JP2012001696A - Method for producing low molecular weight hyaluronic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a low molecular weight hyaluronic acid, particularly a tetrasaccharide low molecular weight hyaluronic acid of high purity, efficiently, safely, and at low cost.SOLUTION: This production method for a low molecular weight hyaluronic acid includes: a process of making hyaluronic acid having a low molecular weight by decomposing hyaluronic acid and/or a salt thereof with an aqueous organic sulfonic acid solution, where the organic sulfonic acid is preferably a compound represented by formula (1): RSOH (1), more preferably at least one type selected from the group consisting of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and toluenesulfonic acid, particularly preferably methanesulfonic acid.

Description

  The present invention relates to a method for producing low molecular weight hyaluronic acid.

  Hyaluronic acid (hereinafter also referred to as “HA”) is a polysaccharide having two or more basic structural molecules, glucuronic acid and N-acetylglucosamine, as repeating structural units (see the following general formula (a)).

Hyaluronic acid having such a structure is widely used in the fields of pharmaceuticals, foods and drinks, and cosmetics due to its high moisturizing function.

  In the general formula (a), when n is 1, that is, the minimum structural unit of hyaluronic acid is referred to as disaccharide hyaluronic acid (hereinafter also referred to as “HA2”). The case where n is 2, that is, the case where two HA2 are combined is called tetrasaccharide hyaluronic acid (hereinafter also referred to as “HA4”). Hyaluronic acid is usually a polymer in which a large number of HA2 are bonded, and has a molecular weight of 500,000 or more.

  In recent years, it has been found that hyaluronic acid having a molecular weight reduced to less than tens of thousands (hereinafter also referred to as “low molecular hyaluronic acid”) exhibits various activities. In particular, it has been known that low molecular weight hyaluronic acids such as HA2 and HA4 exhibit various activities. Examples of the activity of HA4 include suppression of cell death caused by maintaining cells at a high temperature (HA4 increases production of heat shock protein, which is a kind of stress protein, and protects cell death), When added and cultured in the derived cultured cell line PC12, differentiation induction of PC12 cells into nerve cells is caused and neurite extension action is exhibited.

  As described above, it has been found that HA4 exhibits a characteristic and significant action as compared with other low-molecular hyaluronic acid.

  As a method for producing such low-molecular hyaluronic acid, a method of decomposing hyaluronic acid with an enzyme such as hyaluronidase (for example, see Patent Document 1), a method of treating hyaluronic acid with an acid such as hydrochloric acid (for example, Patent Document 2) And 3) are known.

JP 2003-339393 A JP 2009-256683 A JP 2006-265287 A

  However, the method of degrading hyaluronic acid with an enzyme such as hyaluronidase has a problem that the purity is lowered because the enzyme component remains in the low-molecular hyaluronic acid. In order to obtain low-molecular-weight hyaluronic acid with high purity, the production process becomes complicated, for example, a process for removing the enzyme component is required. Furthermore, the price of the enzyme is extremely high, which is economically disadvantageous.

  In addition, the method of treating hyaluronic acid with an acid such as hydrochloric acid has a simple reaction system compared with the method of decomposing with an enzyme, but it is difficult to control the decomposition reaction and the yield of low-molecular hyaluronic acid is low. There is. In addition, in the decomposition reaction with acids such as hydrochloric acid, a large amount of salt is generated by neutralization, so a desalting step is required after decomposition, and with volatile acids such as hydrochloric acid, equipment that prevents corrosion and human damage of peripheral equipment The manufacturing process is complicated.

  Until now, there have been little studies on methods for producing low molecular weight hyaluronic acid, particularly HA2 and HA4. However, as described above, since low molecular hyaluronic acid such as HA4 is expected to be used in the field of medicine, etc., the above problems are solved, and low molecular hyaluronic acid with higher purity is efficiently produced at a low price. Therefore, it is desired to manufacture it safely.

  Accordingly, an object of the present invention is to provide a method for producing a low-molecular hyaluronic acid having a high purity, particularly HA4, efficiently, safely and at a low cost.

