WO2021081999A1 - Low-molecular-weight chondroitin sulfate and preparation method therefor - Google Patents
Low-molecular-weight chondroitin sulfate and preparation method therefor Download PDFInfo
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- WO2021081999A1 WO2021081999A1 PCT/CN2019/115120 CN2019115120W WO2021081999A1 WO 2021081999 A1 WO2021081999 A1 WO 2021081999A1 CN 2019115120 W CN2019115120 W CN 2019115120W WO 2021081999 A1 WO2021081999 A1 WO 2021081999A1
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- chondroitin sulfate
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- 229920001287 Chondroitin sulfate Polymers 0.000 title claims abstract description 163
- 229940059329 chondroitin sulfate Drugs 0.000 title claims abstract description 163
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 title claims abstract description 115
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- -1 chondroitin sulfate disaccharide Chemical class 0.000 claims abstract description 48
- 230000000694 effects Effects 0.000 claims abstract description 18
- 238000009826 distribution Methods 0.000 claims abstract description 16
- 239000002994 raw material Substances 0.000 claims abstract description 15
- 102000018963 Chondroitin Lyases Human genes 0.000 claims abstract description 14
- 108010026719 Chondroitin Lyases Proteins 0.000 claims abstract description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000008439 repair process Effects 0.000 claims abstract description 10
- 235000013305 food Nutrition 0.000 claims abstract description 5
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- 238000006243 chemical reaction Methods 0.000 claims description 38
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
Definitions
- the invention belongs to the technical field of biochemical industry, and specifically relates to a low-molecular-weight chondroitin sulfate and a preparation method thereof.
- Chondroitin sulfate (CS, macromolecular chondroitin sulfate or chondroitin sulfate polysaccharide) is a class of linear polysaccharides containing polyanions, composed of D-glucuronic acid (GlcA) and N-acetyl-D-amino Lactose (GalNAc) is connected by ⁇ -1,3 glycosidic bonds to form disaccharide units, and the disaccharide units are connected by ⁇ -1,4 glycosidic bonds, and sulfuric acid groups are introduced at different positions in the subsequent biosynthesis process. It is widely found in cartilage and connective tissues of various animals.
- CS cardiovascular diseases and oral diseases. field.
- CS is considered to be an effective drug for the treatment of knee osteoarthritis
- in Japan CS is made into oral preparations for joint pain relief, or as eye drops for use Tear supplementation or corneal protection
- CS is sold as a dietary supplement in the United States
- in Australia CS is mostly made into a compound preparation and sold as a nutritional health product
- the 2015 Chinese Pharmacopoeia also includes chondroitin sulfate sodium (CS-Na) and CS -Na tablets and CS-Na capsules, in the category of lowering blood lipids and the treatment of bone and joint diseases.
- CS is a kind of macromolecular substance with diverse biological activities and a wide range of applications, which has high development and utilization value.
- traditional high-molecular-weight CS has high apparent viscosity, complex structure, and it is difficult to pass through cell membranes. In clinical applications, it mainly faces the problems of low bioavailability, poor oral absorption and unstable efficacy.
- the relative molecular mass (Mr) of natural CS is generally in the range of 50-100kDa, and the Mr range of CS produced by different processes and different sources is generally in the range of 10-40kDa. When it is lower than 10kDa, it is called low molecular weight chondroitin sulfate (Low Molecular weight chondroitin sulfate, LMWCS) or chondroitin sulfate oligosaccharide.
- LMWCS Low Molecular weight chondroitin sulfate
- LMWCS is generally prepared by degradation of CS products, mainly including acid hydrolysis, alkaline hydrolysis and enzymatic methods.
- the acid degradation reaction product contains many impurities and is difficult to remove.
- the sulfonic acid groups on the chondroitin sulfate will also fall off to varying degrees, causing environmental pollution.
- enzymatic hydrolysis has mild reaction conditions and less pollution. Manipulation will not cause damage to sulfonic acid groups, which is conducive to industrial production.
- Prior art patent document CN102676613B discloses that the use of hyaluronidase from bovine testis is not highly specific and has low efficiency.
- the cleavage yields a mixture of chondroitin sulfate disaccharides, tetrasaccharides, and hexasaccharides, and separates and prepares three monomers, but Its molecular weights are 521, 1024, 1527, which are different from our low molecular weight chondroitin sulfate in composition and molecular weight.
- the molecular weight of disaccharide is about 379 and 459
- the molecular weight of tetrasaccharide is about 838 and 918.
- the literature has not carried out the pharmacodynamic research of the obtained product.
- Patent CN108070627A discloses the use of chondroitin sulfate AC enzyme, which is costly, and obtains chondroitin sulfate D tetrasaccharide with a specific structure and molecular weight, but its molecular weight is 1078, which is different from our low molecular weight chondroitin sulfate in composition and molecular weight.
- the molecular weight of the tetrasaccharide in our process is about 838 and 918, and the pharmacodynamic research of the obtained product has not been carried out in this document.
- Patent CN103602711B discloses the use of enzyme solution from Arthrobacter sulfoneum after fermentation. The efficiency is low. 1000L of fermentation broth can only catalyze 400kg of product.
- the chondroitin sulfate disaccharide obtained by lysis has a molecular weight of 450-480 and a disaccharide content of 97%. The above is different from our low-molecular-weight chondroitin sulfate in composition and molecular weight.
