Method for temperature-programmed controlled degradation of hyaluronic acid glycosidic bonds

Abstract

The application discloses a method for controllably degrading glycosidic bonds of hyaluronic acid by programmed temperature rising. The method is used for high-molecular-weight hyaluronic acid aqueous solution, and a combination of an oxygen oxidant and an acidic additive is used to controllably cut and degrade glycosidic bonds in the macromolecule through a heating process of programmed temperature rising control system, so as to obtain oligomeric hyaluronic acid. The oligomeric hyaluronic acid prepared by the method has very good stability and no color change after long-time storage. The oligomeric hyaluronic acid prepared by the method can be applied to the fields of cosmetics, food, medicine and the like, and has very strong practical value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological materials, and particularly relates to a method for degrading glycosidic bonds of hyaluronic acid, and particularly relates to a method for degrading glycosidic bonds of hyaluronic acid by programmed temperature control. BACKGROUND

[0002] Hyaluronic acid is a high-molecular polysaccharide produced from a rooster comb or microbial fermentation, and each of two adjacent units of the high-molecular polysaccharide contains a carboxyl group and an amino group (acetylated). The substance has super water absorption capacity and has a good moisturizing effect on the skin surface. However, due to the large molecule, the penetration of the substance into the epidermal cells of the skin is blocked. In order to improve the permeability, the substance needs to be partially degraded. The degraded polysaccharide is oligomeric hyaluronic acid, which has a strong ability to penetrate into cells and can penetrate into the stratum corneum of the skin to achieve the function of deep moisturizing. The oligomeric hyaluronic acid has the functions of activating the immune system of the body, repairing the skin, and playing the roles of anti-inflammation and cell repair. Therefore, the degraded oligomeric hyaluronic acid is an important raw material for cosmetics and medicines.

[0003] Since the hyaluronic acid obtained by fermentation and separation has a large molecular weight and poor penetration effect, it is necessary to shear the high-molecular hyaluronic acid to obtain oligomeric hyaluronic acid. The current related reports cannot effectively control the degradation of high-molecular hyaluronic acid. The present application provides a degradation method under the catalysis of an acidic auxiliary agent by programmed temperature control, which can control the molecular weight of the oligomeric hyaluronic acid. Therefore, the method of the present application has strong practical value. SUMMARY

[0004] The present application provides a method for degrading glycosidic bonds of hyaluronic acid by programmed temperature control. The method can achieve controllable degradation of hyaluronic acid by programmed temperature control, and can obtain precisely controlled molecular weight to prepare small-molecule hyaluronic acid products.

[0005] The object of the present application is achieved at least by one of the following technical solutions.

[0006] A method for degrading glycosidic bonds of hyaluronic acid by programmed temperature control. High-molecular hyaluronic acid produced by fermentation is prepared into an aqueous solution, and then is oxidatively degraded in an acidic auxiliary agent catalytic system. The reaction is controlled by programmed temperature control, and hyaluronic acid with a desired molecular weight can be obtained. The specific steps are as follows:

[0007] (1) High-molecular hyaluronic acid or high-molecular sodium hyaluronate is first prepared into an aqueous solution, and the solution is sterilized at 105-115 DEG C for 20-60 minutes to obtain solution A;

[0008] (2) An oxygen-based oxidizing agent and an acidic auxiliary agent are added to solution A for pre-reaction. The volume ratio of solution A, the oxygen-based oxidizing agent, and the acidic auxiliary agent is (10-100):(5-10):(5-10);

[0009] (3) The high temperature programmed reactor is transferred into the program temperature reactor, and the program temperature is carried out, and after reaching the set stable, the temperature is kept, the hyaluronic acid with the required molecular weight is obtained, and the molecular weight is measured.

[0010] In the above method, in step (1), the molecular weight of the high molecular weight hyaluronic acid or the high molecular weight sodium hyaluronate is above 1.5 million Daltons.