  As a result of intensive studies to solve the above problems, the present inventor has improved the above-described conventional method of decomposing hyaluronic acid, and using organic sulfonic acid for the decomposition of hyaluronic acid, thereby efficiently and safely at low cost. In addition, the present inventors have found that low-molecular-weight hyaluronic acid, particularly HA4, can be obtained with a very high yield and high purity, and the present invention has been completed.

  That is, the present invention is as follows.

  [1] A method for producing low molecular weight hyaluronic acid, comprising the step of decomposing hyaluronic acid and / or a salt thereof with an organic sulfonic acid aqueous solution to lower the molecular weight.

  [2] The method for producing low-molecular hyaluronic acid according to [1], wherein the organic sulfonic acid is a compound represented by the following formula (1).

(In the formula (1), R represents a hydrocarbon group having 1 to 10 carbon atoms.)
[3] The organic sulfonic acid is at least one selected from the group consisting of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and toluenesulfonic acid [1] or [1] [2] The method for producing a low molecular weight hyaluronic acid according to [2].

  [4] The method for producing low-molecular hyaluronic acid according to any one of [1] to [3], wherein the organic sulfonic acid is methanesulfonic acid.

  [5] The method for producing low-molecular hyaluronic acid according to any one of [1] to [4], wherein the low-molecular hyaluronic acid is a low-molecular hyaluronic acid having 4 or less sugars.

  [6] The method for producing low molecular hyaluronic acid according to any one of [1] to [5], wherein the low molecular hyaluronic acid is tetrasaccharide and / or disaccharide low molecular hyaluronic acid.

  [7] The method for producing low-molecular hyaluronic acid according to any one of [1] to [6], wherein the step is performed under heating.

  [8] The method for producing low molecular weight hyaluronic acid according to [7], wherein the heating temperature is in the range of 60 to 80 ° C.

  [9] The method for producing low-molecular hyaluronic acid according to [7], wherein the heating time is 3 to 24 hours.

  [10] The method for producing low-molecular hyaluronic acid according to any one of [1] to [9], wherein the concentration of the organic sulfonic acid aqueous solution is 0.1 to 0.6 N.

  [11] The low amount according to any one of [1] to [10], wherein 1 to 10 parts by mass of the hyaluronic acid and / or a salt thereof is added to 100 parts by mass of the organic sulfonic acid aqueous solution. A method for producing molecular hyaluronic acid.

  According to the method for producing low-molecular hyaluronic acid of the present invention, low-molecular hyaluronic acid, particularly HA4, having high purity and high yield can be obtained efficiently, safely and at low cost.

It is a figure which shows an example of the HPLC analysis result of HA4 obtained in Example 13. It is a figure which shows an example of the HPLC analysis result of the low molecular weight hyaluronic acid produced | generated when a raw material hyaluronic acid is decomposed | disassembled with methanesulfonic acid aqueous solution. It is a figure which shows an example of the HPLC analysis result of the low molecular weight hyaluronic acid produced | generated when a raw material hyaluronic acid is decomposed | disassembled with hydrochloric acid.

  Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. The present invention is not limited to the following description, and various modifications can be made within the scope of the gist thereof.

  The method for producing low-molecular hyaluronic acid according to this embodiment includes a step of decomposing hyaluronic acid and / or a salt thereof with an organic sulfonic acid aqueous solution to reduce the molecular weight.

  In the present embodiment, hyaluronic acid means a polysaccharide having glucuronic acid and N-acetylglucosamine disaccharide as repeating structural units. Moreover, it does not specifically limit as a salt of hyaluronic acid, For example, sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt etc. are mentioned.

  Hyaluronic acid and / or a salt thereof (hereinafter also referred to as “raw hyaluronic acid”), which is a raw material for the production method of the present embodiment, is a polymer having a molecular weight of usually 500,000 or more.

  The raw material hyaluronic acid used in the production method of the present embodiment is generally a biological tissue such as chicken crown, umbilical cord, eyeball, skin, cartilage, or a culture solution obtained by culturing a hyaluronic acid-producing microorganism such as a microorganism belonging to the genus Streptococcus. The raw material is obtained by extraction (further purified as necessary) from these raw materials. As the raw material hyaluronic acid used in the present embodiment, either the crude extract or the purified product may be used.

