PREPARATION METHOD FOR ACETYLATED SODIUM HYALURONATE

Abstract

The present invention relates to a preparation method for acetylated sodium hyaluronate, which pertains to the field of organic chemical synthesis, characterized in that, the preparation method includes the following steps: in the protective atmosphere of inert gas, subjecting a hyaluronate and an acetic anhydride to a catalytic reaction in an organic solvent with a catalyst added, adding a water absorbent during the reaction, obtaining a reaction liquid, subjecting a reaction liquid to post treatment, separation, purification, and drying to obtain a product. A too high or too low degree of substitution is effectively avoided in the present invention, so as to obtain a product with a degree of substitution of acetyl groups ranging from 2.7 to 3.2, with mild reactive materials and reacting conditions, and simple separation and purification steps.

Claims

1. A preparation method for acetylated sodium hyaluronate, wherein the preparation method comprises the following steps: in the protective atmosphere of inert gas, subjecting a hyaluronate and an acetic anhydride to a catalytic reaction in an organic solvent with a catalyst added, adding a water absorbent during the reaction, obtaining a reaction liquid, subjecting a reaction liquid to post treatment, separation, purification, and drying to obtain a product.

2. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein reaction of the hyaluronate and the acetic anhydride is as follows: dissolving the hyaluronate in the organic solvent until a homogeneous phase is formed, adding the water absorbent, decreasing a temperature of the reaction system to 10° C.-−5° C., dropwise adding the acetic anhydride to the reaction system, then dropwise adding a DMAP-toluene solution to the reaction system, after reacting for a certain time, heating the reaction system to 40° C.-70° C. for reacting for a certain time; a mass ratio of the hyaluronate to the acetic anhydride ranges from 1:1 to 1:5, an average molecular weight of the hyaluronate ranges from 5 KDa to 5000 KDa.

3. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein after the catalytic reaction is finished, the post treatment to the reaction liquid is as follows: adding water to the reaction liquid to remove the remaining acetic anhydride, and then filtering the reaction liquid to remove the water absorbent.

4. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein molecular sieve is used as the water absorbent, 4-dimethylaminopyridine is used as the catalyst.

5. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein toluene is used as the organic solvent, a solid-liquid ratio of the hyaluronate in the organic solvent ranges from 1:5-1:20.

6. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein the separation and purification of the reaction liquid is as follows: subjecting the reaction liquid to membrane filtration for purification, wherein water in small amount is added several times during the purification until a fraction is colorless and smells slightly sour.

7. The preparation method for acetylated sodium hyaluronate according to claim 6, wherein an organic membrane or a ceramic membrane is used as the membrane, a pore size of the membrane ranges from 200 KD-5000 KD.

8. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein after the separation and purification, pH of the reaction liquid is adjusted to 5-7 by an alkali solution, and then concentration is performed.

9. The preparation method for acetylated sodium hyaluronate according to claim 8, wherein at least one of a sodium hydroxide solution, a sodium carbonate solution and a sodium bicarbonate solution is used as the alkali solution.

10. The preparation method for acetylated sodium hyaluronate according to claim 1, wherein low-temperature drying is adopted as the drying, and the temperature is 0-25° C.

11. The preparation method for acetylated sodium hyaluronate according to claim 2, wherein after the catalytic reaction is finished, the post treatment to the reaction liquid is as follows: adding water to the reaction liquid to remove the remaining acetic anhydride, and then filtering the reaction liquid to remove the water absorbent.

12. The preparation method for acetylated sodium hyaluronate according to claim 2, wherein molecular sieve is used as the water absorbent, 4-dimethylaminopyridine is used as the catalyst.

13. The preparation method for acetylated sodium hyaluronate according to claim 2, wherein toluene is used as the organic solvent, a solid-liquid ratio of the hyaluronate in the organic solvent ranges from 1:5-1:20.

14. The preparation method for acetylated sodium hyaluronate according to claim 6, wherein after the separation and purification, pH of the reaction liquid is adjusted to 5-7 by an alkali solution, and then concentration is performed.