[0011] In the above method, in step (1), the mass fraction concentration of the high molecular weight hyaluronic acid aqueous solution is 1-5%, and the mass fraction concentration of the high molecular weight sodium hyaluronate aqueous solution is 2-8%.

[0012] In the above method, in step (2), the oxygen-based oxidizing agent is ROOH, wherein R is CH3CO or H; and the acidic auxiliary agent is a Lewis acid.

[0013] In the above method, in step (2), the pre-reaction time is 30-120 minutes, and the pre-reaction temperature is 30-50 DEG C.

[0014] In the above method, in step (3), the program temperature is 1-5 DEG C / min, and the end point temperature is 115-121 DEG C.

[0015] In the above method, in step (3), the temperature keeping time is 30-180 min.

[0016] In the above method, the Lewis acid is a polyhydroxy compound with a molecular formula of C6H8O6, or an organic metal framework compound with a molecular formula of C 24 H 12 ClFe3O 13 The iron atom provides a catalytic center.

[0017] In the above method, in step (2), the concentration of the oxygen-based oxidizing agent is 1-3% w / w, and is not limited thereto.

[0018] In the above method, in step (2), the concentration of the Lewis acid is 0.2-0.4% w / w, and is not limited thereto.

[0019] In the present application, the measuring method of the product is as follows:

[0020] 1) A certain amount of dried hyaluronic acid sample is accurately weighed and placed in a 200ml volumetric flask, and a certain amount of 0.2mol / L sodium chloride solution is added to dissolve, and after there is no bubble in the test solution, the test solution 1 is diluted to the scale with 0.2mol / L sodium chloride solution.

[0021] 2) Take the test solution 1 respectively with 0.2mol / L sodium chloride solution dilution to 0.8 times, 0.6 times and 0.4 times, as test solution 2, test solution 3 and test solution 4, after filtering through No.3 vertical melting glass funnel, use.

[0022] 3) Using viscosity determination method, using Ubbelohde viscometer, the efflux time t 0 of 0.2mol / L sodium chloride solution and the efflux time t 1, t 2, t 3, t 4 of four test solutions are determined at 30±0.1, and the appropriate inner diameter Ubbelohde viscometer is selected.

[0023] 4) The efflux time of 0.2mol / L sodium chloride solution is 200-300 seconds, the sample weight of test solution 1 is adjusted so that the efflux time is 2.0-2.4 times of the efflux time of 0.2mol / L sodium chloride solution. All tests use the same viscometer without reloading the sample, and the difference between the three determination values and the average value should not exceed ±0.35% of the average value. The characteristic viscosity [eta] is calculated by four-point method and least square linear regression, and the linear regression of specific viscosity [eta sp / C, namely (eta r-1) / C] against concentration C is carried out. When the concentration approaches 0, the intercept of the linear regression equation is the characteristic viscosity, and the linear regression coefficient should be not less than 0.95, and the unit is L / g.

[0024] 5) The characteristic viscosity is between 1.00L / g-2.49L / g or 2.50L / g-5.50L / g, calculated according to the dry product.

[0025] 6) The average molecular weight is calculated according to the characteristic viscosity [eta] of the product, and the calculation result should be within the indicated molecular weight range. The average molecular weight is calculated according to the following formula:

[0026]

[0027] Compared with the prior art, the advantages of the present application are:

[0028] In the present application, the high molecular weight hyaluronic acid produced by fermentation is made into an aqueous solution, and then is oxidatively degraded in an acidic catalyst system. Through programmed temperature rising, the hyaluronic acid realizes controllable degradation, and precise control of molecular weight can be achieved to prepare small molecular hyaluronic acid products.

[0029] The oligomeric hyaluronic acid prepared by the present application has very good stability and no color change after long-term storage. The oligomeric hyaluronic acid prepared by the present application can be applied in the fields of cosmetics, food, medicine and the like, and has strong practical value. DETAILED DESCRIPTION

[0030] The application will be further described in conjunction with the specific embodiments of the description. The embodiments are only used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified.