  In the present embodiment, the low molecular weight hyaluronic acid refers to a raw material hyaluronic acid having a low molecular weight and a molecular weight of usually less than tens of thousands.

  The low molecular hyaluronic acid obtained in the production method of the present embodiment is preferably a low molecular hyaluronic acid having 4 or less sugars, and more preferably a tetrasaccharide and / or disaccharide low molecular hyaluronic acid. The tetrasaccharide low-molecular-weight hyaluronic acid (hereinafter also referred to as “HA4”) obtained in the production method of the present embodiment has high purity, and can be suitably used as a pharmaceutical, particularly a therapeutic agent for the nervous system such as the brain.

  The organic sulfonic acid used in the production method of the present embodiment is preferably a compound represented by the following formula (1).

In the above formula (1), R is a hydrocarbon group having 1 to 10 carbon atoms, preferably a hydrocarbon group having 1 to 2 carbon atoms (methyl group, ethyl group), more preferably a carbon atom having 1 carbon atom. Represents a hydrogen group (methyl group).

  The organic sulfonic acid is composed of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, o-toluenesulfonic acid, p-toluenesulfonic acid, m-toluenesulfonic acid, α-naphthalenesulfonic acid, β-naphthalenesulfonic acid. More preferred is at least one selected from the group, and particularly preferred is methanesulfonic acid.

  In the production method of the present embodiment, when the organic sulfonic acid as described above is used, the raw material hyaluronic acid can be appropriately decomposed to have a low molecular weight, and the high-purity low-molecular hyaluronic acid, particularly a tetrasaccharide low-molecular weight Hyaluronic acid can be obtained efficiently with a very high yield.

  The present inventor considers the reason why such an effect is obtained as follows.

  The inventor first focused on the binding relationship of the elements constituting hyaluronic acid, and searched for a substance capable of appropriately cutting the bond and obtaining the target low-molecular hyaluronic acid with high selectivity.

  In order to hydrolyze hyaluronic acid having disaccharides of glucuronic acid and N-acetylglucosamine as repeating structural units with acid to obtain low molecular weight hyaluronic acid, the glycosidic bond on the reducing end side of N-acetylglucosamine is cleaved. However, the glycosidic bond on the reducing end side of glucuronic acid must remain.

  These two glycosidic bonds are physicochemically strong and weak, and the glycosidic bond on the reducing end side of N-acetylglucosamine is more likely to be cleaved. By utilizing this difference in ease of cleavage, hydrolysis under the condition that the reducing end side of N-acetylglucosamine is cut but the reducing end side of glucuronic acid is left uncut, the target low molecular weight hyaluronic acid is obtained. Can be efficiently obtained in high yield.

  Therefore, the acid that decomposes the raw material hyaluronic acid is not an acid that breaks the glycosidic bond on the reducing end side of glucuronic acid, or even destroys the carbon skeleton inside the sugar molecule, but the reducing end side of N-acetylglucosamine. Acids that cleave only the glycosidic bonds are desirable. Furthermore, the ease with which these bonds are cleaved (whether cleaving is facilitated) depends on the type of acid. For example, in the case of hydrolysis with hydrochloric acid, the difference in the ease of cleaving the internal bond of hyaluronic acid It is small depending on the cleavage site, and it is considered that an excessive decomposition reaction is likely to occur, in which not only the glycosidic bond on the reducing end side of N-acetylglucosamine but also other bonds are simultaneously cleaved.

  As a result of intensive investigation of an acid capable of efficiently producing a high-purity low-molecular hyaluronic acid without causing an excessive decomposition reaction like hydrochloric acid, the bond is broken when an organic sulfonic acid is used. It has been found that the difference in the degree of easiness to be performed is large depending on the cleavage site, and the glycosidic bond on the reducing end side of N-acetylglucosamine is easily cleaved, for example, similarly to hydrochloric acid, but other bonds are not easily cleaved.

