CN105949348B - Hyaluronic acid molecular weight grading method - Google Patents
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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Abstract
The invention provides a molecular weight grading method of sodium hyaluronate, which is characterized by comprising the following steps: (1) preparing a sodium hyaluronate solution; (2) adding 4-40% (w/v) of strong base anion exchange resin into the sodium hyaluronate solution in the step (1), and stirring and adsorbing; (3) adding a first strong electrolyte salt solution, standing to resolve the resin, and collecting a first resolving solution; (4) adding a second strong electrolyte salt solution, stirring the desorption resin, and collecting a second desorption solution; (5) adding a third strong electrolyte salt solution and an inorganic alkali solution to regenerate resin, and collecting regenerated liquid; (6) respectively carrying out ethanol precipitation on the first analysis solution, the second analysis solution and the regeneration solution; (7) and (5) drying. The method uses a resin separation technology with low cost and simple operation, adopts static adsorption, and is suitable for large-scale production.
Description
Technical Field
The invention relates to a hyaluronic acid molecular weight grading method.
Background
Hyaluronic Acid (HA), also known as Hyaluronic acid, is a linear macromolecular acidic mucopolysaccharide formed by repeating and alternating D-glucuronic acid and N-acetylglucosamine disaccharide units, the aqueous solution HAs negative charges, the weight-average molecular weight is generally 10-1000 Da, and sodium hyaluronate is in the form of sodium salt of Hyaluronic acid. Currently commercialized HA is generally in the form of Na salt, is a white fibrous or powdery solid, HAs strong hygroscopicity, is soluble in water, and is insoluble in organic solvents. Above 200 ten thousand Da is high molecular weight HA; 100-200 million Da is medium molecular weight HA; 1-100 million Da is low molecular weight HA; less than 1 million is oligomeric HA. The molecular weight of the sodium hyaluronate is closely related to the physiological function of the sodium hyaluronate, and the sodium hyaluronate with different molecular weights has different biological activities. HA molecular weight is an important pharmacological property, and due to the importance of HA molecular weight, the classification of HA molecular weight HAs become a research hotspot for the skilled person.
Patent application CN 102516410 a uses acid-base hydrolysis combination method to prepare sodium hyaluronate with high molecular weight into sodium hyaluronate with different small molecular weights. It is difficult to obtain high molecular weight sodium hyaluronate which has high economic value and medical grade requirement.
The ultrafiltration membrane separation technology such as CN103319627A has high requirements on equipment and high cost, is not suitable for large-scale production and can only obtain hyaluronic acid finished products with two specifications.
Disclosure of Invention
The invention provides a method for grading the molecular weight of sodium hyaluronate. The method adopts a resin separation technology, can be used in the production process of the hyaluronic acid or used for reprocessing the finished hyaluronic acid product, can obtain HA finished products with different molecular weights according to requirements, and can better improve the commercial application value of HA.
In one aspect, the invention provides a method for molecular weight fractionation of sodium hyaluronate, which is characterized by comprising the following steps:
(1) preparing a sodium hyaluronate solution;
(2) adding 4-40% (w/v) of strong base anion exchange resin into the sodium hyaluronate solution in the step (1), and stirring and adsorbing;
(3) adding a first strong electrolyte salt solution, standing to resolve the resin, and collecting a first resolving solution;
(4) adding a second strong electrolyte salt solution, stirring the desorption resin, and collecting a second desorption solution;
(5) adding a third strong electrolyte salt solution and an inorganic alkali solution to regenerate resin, and collecting regenerated liquid;
(6) respectively carrying out ethanol precipitation on the first analysis solution, the second analysis solution and the regeneration solution;
(7) and (5) drying.
The sodium hyaluronate is a high molecular weight polymer, which has negative polyanion charge in aqueous solution, can be adsorbed by strongly basic anion exchange resin, and can be resolved by different resolving methods, such as standing resolving and stirring resolving to obtain sodium hyaluronate finished products with different molecular weight specifications. The first step of the method adopts static standing analysis instead of conventional stirring static analysis, and aims to analyze the high molecular weight sodium hyaluronate adsorbed on the outer surface of the macroporous strong base anion resin; secondly, stirring and static resolving are adopted, and the aim is to resolve the sodium hyaluronate with medium molecular weight adsorbed in the pore diameter of the macroporous strong base anion resin; and thirdly, regenerating, and resolving the sodium hyaluronate with low molecular weight. Explanation of possible mechanisms of stepwise resolution: sodium hyaluronate is a linear macromolecular mucopolysaccharide with polyanion, so that the exchange adsorption behavior of the sodium hyaluronate with macroporous anion resin is determined by: 1. charged; 2. molecular size 3, steric hindrance. The small molecular weight HA can be drilled into the pore size of the macroporous resin and subjected to multi-site exchange adsorption. However, since HA having a high molecular weight cannot enter pores due to its large particle size and HAs a large steric hindrance, it forms weak adsorption on the outer surface of the resin and is easily resolved.