- the molecular weight of disaccharides in our process is about 379 and 459, and the disaccharide content accounts for 42-58%, and the document discloses that the raw material of the product is Chicken cartilage, porcine cartilage and bovine cartilage are specifically used to treat myocarditis.
- the m/Z of 342 and 458 are disaccharides, m/
- the tetrasaccharide with Z of 939 is different from our low molecular weight chondroitin sulfate in composition and molecular weight.
- the molecular weight of disaccharide is about 379 and 459 and the molecular weight of tetrasaccharide is about 838 and 918 in our process.
- the product has only been tested for antioxidant activity, and no other pharmacodynamic studies have been carried out.
- the purpose of the present invention is to overcome the above-mentioned shortcomings in the prior art and provide a new low-molecular-weight chondroitin sulfate and a preparation method thereof.
- a low molecular weight chondroitin sulfate the average molecular weight of the low molecular weight chondroitin sulfate is less than 1000 Daltons, and its molecular weight distribution range is narrow. It is mainly composed of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide. The content of chondroitin sulfate disaccharide is 43 to 60%, and the content of chondroitin sulfate tetrasaccharide is 30 to 45%. The total content of chondroitin sulfate tetrasaccharide is greater than 87%; the general structural formula of the low molecular weight chondroitin sulfate is shown in the following formula I:
- the average molecular weight of the low molecular weight chondroitin sulfate is 590-830 Da, more preferably 677-742 Da.
- chondroitin sulfate disaccharide in the low molecular weight chondroitin sulfate accounts for 48-55%
- the content of chondroitin sulfate tetrasaccharide accounts for 35-40%.
- the second objective of the present invention is to provide the following technical solution: a method for preparing low molecular weight chondroitin sulfate, which includes: enzymatically digesting macromolecular chondroitin sulfate raw materials with chondroitin sulfate lyase to obtain an average molecular weight that can be stably controlled at Low molecular weight chondroitin sulfate products below 1000 Daltons have a narrow molecular weight distribution range.
- the products are mainly chondroitin sulfate disaccharides and chondroitin sulfate tetrasaccharides, and the content of chondroitin sulfate disaccharides accounts for 43-60% ,
- the content of chondroitin sulfate tetrasaccharide is 30-45%, and the sum of the content of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is greater than 87%;
- the general structural formula of the low molecular weight chondroitin sulfate is shown in the following formula I:
- chondroitin sulfate lyase is screened and identified from soil samples, sewage or sludge at coasts, rivers, farmers' markets, slaughterhouses and canteens, and is obtained by optimized expression using Escherichia coli or Bacillus subtilis.
- the technical method involved is to use commercially available common macromolecular chondroitin sulfate as the production raw material, derived from cartilage tissue of terrestrial and marine animals, and the raw material further refers to chicken cartilage, pig cartilage, bovine cartilage or shark bone One or a mixture of several, and more preferably shark bone.
- the operating conditions of the enzymatic hydrolysis reaction are that the added amount of the chondroitin sulfate lyase per liter of fermentation broth is 100-300 U/L, the concentration of the macromolecular chondroitin sulfate raw material is 100-700 g/L, and the enzymatic hydrolysis The time is 6-10h, the enzymatic hydrolysis temperature is 25-35°C, the stirring speed is 100-700rpm, and the enzymatic hydrolysis pH is 6.5-8.5.
- the hydrolyzed liquid after the enzymolysis reaction is deproteinized by using a mixed solvent
- the volume ratio of the hydrolyzed liquid to the mixed solvent is 2 to 5:1
- the volume ratio of dichloromethane and isopropanol in the mixed solvent is 3 to 3 5:1 stir at 100-500rpm for 10-40min, centrifuge at 3000-5000rpm for 10-30min, and take the upper reaction solution.
- hydrolyzed liquid after the enzymatic hydrolysis reaction can also be subjected to ultrafiltration to remove protein to obtain a reaction liquid.
- reaction solution after deproteinization is filtered and sterilized by a 0.22um capsule filter, the reaction solution is added to 8-12 times the volume of absolute ethanol, precipitated with alcohol, and dried in vacuum.
- reaction solution after the protein is removed by means of ultrafiltration is spray-dried after being filtered and sterilized by a 0.22um capsule filter.
- the low-molecular-weight chondroitin sulfate obtained by enzymatic hydrolysis of one of shark bone and chicken cartilage or its mixed bone is relative to the macromolecular chondroitin sulfate of shark bone, at a concentration of 50-100 ⁇ g/mL, to the cartilage damaged by 1 mM hydrogen peroxide.
- Cells have a more obvious repair effect, and the repair rate is between 14% and 23%.
- the low-molecular-weight chondroitin sulfate has applications in the fields of preparing medicines, cosmetics, health products, and foods.
- the present invention has the following advantages:
- the ratio of oligosaccharides of different components is clear: the product quality is stable, and the total content of low molecular weight chondroitin sulfate oligosaccharides including chondroitin sulfate disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 97%
- the products are mainly chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide, the content of chondroitin sulfate disaccharide is 43-60%, the content of chondroitin sulfate tetrasaccharide is 30-45%, and the content of chondroitin sulfate tetrasaccharide is 30-45%.
- the sum of the content of disaccharides and chondroitin sulfate tetrasaccharides is more than 87%.
