A method for preparing low molecular weight sodium hyaluronate

Abstract

The present application relates to the field of sodium hyaluronate preparation, in particular to a preparation method of low molecular weight sodium hyaluronate. Specifically comprising the following steps: stirring and dissolving macromolecular weight sodium hyaluronate powder into water at 45-60 DEG C to obtain a macromolecular weight sodium hyaluronate solution; cooling to 35-45 DEG C, adding a buffer solution and mixing uniformly, then adding papain and hyaluronidase with a mass ratio of (2-3):(3-6) for enzymatic reaction to obtain an enzymatic solution; adding sodium hydroxide solution to the enzymatic solution, adjusting pH to 7.5-9.0, heating to 80-90 DEG C for 20-40 minutes, then filtering to obtain a low molecular weight sodium hyaluronate solution; spray drying, and the process is simple, the conditions are mild, the product structure is not damaged, and the prepared low molecular weight sodium hyaluronate has high purity, uniform molecular weight distribution and good permeability.

Description

Technical Field

[0001] This invention relates to the field of sodium hyaluronate preparation, and specifically to a method for preparing low molecular weight sodium hyaluronate and the low molecular weight sodium hyaluronate itself. Background Technology

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

[0003] Currently, most sodium hyaluronates on the market have molecular weights ranging from several thousand to several million Daltons. Compared to large-molecule sodium hyaluronates, only those with an average molecular weight of 5K-20W Da can penetrate the stratum corneum of the skin, are easily absorbed by the body, and provide long-lasting hydration. Sodium hyaluronates with an average molecular weight below 5000 Da can penetrate deep into the dermis for deep hydration, effectively inhibiting inflammation and repairing cells. Therefore, smaller molecular weight and a more uniform structure are essential for the easy absorption of sodium hyaluronate, resulting in superior performance. Under these circumstances, developing a sodium hyaluronate with a smaller molecular weight and a more uniform structure is of great significance. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a method for preparing low molecular weight sodium hyaluronate. This method is simple, operates under mild conditions, does not damage the product structure, and produces low molecular weight sodium hyaluronate with 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 to deionized water and stir at 45℃~60℃ until completely dissolved to obtain a high molecular weight sodium hyaluronate solution.

[0007] (2) When the solution is cooled to 35~45℃, add buffer solution and mix well. Then add papain and hyaluronidase to carry out enzymatic hydrolysis to obtain enzymatic hydrolysate. The mass ratio of papain to hyaluronidase is (2~3):(3~6).

[0008] (3) Add sodium hydroxide solution to the enzymatic hydrolysate, adjust the pH to 7.5~9.0, heat to 80-90℃ for 20~40 minutes and filter to obtain low molecular weight sodium hyaluronate solution;

[0009] (4) Concentrate and spray dry the low molecular weight sodium hyaluronate solution.

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

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

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

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

[0014] In 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] In a preferred embodiment, in step (2), the pH value of the enzymatic hydrolysis reaction is 7.0~7.2, the temperature is 30~42℃, and the enzymatic hydrolysis time is 6~12 hours.

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

[0017] In a preferred embodiment, in step (4), the inlet temperature of the spray dryer is 120~150℃ and the outlet temperature is 30~55℃.

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

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

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. The preparation method of low molecular weight sodium hyaluronate of the present invention is simple, mild, and does not damage the product structure; moreover, it does not require the use of large amounts of organic solvents and chemical reagents, thus reducing environmental pollution and production costs.

[0022] 2. The method for preparing low molecular weight sodium hyaluronate of the present invention yields sodium hyaluronate with high purity, uniform molecular weight distribution, molecular weight of 2000~5000 Da, water content ≤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, degrading it into low molecular weight hyaluronic acid. The buffer solution, especially sodium chloride, can improve the stability of hyaluronidase and papain, and reduce the influence 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, which helps to enzymatically hydrolyze 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, improving the purity of the product and the proportion of low molecular weight sodium hyaluronate. Attached Figure Description

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

[0025] Figure 2 The infrared spectrum of the low molecular weight sodium hyaluronate prepared in Example 1 of this invention. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

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

[0028] All experimental methods involved are well-known experimental methods.

