Preparation method of low-molecular-weight sodium hyaluronate

Abstract

The invention relates to the field of preparation of sodium hyaluronate, in particular to a preparation method of low-molecular-weight sodium hyaluronate. The method specifically comprises the following steps: stirring and dissolving high-molecular-weight sodium hyaluronate powder into water at 45-60 DEG C to obtain a high-molecular-weight sodium hyaluronate solution; cooling to 35-45 DEG C, adding a buffer solution, uniformly mixing, and then adding papain and hyaluronidase in a mass ratio of (2-3): (3-6) to carry out enzymolysis reaction, so as to obtain enzymatic hydrolysate; adding a sodium hydroxide solution into the enzymatic hydrolysate, adjusting the pH value to 7.5-9.0, heating to 80-90 DEG C, treating for 20-40 minutes, and filtering to obtain a low-molecular-weight sodium hyaluronate solution; and performing spray drying. The preparation method is simple in process, mild in condition and free of damage to the product structure, and the prepared low-molecular-weight sodium hyaluronate is high in purity, uniform in molecular weight distribution and good in permeability.

Description

Technical Field

[0001] The present invention relates to the field of preparation of sodium hyaluronate, and in particular to a preparation method of low molecular weight sodium hyaluronate and low molecular weight sodium hyaluronate. Background Art

[0002] Sodium hyaluronate (HA) is a natural high molecular weight mucopolysaccharide, known as the "natural moisturizing factor". Studies have shown that sodium hyaluronate has other important physiological functions such as improving skin moisture, preventing postoperative adhesion, promoting wound healing, and anti-inflammatory and anti-tumor. Therefore, it is widely used in the medical, cosmetic and food industries.

[0003] At present, the molecular weight of most sodium hyaluronates on the market is around several thousand to several million Daltons. Compared with large-molecule sodium hyaluronate, sodium hyaluronate with an average molecular weight of 5K-20W Da is likely to penetrate into the stratum corneum of the skin, and is easily absorbed by the human body and keeps the skin moisturized for a long time; sodium hyaluronate with an average molecular weight below 5000Da can penetrate into the dermis, deeply moisturize, and truly inhibit inflammation and repair cells. Therefore, sodium hyaluronate with a small molecular weight and uniform structure can be easily absorbed, making its performance more superior. Under this premise, it is of great significance to develop a sodium hyaluronate with a small molecular weight and uniform structure. Summary of the invention

[0004] In view of the above technical problems, the object of the present invention is to provide a method for preparing low molecular weight sodium hyaluronate, which has simple process, mild conditions, no damage to the product structure, and the prepared low molecular weight sodium hyaluronate has high purity, uniform molecular weight distribution and good permeability.

[0005] In a first aspect, the present invention provides a method for preparing low molecular weight sodium hyaluronate, comprising the following steps:

[0006] (1) Add high molecular weight sodium hyaluronate powder into deionized water and stir at 45°C to 60°C until completely dissolved to obtain a high molecular weight sodium hyaluronate solution;

[0007] (2) When the solution is cooled to 35-45°C, a buffer solution is added and mixed evenly, and then papain and hyaluronidase are added to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolyzate, wherein the mass ratio of papain to hyaluronidase is (2-3):(3-6);

[0008] (3) Adding sodium hydroxide solution to the enzymatic hydrolyzate, adjusting the pH to 7.5-9.0, heating to 80-90°C for 20-40 minutes, and filtering to obtain a low molecular weight sodium hyaluronate solution;

[0009] (4) Concentrating and spray-drying the low molecular weight sodium hyaluronate solution.

[0010] As a preferred embodiment, in step (1), the molecular weight of the high molecular weight sodium hyaluronate is 20W~160WDa.

[0011] As a preferred embodiment, in step (2), the mass ratio of papain to hyaluronidase is 2:5.

[0012] As a preferred embodiment, in step (2), the amount of hyaluronidase used is 3-6% of the high molecular weight sodium hyaluronate.