- the low molecular weight chondroitin sulfate obtained by enzymatic hydrolysis of one of shark bone and chicken cartilage or its mixed bone is relative to the macromolecular chondroitin sulfate of shark bone, at a concentration of 50-100 ⁇ g/mL, it can damage the cartilage damaged by 1mM hydrogen peroxide.
- the cells have a more obvious repairing effect, strong repairing ability, and the repairing rate is between 14% and 23%, which can be used to treat joint injuries.
- shark-derived low-molecular-weight chondroitin sulfate has a better repair effect than chicken cartilage, porcine cartilage, bovine cartilage, and mixed bone-derived low-molecular-weight chondroitin sulfate.
- the low-molecular-weight chondroitin sulfate derived from shark bone, chicken cartilage, porcine cartilage, bovine cartilage and mixed bone-derived enzymatic hydrolysis is better than shark bone-derived macromolecular chondroitin sulfate, and has better effects on cartilage damaged by 1mM hydrogen peroxide. Cells have a more obvious repair effect.
- Fig. 1 is the distribution spectrum of oligosaccharides of low molecular weight chondroitin sulfate derived from shark bone in Example 9.
- Fig. 2 is a distribution spectrum of oligosaccharides of low molecular weight chondroitin sulfate derived from shark bone in Example 10.
- Fig. 3 is a distribution spectrum of oligosaccharides of low-molecular-weight chondroitin sulfate derived from shark bone in Example 11.
- Fig. 4 is a distribution spectrum of oligosaccharides of low molecular weight chondroitin sulfate derived from shark bone in Example 12.
- Figure 5 is a graph showing the efficacy and activity detection results of low molecular weight chondroitin sulfate from different sources in Example 14.
- the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
- the chondroitin sulfate lyase is selected and identified from the soil samples, sewage or sludge at the coast, riverside, farmer’s market, slaughterhouse, and canteen preserved in our laboratory, and optimized and expressed by E. coli or Bacillus subtilis.
- the highest enzyme activity can reach 11976.5U/L, contains 998 amino acids, and has a molecular weight of 113KDa.
- the amino acid sequence of this chondroitin sulfate lyase is disclosed in another invention patent filed by our company. Its application date: April 2019 On the 3rd, application number: 201910264385.5, publication date: June 21, 2019, publication number: CN109913437A, all relevant contents of this patent application are incorporated into this patent application.
- r t r U1 +r U2 +r U3 +r U4 +r U5 .
- ru1 the peak response value of component one (hexasaccharide and octasaccharide) in the sample solution
- M W1 is the molecular weight of component one in the sample solution
- r u2 peak response value of component two (tetrasaccharide) in the sample solution
- M W2 is the molecular weight of component two in the sample solution
- r u3 peak response value of component three (tetrasaccharide) in the sample solution
- M W3 is the molecular weight of component three in the sample solution
- r u4 peak response value of component four (disaccharide) in the sample solution
- M W4 is the molecular weight of component four in the sample solution
- r u5 peak response value of component five (disaccharide) in the sample solution
- M W5 is the molecular weight of component five in the sample solution
- r t The sum of the peak response values of component 1, component 2, component 3, component 4, and component 5 in the sample solution.
- chondroitin sulfate obtained by enzymatic hydrolysis of large molecular chondroitin sulfate derived from shark bone
- the component with peak time of 5.879min is mainly The composition of hexasaccharide and octasaccharide, the content is 9.51%; the peak time is 8.632min, the second component is tetrasaccharide, the content is 33.83%; the peak time is 8.966min, the component three is also the tetrasaccharide, the content is 5.07 %; component four with a peak time of 9.846min is a disaccharide with a content of 47.08%; component five with a peak time of 10.786min is also a disaccharide with a content of 2.03%; the component with a peak time of 12.506min Six is a salt peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 97.52%, mainly disaccharides and tetrasaccharides, and the
- the peak time of 5.878min component one is six Sugars and octasaccharides, the content is 9.50%; the second component with a peak time of 8.625min is tetrasaccharides with a content of 33.90%; the component three with a peak time of 8.958min also contains tetrasaccharides with a content of 5.08%;
- the component four with a peak time of 9.838min is a disaccharide with a content of 46.86%; the component five with a peak time of 10.785min is also a disaccharide with a content of 2.13%; the component six with a peak time of 12.505min is a salt Peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 97.47%, mainly disaccharides and tetrasaccharides, of which
- the peak time of 5.879min component one is six
- the content of sugar and octasaccharide is 8.62%
- the second component with a peak time of 8.632min is tetrasaccharide, the content is 30.79%
- the component three with a peak time of 8.966min is also a tetrasaccharide, the content is 4.41%
- the component four with a peak time of 9.872min is a disaccharide with a content of 52.49%
- the component five with a peak time of 10.786min is also a disaccharide with a content of 2.02%
- the component six with a peak time of 12.512min is a salt Peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 98.32%, mainly disaccharides and tetrasaccharides, of
- the first component with a peak time of 5.876 min is hexasaccharides and octasaccharides, and the content is 8.50%; the second component with a peak time of 8.636 min is tetrasaccharides, and the content is 30.59%; component three with a peak time of 8.963min is also a tetrasaccharide with a content of 4.43%; a component four with a peak time of 9.870min is a disaccharide with a content of 52.69%; the group with a peak time of 10.783min Fraction five is also disaccharide with a content of 2.14%; component six with peak time of 12.510min is salt peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 98.35% , Mainly disaccharides and tetrasaccharides
- Example 9 Using bovine cartilage, porcine cartilage, chicken cartilage, and a mixture of chicken cartilage and shark bone, respectively, according to the enzymatic hydrolysis reaction of Example 1, the protein removal process of Example 5, and the alcohol precipitation drying process of Example 9 were obtained. Low molecular weight chondroitin sulfate from different sources.
