JP2587268B2 - Method for producing low-viscosity hyaluronic acid or salt thereof - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing low-viscosity hyaluronic acid or a salt thereof (hereinafter abbreviated as hyaluronic acid (salt)).

(Prior art) Hyaluronic acid is used in joints, vitreous, umbilical cord, cartilage, skin,
It is present as a component in connective tissues such as birds' crests, and plays an important role in tissue flexibility, structure maintenance, regulation of cell metabolism, and the like. In addition, hyaluronic acid is a polymer substance, and its solution has strong viscoelasticity and has a water retention effect, and is therefore widely used as a cosmetic raw material.
Among such hyaluronic acids, those having a particularly low viscosity, when used as cosmetics, give the skin a firmness and a sticky feeling, and have a low viscosity decrease even when held for a long time,
Storage stability is improved.

Conventionally, as a method for industrially obtaining hyaluronic acid (salt), a method of extracting from chicken chicks, vitreous body or umbilical cord of cattle, or a microorganism capable of producing hyaluronic acid (salt) is used as a medium. Production method by fermentation (fermentation method)
Has been done.

A low-viscosity hyaluronic acid (salt) can be obtained by lowering the viscosity of hyaluronic acid (salt) obtained by the extraction method or the fermentation method. An enzymatic decomposition method, which is a method for increasing the viscosity, has been performed.

Factors for lowering the viscosity of hyaluronic acid (salt) obtained by the extraction method or the fermentation method are described in JP-A-58-37001.
Are known.

 The following table shows these factors.

There is a correlation between the intrinsic viscosity and molecular weight of hyaluronic acid, and the lower the molecular weight, the lower the intrinsic viscosity. Therefore,
In order to produce low-viscosity hyaluronic acid (salt), the molecular weight of hyaluronic acid (salt) may be reduced.

Hyaluronidase is an enzyme present in living tissues and has an action of selectively cleaving the β-1-4 glycoside bond between N-acetylglucosamine of hyaluronic acid and glucuronic acid. Therefore, when hyaluronidase is allowed to act on hyaluronic acid, the molecular chain of hyaluronic acid is cut to reduce the molecular weight, and as a result, low-viscosity hyaluronic acid is obtained. The method of lowering the viscosity using hyaluronidase is a method in which only the β-1-4 glycosidic bond of hyaluronic acid is cleaved and other functional groups such as N-acetyl groups are not hydrolyzed. As a method for producing hyaluronic acid (salt), a method using exclusively hyaluronidase has been used.

(Problems to be Solved by the Invention) However, this method requires a high production cost because the enzyme is expensive. Hyaluronidase, an enzyme,
Since it is produced from animal tissues by the extraction method, the titer varies. For this reason, the time required to reduce the viscosity varies depending on the enzyme titer, and the desired low viscosity hyaluronic acid (salt)
There are drawbacks such as that very difficult process control is required to obtain

Further, as can be seen from Table 1 already shown, it is considered that the heat treatment and the alkali treatment are promising as methods for lowering the viscosity. However, when deacetylating chitin to produce chitosan, more than 30% caustic soda solution is used,
Although heat treatment is performed at 0 ° C. for 3 hours, this method not only cuts the β-1-4 glycosidic bond of hyaluronic acid but also cuts the N-acetyl group. For this reason, alkali treatment has not been performed as a method for producing low-viscosity hyaluronic acid (salt).

Therefore, the present invention does not cleave the N-acetyl group and
It provides an easy method of controlling the process by using only inexpensive drugs without using high production cost enzymes to produce low-viscosity hyaluronic acid (salt) industrially at low cost by cutting only 4 glycosidic bonds. The purpose is to do.

(Means for Solving the Problems) In the present invention, an aqueous solution of hyaluronic acid (salt) is added with an acid or alkali having a concentration of 0.1 N or less, and the mixture is uniformly stirred at a temperature of 30 ° C. or higher and lower than 50 ° C. In addition, the molecular weight of hyaluronic acid (salt) is reduced without deacetylation.

The acid is preferably one or more of sulfuric acid, hydrochloric acid and phosphoric acid.

The alkali is preferably one or more of the alkali metal hydroxides.

The intrinsic viscosity of the low-viscosity hyaluronic acid (salt) is preferably 10 dl / g or less.

