dispersion executed in step (a) to obtain a supernatant therefrom; (c) neutralizing the obtained supernatant and applying sodium chlorides and an organic solvent thereto; and (d) centrifuging the solution prepared in step (c) to obtain a precipitate therefrom. The method can be used to easily prepare the hyaluronic acid, which has a low molecular weight and does not cause microbial contamination, by dispersing the hyaluronic acid in the basic functional solvent and reprocessing the supernatant obtained by centrifugation.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing ultra low molecular weight hyaluronic acid,

The present invention relates to a method for producing ultra low molecular weight hyaluronic acid, and more particularly, to a method for producing ultra low molecular weight hyaluronic acid, comprising the steps of: (a) dispersing hyaluronic acid, a salt thereof or hyaluronic acid and a salt thereof in a basic functional medium; (b) centrifuging the dispersion to obtain a supernatant; (c) neutralizing the obtained supernatant and adding sodium chloride and an organic solvent; And (d) centrifuging the solution produced in step (c) to obtain a precipitate. The present invention also relates to a method for producing hyaluronic acid or a salt thereof having a molecular weight of 10,000 or less.

Hyaluronic acid is a polymer with a molecular weight of 500,000 to 13,000,000 Da and exists in nature and belongs to a colorless high viscosity polysaccharide.

The hyaluronic acid is a kind of glycosaminoglycan present in vivo and is a water-soluble, colorless, high viscosity, linear polysaccharide, which was first discovered by Meyer and Palmer from a glass bead blend.

The structure of the hyaluronic acid is the same repeating molecular structure of D-glucuronic acid and N-acetylglucosamine as repeating units, that is, glucuronic acid and N-acetylglucosamine are β-1 , 3-bond and β-1,4-bond are alternately and repeatedly connected.

Hyaluronic acid has various effects and excellent physical properties such as moisturizing effect, lubrication effect to physical friction, and protection against invasion of bacteria and the like, so that cosmetic additives, arthritis treatment agents, surgical aids for ophthalmic surgery and adhesion inhibitors after surgery Cosmetics, medicines, quasi-drugs, and foods. More specifically, the hyaluronic acid has a lubrication effect that smoothes the movement of the body, controls the movement of physiologically active substances, plays a mediator role in inducing differentiation and growth of cells by a specific action with cells, Collagen, elastin, and chondroitin sulfate, and is known to be excellent in the ability to contain water and excellent in viscosity and elasticity

.

Therefore, due to these properties, hyaluronic acid is used as a treatment for degenerative arthritis caused by aging and as a pain relieving agent for ophthalmologic surgery, and as a main ingredient of surgical assistants and dry eye drugs in the ophthalmic surgery, and also to supplement wrinkles and depressed skin tissues And it is also used as an additive for cosmetics and an additive for foods.

It has been reported that the physiological activity or functionality of hyaluronic acid varies depending on the molecular weight. Specifically, the hyaluronic acid of the polymer is used as a space-filler, has a function such as anti-angiogenic activity and immunosuppressive activity, and hyaluronic acid of the intermediate molecule is immunostimulatory, And low molecular weight hyaluronic acid is known to be involved in apoptosis and protein induction.

In the past, interest in polymer hyaluronic acid has been mainly focused on. However, due to the ability of low-molecular hyaluronic acid to absorb in the body, low-molecular hyaluronic acid has been gaining attention as a cosmetic or food for which absorption capacity is required in tissues.

As described above, when hyaluronic acid is used for foods and cosmetics, there is a disadvantage that hyaluronic acid having a high molecular weight is not easily absorbed into the body and absorbed into the skin, thereby failing to fully exhibit its effect. Therefore, it is necessary to develop an efficient method of lowering the molecular weight of the polymer hyaluronic acid or producing a low molecular weight hyaluronic acid from the polymer hyaluronic acid.

Accordingly, the present inventors have made much efforts to develop a method for efficiently producing a low molecular weight hyaluronic acid, particularly an ultra low molecular weight hyaluronic acid having a molecular weight of 10,000 or less, and as a result, it has been found that after a high molecular weight hyaluronic acid is dispersed in a basic functional medium Low molecular weight hyaluronic acid is separated from the precipitate by centrifugation and the supernatant is reprocessed to obtain ultra low molecular weight hyaluronic acid having a molecular weight of 10,000 or less.