- the CCK method for cell viability determination was used to investigate the repair effects of low molecular weight chondroitin sulfate and macromolecular chondroitin sulfate from different sources on damaged chondrocytes.
- the results of the efficacy and activity test are shown in Figure 5, and the results show that it is made from shark bone or chicken.
- the low-molecular-weight chondroitin sulfate obtained by enzymatic hydrolysis of one kind of bone or its mixed bone is relative to the macromolecular chondroitin sulfate (chondroitin sulfate polysaccharide) derived from shark bone, which can damage chondrocytes damaged by 1mM hydrogen peroxide at a concentration of 50-100 ⁇ g/mL Both have a more obvious repairing effect, the repairing ability is between 14% and 23%, and it can be used to treat joint injuries.
- shark-derived low-molecular-weight chondroitin sulfate has a better repair effect than chicken cartilage, porcine cartilage, bovine cartilage, and mixed bone-derived low-molecular-weight chondroitin sulfate.
- the low-molecular-weight chondroitin sulfate derived from shark bone, chicken cartilage, porcine cartilage, bovine cartilage and mixed bone-derived enzymatic hydrolysis is better than shark bone-derived macromolecular chondroitin sulfate, and has better effects on cartilage damaged by 1mM hydrogen peroxide. Cells have a more obvious repair effect.
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Abstract
Disclosed are a low-molecular-weight chondroitin sulfate and a preparation method therefor. Macromolecular chondroitin sulfate is used as a raw material. A low-molecular-weight chondroitin sulfate having an average molecular weight of less than 1000 daltons can be obtained by means of a production process comprising chondroitin sulfate lyase degradation, deproteinization, filtration sterilization, drying, etc. With respect to the low-molecular-weight chondroitin sulfate, the molecular weight distribution range is narrow, the proportion of chondroitin sulfate disaccharide is 43％ to 60％, the proportion of chondroitin sulfate tetrasaccharide is 30％ to 45％, the sum of the contents of the chondroitin sulfate disaccharide and the chondroitin sulfate tetrasaccharide is greater than 87％, the total content of oligosaccharides in the low-molecular-weight chondroitin sulfate is not less than 97％, and the protein content is not more than 0.5％. Compared with common marketed macromolecular chondroitin sulfate, the product has, at a concentration of 50 μg/mL to 100 μg/mL, a more obvious repairing effect on chondrocytes damaged by 1 mM hydrogen peroxide, has a strong repairing ability and a repair rate between 14％ and 23％, and can be used to treat joint injuries, and same is thus an important raw material for medical products, healthcare products, cosmetics, food product, etc.
Description
本发明属于生物化工技术领域，具体涉及一种低分子量硫酸软骨素及其制备方法。The invention belongs to the technical field of biochemical industry, and specifically relates to a low-molecular-weight chondroitin sulfate and a preparation method thereof.
硫酸软骨素(Chondroitin sulfate，CS，即大分子硫酸软骨素或硫酸软骨素多糖)是一类含有聚阴离子的线性多糖，由D-葡糖醛酸(GlcA)和N-乙酰-D-氨基半乳糖(GalNAc)以β-1,3糖苷键连接形成二糖单位，二糖单位之间以β-1,4糖苷键连接，并在后续的生物合成过程中在不同的位置引入硫酸基，其广泛存在于各种动物的软骨和结缔组织中。Chondroitin sulfate (CS, macromolecular chondroitin sulfate or chondroitin sulfate polysaccharide) is a class of linear polysaccharides containing polyanions, composed of D-glucuronic acid (GlcA) and N-acetyl-D-amino Lactose (GalNAc) is connected by β-1,3 glycosidic bonds to form disaccharide units, and the disaccharide units are connected by β-1,4 glycosidic bonds, and sulfuric acid groups are introduced at different positions in the subsequent biosynthesis process. It is widely found in cartilage and connective tissues of various animals.