When a 0.5% aqueous solution of hyaluronic acid (salt) is reacted with stirring at a temperature of 50 ° C in a 0.1 N aqueous caustic soda solution and a 0.1 N diluted aqueous hydrochloric acid solution, the molecular weight of hyaluronic acid (salt) is as shown in Table 2. It changes like

As can be seen from Table 2, 0.1N caustic soda aqueous solution,
Alternatively, when treated with an aqueous solution of hydrochloric acid, the hyaluronic acid (salt) decreases in viscosity over time at 50 ° C.

It is known that N-acetylglucosamines such as hyaluronic acid (salt) are hydrolyzed to an acetyl group to give glycosamine when treated with an acid or an alkali. Therefore, aqueous solutions of hyaluronic acid with various acid or alkali concentrations
After treatment at 7 ° C for 7 hours, sodium hyaluronate 20
The amount of acetic acid in mg was as shown in Table 3.

The results in Table 3 show that under the condition of treatment at 45 ° C. for 7 hours, the deacetylation reaction does not occur if the acid or alkali concentration is 0.1 N or less.

Then, the mixture was treated for 7 hours at a reaction temperature of 0.1 N under the condition of an acid or alkali concentration. As a result, the amount of acetic acid in 20 mg of sodium hyaluronate was as shown in Table 4.

From the results in Table 4, it can be seen that under the condition of treatment for 7 hours at 0.1 N acid or alkali concentration, the deacetylation reaction does not occur at a temperature lower than 50 ° C, preferably at a temperature of 45 ° C or lower. .

The amount of acetic acid in sodium hyaluronate was determined as follows.

Hydrolysis Take exactly 20 mg of sodium hyaluronate in terms of dry matter. 1 ml of 2N hydrochloric acid is added, and a hydrolysis reaction is performed in boiling water for 3 hours. After cooling on ice, add 0.10 ml of propionic acid and add 1 μl
Is subjected to gas chromatography for analysis.

Calibration curve To 1.0 ml of 0.1-0.5% acetic acid (in 2N hydrochloric acid) is added 0.100 ml of propionic acid as an internal standard, and 1 μ of the mixture is subjected to gas chromatography. A calibration curve is prepared from the peak area ratio of acetic acid to propionic acid and the concentration of acetic acid.

Gas chromatography conditions Model: Shimadzu GC-6A Column: Polapack Q Temperature: 170 ° C to 172 ° C Detection: TCD (200 ° C) The theoretical value of the amount of acetic acid in 20 mg of sodium hyaluronate is calculated by the following formula.

As a result of examining the infrared absorption spectrum and the NMR chart of sodium hyaluronate treated with 0.1 N acid or alkali, no difference was observed from the untreated product (blank).

In order to obtain powdery sodium hyaluronate from the treatment liquid after completion of the viscosity reduction treatment, neutralize the treatment liquid to a pH of about 7, then add a small amount of sodium chloride and dissolve it. Ethyl alcohol, methyl alcohol and acetone as solvents are added to precipitate sodium hyaluronate. After the mother liquor is filtered off, it is dried under reduced pressure to obtain a low-viscosity sodium hyaluronate powder.

(Effect of the Invention) By using the method of the present invention, a large amount of hyaluronic acid (salt) can be produced at a very low cost and with good reproducibility, and the N-acetyl group is not cleaved and the β-1-4 glycoside is not cleaved. A low-viscosity hyaluronic acid (salt) having the desired viscosity is obtained by breaking only the bond. Thus, it is possible to provide a method for easily managing a treatment process using an inexpensive drug without using an enzyme having a high production cost.

Example 1 100 g of sodium hyaluronate (intrinsic viscosity = 14.1 dl / g) was added to a 0.1 N hydrochloric acid aqueous solution 20 and dissolved by stirring. Completely dissolved sodium hyaluronate 0.5%
The solution is heated to 45 ° C. while stirring. Thereafter, a viscosity lowering treatment was performed at 45 ° C. for 24 hours with uniform stirring.

After 24 hours, a 6N aqueous sodium hydroxide solution was added to neutralize the aqueous sodium hyaluronate solution. After rapidly cooling the solution temperature to room temperature, sodium chloride was dissolved so that the concentration of sodium chloride became 0.2 mol.

