Preparation process and application of a biohydrogel for sustained release of L-carnosine

Abstract

The present invention relates to the technical field of biohydrogels, and discloses a preparation process and application of a biohydrogel for sustained-release L-carnosine, utilizes N-maleyl-2-aminoethanesulfonic acid and ethylenediamine-modified β-cyclodextrin to undergo amidation reaction, obtains maleylethanesulfonic acid-modified β-cyclodextrin containing alkenyl and sulfonic acid groups, utilizes its alkenyl and N-isopropylacrylamide to carry out polymerization reaction, obtains thermosensitive β-cyclodextrin hydrogel, has hydrophilic sulfonic acid groups, improves the water absorption rate of hydrogel. Utilize the inclusion action of β-cyclodextrin in hydrogel, carry out ultrasound-assisted inclusion process to L-carnosine, obtain the biohydrogel for sustained-release L-carnosine, good biocompatibility, high sustained-release rate of L-carnosine, strong scavenging rate and antioxidant performance of hydrogen peroxide, and has broad application prospects in the fields of drug carriers, drug sustained release, bioengineering, etc.

Description

Technical Field

[0001] The invention relates to the technical field of biohydrogels, in particular to a preparation process and application of a biohydrogel for sustained-release L-carnosine. Background Art

[0002] Thermosensitive hydrogels have the advantages of temperature sensitivity, excellent water absorption, and good biocompatibility, such as N-isopropylacrylamide hydrogel and chitosan thermosensitive hydrogel. They are widely used in the fields of drug sustained release, medical care, bioengineering, etc. For example, the paper "Construction of methylcellulose / glycerol / poly-β-cyclodextrin thermosensitive hydrogel composite system and study on loading hydrophobic drugs Ⅱ" constructed a methylcellulose / glycerol / poly-β-cyclodextrin thermosensitive hydrogel system, and loaded the hydrophobic drug flurbiprofen with poly-β-cyclodextrin, which increased the loading rate of flurbiprofen. It has broad application prospects in drug carriers and sustained release.

[0003] L-carnosine is a small molecule titanium compound containing β-alanine and L-histidine, has excellent antioxidant activity and biological activity, and is widely used in disease treatment, anti-oxidation, anti-aging, anti-corrosion, etc. For example, the document "Preparation of a new CTS boron salt water gel and study on its effect on wood antiseptic" reports that an L-carnosine surfactant was synthesized with N-tert-butyloxycarbonyl-L-histidine, octylamine and tert-butyloxy β-alanine, and then two supramolecular chitosan hydrogels were synthesized with chitosan, which have broad application prospects in the field of antiseptic. The present invention aims to synthesize a new hydrophilic and thermosensitive β-cyclodextrin hydrogel for coating and sustained-release of L-carnosine. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] The invention provides a preparation process and application of a hydrophilic and thermosensitive L-carnosine sustained-release biohydrogel.

[0006] (II) Technical solution

[0007] A preparation process of a biohydrogel for sustained-release of L-carnosine, the preparation process comprising the following steps:

[0008] S1. Add ethylenediamine-modified β-cyclodextrin and N-maleoyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, add N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide, heat to 40-65°C in a nitrogen atmosphere to carry out amidation reaction for 18-36 hours, add acetone after the reaction to precipitate, filter, wash with deionized water and ethanol in turn, and obtain maleoylethanesulfonic acid-modified β-cyclodextrin.

[0009] S2. Add maleylethanesulfonic acid modified β-cyclodextrin and N-isopropylacrylamide to N,N-dimethylformamide, heat to 65-75°C in a nitrogen atmosphere, and drop azobisisobutyronitrile to react for 3-6 hours. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0010] S3, adding β-cyclodextrin hydrogel to deionized water, stirring and swelling, adding L-carnosine, performing ultrasonic treatment inclusion process in an ultrasonic reactor, filtering the solvent, and obtaining a biological hydrogel for sustained release of L-carnosine.

[0011] Preferably, the amount of N-maleyl-2-aminoethanesulfonic acid in S1 is 30-70% of the mass of ethylenediamine-modified β-cyclodextrin.

[0012] Preferably, the amount of N-isopropylacrylamide in S2 is 120-300% of the mass of maleylethanesulfonic acid modified β-cyclodextrin.

[0013] Preferably, the azobisisobutyronitrile in S3 is 0.7-1% of the total mass of the reactants.