[0013] As a preferred embodiment, in step (2), the buffer solution is prepared by adding cysteine ​​hydrochloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride into distilled water and stirring to dissolve.

[0014] As a more preferred embodiment, the mass concentration ratio of cysteine ​​hydrochloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride is (0.05-0.2):(1-3):(4-6):(9-11).

[0015] As a preferred embodiment, in step (2), the pH value of the enzymatic hydrolysis reaction is 7.0-7.2, the temperature is 30-42° C., and the enzymatic hydrolysis time is 6-12 hours.

[0016] As a more preferred embodiment, in the step (2), the temperature of the enzymatic hydrolysis reaction is 37°C.

[0017] As a preferred embodiment, in step (4), the inlet temperature of the spray drying is 120-150°C, and the outlet temperature is 30-55°C.

[0018] As a more preferred embodiment, in the step (4), the outlet temperature of the spray drying is 40°C.

[0019] In a second aspect, the present invention provides low molecular weight sodium hyaluronate obtained by the above preparation method, which has a molecular weight of 2000-5000 Da and a water content of ≤5%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The preparation method of low molecular weight sodium hyaluronate of the present invention has simple process, mild conditions, and no damage to the product structure; and does not require the use of large amounts of organic solvents and chemical reagents, thereby reducing environmental pollution and production costs.

[0022] 2. The method for preparing low molecular weight sodium hyaluronate of the present invention has high purity, uniform molecular weight distribution, a molecular weight of 2000-5000Da, a water content of ≤5%, and excellent performance.

[0023] 3. The preparation method of low molecular weight sodium hyaluronate of the present invention uses papain and hyaluronidase in the enzymatic hydrolysis reaction. Hyaluronidase can specifically hydrolyze the glycosidic bonds in hyaluronic acid and degrade it into low molecular weight hyaluronic acid; the buffer solution, especially sodium chloride, can improve the stability of hyaluronidase and papain and reduce the effects of temperature and pH on enzyme activity; cysteine ​​hydrochloride can improve enzyme activity and shorten the enzymatic hydrolysis reaction time; papain can reduce the viscosity of high molecular weight sodium hyaluronate on the one hand, which helps to enzymatically hydrolyze sodium hyaluronate into lower molecular weight sodium hyaluronate, and on the other hand, it can act on the residual high molecular weight part and possible protein impurities in hyaluronic acid to improve the purity of the product and the proportion of low molecular weight sodium hyaluronate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the standard infrared spectrum of sodium hyaluronate in the European Pharmacopoeia;

[0025] Figure 2 This is the infrared spectrum of the low molecular weight sodium hyaluronate prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods, but does not constitute any limitation to the present invention.

[0027] The chemical drugs involved in the embodiments of the present invention are all commercially available products;

[0028] The experimental methods involved are all well-known experimental methods.

[0029] Example 1

[0030] 0.05 g of cysteine ​​hydrochloride, 1 g of sodium dihydrogen phosphate, 4 g of sodium dihydrogen phosphate and 11 g of sodium chloride were added to 1 L of deionized water, and stirred to dissolve to obtain a buffer solution; 10 g of high molecular weight sodium hyaluronate powder with a molecular weight of 20W~160W Da was added to 500 mL of deionized water, and stirred at 45°C~60°C until completely dissolved to obtain a high molecular weight sodium hyaluronate solution; when the high molecular weight sodium hyaluronate solution was cooled to 35~45°C, 150 mL of buffer solution was added and mixed evenly, and then 0.2 g of papain and 0.5 g of hyaluronidase were added, and the mixture was stirred at a pH of 7.0, a temperature of 30~42°C, and a stirring speed of 280 r / min. The enzymatic hydrolysis reaction was carried out for 12 hours to obtain an enzymatic solution; sodium hydroxide solution was added to the enzymatic solution to adjust the pH to 7.5-9.0, heated to 80-90°C for 20-40 minutes and filtered to obtain a low molecular weight sodium hyaluronate solution; the low molecular weight sodium hyaluronate solution was ultrafiltered and concentrated using a 300Da ultrafiltration membrane to obtain a concentrated solution, which was then spray dried, with the inlet temperature of the spray drying set to 120-150°C and the outlet temperature set to 30-55°C to obtain a low molecular weight sodium hyaluronate powder. The low molecular weight sodium hyaluronate powder was subjected to infrared detection, and the results were as follows: Figure 2 As shown in the infrared spectrum, it can be seen that the sodium hyaluronate obtained by the preparation method of the present invention is consistent with the European Pharmacopoeia standard spectrum ( Figure 1 ), indicating that its structural integrity was not destroyed during the preparation process.