Accordingly, it is an object of the present invention to provide a process for the preparation of (a) dispersing hyaluronic acid, a salt thereof, or hyaluronic acid and a salt thereof in a basic functional medium;

(b) centrifuging the dispersion to obtain a supernatant;

(c) neutralizing the obtained supernatant and adding sodium chloride and an organic solvent; And

(d) centrifuging the solution produced in step (c) to obtain a precipitate. The present invention also provides a method for producing an ultra low molecular weight hyaluronic acid or a salt thereof having a molecular weight of 10,000 or less.

In order to accomplish the above object of the present invention, the present invention provides a method for producing a hyaluronic acid, comprising: (a) dispersing hyaluronic acid, a salt thereof, or hyaluronic acid and a salt thereof in a basic functional medium;

(b) centrifuging the dispersion to obtain a supernatant;

(c) neutralizing the obtained supernatant and adding sodium chloride and an organic solvent; And

(d) centrifuging the solution produced in step (c) to obtain a precipitate. The method comprises the steps of: (a) preparing a solution of a low molecular weight hyaluronic acid having a molecular weight of 10,000 or less;

Hereinafter, the present invention will be described in detail.

(a) dispersing hyaluronic acid, a salt thereof, or hyaluronic acid and a salt thereof in a basic functional medium ;

In the present invention, 'hyaluronic acid' is a polysaccharide having repeating units of glucuronic acid and two sugars of N-acetylglucosamine. The 'hyaluronic acid salt' is not particularly limited, but is preferably a pharmaceutically acceptable salt, and examples thereof include a sodium salt, a potassium salt, a calcium salt, a zinc salt, a magnesium salt and an ammonium salt .

The hyaluronic acid and its salt (hereinafter, also referred to as "raw hyaluronic acid and its salt") which are raw materials of the low molecular weight hyaluronic acid and / or its salt of the present invention are generally used as biological tissues such as blood vessels, umbilical cord, Or a culture solution obtained by culturing a hyaluronic acid-producing microorganism such as a microorganism belonging to the genus Streptococcus (Strepto-cocus) as a raw material, and extracting (or purifying it if necessary) from such a raw material.

As the raw hyaluronic acid and its salt used in the present invention, any of the crude extract and the purified product may be used, but it is preferable that the purity of the purified product, specifically, hyaluronic acid and / or its salt is 90% or more. When raw hyaluronic acid having a purity of 90% or more and a salt thereof is used as a raw material for a cosmetic or food, it is difficult to cause change in color tone or flavor during storage, and thus a stable cosmetic or food can be obtained.

In one embodiment of the present invention, in order to obtain the raw hyaluronic acid, the remaining cells of the hyaluronic acid culture solution are removed by a lamination filtration method, and residual impurities are removed using an ultrafilter. This was treated with an adsorbent such as activated carbon, followed by decolorization and deodorization. After removing the adsorbent, a pure, colorless sterile hyaluronic acid solution was obtained using a filter of 0.22 μM pore size. No pulverization through separate precipitation proceeded.

In the present invention, the step (a) is a step of dispersing hyaluronic acid and / or a salt thereof in a basic functional medium. For example, the raw material hyaluronic acid and / or its salt in powder form may be added to a basic functional medium and stirred, or an organic solvent may be added to a pure hyaluronic acid solution as in the embodiment of the present invention, And then adjusting the pH by adding an alkali substance, followed by stirring.

The alkali material may be used without limitation as a substance that is ionized in a solution to render the solution basic, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, or the like. The amount of the alkali substance to be added is not particularly limited. However, if the amount of the alkali added is too small, the low molecular weight of the hyaluronic acid and / or its salt does not sufficiently progress and the production efficiency lowers. When the amount of the alkali added is too large, It is difficult to stably produce hyaluronic acid. Therefore, in the present invention, it is preferable that the alkaline substance is added so that the pH of the basic functional medium is adjusted to be in the range of 12-14.

In the production method of the present invention, the functional medium refers to a dispersion medium of hyaluronic acid and / or a salt thereof containing water. The medium which can be used for the functional medium is not particularly limited. For example, it is preferable that it is a liquid, has a property of dissolving in water, and can be used in a cosmetic or food manufacturing process. (For example, methanol, ethanol, n-propanol, 2-propanol, etc.), a ketone-based medium (for example, acetone, methyl ethyl ketone, etc.) , Tetrahydrofuran, acetonitrile and the like, and most preferably, ethanol, methanol, isopropyl alcohol, acetone, and mixtures thereof.