[0031] Example 1

[0032] Hyaluronic acid with a molecular weight of 1.5 million Daltons was weighed to prepare a 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added to 95 mL of deionized water, and uniformly shaken to obtain), which was sterilized at 105°C for 30 minutes to obtain solution A; 20 mL of 10% w / w H2O2 and 10 mL of 2% w / w ascorbic acid were added to the solution, and pre-reacted at 50°C for 4 h; the reaction bottle was transferred into a programmed temperature reactor, and programmed temperature heating was performed at 5°C / min, and after reaching the set temperature of 120°C, the temperature was maintained for 2 h to obtain hyaluronic acid with the desired molecular weight, and the molecular weight was determined to be 3000 Daltons.

[0033] Example 2

[0034] Hyaluronic acid with a molecular weight of 1.5 million Daltons was weighed to prepare a 1% w / w aqueous solution (1 g of hyaluronic acid was weighed and added to 99 mL of deionized water, and uniformly shaken to obtain), which was sterilized at 105°C for 30 minutes to obtain solution A; 20 mL of 10% w / w H2O2 and 10 mL of 2% w / w ascorbic acid were added to the solution, and pre-reacted at 50°C for 4 h; the reaction bottle was transferred into a programmed temperature reactor, and programmed temperature heating was performed at 5°C / min, and after reaching the set temperature of 120°C, the temperature was maintained for 2 h to obtain hyaluronic acid with the desired molecular weight, and the molecular weight was determined to be 3000 Daltons.

[0035] Example 3

[0036] Hyaluronic acid with a molecular weight of 1.5 million Daltons was weighed to prepare a 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added to 95 mL of deionized water, and uniformly shaken to obtain), which was sterilized at 105°C for 30 minutes to obtain solution A; 20 mL of 10% w / w H2O2 and 10 mL of 2% w / w ascorbic acid were added to the solution, and pre-reacted at 50°C for 4 h; the reaction bottle was transferred into a programmed temperature reactor, and programmed temperature heating was performed at 5°C / min, and after reaching the set temperature of 120°C, the temperature was maintained for 2 h to obtain hyaluronic acid with the desired molecular weight, and the molecular weight was determined to be 3000 Daltons.

[0037] Example 4

[0038] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added into 95 mL of deionized water, and then stirred to obtain a uniform solution), and then sterilized at 105°C for 30 minutes to obtain solution A; 50 mL of 10% w / w H2O2 and 10 mL of 4% w / w ascorbic acid were added into the solution, and then pre-reacted at 50°C for 4 hours; the reaction bottle was transferred into a programmed temperature reactor, and then programmed temperature reaction was performed at a rate of 5°C / min, and then kept at a set temperature of 120°C for 2 hours, so that hyaluronic acid with a desired molecular weight was obtained, and the molecular weight was determined to be 800 Dalton.

[0039] Example 5

[0040] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added into 95 mL of deionized water, and then stirred to obtain a uniform solution), and then sterilized at 105°C for 30 minutes to obtain solution A; 50 mL of 10% w / w H2O2 and 10 mL of 4% w / w ascorbic acid were added into the solution, and then pre-reacted at 50°C for 4 hours; the reaction bottle was transferred into a programmed temperature reactor, and then programmed temperature reaction was performed at a rate of 5°C / min, and then kept at a set temperature of 120°C for 2 hours, so that hyaluronic acid with a desired molecular weight was obtained, and the molecular weight was determined to be 800 Dalton.

