CN105646733A

CN105646733A - Method for preparing lower-molecular hyaluronic acid through electron beam irradiation

Info

Publication number: CN105646733A
Application number: CN201610072524.0A
Authority: CN
Inventors: 徐宝琨; 杨松山; 温延奎; 孙连柱
Original assignee: SHANDONG VANFORM HIGH ENERGY PHYSICS TECHNOLOGY Co Ltd
Current assignee: SHANDONG VANFORM HIGH ENERGY PHYSICS TECHNOLOGY Co Ltd
Priority date: 2016-02-02
Filing date: 2016-02-02
Publication date: 2016-06-08

Abstract

The invention discloses a new method for preparing lower-molecular hyaluronic acid with a radiation method. The method includes the steps that common-molecular-weight hyaluronic acid serves as a raw material and is irradiated with an electron beam of an industrial high-energy electronic accelerator and degraded, and the product obtained after degradation is dissolved, subjected to ultrafiltration and dried to obtain the lower-molecular hyaluronic acid product. The method is easy to operate, high in yield, stable in performance and small in pollution, and the molecular weight is easy to control. The obtained product has the important physiological functions in multiple aspects such as water holding, osmotic pressure adjusting, wound healing, tumor invasion stopping and cell function adjusting in the body.

Description

A kind of electron beam irradiation prepares the method for low molecular weight hyaluronic acid

Technical field

The present invention relates to the new method using electron beam irradiation method to prepare low molecular weight hyaluronic acid.

Background technology

Hyaluronic acid, has another name called hyaluronic acid, Hyaluronic Acid, abridges (HA). Low-molecular-weight HA then has the irreplaceable biological activity advantage of HA of convenient molecular weight and the feature such as high osmosis and easy absorbability, has good application and development and is worth. Along with the understanding of hyaluronic structure, physicochemical property and physiological function is deepened continuously by people, the continuous maturation of preparation extractive technique, research and develop the HA product producing multiple different purposes at present. Its main application fields includes medicine, cosmetics, health product and the clinical treatment product of ophthalmology, orthopaedics, department of dermatologry. With the HA that animal tissue extracts for raw material, mean molecule quantity (Mr) is typically in 100,000-250 ten thousand Da, and its mean molecule quantity of HA cultivated by fermentable is typically in 1,000,000-400 ten thousand Da. General by having the HA of 100,000-400 ten thousand average molecular weight ranges, it is called the HA of convenient molecular weight. LMW HA (LMWHA) has antitumor, promotes wound healing, promotes bone and the effect such as angiogenesis, immunomodulating, has potential medical application prospect. Generally, the mean molecule quantity Mr of HA is bigger, and its water-retaining property and viscoelasticity are better; Mr is less, and the permeability of water, permeability between tissue, the biological activity that is absorbed by organisms is stronger. Along with the application of HA constantly extends, the HA performance of convenient molecular weight can not meet demand, promotes researcher to obtaining, exploitation has more high molecular and the direction of more LMW HA is developed. Low-molecular-weight HA then has the irreplaceable biological activity advantage of HA of convenient molecular weight and the feature such as high osmosis and easy absorbability, has good application and development and is worth.

The technology of preparing of current low molecular weight hyaluronic acid or its salt: have chemical degradation method, enzymatic isolation method, physical degradation methods, for instance following patent.

1. nano micromolecule hyaluronic acid and preparation method thereof (publication No. CN101507733B), it is that micromolecular technical process includes acid degradation, enzymatic degradation by macromolecules degradation.

2. the preparation method (publication number CN101429255A) of a kind of low-numerator sodium hyaluronate, it is mainly add hydrochloric acid, sodium hydroxide, ethanol in micromolecular technical process by macromolecules degradation.

3. a kind of low molecular weight hyaluronic acid salt, Preparation Method And The Use (publication No. CN103255187B), it is that micromolecular technical process includes by macromolecules degradation: obtained aqueous solution hyaluronic acid enzymatic degradation adds inorganic salt and filtration adds ethanol or ketone makes hyaluronic acid precipitate out dehydration of organic solvent vacuum drying.