[0031] Example 2

[0032] Add 0.4g cysteine ​​hydrochloride, 4g sodium dihydrogen phosphate, 12g disodium hydrogen phosphate and 22g sodium chloride into 2L deionized water, stir and dissolve to obtain a buffer solution; 100g of high molecular weight sodium hyaluronate powder (Da) is added to 4000mL of deionized water, and stirred at 45°C-60°C until completely dissolved to obtain a high molecular weight sodium hyaluronate solution; when the high molecular weight sodium hyaluronate solution is cooled to 45°C, 1250mL of buffer solution is added and mixed evenly, and then 3g of papain and 6g of hyaluronidase are added, and enzymolysis reaction is carried out for 6 hours at a pH value of 7.2, a temperature of 37°C, and a stirring speed of 300r / min to obtain an enzymolysis solution; sodium hydroxide solution is added to the enzymolysis solution, the pH is adjusted to 9.0, and the solution is heated to 80-90°C for 20 minutes and then filtered to obtain a low molecular weight sodium hyaluronate solution; the low molecular weight sodium hyaluronate solution is filtered and concentrated with a 300Da reverse osmosis membrane to obtain a concentrated solution, and then spray dried, and the inlet temperature of the spray drying is set to 120°C and the outlet temperature is set to 55°C to obtain a low molecular weight sodium hyaluronate powder.

[0033] Example 3

[0034] Add 0.1g cysteine ​​hydrochloride, 2g sodium dihydrogen phosphate, 6g disodium hydrogen phosphate and 20g sodium chloride to 1L deionized water, stir and dissolve to obtain a buffer solution; 20g of high molecular weight sodium hyaluronate powder (Da) was added into 1200mL of deionized water, and stirred at 60°C until completely dissolved to obtain a high molecular weight sodium hyaluronate solution; when the high molecular weight sodium hyaluronate solution was cooled to 35°C, 250mL of buffer solution was added and mixed evenly, and then 0.5g of papain and 1.0g of hyaluronidase were added, and the enzymolysis reaction was carried out for 10 hours at a pH value of 7.0, a temperature of 37°C, and a stirring speed of 350r / min to obtain an enzymolysis solution; sodium hydroxide solution was added to the enzymolysis solution to adjust the pH to 9.0, and the solution was heated to 80-90°C for 40 minutes and then filtered to obtain a low molecular weight sodium hyaluronate solution; the low molecular weight sodium hyaluronate solution was concentrated with a 300Da ultrafiltration membrane to obtain a concentrated solution, and then spray-dried, and the inlet temperature of the spray-drying was set to 130°C and the outlet temperature was set to 40°C to obtain a low molecular weight sodium hyaluronate powder.

[0035] Comparative Example 1

[0036] Low molecular weight sodium hyaluronate was prepared using the patented method with publication number CN114181986A: 2 mL / L of hyaluronidase was added to a sodium hyaluronate solution with a molecular weight of 1.5 million Da, the pH of the solution was 7.0, the stirring speed was 370 r / min, the temperature was 45°C, and after reacting for 6 hours, a low molecular weight sodium hyaluronate with a molecular weight of 49,000 Da was prepared. The drying method was vacuum drying at 60°C.