The ratio of water in the functional medium is preferably from 40 to 60% (v / v), most preferably 50% (v / v).

In the process for producing a low molecular weight hyaluronic acid and / or a salt thereof of the present invention, the above-described dispersion step can be carried out under heating. More specifically, the dispersion medium obtained by adding raw hyaluronic acid in powder form and / or a salt thereof to a basic functional medium with stirring can be heated. Or the basic functional medium may be heated in advance, and the raw hyaluronic acid and / or its salt may be added to maintain the temperature.

Here, the heating temperature of the basic functional medium is 50 to 90 占 폚, preferably 55 to 85 占 폚, more preferably 60 to 80 占 폚. By heating the basic functional medium at the above-mentioned temperature range for 1 hour to 10 hours, preferably 1.5 hours to 10 hours, more preferably 2 hours to 8 hours, it is possible to stably lower the molecular weight to the desired molecular weight.

Preferably, after the step (a), the step of cooling the resultant dispersion at 20 DEG C or lower, preferably 10 DEG C or lower, more preferably 4 DEG C or lower may be further included.

(b) centrifuging the dispersion to obtain a supernatant;

In the step (b), the dispersion prepared in the step (a) is centrifuged to separate into a precipitate and a supernatant. That is, in the step (a), hyaluronic acid having already been low-molecular-weight is divided again according to the molecular weight.

In the step (b), the centrifugation is preferably performed at 8000 to 15000 rpm for 1 minute to 10 minutes.

The low molecular weight hyaluronic acid having a molecular weight of 10,000 to 100,000 can be firstly obtained by washing and drying the precipitate obtained through the centrifugal separation in the step (b). According to one embodiment of the present invention, hyaluronic acid having an average molecular weight of about 30,000 to 50,000 can be prepared by washing and drying the precipitate obtained in the step (b).

On the other hand, the low molecular weight hyaluronic acid having a molecular weight of 10,000 or less which is the object of the present invention is dispersed in the supernatant after the centrifugation.

(c) neutralizing the obtained supernatant and then adding sodium chloride and Organic solvent  ;

In the step (c) of the present invention

First, the supernatant obtained in step (b) is neutralized to pH 7-8 using an acidic medium. The kind of the acidic medium used for neutralization is not particularly limited.

Then, an organic solvent is added to the neutralized supernatant by adding 1 to 10% (w / v) of sodium chloride to make a hydrated solution of 60 to 80% (v / v) 80% and the balance being water). This is because it is impossible to precipitate with only the neutralized hyaluronic acid solution and induces the dissolution of hyaluronic acid in the form of ultra-low molecular weight. As a result, it is possible to selectively elute only hyaluronic acid having a molecular weight of 5,000 to 15,000 Da in the form of an ultra-small molecule which has not been precipitated in a 50% aqueous solution.

The organic solvent may be selected from the group consisting of ethanol, methanol, isopropyl alcohol, acetone, and mixtures thereof, but is not limited thereto.

(d) centrifuging the solution produced in step (c) to obtain a precipitate;

When the sodium chloride and the organic solvent are added in the step (c), the solution becomes turbid. In the step (d) of the present invention, the desired ultra-low molecular weight hyaluronic acid can be obtained by centrifuging. The centrifugation may be performed at 8000 to 15000 rpm for 1 to 10 minutes, but is not limited thereto.

According to one embodiment of the present invention, the precipitate obtained in the step (d) was washed and dried, and the molecular weight was measured. As a result, it was confirmed that a low molecular weight hyaluronic acid having an average molecular weight of 5,000 to 7,000 was produced.

The method of converting the low molecular weight hyaluronic acid obtained by the above method into the low molecular weight hyaluronic acid salt and the method of converting the low molecular weight hyaluronic acid salt of the present invention into the low molecular weight hyaluronic acid is not particularly limited and a known method can be used have.

Examples of the method for converting the low molecular weight hyaluronic acid of the present invention into the low molecular weight hyaluronic acid salt include a method of treating with an aqueous acid solution (for example, an aqueous solution of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, etc.). Examples of the method for converting a salt of a low molecular weight hyaluronic acid of the present invention into a low molecular weight hyaluronic acid include an aqueous solution of an alkali (for example, an aqueous solution of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, And the like.