Chemical degradation method main drawback is that and includes acid hydrolysis, basic hydrolysis, and adopting the chemical reagent such as sodium hypochlorite, hydrogen peroxide, course of reaction is complicated, brings difficulty to the purification of product, hyaluronic acid quality and inevitable industrial wastewater discharge, problem of environmental pollution can be affected.

The shortcoming of enzymatic isolation method includes: the palliating degradation degree of wayward molecular weight; The procurement cost of enzyme is high; The functional activity of enzyme preparation product is unstable, causes that subsequent technique condition (temperature, pH value etc.) needs repeatedly to debug, adds production difficulty; Reacted enzyme is also performed to inactivation treatment and separating treatment; Between product batches, physicochemical property is unstable.

Physical degradation methods: including heating, mechanical shear stress, ultraviolet, ultrasound wave etc., product stability is generally relatively low, molecular weight ranges distribution is excessive, obtains the mean molecule quantity product lower than 10000Da extremely difficult. .

Summary of the invention

The present invention is the new method that a kind of radiation method prepares low molecular weight hyaluronic acid. With the hyaluronic acid of conventional molecular weight for raw material, through industry high-energy electron accelerator electron beam irradiation degraded, degraded afterproduct, through dissolving ultrafiltration, obtains low molecular weight hyaluronic acid after drying. This method is simple to operate, and productivity is high, and molecular weight is easily controllable, stable performance, pollutes few. Products obtained therefrom has all many-sides such as water conservation, adjustment osmotic pressure, wound healing, the invasion of prevention tumor and adjustment cell function in vivo and has important physiological function.

Accompanying drawing 1 gives the technological process of this method, is summarized as follows:

From the hyaluronic acid product of Market Selection conventional molecular weight as coarse fodder, molecular weight 100,000��4,000,000 Da. Use industry high-energy electron accelerator electron beam irradiation degraded, ray energy 5.0��10.0MeV, absorbed dose 50��350KGy. This step makes its mean molecule quantity be down to below 10000Da, including other the irradiation bombs such as gamma-rays or X ray that meet the principle of the invention, subsequently into post-processing step, carries out the time of post processing preferably within 10 days after irradiation-induced degradation.

Ultrafiltration: the catabolite of acquisition is configured to the aqueous solution of 5%-20% concentration, the ultrafilter membrane being used below mean molecule quantity 2000Da molecular cut off carries out purifying and concentration, including the aqueous solution being configured to other concentration.

Make dry powder: the liquid after concentration is extracted low molecular weight hyaluronic acid dry powder formulations by spray dryer. Spray dryer inlet temperature 90 to 120 degrees Celsius, outlet temperature 50 to 60 degrees Celsius. The time of post processing is carried out preferably within 10 days after irradiation-induced degradation.

Adopting this kind of pure physical means of electron beam irradiation, reaching to make hyaluronic acid macromolecules degradation, thus obtaining low molecular weight hyaluronic acid product. It is required for adding multiple enzyme preparation, chemical reagent with prior art enzymatic isolation method, chemical degradation method, through multiple complex steps, certainly exists enzyme preparation in product, chemical residue, chemical waste liquid emission problem. Other current physical degradation methods product stability is generally relatively low, and molecular weight ranges distribution is excessive. This method step is simple and direct, and product purity is high, and quality conformance is good, is completely absent enzyme preparation, chemical residue and discharge.

Ultrafiltration: undertaken purifying and concentrating by the ultrafilter membrane of PSPP, uses physical means to reduce impurity further.

From solution, low molecular weight hyaluronic acid product being extracted, use a large amount of ethanol to precipitate with prior art, then drying, grind into powder are compared. This method uses that spray dryer is disposable completes, it is to avoid a large amount of ethanol losses, saves subsequent technique link. The minimizing of step has made equipment needed thereby few, intermediate link loss is few, product yield is high, can reach more than 90%.

Accompanying drawing explanation

Fig. 1 hyaluronic acid irradiation-induced degradation flow chart.

Fig. 2 viscosimetry measures mean molecule quantity.