While uniformly stirring the entire solution, three times the volume of ethyl alcohol was gradually added. The stirring was stopped to precipitate the precipitated sodium hyaluronate. A half amount of the supernatant was removed by decantation, and the same amount of ethyl alcohol as this solution was added, followed by stirring again. After stirring for 30 minutes, the stirring was stopped, and the precipitate of sodium hyaluronate was separated from the mother liquor by suction filtration. The sodium hyaluronate precipitate on the filter cloth was washed again with ethanol and then reduced under reduced pressure.
Dry at 40 ° C for 12 hours to obtain low viscosity sodium hyaluronate
8.4 g were obtained.

The purity of this low-viscosity sodium hyaluronate is 98.7% as dry matter, the intrinsic viscosity is 2.0 dl / g, and the amount of acetic acid in 20 mg is 2.9.
7 mg.

Example 2 100 g of sodium hyaluronate (intrinsic viscosity = 14.1 dl / g) was added to 0.1 N aqueous sodium hydroxide solution 20 and dissolved by stirring.

While stirring the completely dissolved 0.5% sodium hyaluronate solution, the temperature is raised to 45 ° C. After that, with uniform stirring
Low viscosity treatment was performed at 45 ° C for 24 hours. At this time, the intrinsic viscosity of sodium hyaluronate changed with time as follows.

After 24 hours, 6N hydrochloric acid was added to neutralize the aqueous sodium hyaluronate solution. After rapidly cooling the solution temperature to room temperature, sodium chloride was dissolved so that the concentration of sodium chloride became 0.2 mol.

While uniformly stirring the entire solution, three times the volume of ethyl alcohol was gradually added. The stirring was stopped to precipitate the precipitated sodium hyaluronate, and a half of the supernatant was taken out by decantation. The same amount of ethyl alcohol as this solution was added, followed by stirring again. After stirring for 30 minutes, the stirring was stopped, and the precipitate of sodium hyaluronate was separated from the mother liquor by suction filtration. The sodium hyaluronate precipitate on the filter cloth was washed again with ethanol, and then dried under reduced pressure at 40 ° C. for 12 hours to obtain 8.0 g of low viscosity and sodium aluronate.

The purity of this low-viscosity sodium hyaluronate is 98.5% as dry matter, the intrinsic viscosity is 1.9dl / g, and the amount of acetic acid in 20mg is
2.96 mg.

FIG. 1 shows an infrared absorption spectrum of sodium hyaluronate used as a raw material, and FIG. 2 shows an infrared absorption spectrum of sodium hyaluronate subjected to low viscosity treatment.
No difference was observed in the infrared absorption chart.

Comparative Example A viscosity lowering treatment was performed under exactly the same conditions as in Example 2 except that the viscosity lowering treatment was performed using a 0.5 N aqueous solution of soda.

The purity of the obtained sodium hyaluronate was 97.9% as a dried product, the intrinsic viscosity was 1.2 dl / g, and the amount of acetic acid in 20 mg was 2.6.
It was 8 mg, and it was clear that a deacetylation reaction had occurred. The infrared absorption spectrum of the obtained sodium hyaluronate was as shown in FIG.

[Brief description of the drawings]

1 is an infrared absorption spectrum of sodium hyaluronate as a raw material according to an embodiment of the present invention, FIG. 2 is an infrared absorption spectrum of sodium hyaluronate subjected to low viscosity treatment, and FIG. 3 is a hyaluronic acid in a comparative example. It is an infrared absorption spectrum of sodium.

Claims (4)

(57) [Claims] 1. An aqueous solution of hyaluronic acid or a salt thereof, wherein 0.1
A low-viscosity hyaluronic acid or a low-viscosity hyaluronic acid characterized by adding an acid or alkali below the specified temperature and uniformly stirring under conditions of a temperature of 30 ° C. or higher and lower than 50 ° C. to reduce the molecular weight of hyaluronic acid or a salt thereof. A method for producing the salt. 2. The method according to claim 1, wherein the acid is one or more of sulfuric acid, hydrochloric acid and phosphoric acid. 3. The method according to claim 1, wherein the alkali is one or more of alkali metal hydroxides. 4. The method according to claim 1, wherein the intrinsic viscosity of the low-viscosity hyaluronic acid or a salt thereof is 10 dl / g or less.