CN1537868A - Preparation method of sodium hyaluronic acid - Google Patents

Abstract

A process for preparing hyaluronic acid or sodium hyaluronate includes such steps as breaking the cockscomb, deactivating, washing with alcohol or physiological saline, removing isoprotein and fat, enzymolyzing by trypsase, purifying by haloalkylpyridine and depositing in alcohol.

Description

The preparation method of hyaluronate sodium

Technical field

The present invention relates to from animal tissues, extract the preparation method of macromolecule polysaccharide body, more specifically say so and from the animal tissuess such as cockscomb of chicken, extract macromolecule polysaccharide body-hyaluronic preparation method.

Background technology

The present invention is the preparing process of high-purity medical hyaluronate sodium.

Hyaluronic acid is a kind of macromolecule polysaccharide body that is present in the animal connective tissue such as cockscomb, and it is that extracting and refining obtains hyaluronic acid or hyaluronate sodium with good water-retentivity and oilness from animal connective tissue.Be widely used as at present the main raw material of makeup or the viscoelastic agent that ophthalmologic operation is used, the lubricant in orthopaedics joint, and the anti in the gynaecology, surgical operation.In order to reach the purpose that is suitable for above-mentioned each field, just need produce various different molecular weights and be highly purified hyaluronic acid or hyaluronate sodium.Too low as molecular weight, just can not obtain good water-retentivity and oilness, hanging down as purity then can not be as the various auxiliary material in the medical field.

Though the hyaluronic acid of in the past producing or its sodium salt also from animal connective tissue extracting and refining form, but its extraction process is the protein with reticular tissue adopts its decomposition of multiple enzymatic, the complex production process complexity, and the matter acid or the matter acid sodium that decompose gained often take repeatedly to add the halogenated alkyl pyridinium salt, allowing matter acid and pyridinium salt be binding substances precipitates, allow throw out be dissolved in the salt solution then, its annexation and matter acid are dissociated, add water miscible non-solvent again, example: add ethanol etc., the sodium salt as matter acid just folds from solution like this.(USP?4141973.4328803)

Aforesaid method, utilize the plurality of enzymes protein that will coexist to decompose, the Unidasa that contains in the cockscomb (as its energy disintegrant acid of matrix), just need heat inactivation, natural matter acid has higher molecular weight (being generally 150-250 ten thousand), in order to make not excessive descent of molecular weight, and obtain hyaluronic acid or its sodium salt of high-purity medical, aforesaid method just seems and can not satisfy, promptly in the cockscomb reticular tissue, extract under coexisting state at hyaluronic acid and chondroitin sulfate or other mucopolysaccharide in addition, often be mixed with the mucopolysaccharide of other kind in the hyaluronic acid, and the halogenated alkyl pyridinium salt also can generate hyaluronic acid other mucopolysaccharide in addition, when being dissolved in throw out in the salt solution, inevitable also other mucopolysaccharide can being brought in this solution simultaneously gone, and utilizes ethanol often also can separate out other all kinds of mucopolysaccharides when separating out hyaluronate sodium.

Summary of the invention

Weak points such as it is low that the object of the invention overcomes the product purity that existence makes in the prior art, and contain other Crude polysaccharides impurity, and molecular weight is lower.Thereby adopt cockscomb pre-treatment and a trypsinase to add the simple and easy facilities of step such as enzymolysis process, meet the mass production requirement, and can low cost make highly purified hyaluronate sodium.

The present invention adopts following steps to make hyaluronic acid or hyaluronate sodium.

1, inactivation treatment: fresh or freezing cockscomb (as freezing cockscomb then earlier after room temperature was thawed 4 hours at 40-80 ℃, carry out inactivation treatment under 20-40 minute, original enzyme classes in the cockscomb is carried out deactivation.

2, clean pre-treatment: the cockscomb after the above-mentioned deactivation is carried out secondary rub, clean pre-treatment with ethanol, physiological saline then, to remove M-band and fat etc.

3, enzymolysis: stir into pasty state through cleaning pretreated cockscomb adding suitable quantity of water, once add trypsinase and carry out enzymolysis processing at 35-45 ℃.Enzymolysis time is 2-8 hour.

4, purifying: once or once abovely add cetyl pyridinium salt and purify.

5, precipitation: above-mentioned purification thing is dissolved in the dissociation solution of ethanol and physiological saline, obtains pure product with ethanol sedimentation again.