大量研究表明，CS具有降血脂、抗动脉粥样硬化、增强免疫力、抗病毒性肝炎和抗肿瘤等活性，临床中已广泛应用于骨科、眼科、心血管疾病及口腔疾病等医药领域和食品领域。如欧洲风湿病联盟发表的治疗膝骨关节炎的建议中认为CS是一种治疗膝骨关节炎的有效药物；在日本，CS制成口服制剂用于关节止痛，或制成滴眼剂用于泪液补充或角膜保护；美国将CS作为膳食补充剂销售；在澳大利亚，CS大都被制成复合制剂，作为营养保健品销售；2015年版中国药典亦收录了硫酸软骨素钠(CS-Na)、CS-Na片及CS-Na胶囊，类别为降血脂及骨关节疾病治疗药。由此可见，CS是一类生物活性多样、应用广泛的大分子物质，具有很高的开发利用价值。但传统高分子量的CS表观粘度高、结构复杂，不易通过细胞膜，临床应用中主要面临生物利用率低、口服吸收差及疗效不稳定的问题。A large number of studies have shown that CS has the activities of lowering blood lipids, anti-atherosclerosis, enhancing immunity, anti-viral hepatitis and anti-tumor. It has been widely used in clinical medicine and food such as orthopedics, ophthalmology, cardiovascular diseases and oral diseases. field. For example, in the recommendations for the treatment of knee osteoarthritis published by the European Rheumatism Federation, CS is considered to be an effective drug for the treatment of knee osteoarthritis; in Japan, CS is made into oral preparations for joint pain relief, or as eye drops for use Tear supplementation or corneal protection; CS is sold as a dietary supplement in the United States; in Australia, CS is mostly made into a compound preparation and sold as a nutritional health product; the 2015 Chinese Pharmacopoeia also includes chondroitin sulfate sodium (CS-Na) and CS -Na tablets and CS-Na capsules, in the category of lowering blood lipids and the treatment of bone and joint diseases. It can be seen that CS is a kind of macromolecular substance with diverse biological activities and a wide range of applications, which has high development and utilization value. However, traditional high-molecular-weight CS has high apparent viscosity, complex structure, and it is difficult to pass through cell membranes. In clinical applications, it mainly faces the problems of low bioavailability, poor oral absorption and unstable efficacy.
天然CS的相对分子质量(Mr)范围一般为50～100kDa，采用不同工艺和不同来源生产得到的CS的Mr范围一般在10～40kDa，低于10kDa时，则称为低分子量硫酸软骨素(Low molecular weight chondroitin sulfate，LMWCS)或称为硫酸软骨素寡糖。The relative molecular mass (Mr) of natural CS is generally in the range of 50-100kDa, and the Mr range of CS produced by different processes and different sources is generally in the range of 10-40kDa. When it is lower than 10kDa, it is called low molecular weight chondroitin sulfate (Low Molecular weight chondroitin sulfate, LMWCS) or chondroitin sulfate oligosaccharide.
LMWCS一般通过CS产品降解来制备，主要有酸水解法、碱水解法和酶法。酸降解反应产物中杂质较多且不易除去，反应过程中硫酸软骨素上的磺酸基团也会不同程度的脱落，且造成环境污染，相比之下，酶解法反应条件温和，污染小易操控，也不会造成磺酸基团的破坏，有利于工业化生产。LMWCS is generally prepared by degradation of CS products, mainly including acid hydrolysis, alkaline hydrolysis and enzymatic methods. The acid degradation reaction product contains many impurities and is difficult to remove. During the reaction, the sulfonic acid groups on the chondroitin sulfate will also fall off to varying degrees, causing environmental pollution. In contrast, enzymatic hydrolysis has mild reaction conditions and less pollution. Manipulation will not cause damage to sulfonic acid groups, which is conducive to industrial production.
现有技术专利文献CN102676613B公开了使用来自牛睾丸的透明质酸酶特异性不高，效率较低，裂解得到了硫酸软骨素二糖、四糖、六糖混合物并分离制备三个单体，但其分子量分别为521、1024、1527，与我们的低分子量硫酸软骨素构成及分子量均有所区别，如我们工艺中二糖分子量约为379、459和四糖分子量约为838、918，并且该文献未开展所得产物的药效学研究。Prior art patent document CN102676613B discloses that the use of hyaluronidase from bovine testis is not highly specific and has low efficiency. The cleavage yields a mixture of chondroitin sulfate disaccharides, tetrasaccharides, and hexasaccharides, and separates and prepares three monomers, but Its molecular weights are 521, 1024, 1527, which are different from our low molecular weight chondroitin sulfate in composition and molecular weight. For example, in our process, the molecular weight of disaccharide is about 379 and 459, and the molecular weight of tetrasaccharide is about 838 and 918. The literature has not carried out the pharmacodynamic research of the obtained product.
专利CN108070627A公开了使用硫酸软骨素AC酶，成本较高，得到了特定结构和分子量的硫酸软骨素D四糖，但其分子量为1078，与我们的低分子量硫酸软骨素构成及分子量均有所区别，我们工艺中四糖的分子量约为838、918，并且该文献未开展所得产物的药效学研究。Patent CN108070627A discloses the use of chondroitin sulfate AC enzyme, which is costly, and obtains chondroitin sulfate D tetrasaccharide with a specific structure and molecular weight, but its molecular weight is 1078, which is different from our low molecular weight chondroitin sulfate in composition and molecular weight. , The molecular weight of the tetrasaccharide in our process is about 838 and 918, and the pharmacodynamic research of the obtained product has not been carried out in this document.
专利CN103602711B公开了使用来自食砜节杆菌发酵后的酶液，效率较低，1000L发酵液只能催化400kg产品，裂解得到的硫酸软骨素二糖，其分子量450～480，二糖含量在97％以上，与我们的低分子量硫酸软骨素构成及分子量均有所区别，如我们工艺中二糖分子量约为379、459且二糖含量占比为42～58％，并且该文献公开所得产品原料是鸡软骨、猪软骨与牛软骨，而且是特定用于治疗心肌炎。Patent CN103602711B discloses the use of enzyme solution from Arthrobacter sulfoneum after fermentation. The efficiency is low. 1000L of fermentation broth can only catalyze 400kg of product. The chondroitin sulfate disaccharide obtained by lysis has a molecular weight of 450-480 and a disaccharide content of 97%. The above is different from our low-molecular-weight chondroitin sulfate in composition and molecular weight. For example, the molecular weight of disaccharides in our process is about 379 and 459, and the disaccharide content accounts for 42-58%, and the document discloses that the raw material of the product is Chicken cartilage, porcine cartilage and bovine cartilage are specifically used to treat myocarditis.