The method for measuring the molecular weight of the low molecular weight hyaluronic acid and / or its salt of the present invention is not particularly limited. For example, a method of obtaining an intrinsic viscosity at a kinetic viscosity, a method of converting the intrinsic viscosity to a molecular weight, .

In one embodiment of the present invention, the molecular weight of the low molecular weight hyaluronic acid and / or its salt is measured by obtaining the intrinsic viscosity at the kinetic viscosity and converting the intrinsic viscosity to the molecular weight.

The present invention can provide a method for easily producing a low molecular weight hyaluronic acid free from microbial contamination by reprocessing a supernatant obtained by dispersing hyaluronic acid in a basic functional solvent and then centrifuging. Further, pure hyaluronic acid in the form of ultra-low molecular weight can be selectively purified from the culture medium and purified.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Preparation of sterile pure hyaluronic acid (HA) solution

For this cultivation, Streptococcus dysgalactii ID9103 strain was inoculated into 3% Todd-Hewitt broth sterilized liquid medium (heart infusion 0.31%, neopeptone 2%, dextropse 0.2%, sodium chloride 0.2%, disodium phosphate 0.04% sodium carbonate 0.25%; BD) and cultured at 37 ° C for 6 hours. This was again inoculated into the same sterilized culture medium, cultured for 20 hours in an intermediate culture, and then inoculated into a 3 L sterilized culture broth in a 5 L fermenter. The culture media consisted of glucose 6%, yeast extract 0.5%, casein pepton 2%, glutamin 0.06%, and gluconic acid 0.1%. 3 L of a 5 g / L culture medium having a molecular weight of 2,000,000 Da, which had been completely cultured, was centrifuged to remove the cells. 2% diatomaceous earth of the filtered culture was added and stirred for 20 minutes. The mixed solution was filtered in a layered manner using a 1.5 [mu] M pore size filter (Hyundai KD-7).

The filtrate was filtered through an ultrafilter cassette to remove impurities. At this time, the filtration membrane used a form of removing less than 50,000 molecular weight. The circulation speed was set so as not to exceed the basic pressure of 2 bar and the osmotic pressure of 1.5 bar. In order to remove impurities, the purity of the hyaluronic acid solution was increased by continuously feeding the third distilled water until the conductivity became 200 mS / cm ² .

Through the above process, 2% activated carbon was added to the solution whose conductivity reached the standard, and the mixture was stirred for 12 hours or longer to remove the remaining endotoxin and color and odor. Thereafter, the adsorbent was removed by filtration again in a stacked manner, and then filtered with a sterilizing filter (pore size 0.22 μM) to obtain a sterilized pure HA solution.

≪ Example 2 >

Preparation of low molecular weight hyaluronic acid

≪ Example 2-1 >

To the HA aqueous solution obtained in Example 1, 1% (w / v) sodium hydroxide solution was added. Ethanol was added thereto to make HA solution of 1% sodium hydroxide and 50% ethanol (v / v). The mixture was stirred at 65 DEG C for 3 hours and then allowed to cool.

The resulting mixture was centrifuged at 10,000 rpm for 5 minutes and the resulting precipitate A was stored separately. The supernatant was neutralized to pH 7 by adding an acidic medium, and then 6% (w / v) sodium chloride was added thereto.

When the filtrate is stirred, an equal volume of ethanol is added to make a 70% aqueous solution (v / v), whereby the solution becomes turbid (that is, the ratio of ethanol: water is 70:30). This was centrifuged at 10,000 rpm for 5 minutes to obtain a precipitate B. The obtained precipitate B was washed once with 70% ethanol and dried at 45 ° C. On the other hand, the precipitate B was dissolved in 80% ethanol, the solvent was removed as much as possible, and dried at 45 ° C.

The dried hyaluronic acid powders A and B were dissolved in water without endotoxin, respectively, and the intrinsic viscosity and endotoxin content for the standard confirmation test were measured, and HA purity was measured by the carbazole method.