[0014] Preferably, the amount of L-carnosine in S3 is 20-50% of the mass of the β-cyclodextrin hydrogel.

[0015] Preferably, the ultrasonic treatment time in S3 is 30-120 min, the temperature is 30-45° C., and the ultrasonic frequency is 20-30 KHz.

[0016] Preferably, the preparation method of the ethylenediamine-modified β-cyclodextrin is:

[0017] Add β-cyclodextrin to a sodium hydroxide aqueous solution with a concentration of 0.4-0.7%, stir to dissolve, then add an acetonitrile solution of p-toluenesulfonyl chloride, stir to react for 4-8 hours, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, dropwise add a dilute hydrochloric acid solution to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to ethylenediamine, heat to 65-80°C to react for 5-8 hours, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol to obtain ethylenediamine-modified β-cyclodextrin.

[0018] Preferably, the preparation method of the N-maleyl-2-aminoethanesulfonic acid is:

[0019] Taurine and maleic anhydride are added to distilled water under an ice water bath, and then sodium hydroxide is added dropwise to adjust the pH value of the solution to 9, and the reaction is stirred for 3-8 hours. After the reaction, the solution is allowed to stand for stratification, and the lower layer of the solution is collected. Then hydrochloric acid solution is added dropwise to adjust the pH value to 2. Crystals are precipitated at room temperature, filtered, and washed with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt. Then, N-maleyl-2-aminoethanesulfonic acid sodium salt is added to a hydrochloric acid solution with a concentration of 5-20% for acidification, the solvent is filtered, and the solution is washed with acetone to obtain N-maleyl-2-aminoethanesulfonic acid. The reaction formula is as follows:

[0020]

[0021] (III) Beneficial technical effects

[0022] By using N-maleyl-2-aminoethanesulfonic acid and ethylenediamine modified β-cyclodextrin to undergo amidation reaction, maleylethanesulfonic acid modified β-cyclodextrin containing alkenyl and sulfonic acid groups is obtained, and its alkenyl group is polymerized with N-isopropylacrylamide to obtain a thermosensitive β-cyclodextrin hydrogel with hydrophilic sulfonic acid groups, which improves the water absorption rate of the hydrogel. By using the inclusion effect of β-cyclodextrin in the hydrogel, L-carnosine is subjected to ultrasound-assisted inclusion process to obtain a biological hydrogel for sustained release of L-carnosine, which has good biocompatibility, high sustained release rate of L-carnosine, strong scavenging rate and antioxidant performance of hydrogen peroxide, and has broad application prospects in the fields of drug carriers, drug sustained release, bioengineering, etc. DETAILED DESCRIPTION

[0023] Example 1

[0024] (1) Add 3 g of β-cyclodextrin to a 0.5% sodium hydroxide aqueous solution, stir to dissolve, then add 4 g of p-toluenesulfonyl chloride in acetonitrile solution, stir to react for 8 h, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, dropwise add dilute hydrochloric acid solution to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to 10 mL of ethylenediamine, heat to 65° C. to react for 6 h, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol, and obtain ethylenediamine-modified β-cyclodextrin.

[0025] (2) Add 6.2 g of taurine and 5.6 g of maleic anhydride to distilled water under an ice water bath, then add sodium hydroxide dropwise to adjust the pH value of the solution to 9, stir and react for 8 h, let stand to separate the layers after the reaction, collect the lower layer of the solution, then add hydrochloric acid solution dropwise to adjust the pH value to 2, precipitate crystals at room temperature, filter, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt, then add N-maleyl-2-aminoethanesulfonic acid sodium salt to a 20% hydrochloric acid solution for acidification, filter the solvent, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

[0026] (3) Add 5 g of ethylenediamine-modified β-cyclodextrin and 3 g of N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, add 3.2 g of N,N-dicyclohexylcarbodiimide and 1.4 g of N-hydroxysuccinimide, heat to 65° C. in a nitrogen atmosphere for 18 h to carry out an amidation reaction, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin.

[0027] (4) Add 5 g of maleylethanesulfonic acid-modified β-cyclodextrin and 12 g of N-isopropylacrylamide to N,N-dimethylformamide, heat to 70 °C in a nitrogen atmosphere, and drop 0.12 g of azobisisobutyronitrile. React for 4 h. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0028] (5) Add 5 g of β-cyclodextrin hydrogel to deionized water, stir and swell, then add 1 g of L-carnosine, and carry out ultrasonic treatment inclusion process in an ultrasonic reactor. The ultrasonic treatment time is 30 min, the temperature is 30° C., and the ultrasonic frequency is 20 kHz. Filter the solvent to obtain a biohydrogel that slowly releases L-carnosine.