[0029] Example 1

[0030] 0.05 g of cysteine ​​hydrochloride, 1 g of sodium dihydrogen phosphate, 4 g of disodium hydrogen phosphate, and 11 g of sodium chloride were added to 1 L of deionized water and stirred until dissolved to obtain a buffer solution. 10 g of high molecular weight sodium hyaluronate powder (20 W~160 W Da) was added to 500 mL of deionized water and stirred at 45℃~60℃ until completely dissolved to obtain a high molecular weight sodium hyaluronate solution. When the high molecular weight sodium hyaluronate solution cooled to 35~45℃, 150 mL of the buffer solution was added and mixed thoroughly. Then, 0.2 g of papain and 0.5 g of hyaluronidase were added. The mixture was then subjected to the following conditions: pH 7.0, temperature 30~42℃, and stirring speed 280 r / min. Enzymatic hydrolysis was performed for 12 hours to obtain the enzymatic hydrolysate. Sodium hydroxide solution was added to the hydrolysate to adjust the pH to 7.5-9.0. The solution was heated to 80-90℃ for 20-40 minutes and then filtered to obtain a low molecular weight sodium hyaluronate solution. This low molecular weight sodium hyaluronate solution was concentrated by ultrafiltration using a 300 Da ultrafiltration membrane to obtain a concentrate. This concentrate was then spray-dried at an inlet temperature of 120-150℃ and an outlet temperature of 30-55℃ to obtain low molecular weight sodium hyaluronate powder. Infrared spectroscopy was performed on the low molecular weight sodium hyaluronate powder, and the results are as follows... Figure 2 As shown in the infrared spectrum, the sodium hyaluronate obtained by the preparation method of this invention has a similar spectrum to the European Pharmacopoeia standard spectrum (…). Figure 1 The consistency indicates that its structural integrity was not compromised during the preparation process.

[0031] Example 2

[0032] Add 0.4 g cysteine ​​hydrochloride, 4 g sodium dihydrogen phosphate, 12 g disodium hydrogen phosphate, and 22 g sodium chloride to 2 L of deionized water and stir to dissolve to obtain a buffer solution; [The remaining text appears to be unrelated and possibly machine-translated gibberish. It has been omitted from the translation.] 100g of high molecular weight sodium hyaluronate powder (Da) was added to 4000mL of deionized water and stirred at 45℃-60℃ until completely dissolved to obtain a high molecular weight sodium hyaluronate solution. After the high molecular weight sodium hyaluronate solution cooled to 45℃, 1250mL of buffer solution was added and mixed thoroughly. Then, 3g of papain and 6g of hyaluronidase were added, and the enzymatic hydrolysis reaction was carried out for 6 hours at a pH of 7.2, a temperature of 37℃, and a stirring speed of 300r / min to obtain an enzymatic hydrolysate. Sodium hydroxide solution was added to the enzymatic hydrolysate to adjust the pH to 9.0, and the mixture was heated to 80-90℃ for 20 minutes and then filtered to obtain a low molecular weight sodium hyaluronate solution. The low molecular weight sodium hyaluronate solution was concentrated by filtration through a 300Da reverse osmosis membrane to obtain a concentrate, which was then spray-dried at an inlet temperature of 120℃ and an outlet temperature of 55℃ to obtain 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 of deionized water and stir to dissolve to obtain a buffer solution; [The remaining text appears to be unrelated and possibly machine-translated gibberish. It has been omitted from the translation.] 20g of high molecular weight sodium hyaluronate powder (Da) was added to 1200mL of deionized water and stirred at 60℃ until completely dissolved to obtain a high molecular weight sodium hyaluronate solution. After the high molecular weight sodium hyaluronate solution cooled to 35℃, 250mL of buffer solution was added and mixed thoroughly. Then, 0.5g of papain and 1.0g of hyaluronidase were added. Enzymatic hydrolysis was carried out for 10 hours at pH 7.0, temperature 37℃, and stirring speed 350r / min to obtain an enzymatic hydrolysate. Sodium hydroxide solution was added to the enzymatic hydrolysate to adjust the pH to 9.0, and the solution was heated to 80-90℃ for 40 minutes and then filtered to obtain a low molecular weight sodium hyaluronate solution. The low molecular weight sodium hyaluronate solution was concentrated using a 300Da ultrafiltration membrane to obtain a concentrate, which was then spray-dried at an inlet temperature of 130℃ and an outlet temperature of 40℃ to obtain low molecular weight sodium hyaluronate powder.

[0035] Comparative Example 1

[0036] Low molecular weight sodium hyaluronate was prepared using the method described in patent publication 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℃, and the reaction was carried out for 6 hours to obtain low molecular weight sodium hyaluronate with a molecular weight of 49,000 Da. Drying was performed under vacuum at 60℃.

[0037] Comparative Example 2

[0038] Add 0.1g cysteine ​​hydrochloride, 2g sodium dihydrogen phosphate, 6g disodium hydrogen phosphate, and 20g sodium chloride to 1L of deionized water and stir to dissolve to obtain a buffer solution; [The remaining text appears to be unrelated and possibly machine-translated gibberish. It has been omitted from the translation.] 20g of high molecular weight sodium hyaluronate powder (Da) was added to 1200mL of deionized water and stirred at 60℃ until completely dissolved to obtain a high molecular weight sodium hyaluronate solution. After the high molecular weight sodium hyaluronate solution cooled to 35℃, 250mL of buffer solution was added and mixed thoroughly. Then, 1.0g of hyaluronidase was added, and the enzymatic hydrolysis reaction was carried out for 10 hours at a pH of 7.0, a temperature of 37℃, and a stirring speed of 350r / min to obtain an enzymatic hydrolysate. Sodium hydroxide solution was added to the enzymatic hydrolysate to adjust the pH to 9.0, and the mixture was heated to 80-90℃ for 40 minutes and then filtered to obtain a low molecular weight sodium hyaluronate solution. The low molecular weight sodium hyaluronate solution was concentrated by filtration through a 500Da nanofiltration membrane to obtain a concentrate, which was then spray-dried at an inlet temperature of 130℃ and an outlet temperature of 40℃ to obtain low molecular weight sodium hyaluronate powder.