  Organic sulfonic acids are usually used as catalysts for amino acid degradation and chemical synthesis reactions, and are themselves highly reactive and have a chromosomal mutation effect on cells. When such an organic sulfonic acid is used for hydrolysis of the raw material hyaluronic acid, the bond is appropriately broken as described above. Therefore, excessive decomposition is unlikely to occur compared with hydrochloric acid, and high-purity low-molecular hyaluronic acid is extremely high. It is thought that it can be efficiently obtained in a yield. In particular, hydrolyzing hyaluronic acid using methanesulfonic acid suppresses excessive decomposition compared to the case of using other strong acids such as hydrochloric acid, thereby producing highly pure low molecular weight hyaluronic acid in a very high yield. It is thought that it can be obtained efficiently.

  Furthermore, organic sulfonic acids are generally non-volatile and are less likely to vaporize when heated. Therefore, the method using organic sulfonic acid is highly safe in the work environment because the risk of corrosion of peripheral equipment and human damage due to vaporized acid is reduced compared to the conventional method using volatile acid such as hydrochloric acid. No special equipment is required.

  In the manufacturing method according to the present embodiment, the organic sulfonic acid described above is usually used in the decomposition step as an aqueous solution. The concentration of the organic sulfonic acid aqueous solution is preferably 0.1 to 0.6N, more preferably 0.3 to 0.6N, and particularly preferably 0.3N. When the concentration of the organic sulfonic acid aqueous solution is within the above range, a high-purity low-molecular hyaluronic acid tends to be efficiently obtained.

  The addition amount of the raw material hyaluronic acid is preferably 1 to 10 parts by mass, more preferably 1 to 5 parts by mass, and more preferably 5 parts by mass with respect to 100 parts by mass of the organic sulfonic acid aqueous solution. Particularly preferred. When the addition amount of the raw material hyaluronic acid is within the above range, a high-purity low-molecular hyaluronic acid tends to be obtained in a high yield.

  In the manufacturing method according to the present embodiment, the decomposition process of the raw material hyaluronic acid is preferably performed under heating. The heating temperature is preferably in the range of 60 to 80 ° C, more preferably 70 to 80 ° C, and particularly preferably 80 ° C. Furthermore, the heating time is preferably 3 to 24 hours, more preferably 13 to 22 hours, and particularly preferably 18 hours.

  When the decomposition process of the raw material hyaluronic acid is performed under such heating, a high-purity low-molecular hyaluronic acid tends to be efficiently obtained in a shorter time.

  The method for producing low molecular hyaluronic acid according to this embodiment may further include a separation / purification step. As the separation / purification step, a known separation / purification step can be employed, for example, a step of separation / purification using an ion exchange resin column, a step of separation / purification using a gel filtration column, Examples include a step of separation / purification using a filtration membrane, a step of separation / purification using a solvent such as a sodium chloride aqueous solution, hydrochloric acid, and purified water.

  By performing such a separation / purification step, the target low-molecular hyaluronic acid can be obtained with higher purity.

  The method for producing low molecular weight hyaluronic acid according to the present embodiment may further include other steps such as a concentration step and a drying step. These steps are not particularly limited, and known steps can be employed.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

[Example 1]
Below, the example of manufacture of HA4 in an industrial scale by the manufacturing method of this invention is shown.

As a raw material hyaluronic acid, hyaluronic acid sodium salt (manufactured by Baoding Sino-chem Industry, product name: Sodium hyaluronate, molecular weight: 1.25 × 10 ⁶ dalton) was used. Methanesulfonic acid was used as the organic sulfonic acid.

  In a reaction vessel, the sodium hyaluronate (16 kg) and a 0.3 N aqueous methanesulfonic acid solution (320 liters) were added and stirred at 80 ° C. for 4 hours to decompose the sodium hyaluronate. A reaction solution containing low molecular weight hyaluronic acid was obtained.

  The yield of HA4 in the obtained reaction solution was measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100).