期刊文献《酶法制备硫酸软骨素寡糖及其抗氧化活性》食品工业科技，2017,13,48～52.公开了使用来自产酶菌株Acinetobacter sp.C26发酵后的硫酸软骨素酶(分子量76kDa)，催化效率低，报道的反应浓度仅为2％，寡糖的比例并未进行分析；裂解得到了硫酸软骨素二糖、四糖，m/Z为342和458的是二糖，m/Z为939的是四糖，与我们的低分子量硫酸软骨素构成及分子量均有所区别，如我们工艺中二糖分子量约为379、459和四糖分子量约为838、918，并且该文献所得产品只进行了抗氧化活性实验，未开展其它药效学研究。The journal document "Enzymatic Preparation of Chondroitin Sulfate Oligosaccharide and Its Antioxidant Activity" Food Industry Science and Technology, 2017, 13, 48-52. It discloses the use of chondroitin sulfate (molecular weight 76kDa) fermented from the enzyme-producing strain Acinetobacter sp.C26 ), the catalytic efficiency is low, the reported reaction concentration is only 2%, and the proportion of oligosaccharides has not been analyzed; chondroitin sulfate disaccharide and tetrasaccharide are obtained by cleavage. The m/Z of 342 and 458 are disaccharides, m/ The tetrasaccharide with Z of 939 is different from our low molecular weight chondroitin sulfate in composition and molecular weight. For example, the molecular weight of disaccharide is about 379 and 459 and the molecular weight of tetrasaccharide is about 838 and 918 in our process. The product has only been tested for antioxidant activity, and no other pharmacodynamic studies have been carried out.
因此国内外对低分子量硫酸软骨素药效学方面的研究不太充分，特别是对主成分为硫酸软骨素二糖和硫酸软骨素四糖，且二糖和四糖含量控制在一定范围内，平均分子量可稳定地控制在低于1000道尔顿且其分子量分布范围窄的，用于治疗关节损伤的低分子量硫酸软骨素有待进一步研究和确认，从而填补国内外该领域的研究空白。Therefore, research on the pharmacodynamics of low molecular weight chondroitin sulfate at home and abroad is not sufficient, especially for the main components of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide, and the content of disaccharide and tetrasaccharide is controlled within a certain range. The average molecular weight can be stably controlled below 1000 Daltons and its molecular weight distribution range is narrow. The low molecular weight chondroitin sulfate for the treatment of joint damage needs further research and confirmation, so as to fill the research gap in this field at home and abroad.
发明内容Summary of the invention
本发明的目的在于克服现有技术中存在的上述不足之处，提供了一种新的低分子量硫酸软骨素及其制备方法。The purpose of the present invention is to overcome the above-mentioned shortcomings in the prior art and provide a new low-molecular-weight chondroitin sulfate and a preparation method thereof.
为了实现上述目的，本发明的目的之一是提供了如下的技术方案：一种低分子量硫酸软骨素：所述低分子量硫酸软骨素的平均分子量低于1000道尔顿，其分子量分布范围窄，主要以硫酸软骨素二糖和硫酸软骨素四糖为主，硫酸软骨素二糖含量占比为43～60％，硫酸软骨素四糖含量占比为30～45％，硫酸软骨素二糖和硫酸软骨素四糖含量之和大于87％；所述低分子量硫酸软骨素的结构通式如下式I所示：In order to achieve the above objective, one of the objectives of the present invention is to provide the following technical solutions: a low molecular weight chondroitin sulfate: the average molecular weight of the low molecular weight chondroitin sulfate is less than 1000 Daltons, and its molecular weight distribution range is narrow. It is mainly composed of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide. The content of chondroitin sulfate disaccharide is 43 to 60%, and the content of chondroitin sulfate tetrasaccharide is 30 to 45%. The total content of chondroitin sulfate tetrasaccharide is greater than 87%; the general structural formula of the low molecular weight chondroitin sulfate is shown in the following formula I:
式I：n＝0～5；R
1,R
2,R
3＝-H或-SO
3Na。
Formula I: n=0 to 5; R 1 , R 2 , R 3 =-H or -SO 3 Na.
进一步的，所述低分子量硫酸软骨素的平均分子量为590～830Da，进一步优选为677～742Da。Further, the average molecular weight of the low molecular weight chondroitin sulfate is 590-830 Da, more preferably 677-742 Da.
进一步的，所述低分子量硫酸软骨素中硫酸软骨素二糖含量占比为48～55％，硫酸软骨素四糖含量占比为35～40％。Further, the content of chondroitin sulfate disaccharide in the low molecular weight chondroitin sulfate accounts for 48-55%, and the content of chondroitin sulfate tetrasaccharide accounts for 35-40%.