15. The preparation method for acetylated sodium hyaluronate according to claim 14, wherein at least one of a sodium hydroxide solution, a sodium carbonate solution and a sodium bicarbonate solution is used as the alkali solution.

Description

DESCRIPTION OF THE EMBODIMENTS

[0020] Principle and characteristics of the present invention are described in the following, and the examples listed are for illustrating of the present invention only, and are not intended to limit the scope of the present invention.

Example 1

[0021] A preparation method for acetylated sodium hyaluronate in the present example included the following steps:

[0022] (1) In the protective atmosphere of nitrogen, 50.00 g HA with a molecular weight of 10 KDa was weighed in a 1000 mL reaction flask, and an anhydrous solvent, 250 mL toluene, was added followed by increasing the temperature to 60° C. with stirring, until they were all dissolved to form a homogeneous phase. Molecular sieve was added for absorbing water in the reaction. Then, the reaction system was cooled to −5° C. 50 mL acetic anhydride was added to the reaction liquid, and then 0.25 g DMAP dissolved in 4 mL toluene solution was dropwise added to the reaction liquid followed by one-hour reaction at room temperature. The reaction was then heated to 40° C. and lasted for 4 hours to obtain a faint yellow turbid liquid.

[0023] (2) Water in the same volume was added to remove the remaining acetic anhydride, and then the reaction liquid was filtered to remove the molecular sieve.

[0024] (3) The reaction liquid was purified by membrane filtration, water in small amount was added several times during the purification until a fraction was colorless and smelt slightly sour, and the reaction liquid after the last purification had a volume of 200 mL.

[0025] (4) The reaction liquid was adjusted by sodium hydroxide to have a pH ranging from 5-7, and then was subjected to concentration.

[0026] (5) Drying was performed to obtain 39.01 g acetylated sodium hyaluronate, with a yield of 60%.

[0027] The obtained product had a crystalline shape, and was determined by a hydroxylamine colorimetric method to have a degree of substitution of acetyl groups being 2.9, which was within the acceptable range of 2.6-3.6.

[0028] Particularly, the yield μ=401.3*m.sub.2/m.sub.1*(401.3+(43−1)*DS);

[0029] wherein m.sub.1 represents a mass of the fed HA, m.sub.2 represents a mass of the obtained AcHA, and DS represents the degree of substitution of acetyl groups.

Example 2

[0030] A preparation method for acetylated sodium hyaluronate in the present example included the following steps:

[0031] (1) In the protective atmosphere of nitrogen, 30.00 g HA with a molecular weight of 1000 KDa was weighed in a 1000 mL reaction flask, and an anhydrous solvent, 300 mL toluene, was added followed by increasing the temperature to 70° C. with stirring (increasing the temperature was to dissolve the sodium hyaluronate, the greater the molecular weight, the harder the dissolution), until they were all dissolved to form a homogeneous phase. Molecular sieve was added for absorbing water in the reaction. Then, the reaction system was cooled to 0° C. 90 mL acetic anhydride was added to the reaction liquid, and then 0.2 g DMAP dissolved in 4 mL toluene solution was dropwise added to the reaction liquid followed by one-hour reaction at room temperature. The reaction was then heated to 50° C. and lasted for 12 hours to obtain a faint yellow turbid liquid.

[0032] (2) Water in the same volume was added to remove the remaining acetic anhydride, and then the reaction liquid was filtered to remove the molecular sieve.

[0033] (3) The reaction liquid was purified by membrane filtration, water in small amount was added several times during the purification until a fraction was colorless and smelt slightly sour, and the reaction liquid after the last purification had a volume of approximately 500 mL.

[0034] (4) The reaction liquid was adjusted by sodium carbonate to have a pH ranging from 5-7, and then was subjected to concentration.

[0035] (5) Drying was performed to obtain 17.5 g acetylated sodium hyaluronate, with a yield of 58%.