[Example 2]
In the same manner as in Example 1, the sodium hyaluronate was decomposed to obtain a reaction solution containing low molecular weight hyaluronic acid, and the yield of HA4 in the reaction solution was measured.

  The average yield of HA4 obtained in Example 1 and Example 2 is shown in Table 1 below.

[Example 3]
Except that the thermal decomposition time was changed from 4 hours to 5 hours, the reaction mixture containing low molecular weight hyaluronic acid was obtained by decomposing the hyaluronic acid sodium salt in the same manner as in Example 1 to obtain HA4 in the reaction solution. The yield was measured. The average yield of HA4 obtained in Example 3 is shown in Table 1 below.

[Example 4]
Except that the thermal decomposition time was changed from 4 hours to 5.5 hours, the reaction mixture containing the low molecular weight hyaluronic acid was obtained by decomposing the hyaluronic acid sodium salt in the same manner as in Example 1. The yield of HA4 was measured.

[Example 5]
In the same manner as in Example 4, the sodium hyaluronate was decomposed to obtain a reaction solution containing low molecular weight hyaluronic acid, and the yield of HA4 in the reaction solution was measured.

  The average yield of HA4 obtained in Example 4 and Example 5 is shown in Table 1 below.

[Example 6]
Except that the thermal decomposition time was changed from 4 hours to 6.5 hours, in the same manner as in Example 1, the sodium hyaluronate was decomposed to obtain a reaction solution containing low molecular weight hyaluronic acid. The yield of HA4 was measured.

[Examples 7 to 11]
In the same manner as in Example 6, the hyaluronic acid sodium salt was decomposed to obtain a reaction solution containing low molecular weight hyaluronic acid, and the yield of HA4 in the reaction solution was measured.

  The average value of the yield of HA4 obtained in Examples 6 to 11 is shown in Table 1 below.

[Example 12]
The reaction solution containing low molecular weight hyaluronic acid was obtained by decomposing the hyaluronic acid sodium salt in the same manner as in Example 1 except that the heat decomposition time was changed from 4 hours to 19.87 hours. The yield of HA4 was measured. The average yield of HA4 obtained in Example 12 is shown in Table 1 below.

[Example 13]
About the reaction liquid obtained in Examples 3-6, the following processes were performed independently, respectively.

  First, the reaction solution was cooled and then passed through an anion exchange resin (Cl type: Dowex 1 × 4) column to adsorb the methanesulfonic acid.

  The reaction solution containing low molecular weight hyaluronic acid passed through the anion exchange resin column was diluted 5 times or more with purified water, and the pH was adjusted to 6 with an aqueous sodium hydroxide solution. The pH-adjusted reaction solution was passed through an anion exchange resin (Cl type: Dowex 1 × 4) column to adsorb low molecular weight hyaluronic acid.

  Next, a 40 mM sodium chloride aqueous solution having a volume 5 times that of the column was passed through the anion exchange resin column to wash impurities. Further, a sodium chloride aqueous solution whose concentration was linearly increased from 40 mM to 100 mM was passed through the anion exchange resin column to sequentially elute low molecular weight hyaluronic acid.

  The fraction of HA4 was collected from the eluted solution containing low molecular weight hyaluronic acid, and the pH was adjusted to 4.2 to 4.7 with hydrochloric acid. The pH-adjusted solution was passed through an anion exchange resin (Cl type: BioRad High Q) column to adsorb HA4.

  The impurities were washed by flowing 0.1 mM hydrochloric acid through the anion exchange resin (Cl type: BioRad High Q) column, and then 0.5M sodium chloride aqueous solution was flowed to elute HA4. The obtained eluate was concentrated by a rotary evaporator.

  The obtained concentrated solution was passed through a gel filtration column (BioGel P2) whose pH was adjusted to 4 to 5 with dilute hydrochloric acid and eluted with purified water to obtain desalted HA4.

  Prior to performing ultrafiltration, which is the final purification step described below, the desalted HA4 derived from the reaction liquid obtained in Examples 3 to 6 was mixed to form one lot.