In some embodiments, the first strong electrolyte salt, the second strong electrolyte salt, and the third strong electrolyte salt are each independently a halide salt, a sulfate salt, and a nitrate salt.
In some embodiments, the inorganic base is a strong base.
In some embodiments, the first strong electrolyte salt, the second strong electrolyte salt, and the third strong electrolyte salt are each independently NaCl, CaCl, or CaCl2、ZnCl2、MgCl2、Na2SO4、MgSO4Or NaNO3。
In some embodiments, the inorganic base is NaOH or KOH.
In some embodiments, the first strong electrolyte salt, the second strong electrolyte salt, and the third strong electrolyte salt are each independently NaCl, CaCl, or CaCl2、ZnCl2、Na2SO4、MgCl2、MgSO4Or NaNO3(ii) a The inorganic base is NaOH or KOH.
In some embodiments, the sodium hyaluronate of step (1) has a molecular weight of 50 to 95 ten thousand Da.
In some embodiments, the sodium hyaluronate solution of step (1) has a concentration of 1 to 10 g/L.
In some embodiments, the step (1) sodium hyaluronate solution is an aqueous solution.
In some embodiments, the molecular weight of the sodium hyaluronate in step (1) is 50 to 95 ten thousand Da, which is an aqueous solution with a solution concentration of 1 to 10 g/L.
In some embodiments, the stirring adsorption in step (2) is performed at room temperature.
In some embodiments, room temperature refers to 15 ℃ to 40 ℃.
In another embodiment, room temperature refers to 20 ℃ to 35 ℃.
In some embodiments, the adsorption is stirred for 3-24 hours in step (2).
In other embodiments, the adsorption with agitation in step (2) is for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 hours.
In some embodiments, the first strong electrolyte salt concentration in step (3) is 0.05-2M.
In some embodiments, the first strong electrolyte salt solution in step (3) is an aqueous solution.
In some embodiments, the resolution in step (3) is allowed to stand for 3 to 8 hours.
In some embodiments, the first strong electrolyte salt solution in step (3) is an aqueous solution with a concentration of 0.05-2M, and is allowed to stand for 3-8 hours.
In some embodiments, the second strong electrolyte salt concentration in step (4) is 0.05-2M.
In some embodiments, the second strong electrolyte salt solution in step (4) is an aqueous solution.
In some embodiments, the agitation in step (4) is resolved for 3-24 hours.
In some embodiments, the second strong electrolyte salt solution in step (4) is an aqueous solution with a concentration of 0.05-2M, and is stirred and resolved for 3-24 hours
In some embodiments, the third strong electrolyte salt concentration in step (5) is 0.05-2M.
In some embodiments, the third strong electrolyte salt solution in step (5) is an aqueous solution.
In some embodiments, the concentration of inorganic base in step (5) is 0.1 to 0.5M.
In some embodiments, the inorganic base solution in step (5) is an aqueous solution.
In some embodiments, the regeneration in step (5) is for 3-24 hours.
In some embodiments, the third strong electrolyte salt solution in step (5) is an aqueous solution with a concentration of 0.05-2M and the inorganic base solution is an aqueous solution with a concentration of 0.1-0.5M, and is regenerated for 3-8 hours.
The sodium hyaluronate polymer with different molecular weight specifications can be replaced from the resin by taking different salt concentrations as ion exchange analytic agents, and sodium hyaluronate finished products with different molecular weight specifications can be obtained.
In some embodiments, step (6) is precipitated with 1.5-2 volumes of 95% ethanol.
In some embodiments, the present invention provides a method for molecular weight fractionation of sodium hyaluronate, comprising the steps of:
(a) preparing a sodium hyaluronate solution with the molecular weight of 50-95 ten thousand Da, wherein the concentration of the solution is 1-10 g/L;
(b) adding 4-40% (w/v) of strong base anion exchange resin into the sodium hyaluronate solution of step (a), and stirring and adsorbing at room temperature for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 hours;
(c) adding 0.05-2M of first strong electrolyte salt aqueous solution, standing for 3-8 hours to resolve the resin, and collecting first resolution solution;
(d) adding 0.05-2M of second strong electrolyte salt aqueous solution, stirring the desorption resin for 3-24 hours, and collecting second desorption solution;
(e) adding 0.05-2M of third strong electrolyte salt aqueous solution and 0.1-0.5M of inorganic alkaline aqueous solution to regenerate resin for 3-24 hours, and collecting regenerated liquid;
(f) precipitating the first analysis solution, the second analysis solution and the regeneration solution with 1.5-2 times volume of 95% ethanol;
(g) and (5) drying.