[0036] The obtained product had a crystalline shape, and was determined by a hydroxylamine colorimetric method to have a degree of substitution of acetyl groups being 2.7, which was within the acceptable range of 2.6-3.6.

Example 3

[0037] A preparation method for acetylated sodium hyaluronate in the present example included the following steps:

[0038] (1) In the protective atmosphere of nitrogen, 20.00 g HA with a molecular weight of 200 KDa was weighed in a 1000 mL reaction flask, and an anhydrous solvent, 400 mL toluene, was added followed by increasing the temperature to 70° C. with stirring, until they were all dissolved to form a homogeneous phase. Molecular sieve was added for absorbing water in the reaction. Then, the reaction system was cooled to −5° C. 100 mL acetic anhydride was added to the reaction liquid, and then 0.24 g DMAP dissolved in 4 mL toluene solution was dropwise added to the reaction liquid followed by one-hour reaction at room temperature. The reaction was then heated to 70° C. and lasted for 48 hours to obtain a faint yellow turbid liquid.

[0039] (2) Water in the same volume was added to remove the remaining acetic anhydride, and then the reaction liquid was filtered to remove the molecular sieve.

[0040] (3) The reaction liquid was purified by membrane filtration, water in small amount was added several times during the purification until a fraction was colorless and smelt slightly sour, and the reaction liquid after the last purification had a volume of 1000 mL.

[0041] (4) The reaction liquid was adjusted by sodium bicarbonate to have a pH ranging from 5-7, and then was subjected to concentration.

[0042] (5) Drying was performed to obtain 13.8 g acetylated sodium hyaluronate, with a yield of 52%.

[0043] The obtained product had a crystalline shape, and was determined by a hydroxylamine colorimetric method to have a degree of substitution of acetyl groups being 3.17, which was within the acceptable range of 2.6-3.6.

Comparative Example 1

[0044] The preparation method was the same as that in Example 1. The difference lies in that water absorbent was not added in the process, and a degree of substitution for acetylated HA was 2.21.

[0045] The results show that acylation reaction occurred during the reaction process with relatively low degree of substitution. The reason is that sodium hyaluronate has a relatively strong moisture-retaining ability, and the water therein cannot be completely removed without destroying its structure. Sodium hyaluronate in normal cosmetic grade has a loss on drying ranging from 6% to 9%. Acetic anhydride would be subjected to the hydrolysis reaction with the water brought in the reaction system by sodium hyaluronate, thus generating acetic acid and consuming a portion of acetic anhydride. Such acylation reaction is a reversible esterification reaction wherein hydrogens on the hydroxyl group is acylated and substituted. The water in the reaction system would facilitate a reverse reaction in later period, thus affecting the degree of substitution.

Comparative Example 2

[0046] The preparation method was the same as that in Example 1. The difference lies in that water absorbent was replaced by concentrated sulfuric acid, and the result is that the water absorbent had a rather great effect on the pH value of the reaction system and even participated the reaction directly.

Comparative Example 3

[0047] The preparation method was the same as that in Example 1. The difference lies in that water absorbent was replaced by soda lime, and the result is that the water absorbent had a rather great effect on the pH value of the reaction system and even participated the reaction directly.

Comparative Example 4

[0048] The preparation method was the same as that in Example 1. The difference lies in that water absorbent was replaced by anhydrous sodium sulfate, and the result is that the anhydrous sodium sulfate became caked mass after absorbing water and thus affecting the stirring.

Comparative Example 5

[0049] The preparation method was the same as that in Example 1. The difference lies in that the temperature in step (1) was controlled to room temperature and the other conditions remained the same. The results show that it took a very long time to dissolve the sodium hyaluronate, the reaction system was not cooled when dropwise adding acetic anhydride and floccule was generated at the adding point. Then heat released in the reaction system partially and more floccule was generated at the adding point. Upon collection and determination, the floccule is acetylated sodium hyaluronate having a too high degree of substitution being close to 4. The floccule was no long soluble in water and was not hydrophilic, which was unqualified.