[0041] Example 6

[0042] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added into 95 mL of deionized water, and then stirred to obtain a uniform solution), and then sterilized at 105°C for 30 minutes to obtain solution A; 50 mL of 10% w / w H2O2 and 10 mL of 4% w / w ascorbic acid were added into the solution, and then pre-reacted at 50°C for 4 hours; the reaction bottle was transferred into a programmed temperature reactor, and then programmed temperature reaction was performed at a rate of 5°C / min, and then kept at a set temperature of 120°C for 2 hours, so that hyaluronic acid with a desired molecular weight was obtained, and the molecular weight was determined to be 800 Dalton.

[0043] Example 7

[0044] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added into 95 mL of deionized water, and then stirred to obtain a uniform solution), and then sterilized at 105°C for 30 minutes to obtain solution A; 50 mL of 10% w / w H2O2 and 10 mL of 4% w / w ascorbic acid were added into the solution, and then pre-reacted at 50°C for 4 hours; the reaction bottle was transferred into a programmed temperature reactor, and then programmed temperature reaction was performed at a rate of 5°C / min, and then kept at a set temperature of 120°C for 2 hours, so that hyaluronic acid with a desired molecular weight was obtained, and the molecular weight was determined to be 800 Dalton.

[0045] Example 8

[0046] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added with 95 mL of deionized water and shaken to be uniform), sterilized at 105 ℃ for 30 min to obtain solution A; 50 mL of 10% w / w CH3COOOH and 10 mL of 2% w / w ascorbic acid were added into the solution and pre-reacted at 50 ℃ for 4 h; the reaction bottle was transferred into a programmed temperature reactor and programmed temperature was carried out at 5 ℃ / min, and after reaching the set 105 ℃, it was kept for 2 h to obtain hyaluronic acid with the required molecular weight, and the molecular weight was determined to be 2800 Dalton.

[0047] Example 9

[0048] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added with 95 mL of deionized water and shaken to be uniform), sterilized at 105 ℃ for 30 min to obtain solution A; 20 mL of 10% w / w CH3COOOH and 0.2 g of Lewis acid C 24 H 12 ClFe3O 13 were added into the solution and pre-reacted at 50 ℃ for 4 h; the reaction bottle was transferred into a programmed temperature reactor and programmed temperature was carried out at 5 ℃ / min, and after reaching the set 105 ℃, it was kept for 2 h to obtain hyaluronic acid with the required molecular weight, and the molecular weight was determined to be 3200 Dalton.

[0049] Example 10

[0050] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added with 95 mL of deionized water and shaken to be uniform), sterilized at 105 ℃ for 30 min to obtain solution A; 10 mL of 10% w / w CH3COOOH and 0.4 g of Lewis acid C 24 H 12 ClFe3O 13 were added into the solution and pre-reacted at 50 ℃ for 2 h; the reaction bottle was transferred into a programmed temperature reactor and programmed temperature was carried out at 5 ℃ / min, and after reaching the set 105 ℃, it was kept for 2 h to obtain hyaluronic acid with the required molecular weight, and the molecular weight was determined to be 3500 Dalton.

[0051] Example 11

[0052] Hyaluronic acid with molecular weight of 1.5 million Dalton was weighed and prepared into 5% w / w aqueous solution (5 g of hyaluronic acid was weighed and added with 95 mL of deionized water and shaken to be uniform), sterilized at 105 ℃ for 30 min to obtain solution A; 10 mL of 10% w / w CH3COOOH and 0.2 g of Lewis acid C24 H 12 ClFe3O 13 , 50℃ pre-reaction 2h; the reaction bottle is transferred into the programmed temperature reactor, programmed temperature 5℃ / min, reaches the set 121℃, and keeps temperature for 2h, to obtain the hyaluronic acid with the required molecular weight, and the molecular weight is determined to be 1000 Dalton.