In some embodiments, the methods provided herein further comprise: before stirring and adsorbing, the pH value of the sodium hyaluronate solution is adjusted to 5-7 by acid or alkali.
The method for grading the molecular weight of the sodium hyaluronate uses a resin separation technology with low cost and simple operation, adopts static adsorption and is suitable for large-scale production; particularly, the molecular weight of hyaluronic acid of 50-95 ten thousand Da can be classified into the molecular weight required by medical application and cosmetic application, and sodium hyaluronate finished products with 3 different molecular weight specifications can be obtained, so that the commercial value of the sodium hyaluronate finished products is fully improved.
The term "strong electrolyte salt" as used herein is present in the solution entirely in the form of ions, i.e., there are no "molecules" of electrolyte.
The term "solution" as used herein refers to any solution suitable for use in the methods of the present invention, including single solvent solutions or mixed solvent solutions. In some embodiments an aqueous solution.
The term "halide salt" as used herein refers to a strong electrolyte type halide salt that exists in solution all in the form of ions.
The term "sulfate" as used herein refers to a strong electrolyte type sulfate that exists in solution in the form of ions throughout.
The term "nitrate" as used herein refers to a strong electrolyte form of nitrate that exists entirely in the form of ions in solution.
The term "strong base" as used herein refers to the alkali and partial alkaline earth metal corresponding bases.
The following acronyms are used throughout the invention:
w/v mass to volume ratio
NaCl sodium chloride
CaCl2Calcium chloride
ZnCl2Zinc chloride
Na2SO4Sodium sulfate
NaNO3Sodium nitrate
NaOH sodium hydroxide
KOH potassium hydroxide
Da Dalton
g/L grams per liter
M mol per liter
Detailed Description
The embodiment of the invention discloses a method for grading the molecular weight of sodium hyaluronate. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the product and method described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the following examples are provided to illustrate the molecular weight fractionation method of sodium hyaluronate.
Example 1
Taking 1.0g of a sodium hyaluronate sample with molecular weight of 74.7 ten thousand Da, dissolving in 600mL of purified water to obtain a sodium hyaluronate solution with concentration of 1.67g/L, adjusting the pH value of the sodium hyaluronate solution to 6.5 by using 1mol/L NaOH or HCl, adding 40.0g of strong base anion exchange resin, and stirring and adsorbing for 8 hours. Filtering the solution to obtain the resin. The desorption process is divided into 3 stages: firstly, adding 2M NaCl 600mL solution, standing and resolving for 3h, stirring once every 1h, and then filtering to obtain a first resolving solution; then adding 600mL of 2M NaCl solution, stirring and resolving for 3h, stirring at the rotating speed of 100rpm, and filtering to obtain a second resolving solution; and finally, adding 600mL of 2M NaCl +0.1M NaOH solution, stirring and regenerating for 3h, and filtering to obtain a regenerated solution. And precipitating the first analytic solution, the second analytic solution and the regenerated solution respectively by using 2 times of 95% ethanol, and performing vacuum drying to obtain 3 sodium hyaluronate finished products with different molecular weights.
The molecular weight is measured by adopting GPC gel chromatography, and the yield of the sodium hyaluronate molecular weight of the first analysis solution is 25.18 percent when the sodium hyaluronate molecular weight is 147 ten thousand Da; the molecular weight of the sodium hyaluronate of the second analysis solution is 106 ten thousand Da, and the yield is 23.05 percent; the molecular weight of the sodium hyaluronate regenerated solution is 35.8 ten thousand Da, and the yield is 45.31%.
Example 2
Taking 1.0g of a sodium hyaluronate sample with molecular weight of 74.7 ten thousand Da, dissolving in 600mL of purified water to obtain a sodium hyaluronate solution with concentration of 1.67g/L, adjusting the pH value of the sodium hyaluronate solution to 6.5 by using 1mol/L NaOH or HCl, adding 40.0g of strong base anion exchange resin, stirring and adsorbing for 8 hours. Filtering the solution to obtain the resin. The desorption process is divided into 3 stages: firstly, adding 600mL of 0.1M NaCl solution, standing and resolving for 3h, stirring once every 1h, and then filtering to obtain a first resolving solution; then adding 600mL of 2M NaCl solution, stirring and resolving for 3h, stirring at the rotating speed of 100rpm, and filtering to obtain a second resolving solution; and finally, adding 600mL of 2M NaCl +0.1M NaOH solution, stirring and regenerating for 3h, and filtering to obtain a regenerated solution. And precipitating the first analytic solution, the second analytic solution and the regenerated solution respectively by using 2 times of 95% ethanol, and performing vacuum drying to obtain 3 sodium hyaluronate finished products with different molecular weights.
