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CN-121975889-A - Preparation method and application of chondroitin sulfate oligosaccharide and nerve repair release preparation

CN121975889ACN 121975889 ACN121975889 ACN 121975889ACN-121975889-A

Abstract

The invention relates to the technical field of biological enzymolysis, and discloses a preparation method and application of chondroitin sulfate oligosaccharide. The preparation method comprises the steps of carrying out freezing treatment and thawing treatment on cartilage raw materials, and then mixing and carrying out enzymolysis on the cartilage raw materials with compound enzymes, wherein the compound enzymes are chondroitinase ABC and hyaluronidase, and the mass ratio of the chondroitinase ABC to the hyaluronidase is 2-4:1. In addition, the invention also provides a nerve repair release preparation. The chondroitin sulfate oligosaccharide prepared by the preparation method has the advantages of narrow molecular weight distribution, high purity, no chemical reagent residue and good application in the field of medicine.

Inventors

  • GUO DONGSHENG
  • LIU XIAO
  • TONG LINGLING
  • JIA HUITING
  • SHU JUNFENG

Assignees

  • 南京师范大学

Dates

Publication Date
20260505
Application Date
20260114

Claims (10)

  1. 1. A preparation method of chondroitin sulfate oligosaccharide is characterized by comprising the steps of freezing and thawing a cartilage raw material, and then mixing and carrying out enzymolysis on the cartilage raw material with compound enzymes, wherein the compound enzymes are chondroitinase ABC and hyaluronidase, and the mass ratio of the chondroitinase ABC to the hyaluronidase is 2-4:1.
  2. 2. The method according to claim 1, wherein the amount of the complex enzyme is 0.004-0.006g relative to 1g of cartilage material.
  3. 3. The method according to claim 1, wherein the conditions for the enzymatic hydrolysis comprise a pH of 7-7.5, a temperature of 35-39 ℃ and a time of 3.5-4.5 hours.
  4. 4. A production method according to any one of claims 1to 3, wherein the number of repetitions of the freezing treatment and thawing treatment is 2 to 4 times; Preferably, the freezing treatment condition comprises a temperature of minus 90 ℃ to minus 70 ℃ and a time of 10-14h, and the thawing treatment condition comprises a temperature of 20-30 ℃ and a time of 4-8h.
  5. 5. The method according to any one of claims 1 to 3, wherein the step of preparing the cartilage material comprises crushing the cartilage and washing the crushed cartilage, The cartilage is at least one of shark cartilage, pig cartilage and squid cartilage, preferably shark cartilage.
  6. 6. The method according to any one of claims 1 to 3, wherein the method further comprises purifying the product obtained by the enzymolysis; preferably, the step of purifying comprises dialyzing the supernatant after centrifugation; preferably, the ultrafiltration membrane used for the dialysis has a molecular weight cut-off of 1800-2500Da.
  7. 7. Use of chondroitin sulfate oligosaccharide prepared by the preparation method of any one of claims 1 to 6 in nerve repair release products.
  8. 8. A nerve repair release preparation, which is characterized in that the preparation contains neurotrophic factors, a slow release carrier and chondroitin sulfate oligosaccharides prepared by the preparation method according to any one of claims 1 to 6.
  9. 9. The nerve repair release preparation according to claim 8, wherein the content of the chondroitin sulfate oligosaccharide is 800 to 1200 parts by mass and the content of the sustained-release carrier is 6000 to 10000 parts by mass, based on 1 part by mass of the content of the neurotrophic factor, and/or, The slow release carrier is segmented copolymer PLGA-PEG-PLGA and/or segmented copolymer PLGA-b-PEG, preferably, the molecular weight of the slow release carrier is 5000-8000Da; The neurotrophic factor is nerve growth factor and/or brain-derived neurotrophic factor.
  10. 10. The nerve repair release formulation of claim 8 or 9, further comprising a lyoprotectant; Preferably, in the nerve repair release preparation, the content of the lyoprotectant is 8-12wt%, and/or, The freeze-drying protective agent is mannitol.