[0053] Example 12

[0054] The hyaluronic acid with the molecular weight of 1.5 million Dalton is weighed and prepared into a 2% w / w aqueous solution (2g of hyaluronic acid is weighed and added into 98mL of deionized water, and shaken to obtain uniformity), sterilized at 105℃ for 30 minutes to obtain solution A; 50mL of 10% w / w CH3COOOH and 0.2g of Lewis acid C are added into the solution, and pre-reacted at 50℃ for 4h; the reaction bottle is transferred into the programmed temperature reactor, programmed temperature 5℃ / min, reaches the set 121℃, and keeps temperature for 2h, to obtain the hyaluronic acid with the required molecular weight, and the molecular weight is determined to be 820 Dalton. 24 H 12 ClFe3O 13 , 50℃ pre-reaction 2h; the reaction bottle is transferred into the programmed temperature reactor, programmed temperature 5℃ / min, reaches the set 121℃, and keeps temperature for 2h, to obtain the hyaluronic acid with the required molecular weight, and the molecular weight is determined to be 1000 Dalton.

[0055] Example 13

[0056] 2g of sodium hyaluronate with the molecular weight of 1.5 million Dalton is weighed and added into 98mL of deionized water, shaken to mix uniformly, and sterilized at 121℃ for 30 minutes to obtain solution A; 50mL of 10% w / w CH3COOOH and 10mL of 2% w / w ascorbic acid are added into the solution, pre-reacted at 50℃ for 4h; the reaction bottle is transferred into the programmed temperature reactor, programmed temperature 5℃ / min, reaches the set 115℃, and keeps temperature for 180min, to obtain the sodium hyaluronate with the required small molecular weight, and the molecular weight is determined to be 3000 Dalton.

[0057] It should be understood that the above detailed description of the technical solutions of the present application by means of the optimization examples is illustrative rather than limiting, and the specific embodiments of the present application cannot be considered to be limited thereto, and for ordinary skilled persons in the technical field to which the present application belongs, the technical solutions recorded in each embodiment can be modified or some technical features can be replaced equivalently without departing from the concept of the present application, and all of them shall be considered to belong to the patent protection scope determined by the claims submitted by the present application.

[0058] The above embodiments of the present application are merely used for clearly illustrating the present application, but not for limiting the present application. Based on the above description, any modification, equivalent replacement and improvement made by those skilled in the art should be included in the protection scope of the present application.

Claims

1. A method for controlled degradation of hyaluronic acid glycosidic bonds via programmed temperature rise, characterized in that, Includes the following steps: (1) First, prepare an aqueous solution of high molecular weight hyaluronic acid or high molecular weight sodium hyaluronate, and sterilize it at 105-115℃ for 20-60 minutes to obtain solution A; (2) Add an oxygen-based oxidant and an acidic auxiliary agent to solution A for pre-reaction; the oxygen-based oxidant is peracetic acid (CH3COOOH), and the acidic auxiliary agent is an organometallic framework compound with the molecular formula C 24 H 12 ClFe3O 13 Iron atoms provide catalytic centers; the volume ratio of solution A, oxygen-based oxidant and acidic auxiliary agent is (10-100):(5-10):(5-10); the concentration of oxygen-based oxidant is 1%~3% w / w; the concentration of acidic auxiliary agent is 0.2%~0.4% w / w; (3) transfer to a programmed temperature reactor, perform programmed temperature rise at a rate of 1-5℃ / min, the final temperature is 115-121℃, after reaching the set temperature, keep warm for 30-180min, obtain hyaluronic acid with the required molecular weight, and determine the molecular weight.

2. The method according to claim 1, characterized in that, In step (1), the molecular weight of the high molecular weight hyaluronic acid or high molecular weight sodium hyaluronate is above 1.5 million Daltons.

3. The method according to claim 1, characterized in that, In step (1), the mass fraction concentration of the high molecular weight hyaluronic acid aqueous solution is 1%~5%; the mass fraction concentration of the high molecular weight sodium hyaluronate aqueous solution is 2-8%.

4. The method according to claim 1, characterized in that, In step (2), the pre-reaction time is 30-120 minutes; the pre-reaction temperature is 30-50℃.

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