The molecular weight was measured by GPC gel chromatography, and the sodium hyaluronate molecular weight of the first analysis solution was 98.8 ten thousand Da yield 7.1%, the sodium hyaluronate molecular weight of the second analysis solution was 132 ten thousand Da yield 37.87%, and the sodium hyaluronate molecular weight of the regenerated solution was 29.8 ten thousand Da yield 40.31%.
Example 3
Taking 1.0g of sodium hyaluronate sample with the molecular weight of 92.5 ten thousand Da, dissolving in 600mL of purified water to obtain a sodium hyaluronate solution with the concentration of 1.67g/L and the pH value of 5.65. 40.0g of strong base anion exchange resin is added into the solution, and the solution is stirred and adsorbed for 8 hours. Filtering the solution to obtain the resin. The desorption process is divided into 3 stages: firstly, adding 600mL of 2M NaCl solution, standing and resolving for 3h, stirring once every 1h, and then filtering to obtain a first resolving solution; then adding 600mL of 2M NaCl solution, stirring and resolving for 3h, stirring at the rotating speed of 100rpm, and filtering to obtain a second resolving solution; and finally, adding 600mL of 2M NaCl +0.1M NaOH solution, stirring and regenerating for 3h, and filtering to obtain a regenerated solution. And precipitating the first analytic solution, the second analytic solution and the regenerated solution respectively by using 2 times of 95% ethanol, and performing vacuum drying to obtain 3 sodium hyaluronate finished products with different molecular weights.
The molecular weight was measured by GPC gel chromatography, the molecular weight of sodium hyaluronate of the first analytical solution was 169.7 ten thousand Da yield 26.5%, the molecular weight of sodium hyaluronate of the second analytical solution was 110 ten thousand Da yield 22.15%, and the molecular weight of sodium hyaluronate of the regenerated solution was 45.8 ten thousand Da yield 41.65%.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (2)
1. A method for grading the molecular weight of sodium hyaluronate is characterized by comprising the following steps:
(1) preparing a sodium hyaluronate solution with the molecular weight of 50-95 ten thousand Da, wherein the concentration of the solution is 1-10 g/L;
(2) adding 4-40% w/v of strong base anion exchange resin into the sodium hyaluronate solution of step (1), and stirring and adsorbing at room temperature for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 hours;
(3) adding a first strong electrolyte salt solution, standing to resolve the resin, and collecting a first resolving solution;
(4) adding a second strong electrolyte salt solution, stirring the desorption resin, and collecting a second desorption solution;
(5) adding a third strong electrolyte salt solution and inorganic alkali solution to regenerate resin, stirring and resolving, and collecting regenerated liquid;
(6) respectively carrying out ethanol precipitation on the first analysis solution, the second analysis solution and the regeneration solution;
(7) drying;
in the step (3), the first strong electrolyte salt solution is an aqueous solution with the concentration of 0.05-2M, and is statically resolved for 3-8 hours;
in the step (4), the second strong electrolyte salt solution is an aqueous solution with the concentration of 0.05-2M, and is stirred and analyzed for 3-24 hours;
in the step (5), the third strong electrolyte salt solution is an aqueous solution with the concentration of 0.05-2M, the inorganic alkali solution is an aqueous solution with the concentration of 0.1-0.5M, and the regeneration is carried out for 3-24 hours;
the ethanol precipitation in the step (6) is 1.5 to 2 times of 95 percent ethanol precipitation by volume;
wherein the first strong electrolyte salt, the second strong electrolyte salt and the third strong electrolyte salt are respectively and independently NaCl and CaCl2、ZnCl2、Na2SO4、MgCl2Or NaNO3(ii) a The inorganic base is NaOH or KOH.
2. The method as claimed in claim 1, further comprising: before stirring and adsorbing, the pH value of the sodium hyaluronate solution is adjusted to 5-7 by acid or alkali.
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| CN107582424A (en) * | 2017-10-11 | 2018-01-16 | 广州青葱贸易有限公司 | A kind of Sodium Hyaluronate mixture and application thereof |
| CN108383927B (en) * | 2018-04-11 | 2021-06-08 | 南方科技大学 | Chondroitin sulfate magnesium and preparation method thereof |
| CN109517012B (en) * | 2018-11-13 | 2020-05-22 | 华熙生物科技股份有限公司 | Preparation method of hyaluronic acid oligosaccharide |
| CN115873144B (en) * | 2022-12-28 | 2024-04-16 | 水羊化妆品制造有限公司 | Acetylated small-molecule sodium hyaluronate and preparation method and application thereof |
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