本发明的目的之二是提供了如下技术方案：一种低分子量硫酸软骨素的制备方法，包括：将大分子硫酸软骨素原料经过硫酸软骨素裂解酶的酶解得到平均分子量可稳定地控制在低于1000道尔顿的低分子量硫酸软骨素产物，其分子量分布范围窄，产物主要以硫酸软骨素二糖和硫酸软骨素四糖为主，硫酸软骨素二糖含量占比为43～60％，硫酸软骨素四糖含量占比为30～45％，硫酸软骨素二糖和硫酸软骨素四糖含量之和大于87％；所述低分子量硫酸软骨素的结构通式如下式I所示：The second objective of the present invention is to provide the following technical solution: a method for preparing low molecular weight chondroitin sulfate, which includes: enzymatically digesting macromolecular chondroitin sulfate raw materials with chondroitin sulfate lyase to obtain an average molecular weight that can be stably controlled at Low molecular weight chondroitin sulfate products below 1000 Daltons have a narrow molecular weight distribution range. The products are mainly chondroitin sulfate disaccharides and chondroitin sulfate tetrasaccharides, and the content of chondroitin sulfate disaccharides accounts for 43-60% , The content of chondroitin sulfate tetrasaccharide is 30-45%, and the sum of the content of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is greater than 87%; the general structural formula of the low molecular weight chondroitin sulfate is shown in the following formula I:
式I：n＝0～5；R
1,R
2,R
3＝-H或-SO
3Na。
Formula I: n=0 to 5; R 1 , R 2 , R 3 =-H or -SO 3 Na.
进一步的，所述硫酸软骨素裂解酶从海岸边、河边、农贸市场、屠宰厂和食堂处的土样、污水或淤泥中筛选、鉴定，并用大肠杆菌或枯草芽孢杆菌优化表达得到。Further, the chondroitin sulfate lyase is screened and identified from soil samples, sewage or sludge at coasts, rivers, farmers' markets, slaughterhouses and canteens, and is obtained by optimized expression using Escherichia coli or Bacillus subtilis.
进一步的，所涉及的技术方法是利用市售普通大分子硫酸软骨素为生产原料，来源于陆生和海洋动物的软骨组织，所述原料进一步指鸡软骨，猪软骨，牛软骨或鲨鱼骨中的一种或几种混合，更进一步优选为鲨鱼骨。Further, the technical method involved is to use commercially available common macromolecular chondroitin sulfate as the production raw material, derived from cartilage tissue of terrestrial and marine animals, and the raw material further refers to chicken cartilage, pig cartilage, bovine cartilage or shark bone One or a mixture of several, and more preferably shark bone.
进一步的，酶解反应的操作条件为，所述硫酸软骨素裂解酶相对每升发酵液的添加量为100～300U/L，大分子硫酸软骨素原料的浓度为100～700g/L，酶解时间为6～10h，酶解温度25～35℃，搅拌转速为100～700rpm，酶解pH为6.5～8.5。Further, the operating conditions of the enzymatic hydrolysis reaction are that the added amount of the chondroitin sulfate lyase per liter of fermentation broth is 100-300 U/L, the concentration of the macromolecular chondroitin sulfate raw material is 100-700 g/L, and the enzymatic hydrolysis The time is 6-10h, the enzymatic hydrolysis temperature is 25-35°C, the stirring speed is 100-700rpm, and the enzymatic hydrolysis pH is 6.5-8.5.
进一步的，所述酶解反应后的水解液利用混合溶剂脱除蛋白，水解液与混合溶剂的体积比为2～5:1，混合溶剂中二氯甲烷和异丙醇的体积比为3～5:1，100～500rpm搅拌10～40min，3000～5000rpm离心10～30min，取上层反应液。Further, the hydrolyzed liquid after the enzymolysis reaction is deproteinized by using a mixed solvent, the volume ratio of the hydrolyzed liquid to the mixed solvent is 2 to 5:1, and the volume ratio of dichloromethane and isopropanol in the mixed solvent is 3 to 3 5:1, stir at 100-500rpm for 10-40min, centrifuge at 3000-5000rpm for 10-30min, and take the upper reaction solution.
进一步的，所述酶解反应后的水解液还可以通过超滤的方式脱除蛋白得到反应液。Further, the hydrolyzed liquid after the enzymatic hydrolysis reaction can also be subjected to ultrafiltration to remove protein to obtain a reaction liquid.
进一步的，所述脱除蛋白后的上层反应液，经过0.22um囊式滤芯过滤除菌，将反应液加入到8～12倍体积量的无水乙醇中，醇沉，真空干燥。Further, the upper layer reaction solution after deproteinization is filtered and sterilized by a 0.22um capsule filter, the reaction solution is added to 8-12 times the volume of absolute ethanol, precipitated with alcohol, and dried in vacuum.
进一步的，所述通过超滤的方式脱除蛋白后的反应液，经过0.22um囊式滤芯过滤除菌后进行喷雾干燥。Further, the reaction solution after the protein is removed by means of ultrafiltration is spray-dried after being filtered and sterilized by a 0.22um capsule filter.
进一步的，由鲨鱼骨和鸡软骨中的一种或其混合骨酶解得到的低分子量硫酸软骨素相对鲨鱼骨的大分子硫酸软骨素，在50～100μg/mL浓度内对1mM双氧水损伤的软骨细胞有更明显的修复作用，修复率在14％～23％之间。Further, the low-molecular-weight chondroitin sulfate obtained by enzymatic hydrolysis of one of shark bone and chicken cartilage or its mixed bone is relative to the macromolecular chondroitin sulfate of shark bone, at a concentration of 50-100 μg/mL, to the cartilage damaged by 1 mM hydrogen peroxide. Cells have a more obvious repair effect, and the repair rate is between 14% and 23%.