[0029] Example 2

[0030] (1) Add 3 g of β-cyclodextrin to a 0.5% sodium hydroxide aqueous solution, stir to dissolve, then add 4 g of p-toluenesulfonyl chloride in acetonitrile solution, stir to react for 4 h, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, dropwise add dilute hydrochloric acid solution to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to 10 mL of ethylenediamine, heat to 70° C. to react for 6 h, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol, and obtain ethylenediamine-modified β-cyclodextrin.

[0031] (2) Add 6.2 g of taurine and 5.6 g of maleic anhydride to distilled water under an ice water bath, then add sodium hydroxide dropwise to adjust the pH of the solution to 9, stir and react for 3 h, let stand to separate the layers after the reaction, collect the lower layer of the solution, then add hydrochloric acid solution dropwise to adjust the pH to 2, precipitate crystals at room temperature, filter, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt, then add N-maleyl-2-aminoethanesulfonic acid sodium salt to a 5% hydrochloric acid solution for acidification, filter the solvent, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

[0032] (3) Add 5 g of ethylenediamine-modified β-cyclodextrin and 3.5 g of N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, then add 4 g of N,N-dicyclohexylcarbodiimide and 1.7 g of N-hydroxysuccinimide, heat to 65° C. in a nitrogen atmosphere to carry out an amidation reaction for 36 h, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin.

[0033] (4) Add 5 g of maleylethanesulfonic acid-modified β-cyclodextrin and 12 g of N-isopropylacrylamide to N,N-dimethylformamide, heat to 70 °C in a nitrogen atmosphere, and drop 0.15 g of azobisisobutyronitrile. React for 5 h. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0034] (5) Add 5 g of β-cyclodextrin hydrogel to deionized water, stir and swell, then add 1.5 g of L-carnosine, and carry out ultrasonic treatment inclusion process in an ultrasonic reactor. The ultrasonic treatment time is 120 min, the temperature is 45° C., and the ultrasonic frequency is 20 kHz. Filter the solvent to obtain a biohydrogel that slowly releases L-carnosine.

[0035] Example 3

[0036] (1) Add 3 g of β-cyclodextrin to a 0.5% sodium hydroxide aqueous solution, stir to dissolve, then add 4 g of p-toluenesulfonyl chloride in acetonitrile solution, stir to react for 8 h, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, add dilute hydrochloric acid solution dropwise to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to 10 mL of ethylenediamine, heat to 70° C. to react for 6 h, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol to obtain ethylenediamine-modified β-cyclodextrin.

[0037] (2) Add 6.2 g of taurine and 5.6 g of maleic anhydride to distilled water under an ice water bath, then add sodium hydroxide dropwise to adjust the pH value of the solution to 9, stir and react for 4 h, let stand to separate the layers after the reaction, collect the lower layer of the solution, then add hydrochloric acid solution dropwise to adjust the pH value to 2, precipitate crystals at room temperature, filter, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt, then add N-maleyl-2-aminoethanesulfonic acid sodium salt to a 10% hydrochloric acid solution for acidification, filter the solvent, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

[0038] (3) Add 5 g of ethylenediamine-modified β-cyclodextrin and 1.5 g of N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, then add 1.8 g of N,N-dicyclohexylcarbodiimide and 0.8 g of N-hydroxysuccinimide, heat to 40° C. in a nitrogen atmosphere to carry out an amidation reaction for 18 h, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin.

[0039] (4) Add 5 g of maleylethanesulfonic acid-modified β-cyclodextrin and 8 g of N-isopropylacrylamide to N,N-dimethylformamide, heat to 70 °C in a nitrogen atmosphere, and drop 0.2 g of azobisisobutyronitrile. React for 4 h. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0040] (5) Add 5 g of β-cyclodextrin hydrogel to deionized water, stir and swell, then add 2.5 g of L-carnosine, and carry out ultrasonic treatment inclusion process in an ultrasonic reactor. The ultrasonic treatment time is 120 min, the temperature is 45° C., and the ultrasonic frequency is 30 kHz. Filter the solvent to obtain a biohydrogel that slowly releases L-carnosine.