Beneficial effect of the present invention:

1, the present invention increases the inactivation treatment of cockscomb, with the various enzyme-deactivatings that contain in the cockscomb, makes it not participate in further enzymolysis, thereby has improved the molecular weight and the purity of the finished product and avoided the complicated of subsequent disposal.

2, adopt once tryptic interpolation enzymolysis process, make it more perfect when enzymolysis.

3, adopt the method for cetyl pyridinium salt to purify, i.e. primary sedimentation.

4, utilize CPC purification thing different to the liberation degree of salt solution, separate with filtering method, can effectively remove impurity, thereby obtain high-purity thing according to its difference with the ethanol sedimentation thing.

5, the step of the present invention's use can make Crude polysaccharides and other impurity in the product be non-molten state, thereby simple method is removed, and makes whole process of production become easy and simple to handle.Cost also lowland is produced the high curable product-hyaluronate sodium of purity.

Embodiment

Below the present invention is described in detail.

1, inactivation treatment:

Get fresh cockscomb or freezing cockscomb (below 20 ℃), fresh cockscomb is cleaned or freezing cockscomb normal temperature flowing water slowly thawed 4-6 hour, can flood 20-40mim in 40-80 ℃ of hot water as required, rubs 2 times with shaking the meat machine then, and cockscomb is 3 * 3mm shape.

2, clean pre-treatment:

The much grains of cockscomb after the deactivation are soaked in the mixed solution of ethanol and physiological saline, and this mixed solution is 2-8 a times of cockscomb amount, and the best is 3-5 times, stirs after 0.5-1 hour, and water is clean slightly, removes M-band and fat etc., and filter is taken out only.

3, enzymolysis:

The clear water of the cockscomb weight that interpolation 1-3 doubly measures, the best is 2 times, allow it become pasty state, once add trypsinase in pasty state, its content is the 0.12-0.4% (weight ratio) of this enzymolysis solution, and the best is 0.2%, treatment condition are 35-45 ℃, enzymolysis time is 2-8 hour, and the best is 4-6 hour, utilizes this pasty state product just to filter through enzymolysis and can obtain the hyaluronic aqueous solution.

4, purifying:

The above-mentioned aqueous solution is suitably added distilled water, salt and 95% ethanol, and then add cetyl pyridinium salt, and the carbon atom of alkyl is 12-18, halogen is comparatively desirable with chlorine, bromine, iodine, the best is a chlorine, and is promptly suitable with chloro-hexadecane yl pyridines salt (CPC).The addition of CPC is good with the 1.0-3.0% of cockscomb weight generally, and ideal is about 1.5-2.0%, and it is simple and convenient to utilize CPC to purify.

5, precipitation:

Utilize throw out that aforesaid method produces after filtration, this throw out can be dissolved in and contain in the alcoholic acid salt solution, concentration of ethanol is 15-40%, the concentration of salt is 0.1M-1M, optimal period is 0.2-0.5M, as adopt the present invention to precipitate-dissolve, when implementing more than 1 or 2 time, after 0.2-0.5M once, this common salt concn is a benchmark with the weight of dissociation solution, dissociation temperature generally is 20-60 ℃, answer agitation as appropriate when dissociating, be cooled to room temperature, basic dissolving has till the small amount of precipitate slightly, remove the small amount of precipitate thing with filter method, and then add 1.5-3.0 95% ethanol of liquid measure that doubly dissociates, and refining through sedimentation and filtration, should be able to obtain ideal medical hyaluronic acid or its sodium salt.

Embodiment 1:

-20 ℃ of 22kg cockscombs that freeze to preserve in 2 months were slowly thawed 4 hours in 20 ℃ of clear water, then this cockscomb is flooded 30mim in 80 ℃ of hot water, utilize mincer repeatedly secondary rub into cockscomb grain about 3m/m, then the mixed solution dipping of 95% ethanol, clear water and the 1M NaCl of 1.5 times of amounts of much usefulness of cockscomb is cleaned (95% ethanol: H ₂O=40: 60), the clear water that adds 2.5 times then, be mixed with 80kg cockscomb pasty state, add trypsinase 140g (the blue season development in science and technology in Shanghai company limited 1: 250), 45 ℃ ± 2 ℃, carried out enzyme and handle in 4 hours, after the enzymolysis processing, in mashed prod, add distilled water 80L and 0.2M NaCl, 10%CPC 3600ml while stirring, and be heated to 45 ℃, produce throw out.