[0039] Comparative Example 3

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

[0041] The low molecular weight sodium hyaluronate obtained in the above examples and comparative examples was determined by gel permeation chromatography, and physical testing of sodium hyaluronate was performed according to the current version of the test standard "QB / T 4416-2012 Sodium Hyaluronate for Cosmetic Raw Materials". The moisturizing properties of hyaluronic acid were tested according to the testing methods for moisturizing properties in QB / T4256-2011 "Guidelines for Evaluation of Moisturizing Efficacy of Cosmetics", and the permeability of hyaluronic acid was tested according to the testing methods for permeability in GB / T27818-2011 "In vitro Test Methods for Skin Absorption of Chemicals". 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 value 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 550nm transmittance 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 methods of Examples 1-3 of this invention has an average molecular weight between 2000 and 5000 Da, a water content of ≤5%, and high transmittance. Comparative Example 1 prepared low molecular weight sodium hyaluronate according to the patent method published in CN114181986A and vacuum-dried it at 60°C, resulting in an average molecular weight of 4.9 W Da. Even without the experimental parameters of this invention, the molecular weight of the sodium hyaluronate obtained was still relatively large. Comparative Example 2, which did not add papain during the enzymatic hydrolysis reaction, obtained sodium hyaluronate with a molecular weight of around 8600, and its transmittance also decreased. This indicates that papain plays an auxiliary role in the enzymatic hydrolysis of sodium hyaluronate. This may be because papain can reduce the viscosity of the high molecular weight sodium hyaluronate solution, which is beneficial for promoting the dispersion of various fragments in the system and facilitates the enzymatic hydrolysis of high molecular weight sodium hyaluronate by hyaluronidase into lower molecular weight sodium hyaluronate. On the other hand, it can act on the residual high molecular weight portion and possible protein impurities in hyaluronic acid. In addition, after enzymatic hydrolysis, papain also helps to inactivate hyaluronidase under alkaline conditions. In Comparative Example 3, the outlet temperature of the spray drying was set too high, which increased the moisture content of the finished product.

[0045] Table 2

[0046] Group molecular weight Moisturizing properties - skin hydration Permeability - Skin Absorption Rate 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] As shown in Table 2, the permeability decreases with increasing degradation and molecular weight of hyaluronic acid. Specifically, the permeability of the small-molecule hyaluronic acid produced in Examples 1-3 is over 72%, significantly higher than the comparative example. Regarding moisturizing properties, a slight decrease in moisturizing effect occurs with increasing molecular weight, but the difference is not significant. This confirms that sodium hyaluronate with an average molecular weight between 5K and 20W Da can penetrate the stratum corneum of the skin, is easily absorbed, and provides long-lasting hydration. Only sodium hyaluronate with an average molecular weight below 5000 Da can penetrate into the dermis and be absorbed by the skin. The addition of papain as a coenzyme in Examples 1-3 improves the moisturizing properties of the product, which also helps to enhance the effectiveness of subsequent skincare products.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing low molecular weight sodium hyaluronate, characterized by, Includes the following steps: (1) Add high molecular weight sodium hyaluronate powder with a molecular weight of 20W~160W Da to deionized water and stir at 45℃~60℃ until completely dissolved to obtain a high molecular weight sodium hyaluronate solution. (2) When the solution is cooled to 35~45℃, add buffer solution and mix well. Then add papain and hyaluronidase to carry out enzymatic hydrolysis to obtain enzymatic hydrolysate. The mass ratio of papain to hyaluronidase is (2~3):(3~6). The buffer solution is prepared by adding cysteine ​​hydrochloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride to distilled water and stirring to dissolve; 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). (3) Add sodium hydroxide solution to the enzymatic hydrolysate, adjust the pH to 7.5~9.0, heat to 80-90℃ for 20~40 minutes and filter to obtain low molecular weight sodium hyaluronate solution; (4) Concentrate the low molecular weight sodium hyaluronate solution and spray dry it; the inlet temperature of the spray dryer is 120~150℃, and the outlet temperature is 30~55℃. The obtained sodium hyaluronate has a molecular weight of 2000~5000 Da and a water content of ≤5%.

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

5.

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

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

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