[0041] Example 4

[0042] (1) Add 3 g of β-cyclodextrin to a 0.4% sodium hydroxide aqueous solution, stir to dissolve, then add 4 g of acetonitrile solution containing p-toluenesulfonyl chloride, stir to react for 4 h, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, add dilute hydrochloric acid solution dropwise to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to 10 mL of ethylenediamine, heat to 65° C. to react for 5 h, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol, and obtain ethylenediamine-modified β-cyclodextrin.

[0043] (2) Add 6.2 g of taurine and 5.6 g of maleic anhydride to distilled water under an ice water bath, then add sodium hydroxide dropwise to adjust the pH value of the solution to 9, stir and react for 5 h, let stand to separate the layers after the reaction, collect the lower layer of the solution, then add hydrochloric acid solution dropwise to adjust the pH value to 2, precipitate crystals at room temperature, filter, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt, then add N-maleyl-2-aminoethanesulfonic acid sodium salt to a 10% hydrochloric acid solution for acidification, filter the solvent, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

[0044] (3) Add 5 g of ethylenediamine-modified β-cyclodextrin and 3.5 g of N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, then add 3.5 g of N,N-dicyclohexylcarbodiimide and 1.2 g of N-hydroxysuccinimide, heat to 65° C. in a nitrogen atmosphere to carry out an amidation reaction for 18 h, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin.

[0045] (4) Add 5 g of maleylethanesulfonic acid-modified β-cyclodextrin and 6 g of N-isopropylacrylamide to N,N-dimethylformamide, heat to 65 °C in a nitrogen atmosphere, and drop 0.08 g of azobisisobutyronitrile. React for 3 h. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0046] (5) Add 5 g of β-cyclodextrin hydrogel to deionized water, stir and swell, then add 2 g of L-carnosine, and carry out ultrasonic treatment inclusion process in an ultrasonic reactor. The ultrasonic treatment time is 90 min, the temperature is 40° C., and the ultrasonic frequency is 30 kHz. Filter the solvent to obtain a biohydrogel that slowly releases L-carnosine.

[0047] Example 5

[0048] (1) Add 3 g of β-cyclodextrin to a 0.7% sodium hydroxide aqueous solution, stir to dissolve, then add 4 g of p-toluenesulfonyl chloride in acetonitrile solution, stir to react for 8 h, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, dropwise add dilute hydrochloric acid solution to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to 10 mL of ethylenediamine, heat to 80° C. to react for 8 h, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol, and obtain ethylenediamine-modified β-cyclodextrin.

[0049] (2) Add 6.2 g of taurine and 5.6 g of maleic anhydride to distilled water under an ice water bath, then add sodium hydroxide dropwise to adjust the pH value of the solution to 9, stir and react for 5 h, let stand to separate the layers after the reaction, collect the lower layer of the solution, then add hydrochloric acid solution dropwise to adjust the pH value to 2, precipitate crystals at room temperature, filter, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt, then add N-maleyl-2-aminoethanesulfonic acid sodium salt to a 5% hydrochloric acid solution for acidification, filter the solvent, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

[0050] (3) Add 5 g of ethylenediamine-modified β-cyclodextrin and 1.5 g of N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, add 2.5 g of N,N-dicyclohexylcarbodiimide and 1.2 g of N-hydroxysuccinimide, heat to 55° C. in a nitrogen atmosphere to carry out an amidation reaction for 24 h, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin.

[0051] (4) Add 5 g of maleylethanesulfonic acid-modified β-cyclodextrin and 10 g of N-isopropylacrylamide to N,N-dimethylformamide, heat to 75 °C in a nitrogen atmosphere, and drop 0.15 g of azobisisobutyronitrile. React for 4 h. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0052] (5) Add 5 g of β-cyclodextrin hydrogel to deionized water, stir and swell, then add 2 g of L-carnosine, and carry out ultrasonic treatment inclusion process in an ultrasonic reactor. The ultrasonic treatment time is 30 min, the temperature is 30° C., and the ultrasonic frequency is 30 kHz. Filter the solvent to obtain a biohydrogel that slowly releases L-carnosine.

[0053] Example 6

[0054] (1) Add 3 g of β-cyclodextrin to a 0.7% sodium hydroxide aqueous solution, stir to dissolve, then add 4 g of p-toluenesulfonyl chloride in acetonitrile solution, stir to react for 8 h, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, dropwise add dilute hydrochloric acid solution to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to 10 mL of ethylenediamine, heat to 65° C. to react for 8 h, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol, and obtain ethylenediamine-modified β-cyclodextrin.