Wherein: 1, industrial electronic accelerates machine 2, configuration aqueous solution 3, ultrafiltration 4, spray dryer

Detailed description of the invention

The following example further illustrates the present invention, but invention is not limited to this.

The hyaluronic acid dry powder raw material 50 grams that embodiment 1 adopts, it is 1.5 �� 10 that viscosimetry measures average relative molecular mass (Mr)⁶Da, under 23 DEG C of room temperatures, the electron beam produced with industry high-energy electron accelerator 1 carries out radiation treatment. The energy of electron beam is 10MeV, and the irradiation dose of hyaluronic acid raw material is 100KGy. After irradiation after 68 hours, sample is configured to 10% aqueous solution 2, removes small molecular weight impurity by retaining the ultrafilter membrane 3 of 8000Da and concentrate, this purified concentration liquid is adopted spray dryer 4 powder process, inlet temperature 100 DEG C, outlet temperature 50 DEG C. Collect hyaluronic acid power-product 45.7 grams, product yield 91.4% altogether. Employing viscosimetry is measured, and the average relative molecular mass (Mr) of product is 2.1 �� 10⁴��

Embodiment 2: the hyaluronic acid dry powder raw material of employing 40 grams, it is 1.3 �� 10 that viscosimetry measures average relative molecular mass (Mr)⁶Da. Under 18 DEG C of room temperatures, the electron beam produced with industry high-energy electron accelerator 1 carries out radiation treatment. The energy of electron beam is 10MeV, and the irradiation dose of hyaluronic acid raw material is 300KGy. After irradiation after 105 hours, sample is configured to 10% aqueous solution 2, removes small molecular weight impurity by retaining the ultrafilter membrane 3 of 3000Da and concentrate, this purified concentration liquid is adopted spray dryer 4 powder process, inlet temperature 110 DEG C, outlet temperature 55 DEG C. Collect hyaluronic acid power-product 37.1 grams, product yield 92.8% altogether. Employing viscosimetry is measured, and the average relative molecular mass (Mr) of product is 5.0 �� 10³Da��

Claims

1. the method that electron beam irradiation prepares low molecular weight hyaluronic acid, mainly has radiation degradation preparation and two steps of post processing:

, hyaluronan molecule amount molecular weight 100,000��4,000,000 Da, use high-energy electron accelerator produce electron beam carry out irradiation-induced degradation, ray energy 5.0��10.0MeV, absorbed dose 50��350KGy, obtain low molecular weight hyaluronic acid;

(2), 1., the catabolite of acquisition is configured to aqueous solution, undertaken purifying and concentration by the ultrafilter membrane of PSPP;

2., the aqueous solution after concentration is extracted low molecular weight hyaluronic acid dry powder formulations by spray dryer.

2. the method that a kind of electron beam irradiation as claimed in claim 1 prepares low molecular weight hyaluronic acid, electron beam irradiation includes other irradiation bombs such as gamma-rays or X ray.

3. the method that a kind of electron beam irradiation as claimed in claim 1 prepares low molecular weight hyaluronic acid, it is thus achieved that hyaluronic acid mean molecule quantity is down to below 10000Da.

4. the method that a kind of electron beam irradiation as claimed in claim 1 prepares low molecular weight hyaluronic acid, catabolite configuration concentration of aqueous solution is 5%-20%.

5. catabolite configuration concentration as claimed in claim 4 is 5%-20% aqueous solution, and the ultrafilter membrane being used below mean molecule quantity 2000Da molecular cut off carries out purifying and concentration.

6. the method that a kind of electron beam irradiation as claimed in claim 1 prepares low molecular weight hyaluronic acid, catabolite configuration aqueous solution is preferably 3000DA and 8000DA by the ultrafilter membrane of molecular cut off.

7. the method that a kind of electron beam irradiation as claimed in claim 1 prepares low molecular weight hyaluronic acid, spray dryer inlet temperature 90 to 120 DEG C, outlet temperature 50 to 60 DEG C.

8. the method that a kind of electron beam irradiation as claimed in claim 1 prepares low molecular weight hyaluronic acid, carries out the time of post processing preferably within 10 days after irradiation-induced degradation.