Description

Preparation method and application of chondroitin sulfate oligosaccharide and nerve repair release preparation Technical Field The invention relates to the technical field of biological enzymolysis, in particular to a preparation method and application of chondroitin sulfate oligosaccharide. In addition, the invention also relates to a nerve repair release preparation containing the chondroitin sulfate oligosaccharide prepared by the preparation method. Background Chondroitin Sulfate (CS) is a long-chain polysaccharide consisting of glucuronic acid and N-acetylgalactosamine, and the oligosaccharide form (CS-OS) has small molecular weight and high bioavailability, and shows biological activity superior to that of the long-chain CS in the fields of nerve repair, anti-inflammation and the like, so that the Chondroitin Sulfate (CS) becomes a research hot spot in the field of biological medicine. In the field of nerve repair, the traditional medicine for treating central nerve injury diseases such as spinal cord injury, cerebral infarction sequelae and the like is difficult to cross blood brain barrier, has short acting time and limited repair effect, and clinical requirements are not satisfied, and the CS-OS has the potential of crossing the blood brain barrier due to the small molecular characteristics, but the CS-OS prepared by the traditional CS-OS preparation process has the problems of wide molecular weight distribution, low purity, residual chemical reagent and the like, so that medical-grade application of the CS-OS is limited. Disclosure of Invention The invention aims to solve the problems of wide molecular weight distribution, low purity and chemical reagent residue of CS-OS prepared by a CS-OS preparation process in the prior art, and provides a preparation method and application of chondroitin sulfate oligosaccharide and a nerve repair release preparation. In order to achieve the aim, the first aspect of the invention provides a preparation method of chondroitin sulfate oligosaccharide, which comprises the steps of freezing and thawing cartilage raw materials, and then mixing and carrying out enzymolysis on the cartilage raw materials with complex enzymes, wherein the complex enzymes are chondroitinase ABC and hyaluronidase, and the mass ratio of the chondroitinase ABC to the hyaluronidase is 2-4:1. Preferably, the complex enzyme is used in an amount of 0.004-0.006g relative to 1g of cartilage material. Preferably, the enzymolysis conditions comprise pH of 7-7.5, temperature of 35-39deg.C and time of 3.5-4.5 hr. Preferably, the number of repetitions of the freezing and thawing treatment is 2 to 4. Preferably, the freezing treatment condition comprises a temperature of minus 90 ℃ to minus 70 ℃ and a time of 10-14h, and the thawing treatment condition comprises a temperature of 20-30 ℃ and a time of 4-8h. Preferably, the cartilage material is prepared by crushing cartilage and washing. Preferably, the cartilage in the cartilage material is at least one selected from shark cartilage, pig cartilage and squid cartilage, and more preferably shark cartilage. Preferably, the preparation method further comprises the step of purifying a product obtained by the enzymolysis. Further preferably, the step of purifying comprises dialyzing the supernatant after centrifugation. More preferably, the ultrafiltration membrane used for the dialysis has a molecular weight cut-off of 1800-2500Da. The second aspect of the invention provides an application of the chondroitin sulfate oligosaccharide prepared by the preparation method in a nerve repair release product. The third aspect of the invention provides a nerve repair release preparation, which contains neurotrophic factors, a slow release carrier and chondroitin sulfate oligosaccharides prepared by the preparation method. Preferably, the content of the chondroitin sulfate oligosaccharide is 800-1200 parts by mass and the content of the slow-release carrier is 6000-10000 parts by mass based on 1 part by mass of the content of the neurotrophic factor. Preferably, the slow release carrier is block copolyester PLGA-PEG-PLGA and/or block copolymer PLGA-b-PEG. Further preferably, the molecular weight of the slow release carrier is 5000-8000Da. Preferably, the neurotrophic factor is nerve growth factor NGF and/or brain derived neurotrophic factor BDNF. Preferably, the preparation also contains a lyoprotectant. Preferably, in the nerve repair release preparation, the content of the lyoprotectant is 8-12wt%. Preferably, the lyoprotectant is mannitol. According to the preparation method provided by the technical scheme, the cartilage raw material is subjected to freezing treatment and thawing treatment, and then is subjected to enzymolysis by using the compound enzyme of the chondroitinase ABC and the hyaluronidase with the mass ratio of 2-4:1, so that the prepared chondroitin sulfate oligosaccharide has narrower molecular weight distribution, higher purity and no chemical rea