[0037] Comparative Example 2

[0038] Add 0.1g cysteine ​​hydrochloride, 2g sodium dihydrogen phosphate, 6g disodium hydrogen phosphate and 20g sodium chloride to 1L deionized water, stir and dissolve to obtain a buffer solution; 20g of high molecular weight sodium hyaluronate powder (Da) was added into 1200mL of deionized water, and stirred at 60°C until completely dissolved to obtain a high molecular weight sodium hyaluronate solution; when the high molecular weight sodium hyaluronate solution was cooled to 35°C, 250mL of buffer solution was added and mixed evenly, and then 1.0g of hyaluronidase was added, and under the conditions of pH 7.0, temperature 37°C and stirring speed 350r / min, enzymolysis reaction was carried out for 10 hours to obtain an enzymolysis solution; sodium hydroxide solution was added to the enzymolysis solution, the pH was adjusted to 9.0, heated to 80-90°C for 40 minutes, and then filtered to obtain a low molecular weight sodium hyaluronate solution; the low molecular weight sodium hyaluronate solution was filtered and concentrated with a 500Da nanofiltration membrane to obtain a concentrated solution, and then spray dried, and the inlet temperature of the spray drying was set to 130°C and the outlet temperature was set to 40°C to obtain a low molecular weight sodium hyaluronate powder.

[0039] Comparative Example 3

[0040] The inlet temperature of the spray drying was set to 160°C and the outlet temperature was set to 60°C. The other preparation methods were the same as those in Example 3.

[0041] The low molecular weight sodium hyaluronate obtained in the above examples and comparative examples was tested for average molecular weight by gel chromatography, and physical testing of sodium hyaluronate was performed with reference to the current version of the inspection standard "QB / T 4416-2012 Sodium hyaluronate for cosmetic raw materials". The moisturizing property of hyaluronic acid was tested according to the moisturizing property testing method in QB / T4256-2011 "Guidelines for the Evaluation of Moisturizing Efficacy of Cosmetics", and the permeability of hyaluronic acid was tested according to the permeability determination method in GB / T27818-2011 "In vitro test method for skin absorption of chemicals", and the results are shown in Tables 1 and 2.

[0042] Table 1

[0043] project Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Molecular weight / Da 4500 2800 3500 4.9W 8600 / Moisture content ≤5% ≤5% ≤5% 18% ≤5% 10% pH 7.0 6.9 6.9 7.3 7.1 6.8 Appearance White powder White powder White powder White powder White powder White powder Transmittance 550nm 99.8% 99.6% 99.8% 98.0% 98.3% 99.1%

[0044] As shown in Table 1, the low molecular weight sodium hyaluronate obtained by the preparation method of Examples 1 to 3 of the present invention has an average molecular weight of 2000 to 5000Da, a water content of ≤5%, and a high light transmittance; Comparative Example 1 prepares low molecular weight sodium hyaluronate according to the patent method with publication number CN114181986A, and vacuum-dries at 60°C, with an average molecular weight of 4.9W Da, and the molecular weight of the sodium hyaluronate obtained under the experimental parameters of the present invention is still large; and Comparative Example 2 does not add papain in the enzymatic hydrolysis reaction, and the molecular weight of the obtained sodium hyaluronate is about 8600, and the light transmittance will also deteriorate, which also shows that papain plays an auxiliary role in the enzymatic hydrolysis of sodium hyaluronate. The reason may be that papain can reduce the viscosity of the high molecular weight sodium hyaluronate solution, which is conducive to promoting the dispersibility of each fragment in the system, and is conducive to hyaluronidase enzymolysis of the macromolecular sodium hyaluronate into lower molecular weight sodium hyaluronate. On the other hand, it can act on the residual high molecular weight part and possible protein impurities in hyaluronic acid. In addition, after enzymatic hydrolysis, under alkaline conditions, papain also helps to inactivate hyaluronidase. In Comparative Example 3, the outlet temperature of spray drying is set too high, which increases the water content of the finished product.