  The lot was passed through a cation exchange resin (acid type: Dowex 50W x 4) column to adsorb impurities, and further filtered through an ultrafiltration membrane (exclusion molecular weight: 5,000 to 10,000 daltons). The obtained filtrate was concentrated with a rotary evaporator and dried to obtain powdered HA4.

  The obtained HA4 was analyzed by high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100), and the purity was 98.89%.

[Example 14]
Powdered HA4 was obtained in the same manner as in Example 13 except that the reaction solutions obtained in Examples 7 to 10 were used. The obtained HA4 was analyzed by high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100), and the purity was 99.14%.

[Example 15]
Powdered HA4 was obtained in the same manner as in Example 13 except that the reaction solution obtained in Example 11 was used alone. The obtained HA4 was analyzed by high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100), and the purity was 99.60%.

  As described above, the three lots of HA4 obtained in Examples 13 to 15 were analyzed by HPLC. As a result, the purity of the lot consisting only of the intermediate product with the heating time set to the optimum condition was 99% or more. The HPLC analysis result is shown in FIG.

[Example 16]
As a raw material hyaluronic acid, hyaluronic acid sodium salt (manufactured by Baoding Sino-chem Industry, product name: Sodium hyaluronate, molecular weight: 1.25 × 10 ⁶ dalton) was used. Methanesulfonic acid was used as the organic sulfonic acid.

  The hyaluronic acid sodium salt and 0.1N methanesulfonic acid aqueous solution were added to the reactor, and the mixture was stirred at 80 ° C. for 12 to 22 hours, whereby the hyaluronic acid sodium salt was decomposed to give low molecular weight hyaluronic acid. A reaction solution containing was obtained. The concentration of the sodium hyaluronate in the methanesulfonic acid aqueous solution was 5%.

  Concentrations of HA4, HA4 partial degradation products, HA2 and monosaccharide degradation products in the obtained reaction solution were measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 2 below.

[Example 17]
A reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Example 16 except that the concentration of the methanesulfonic acid aqueous solution was changed to 0.2N.

  Concentrations of HA4, HA4 partial degradation products, HA2 and monosaccharide degradation products in the obtained reaction solution were measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 3 below.

[Example 18]
A reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Example 16 except that the concentration of the methanesulfonic acid aqueous solution was changed to 0.3N.

  Concentrations of HA4, HA4 partial degradation products, HA2 and monosaccharide degradation products in the obtained reaction solution were measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 4 below.

[Example 19]
A reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Example 16 except that the concentration of the methanesulfonic acid aqueous solution was changed to 0.4N.

  Concentrations of HA4, HA4 partial degradation products, HA2 and monosaccharide degradation products in the obtained reaction solution were measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 5 below.

[Example 20]
A reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Example 16 except that the concentration of the methanesulfonic acid aqueous solution was changed to 0.5N.

  Concentrations of HA4, HA4 partial degradation products, HA2 and monosaccharide degradation products in the obtained reaction solution were measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 6 below.

[Comparative Example 1]
A reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Example 16 except that the 0.1 N methanesulfonic acid aqueous solution was changed to 1.0 N hydrochloric acid.

  Concentrations of HA4, HA4 partial degradation products, HA2 and monosaccharide degradation products in the obtained reaction solution were measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 7 below.

[Example 21]
As the raw material hyaluronic acid, hyaluronic acid (manufactured by Food Chemifa Co., Ltd., product name: hyaluronic acid FCH-SU, cosmetic grade molecular weight of 100,000 or less) was used. Methanesulfonic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the organic sulfonic acid.

  In the reactor, the hyaluronic acid and 0.3N methanesulfonic acid aqueous solution (3 liters) were added and stirred at 80 ° C. for 22 hours, whereby the hyaluronic acid was thermally decomposed to produce low molecular weight hyaluronic acid. A reaction solution containing was obtained. The concentration of the hyaluronic acid in the methanesulfonic acid aqueous solution was 5%.

  The concentration of HA4 in the obtained reaction solution was measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 9 below.