[0055] (2) Add 6.2 g of taurine and 5.6 g of maleic anhydride to distilled water under an ice water bath, then add sodium hydroxide dropwise to adjust the pH value of the solution to 9, stir and react for 5 h, let stand to separate the layers after the reaction, collect the lower layer of the solution, then add hydrochloric acid solution dropwise to adjust the pH value to 2, precipitate crystals at room temperature, filter, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt, then add N-maleyl-2-aminoethanesulfonic acid sodium salt to a 10% hydrochloric acid solution for acidification, filter the solvent, and wash with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

[0056] (3) Add 5 g of ethylenediamine-modified β-cyclodextrin and 1.8 g of N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, then add 2.2 g of N,N-dicyclohexylcarbodiimide and 1.7 g of N-hydroxysuccinimide, heat to 65° C. in a nitrogen atmosphere to carry out an amidation reaction for 18 h, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin.

[0057] (4) Add 5 g of maleylethanesulfonic acid-modified β-cyclodextrin and 15 g of N-isopropylacrylamide to N,N-dimethylformamide, heat to 75 °C in a nitrogen atmosphere, and drop 0.2 g of azobisisobutyronitrile. React for 6 h. After the reaction, cool and add acetone to precipitate, filter, wash with ethanol and dry to obtain β-cyclodextrin hydrogel.

[0058] (5) Add 5 g of β-cyclodextrin hydrogel to deionized water, stir and swell, then add 1.5 g of L-carnosine, and carry out ultrasonic treatment inclusion process in an ultrasonic reactor. The ultrasonic treatment time is 120 min, the temperature is 30° C., and the ultrasonic frequency is 30 kHz. Filter the solvent to obtain a biohydrogel that slowly releases L-carnosine.

[0059] Water absorption test: The β-cyclodextrin hydrogel was dried and weighed, then added to deionized water and soaked at different temperatures for 12 h. After being taken out, the surface water was drained and weighed to test the water absorption rate Q.

[0060] Water absorption rate Q = (m-m0) / m0. m is the mass of the hydrogel after absorbing water, and m0 is the mass of the hydrogel before absorbing water.

[0061]

[0062] Determination of L-carnosine encapsulation rate: The biohydrogel for sustained-release L-carnosine prepared in each example was dried to remove moisture, then weighed, and the L-carnosine encapsulation rate was calculated.

[0063] Covering rate = (m-m0) / m×100%. m is the mass of the sustained-release L-carnosine biohydrogel after drying and removing water, and m0 is the mass of the β-cyclodextrin hydrogel before L-carnosine inclusion (5 g)

[0064] Coverage rate (%) Example 1 14.3 Example 2 19.8 Example 3 30.6 Example 4 22.3 Example 5 23.0 Example 6 16.7

[0065] Sustained release performance test: The biohydrogels for sustained release of L-carnosine prepared in each example were dried and weighed, then added to PBS and soaked at 37°C for different time periods to sustain the release of L-carnosine. After being taken out, they were dried to remove the water in the hydrogels, weighed, and the sustained release rate was tested.

[0066] Sustained release rate W = (m0-m) / m0×100%. Water absorption rate Q = (m-5) / (m0-5). m0 is the mass of the hydrogel after drying before sustained release, and m is the mass of the hydrogel after sustained release and drying.

[0067]

[0068] Hydrogen peroxide clearance test: An aqueous solution of a biohydrogel for sustained release of L-carnosine was prepared at a concentration of 2 mg / mL.

[0069] The hydrogen peroxide solution was titrated with PBS buffer to a concentration of 0.5%, and then 2 mL of 0.5% hydrogen peroxide solution was added to 2 mL of aqueous solutions of bio-hydrogels of different concentrations, and the absorbance A was measured at 230 nm after mixing evenly at room temperature; then 2 mL of 0.5% hydrogen peroxide solution was added to 2 mL of distilled water, and the absorbance A1 was measured at 230 nm after mixing evenly at room temperature; 2 mL of distilled water was added to 2 mL of aqueous solutions of bio-hydrogels, and the absorbance A2 was measured at 230 nm after mixing evenly at room temperature.

[0070] The clearance rate W of hydrogen peroxide was determined, W = [1-(A-A2)÷A1]×100%.