After the throw out filtration that obtains, dissolve with 0.4M NaCl dissociation solution 60L once more and dissociate, and then add 120 L95% ethanol, allow it produce throw out, this throw out through dissolution filter, degerming, clean, the vacuum-drying of ethanol, is obtained smart powder 75.5g.

Embodiment 2

-20 ℃ of 36kg cockscombs that freeze to preserve in 2 months were slowly thawed 4 hours in 20 ℃ of clear water, then this cockscomb is flooded 30mim in 80 ℃ of hot water, utilize mincer repeatedly secondary rub into cockscomb grain about 3m/m, then with 95% ethanol of 3 times of amounts of much usefulness of cockscomb, the mixing scavenging solution of clear water and 1M NaCl soaks clean, and then add 2 times clear water, be mixed with 108kg cockscomb pasty state, add trypsinase 340g (the blue season development in science and technology in Shanghai company limited 1: 250), 45 ℃ ± 2 ℃, carrying out enzyme in 4 hours handles, after the enzymolysis processing, adding distilled water 120L and 0.2M NaCl dilution in mashed prod while stirring stirs, and then add 10%CPC 5700ml, and be heated to 45 ℃, produce throw out.

After the throw out filtration that obtains, dissociate with 0.4M NaCl dissociation solution 160L once more, and then add 250L 95% ethanol, allow it produce throw out, this throw out through dissolution filter, degerming, clean, the vacuum-drying of ethanol, is obtained smart powder 130g.

Reference examples:

-20 ℃ of 22kg cockscombs that freeze to preserve in 2 months were slowly thawed 4 hours in 20 ℃ of clear water, utilize then mincer repeatedly secondary rub into cockscomb grain about 3m/m, then with much clear water that adds 2.5 times of amounts of cockscomb, be mixed with 80kg cockscomb pasty state, add stomach en-600g respectively, trypsinase 60g carries out enzymolysis, then respectively with 38 ℃ ± 2 ℃ gastric enzyme enzymolysis 2 hours and 45 ℃ ± 2 ℃ pancreatin enzymolysis 2 hours, utilize that the foregoing description 1 identical method is dissolved, filtration, degerming, ethanol are cleaned, vacuum-drying, obtain smart powder 68g.

Following table is the HA-Na quality produced of embodiment 1,2 and reference examples and the contrast situation of yield.

	The cockscomb amount	????GA	Molecular weight	Protein content	????260nm	????280nm	Smart grain weight amount	Yield
									Embodiment 1	??22kg	???45.1	2,800,000	????0.11	????0.19	????0.12	????78.5g	????3.57％
Embodiment 2	??36kg	???47.3	2,890,000	????0.10	????0.15	????0.29	????130g	????3.61％
									Reference examples	??22kg	???46.0	1,650,000	????0.12	????0.14	????0.36	????68g	????3.0％

Remarks:

1, molecular weight: sample is diluted with physiological saline, measure its limiting viscosity with Ubbelohde viscometer 25 ℃ ± 0.1, ([η]/0.0361/0.76) calculates its molecular weight by M=then.

2, uv-absorbing 260nm, 280nm utilize 752 ultraviolet spectrophotometers to measure.

Claims (7)

1, a kind ofly prepare the method for hyaluronate, it is characterized in that this method may further comprise the steps by cockscomb:

1., the inactivation treatment of organized enzyme in the cockscomb;

2., clean pre-treatment;

3., enzymolysis;

4., adopt haloalkane pyridinium salt purifying;

5., ethanol sedimentation.

2, according to the method for preparing hyaluronate of claim 1, it is characterized in that the cockscomb organized enzyme 40-80 ℃ of deactivation, inactivation time is 20-40 minute.

3, according to the method for preparing hyaluronate of claim 1, adopt when it is characterized in that enzymolysis and once add trypsinase.

4, according to the method for preparing hyaluronate of claim 1, it is characterized in that hydrolysis temperature is 35-45 ℃, enzymolysis time is 2-8 hour.

5, according to the method for preparing hyaluronate of claim 1, it is characterized in that adopting haloalkane pyridinium salt purifying, wherein the carbon atom of alkyl is C ₁₂-C ₁₈, halogen is chlorine, bromine, iodine.

6, according to the method for preparing hyaluronate of claim 5, the carbon atom that it is characterized in that the pyridinium salt of haloalkane is 16, and halogen is that the chloro-hexadecane pyridinium salt of chlorine is better.

7,, it is characterized in that precipitating employing and contain the ethanol that Jie doubly measures from liquid 1.5-2.5 according to the method for preparing hyaluronate of claim 1.