[0045] Table 2

[0046] Group Molecular weight Moisture retention – water content of the skin Permeability - Skin Absorption Example 1 4500 42.8% 72% Example 2 2800 39.9% 75.1% Example 3 3500 40.7% 73.4% Comparative Example 1 4.9W 40.9% 49% Comparative Example 2 8600 37.1% 69.3%

[0047] From the data in Table 2, it can be seen that as the degree of degradation of hyaluronic acid increases and the molecular weight increases, the permeability decreases. In particular, the permeability of the small molecule hyaluronic acid produced by the present invention in Examples 1 to 3 is above 72%, which is much higher than that of the comparative example; and relative to moisture retention, the molecular weight gradually increases, and the moisture retention is slightly less, but the difference is not great, which also confirms that sodium hyaluronate with an average molecular weight of 5K-20W Da can enter the stratum corneum of the skin, is easily absorbed by the human body and has long-lasting moisture retention; and only sodium hyaluronate with an average molecular weight below 5000Da can penetrate into the dermis and be absorbed by the skin. Examples 1 to 3 add papain as an auxiliary enzyme, thereby improving the moisture retention of the product, which also helps to improve the use effect of subsequent skin care products.

[0048] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing low molecular weight sodium hyaluronate, characterized in that: The following steps are involved: (1) Add high molecular weight sodium hyaluronate powder into deionized water and stir at 45°C to 60°C until completely dissolved to obtain a high molecular weight sodium hyaluronate solution; (2) When the solution is cooled to 35-45°C, a buffer solution is added and mixed evenly, and then papain and hyaluronidase are added to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolyzate, wherein the mass ratio of papain to hyaluronidase is (2-3):(3-6); (3) Adding sodium hydroxide solution to the enzymatic hydrolyzate, adjusting the pH to 7.5-9.0, heating to 80-90° C. for 20-40 minutes, and filtering to obtain a low molecular weight sodium hyaluronate solution; (4) Concentrating the low molecular weight sodium hyaluronate solution and spray drying it.

2. The method for preparing low molecular weight sodium hyaluronate according to claim 1, characterized in that: In the step (1), the molecular weight of the high molecular weight sodium hyaluronate is 20W~160W Da.

3. The method for preparing low molecular weight sodium hyaluronate according to claim 1, characterized in that: In the step (2), the mass ratio of papain to hyaluronidase is 2:

5.

4. The method for preparing low molecular weight sodium hyaluronate according to claim 1, characterized in that: In step (2), the buffer solution is prepared by adding cysteine ​​hydrochloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride into distilled water and stirring to dissolve.

5. The method for preparing low molecular weight sodium hyaluronate according to claim 4, characterized in that: The mass concentration ratio of cysteine ​​hydrochloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride is (0.05-0.2):(1-3):(4-6):(9-11).

6. The method for preparing low molecular weight sodium hyaluronate according to claim 1, characterized in that: In the step (2), the pH value of the enzymatic hydrolysis reaction is 7.0-7.2, the temperature is 30-42° C., and the enzymatic hydrolysis time is 6-12 hours.

7. The method for preparing low molecular weight sodium hyaluronate according to claim 1, characterized in that: In the step (4), the inlet temperature of the spray drying is 120-150°C, and the outlet temperature is 30-55°C.

8. The method for preparing low molecular weight sodium hyaluronate according to claim 1 or 7, characterized in that: In the step (4), the outlet temperature of the spray drying is 40°C.

9. The low molecular weight sodium hyaluronate obtained by the preparation method according to any one of claims 1 to 8, characterized in that: The molecular weight of sodium hyaluronate is 2000~5000Da, and the water content is ≤5%.

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