[0071]

[0072]

[0073] The scavenging rate of hydrogen peroxide by the biohydrogel with sustained release of L-carnosine reached 63.2-86.8%.

Claims

1. A preparation process of a biohydrogel for sustained-release L-carnosine, characterized in that: The preparation process comprises the following steps: S1. Add ethylenediamine-modified β-cyclodextrin and N-maleyl-2-aminoethanesulfonic acid to N,N-dimethylformamide, stir to dissolve, add N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide, heat to 40-65° C. in a nitrogen atmosphere to carry out an amidation reaction for 18-36 hours, add acetone after the reaction to precipitate, filter, and wash with deionized water and ethanol in turn to obtain maleylethanesulfonic acid-modified β-cyclodextrin; S2, adding maleylethanesulfonic acid modified β-cyclodextrin and N-isopropylacrylamide to N,N-dimethylformamide, heating to 65-75°C in a nitrogen atmosphere, and adding azobisisobutyronitrile dropwise, reacting for 3-6 hours, cooling after the reaction and adding acetone to precipitate, filtering, washing with ethanol and drying to obtain β-cyclodextrin hydrogel; S3, adding β-cyclodextrin hydrogel to deionized water, stirring and swelling, adding L-carnosine, performing ultrasonic treatment inclusion process in an ultrasonic reactor, filtering the solvent, and obtaining a biological hydrogel for sustained release of L-carnosine.

2. The preparation technology of the biohydrogel of sustained-release L-carnosine according to claim 1, characterized in that: The amount of N-maleyl-2-aminoethanesulfonic acid in S1 is 30-70% of the mass of ethylenediamine-modified β-cyclodextrin.

3. The preparation technology of the biohydrogel of sustained-release L-carnosine according to claim 1, characterized in that: The amount of N-isopropylacrylamide in S2 is 120-300% of the mass of maleylethanesulfonic acid modified β-cyclodextrin.

4. The preparation technology of the biohydrogel of sustained-release L-carnosine according to claim 1, characterized in that: The azobisisobutyronitrile in S3 accounts for 0.7-1% of the total mass of the reactants.

5. The preparation process of the biohydrogel of sustained-release L-carnosine according to claim 1, characterized in that: The amount of L-carnosine in S3 is 20-50% of the mass of the β-cyclodextrin hydrogel.

6. The preparation process of the biohydrogel for sustained-release L-carnosine according to claim 1, characterized in that: The ultrasonic treatment time in S3 is 30-120 min, the temperature is 30-45° C., and the ultrasonic frequency is 20-30 KHz.

7. The preparation process of the biohydrogel for sustained-release L-carnosine according to claim 1, characterized in that: The preparation method of the ethylenediamine-modified β-cyclodextrin is: Add β-cyclodextrin to a sodium hydroxide aqueous solution with a concentration of 0.4-0.7%, stir to dissolve, then add an acetonitrile solution of p-toluenesulfonyl chloride, stir to react for 4-8 hours, do not add sodium hydroxide aqueous solution during the reaction, adjust the pH of the reaction solution to 13, dropwise add a dilute hydrochloric acid solution to neutralize after the reaction, filter, wash the intermediate product with ethanol, then add the intermediate product to ethylenediamine, heat to 65-80°C to react for 5-8 hours, add acetone to precipitate after the reaction, filter the solvent, wash with ethanol to obtain ethylenediamine-modified β-cyclodextrin.

8. The preparation process of the biohydrogel for sustained-release L-carnosine according to claim 1, characterized in that: The preparation method of the N-maleyl-2-aminoethanesulfonic acid is: Taurine and maleic anhydride are added to distilled water under an ice water bath, and then sodium hydroxide is added dropwise to adjust the pH value of the solution to 9, and the reaction is stirred for 3-8 hours. After the reaction, the solution is allowed to stand for stratification, and the lower layer of the solution is collected. Then hydrochloric acid solution is added dropwise to adjust the pH value to 2. Crystals are precipitated at room temperature, filtered, and washed with acetone to obtain N-maleyl-2-aminoethanesulfonic acid sodium salt. Then, the N-maleyl-2-aminoethanesulfonic acid sodium salt is added to a hydrochloric acid solution with a concentration of 5-20% for acidification, the solvent is filtered, and the solution is washed with acetone to obtain N-maleyl-2-aminoethanesulfonic acid.

Images