[Comparative Example 2]
A reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Example 21 except that the conditions of the acid to be added and the heating conditions were changed as shown in Table 8 below. The concentration of HA4 in the obtained reaction solution was measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 8 below.

[Comparative Example 3]
As shown in Table 8 below, a reaction solution containing low molecular weight hyaluronic acid was obtained in the same manner as in Comparative Example 2 except that the concentration of hyaluronic acid in the methanesulfonic acid aqueous solution was changed to 10% by mass. The concentration of HA4 in the obtained reaction solution was measured using high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100). The measurement results are shown in Table 8 below.

[Comparative Example 4]
A purification step was performed in the same manner as in Example 13 except that the reaction solution obtained in Comparative Example 3 was used to obtain powdered HA4. The obtained HA4 was analyzed by high performance liquid chromatography (HPLC, manufactured by Agilent Technologies, model number: Agilent 1100), and the purity was 96.1%.

  From the above results, it was found that when hydrolyzing the raw material hyaluronic acid with hydrochloric acid, there are many decomposition products of the sugar itself due to excessive decomposition. These decomposition products become impurities in the subsequent purification process of low-molecular hyaluronic acid, and reduce the purity of the target low-molecular hyaluronic acid after purification. Therefore, when the raw material hyaluronic acid was hydrolyzed with hydrochloric acid, the final HA4 purity was about 96% even if the purification step was performed as described above.

  On the other hand, when the raw material hyaluronic acid is hydrolyzed with an aqueous sulfonic acid solution, sugar decomposition products due to excessive decomposition can be reduced as compared with the conventional method using hydrochloric acid, and the low product obtained by separation and purification with a column or the like after the decomposition step. It is believed that the purity and yield of molecular hyaluronic acid, especially HA4, was better than when it was decomposed with hydrochloric acid.

  The method for producing low-molecular hyaluronic acid of the present invention can obtain low-molecular hyaluronic acid, particularly HA4, which is efficient, safe and low-cost, and has a very high yield and high purity. Such high-purity low-molecular hyaluronic acid can be effectively used in a wide range of fields such as pharmaceuticals, foods and drinks, and cosmetics.

Claims (11)

  A method for producing low molecular weight hyaluronic acid, comprising a step of decomposing hyaluronic acid and / or a salt thereof with an organic sulfonic acid aqueous solution to lower the molecular weight. The said organic sulfonic acid is a compound represented by following formula (1), The manufacturing method of the low molecular hyaluronic acid of Claim 1 characterized by the above-mentioned.
(In the formula (1), R represents a hydrocarbon group having 1 to 10 carbon atoms.)   The organic sulfonic acid is at least one selected from the group consisting of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and toluenesulfonic acid. A method for producing low-molecular hyaluronic acid.   The method for producing low-molecular hyaluronic acid according to any one of claims 1 to 3, wherein the organic sulfonic acid is methanesulfonic acid.   The method for producing low-molecular hyaluronic acid according to any one of claims 1 to 4, wherein the low-molecular hyaluronic acid is a low-molecular hyaluronic acid having 4 or less sugars.   The method for producing low-molecular hyaluronic acid according to any one of claims 1 to 5, wherein the low-molecular hyaluronic acid is tetrasaccharide and / or disaccharide low-molecular hyaluronic acid.   The said process is performed under a heating, The manufacturing method of the low molecular weight hyaluronic acid as described in any one of Claims 1-6 characterized by the above-mentioned.   The said heating temperature is the range of 60-80 degreeC, The manufacturing method of the low molecular hyaluronic acid of Claim 7 characterized by the above-mentioned.   The method for producing low molecular weight hyaluronic acid according to claim 7, wherein the heating time is 3 to 24 hours.   The method for producing low-molecular hyaluronic acid according to any one of claims 1 to 9, wherein the concentration of the aqueous organic sulfonic acid solution is 0.1 to 0.6N.   The low-molecular-weight hyaluronic acid according to any one of claims 1 to 10, wherein 1 to 10 parts by mass of the hyaluronic acid and / or a salt thereof is added to 100 parts by mass of the organic sulfonic acid aqueous solution. Manufacturing method.