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US-12624128-B2 - Low molecular weight chondroitin sulfate, composition, preparation method and use thereof

US12624128B2US 12624128 B2US12624128 B2US 12624128B2US-12624128-B2

Abstract

The invention relates to a low molecular weight sulfate chondroitin and a preparation method thereof. A low molecular weight chondroitin sulfate with the average molecular weight of less than 1000 Dalton can be obtained by a production process of chondroitin sulfate lyase degradation, deproteinization, filtration and sterilization and drying using macromolecular sulfate chondroitin as a raw material. The low molecular weight Chondroitin sulfate has a narrow molecular weight distribution range, the ratio of chondroitin sulfate disaccharide is 43˜60% and the ratio of chondroitin sulfate tetrasaccharide is 30˜45%, the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%, the total oligosaccharide content of low molecular weight chondroitin sulfate is more than 97% and the protein content is less than 0.5%; Compared with the general market macromolecule chondroitin sulfate, the product has more remarkable repair effect at the concentration of 50˜100 μg/mL on chondrocytes damaged by 1 mM hydrogen peroxide, with strong repair ability and repair rate of 14%˜23%. The low molecular weight chondroitin sulfate can be used to treat joint injury and is an important raw material for medical products, health care products, cosmetics and food.

Inventors

  • Haoning Zhang
  • Song Chen
  • Bo Jin
  • Yonggang Xu
  • Chuangen TANG

Assignees

  • Nanjing Hanxin Pharmaceutical Technology Co., Ltd.

Dates

Publication Date
20260512
Application Date
20221230
Priority Date
20200930

Claims (20)

  1. 1 . A composition containing 50 mg˜800 mg of low molecular weight chondroitin sulfate, wherein the average molecular weight of the low molecular weight chondroitin sulfate is less than 1000 Dalton, comprising chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as main components, of which the content of chondroitin sulfate tetrasaccharide is about 30%˜about 45% and the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%; and wherein the general formula of the structure of the low molecular weight chondroitin sulfate is shown in the following formula I: wherein n=0˜5, and n is an integer, R 1 , R 2 , R 3 =—H or —SO 3 Na.
  2. 2 . The composition according to claim 1 , wherein the average molecular weight of the low molecular weight chondroitin sulfate is 590˜830 Da.
  3. 3 . A method for preparing the composition according to claim 1 , the method comprising: depolymerizing macromolecular chondroitin sulfate as raw material with chondroitin sulfate lyase through enzymatic depolymerization to obtain a mixture; obtaining from the mixture a low molecular weight chondroitin sulfate product with the average molecular weight stably controlled to less than 1000 Dalton; wherein the low molecular weight chondroitin sulfate comprising chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide as main components, of which the content of chondroitin sulfate tetrasaccharide is about 30%˜about 45% and the sum of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is more than 87%; and wherein the general formula of the structure of the low molecular weight chondroitin sulfate is shown in the following formula I: wherein n=0˜5, and n is an integer, R 1 , R 2 , R 3 =—H or —SO 3 Na.
  4. 4 . The method for preparing the composition according to claim 3 , wherein the chondroitin sulfate lyase is obtained by the following steps: screening and identifying the chondroitin sulfate lyase from: soil samples, sewage or silt from coastal areas, river banks, farmers' markets, slaughterhouses and dining halls, and expressing the chondroitin sulfate lyase in Escherichia coli or Bacillus subtilis.
  5. 5 . The method for preparing the composition according to claim 3 , wherein the macromolecule chondroitin sulfate as raw material is derived from the cartilaginous tissue of terrestrial and marine animals selected from one or more of chicken cartilage, pig cartilage, bovine cartilage or shark bone.
  6. 6 . The method for preparing the composition according to claim 5 , wherein the macromolecule chondroitin sulfate as raw material is derived from shark bone.
  7. 7 . The method for preparing the composition according to claim 3 , wherein the addition amount of the chondroitin sulfate lyase relative to fermentation broth per liter is about 100˜300 U/L.
  8. 8 . The method for preparing the composition according to claim 3 , wherein the concentration of the macromolecule chondroitin sulfate as raw material is about 100˜700 g/L.
  9. 9 . The method for preparing the composition according to claim 3 , wherein the time of enzymatic depolymerization is about 6˜40 h.
  10. 10 . The method for preparing the composition according to claim 3 , wherein the temperature of enzymatic depolymerization is about 25˜35° C.
  11. 11 . The method for preparing the composition according to claim 3 , wherein the pH of the enzymatic depolymerization is about 6.5˜8.5.
  12. 12 . The method for preparing the composition according to claim 3 , wherein enzymatic protein is removed from the mixture by mixed solvents after enzymolysis reaction in a reaction, in which the volume ratio of the mixture to mixed solvents is 2˜5:1, and the volume ratio of dichloromethane and isopropyl alcohol in the mixed solvents is 3˜5:1; and wherein the reaction is stirred at 100˜500 rpm for 10˜40 min, centrifuged at 3000˜5000 rpm for 10˜30 min, and the top layer of the reaction solution is taken.
  13. 13 . The method for preparing the composition according to claim 3 , wherein the enzymatic protein is removed from the mixture after enzymolysis reaction by ultrafiltration to obtain reaction solution.
  14. 14 . The method for preparing the composition according to claim 12 , wherein the upper reaction solution is filtered and sterilized through a 0.22 μm capsule filter after removing the enzymatic protein, and then the reaction solution is added into 8˜12 times volume of anhydrous ethanol for precipitation and dried in vacuum.
  15. 15 . The method for preparing the composition of according to claim 13 , wherein the reaction solution is filtered and sterilized through a 0.22 μm capsule filter after removing the enzymatic protein and then dried in spray.
  16. 16 . The composition according to claim 1 , wherein compared with the macromolecule chondroitin sulfate from shark bone, the low molecular weight chondroitin sulfate obtained by enzymatic depolymerization from one or more of shark bone and chicken cartilage has the repair rate is 14%˜23% at the concentration of 50-100 μg/mL on chondrocytes damaged by 1 mM hydrogen peroxide; or the low molecular weight chondroitin sulfate with specific content range of disaccharide and tetrasaccharide mentioned in claim 1 in the concentration range of 50˜1600 μg/mL repair chondrocytes damaged by 1 mM hydrogen peroxide, and the repair rate is 20%˜62.4%.
  17. 17 . The composition containing the low molecular weight chondroitin sulfate according to claim 1 , wherein the composition contains glucosamine.
  18. 18 . The composition containing the low molecular weight chondroitin sulfate according to claim 17 , wherein the glucosamine is glucosamine hydrochloride, glucosamine sulfate or a mixture thereof.
  19. 19 . The composition containing the low molecular weight chondroitin sulfate according to claim 1 , wherein the hydrolyzed chondroitin sulfate is a mixture of hydrolyzed chondroitin sulfate with various molecular weights.
  20. 20 . The composition containing the low molecular weight chondroitin sulfate according to claim 1 , wherein the composition comprises pharmaceutically acceptable excipients selected from fillers, disintegrants, adhesives, odorants, lubricants and film coating agents.