In the above steps, 1.65 g of low molecular weight hyaluronic acid (precipitate A) and 0.32 g of ultra low molecular weight hyaluronic acid (precipitate B) were obtained respectively as white fine powder. The intrinsic viscosity values using a Uberto viscometer were 0.1353 and 0.0292, respectively, and the calculated molecular weights were 41,000 Da and 5,700 Da. The purity by the analysis of carbazole was 101.8 and 100.6%, and the amount of endotoxin remaining was 0.0327 and 0.0458 EU per mg of hyaluronic acid. The carbazole method used in the present invention was (T.bitter, Anal. Biochem, 1962, 4, 330-334), and the endotoxin measurement was performed by Limulus Amebocyte Lysate (LAL) Reagent of Charles River Laboratories Korea. For molecular weight, the molecular weight is calculated based on the Mark-Houwink relation. (JF Voeks, Estimation of the parameters in the Mark-Houwink equation, Journal of Polymer Science, 1959).

< Example  2-2>

To the HA aqueous solution obtained in Example 1, 2% (w / v) sodium hydroxide solution was added. Ethanol was added thereto to prepare HA solution of 2% sodium hydroxide and 50% ethanol (v / v). The mixture was stirred at 65 DEG C for 3 hours and then allowed to cool.

The resulting mixture was centrifuged at 10,000 rpm for 5 minutes, and the resulting precipitate A was separately stored. The filtrate was neutralized to pH 7 by adding an acidic medium, and then 6% (w / v) sodium chloride was added thereto.

While the filtrate was stirred, an equal volume of 95% ethanol was added to make a 70% aqueous solution, followed by centrifugation at 10,000 rpm for 5 minutes to obtain a precipitate B. The obtained precipitate B was washed once with 70% ethanol and dried at 45 ° C. On the other hand, the precipitate B was dissolved in 80% ethanol, the solvent was removed as much as possible, and dried at 45 ° C.

The dried hyaluronic acid powders A and B were dissolved in water without endotoxin, respectively, and the intrinsic viscosity and endotoxin content for the standard confirmation test were measured, and HA purity was measured by the carbazole method.

In the above steps, 1.17 g and 0.59 g of a white fine powder of ultralow molecular hyaluronic acid were obtained, respectively. The intrinsic viscosity values using the Uberto viscometer were 0.1121 and 0.0332, respectively, and the calculated molecular weights were 32,000 Da and 6,700 Da. The purity by the analysis of carbazole was 102.0 and 100.2%, and the amount of endotoxin remaining was 0.0378 and 0.0571 EU per mg of hyaluronic acid.

< Example  3>

Depending on manufacturing conditions Low molecular weight  Production of hyaluronic acid

(I) the temperature and time at which hyaluronic acid is dispersed in the basic aqueous medium, (ii) the temperature and time at which the hyaluronic acid is dispersed in the basic aqueous medium, and (Iii) the concentration of sodium hydroxide to be treated was measured to determine the molecular weight of hyaluronic acid produced.

The results are shown in Tables 1 to 3 below.

The present invention provides a method for easily producing a low molecular weight hyaluronic acid free from microbial contamination by reprocessing a supernatant obtained by dispersing hyaluronic acid in a basic functional solvent and then centrifuging the resultant solution, thus being highly industrially applicable.

Claims (9)

(a) dispersing hyaluronic acid, a salt thereof, or hyaluronic acid and a salt thereof in a basic functional medium to prepare a dispersion;
(b) centrifuging the dispersion to obtain a supernatant;
(c) neutralizing the obtained supernatant and adding sodium chloride and an organic solvent; And
(d) centrifuging the solution produced in step (c) to obtain a precipitate. The method of producing hyaluronic acid or its salt having a molecular weight of 10,000 or less.
The process according to claim 1, wherein the basic functional medium has a pH of from 12 to 14.
The method according to claim 1, wherein the ratio of water in the functional medium is 40 to 60% (v / v).
The method according to claim 1, wherein the medium used as the functional medium is any one selected from the group consisting of ethanol, methanol, isopropyl alcohol, acetone, and mixtures thereof.
The method according to claim 1, wherein the step (a) is carried out while stirring at a temperature of 50 to 90 ° C.
The method according to claim 1, wherein the centrifugal separation in steps (b) and (d) is performed at 8000 to 15000 rpm for 1 to 10 minutes.
The method according to claim 1, wherein the organic solvent is any one selected from the group consisting of ethanol, methanol, isopropyl alcohol, acetone, and mixtures thereof.
The method of claim 1, further comprising washing the precipitate after step (d) and drying the precipitate.
The process for producing hyaluronic acid or a salt thereof according to claim 1, wherein the molecular weight is 2000 to 8000.