Description

BACKGROUND Technical Field The invention relates to the technical field of biochemistry, in particular to a low molecular weight chondroitin sulfate, composition, preparation method and use thereof. Description of Related Art Chondroitin sulfate (CS, namely macromolecular chondroitin sulfate or chondroitin sulfate polysaccharide) is a class of linear polysaccharide containing polyanion. D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) are linked by (β-1,3 glycosidic bond to form disaccharide units, which are linked to each other by (β-1,4 glycosidic bond, and sulphate groups are introduced at different positions in the subsequent biosynthesis process. Chondroitin sulfate widely exists in the cartilage and connective tissue in various animals. A large number of studies have shown that CS has the activities of lowering blood lipid, anti-atherosclerosis, enhancing immunity, anti-viral hepatitis and anti-tumor, and has been clinically widely used in the medicine and food fields for orthopedics, ophthalmology, cardiovascular diseases and oral diseases. For example, in the recommendation for the treatment of knee osteoarthritis published by the European League of Rheumatism, CS is considered to be an effective drug for the treatment of knee osteoarthritis; in Japan, CS is prepared as an oral preparation for joint pain relief, or as an eye drop for tear-filling or corneal protection; in the United States, CS is marketed as a dietary supplement; in Australia, CS is mostly prepared into composite preparations and marketed as nutritional supplements; Sodium chondroitin sulfate (CS-Na), CS-Na tablets and CS-Na capsules are also collected in the 2015 edition of the Chinese Pharmacopoeia, and they are classified as drugs for reducing blood lipid and treating bone and joint diseases. Therefore, CS, as a kind of macromolecular substance with diverse biological activities and wide application, has valuable development and utilization value. However, the traditional high molecular weight CS has high apparent viscosity, complex structure and is not easy to pass through the cell membrane. In clinical application, it is mainly faced with the problems of low bioavailability, poor oral absorption and unstable efficacy. The relative molecular weight (Mr) of natural CS is generally 50-100 kDa, and the Mr range of CS prepared by different processes and sources is generally 10-40 kDa. When the Mr is lower than 10 kDa, it is called as low molecular weight chondroitin sulfate (LMWCS) or chondroitin sulfate oligosaccharide (CSO). LMWCS is usually prepared by degradation of CS products, including acid hydrolysis, alkaline hydrolysis and enzymatic depolymerization methods. There are many impurities in the reaction products by acid degradation and they are not easy to be removed. The sulfonic groups on the chondroitin sulfate will also fall off to varying degrees during the reaction, and cause environmental pollution. In contrast, the reaction conditions of enzymatic depolymerization are mild, and thus the pollution is reduced and easilycontrolled, and enzymatic depolymerization will not cause the destruction of sulfonic acid group, which is conducive to industrial production. According to the prior art of a patent literature CN102676613B, it discloses that the hyaluronidase from bovine testicles has low specificity and low efficiency, and the mixture of chondroitin sulfate disaccharide, tetrasaccharide and hexasaccharide are obtained and then separated into three monomers with molecular weight of 521 Da, 1024 Da and 1527 Da, respectively, which are different in composition and molecular weight from low molecular weight chondroitin sulfate in the present invention. The molecular weight of disaccharide in the present process is about 379 Da and about 459 Da, and the molecular weight of tetrasaccharide is about 838 Da and about 918 Da. In addition, no pharmacodynamic studies of the obtained product have been carried out in the patent literature. The patent application CN108070627A discloses a use of chondroitin sulfate AC enzyme with a high cost to obtain chondroitin sulfate D tetrasaccharide with a specific structure and molecular weight, but its molecular weight is 1078 Da which is different from the present low molecular weight chondroitin sulfate tetrasaccharide of about 838 Da and about 918 Da. In addition, no pharmacodynamic studies of the obtained product have been carried out in the patent application. The patent CN103602711B discloses a use of enzyme liquid upon fermentation from Bacteria sulfolifolia, which efficiency is low, 1000 L fermentation liquid can only catalyze 400 kg products, the resulting chondroitin sulfate disaccharide has a molecular weight of 450-480 Da, and the content of disaccharide is more than 97%. Different from low molecular weight chondroitin sulfate in composition and molecular weight of the present invention, the molecular weight of disaccharide in the present process is about 379 Da and abo