进一步的，所述低分子量硫酸软骨素具有在制备医药品、化妆品、保健品和食品等领域方面的应用。Further, the low-molecular-weight chondroitin sulfate has applications in the fields of preparing medicines, cosmetics, health products, and foods.
本发明相对现有技术，具有以下优势：Compared with the prior art, the present invention has the following advantages:
1、利用从海岸边、河边、农贸市场、屠宰厂和食堂处的土样、污水或淤泥中筛选、鉴定，并用大肠杆菌或枯草芽孢杆菌优化表达得到的硫酸软骨素裂解酶催化法酶解，特异性好，酶活更高；可稳定地得到平均分子量小于1000道尔顿的低分子量硫酸软骨素，尤其是平均分子量为590～830Da的低分子量硫酸软骨素，分子量分布范围窄。1. Use chondroitin sulfate lyase catalyzed enzymatic hydrolysis from soil samples, sewage or sludge from coasts, rivers, farmer's markets, slaughterhouses and canteens, and optimized expression by Escherichia coli or Bacillus subtilis. , Good specificity, higher enzyme activity; Low molecular weight chondroitin sulfate with an average molecular weight of less than 1000 Daltons can be stably obtained, especially low molecular weight chondroitin sulfate with an average molecular weight of 590 to 830 Da, with a narrow molecular weight distribution range.
2、利用溶剂法或超滤方法脱除蛋白，蛋白的含量不超过0.5％。2. Use solvent method or ultrafiltration method to remove protein, and the protein content should not exceed 0.5%.
3、100L发酵液催化超过400kg大分子硫酸软骨素，生产周期短，效率高，适于工业化放大使用。3. 100L fermentation broth catalyzes over 400kg of macromolecular chondroitin sulfate, with short production cycle and high efficiency, which is suitable for industrialized amplification.
4、通过LC-MS分析明确了不同组分寡糖的比例：产品质量稳定，包括硫酸软骨素二糖、四糖、六糖和八糖的低分子量硫酸软骨素的寡糖总含量在97％以上，其中产物主要以硫酸软骨素二糖和硫酸软骨素四糖为主，硫酸软骨素二糖含量占比为43～60％，硫酸软骨素四糖含量占比为30～45％，硫酸软骨素二糖和硫酸软骨素四糖含量之和在87％以上。4. Through LC-MS analysis, the ratio of oligosaccharides of different components is clear: the product quality is stable, and the total content of low molecular weight chondroitin sulfate oligosaccharides including chondroitin sulfate disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 97% Above, the products are mainly chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide, the content of chondroitin sulfate disaccharide is 43-60%, the content of chondroitin sulfate tetrasaccharide is 30-45%, and the content of chondroitin sulfate tetrasaccharide is 30-45%. The sum of the content of disaccharides and chondroitin sulfate tetrasaccharides is more than 87%.
5、由鲨鱼骨和鸡软骨中的一种或其混合骨酶解得到的低分子量硫酸软骨素相对鲨鱼骨的大分子硫酸软骨素，在50～100μg/mL浓度内，对1mM双氧水损伤的软骨细胞均 有更明显的修复作用，修复能力强，修复率在14％～23％之间，可用于治疗关节损伤。其中鲨鱼源的低分子量硫酸软骨素修复效果优于鸡软骨、猪软骨、牛软骨和混骨来源的低分子量硫酸软骨素。在50μg/mL浓度时，由鲨鱼骨、鸡软骨、猪软骨、牛软骨和混骨来源酶解得到的低分子量硫酸软骨素均比鲨鱼骨来源的大分子硫酸软骨素，对1mM双氧水损伤的软骨细胞有更明显的修复作用。5. The low molecular weight chondroitin sulfate obtained by enzymatic hydrolysis of one of shark bone and chicken cartilage or its mixed bone is relative to the macromolecular chondroitin sulfate of shark bone, at a concentration of 50-100μg/mL, it can damage the cartilage damaged by 1mM hydrogen peroxide. The cells have a more obvious repairing effect, strong repairing ability, and the repairing rate is between 14% and 23%, which can be used to treat joint injuries. Among them, shark-derived low-molecular-weight chondroitin sulfate has a better repair effect than chicken cartilage, porcine cartilage, bovine cartilage, and mixed bone-derived low-molecular-weight chondroitin sulfate. At a concentration of 50μg/mL, the low-molecular-weight chondroitin sulfate derived from shark bone, chicken cartilage, porcine cartilage, bovine cartilage and mixed bone-derived enzymatic hydrolysis is better than shark bone-derived macromolecular chondroitin sulfate, and has better effects on cartilage damaged by 1mM hydrogen peroxide. Cells have a more obvious repair effect.
图1是实施例9中鲨鱼骨来源的低分子量硫酸软骨素的寡糖分布谱图。Fig. 1 is the distribution spectrum of oligosaccharides of low molecular weight chondroitin sulfate derived from shark bone in Example 9.
图2是实施例10中鲨鱼骨来源的低分子量硫酸软骨素的寡糖分布谱图。Fig. 2 is a distribution spectrum of oligosaccharides of low molecular weight chondroitin sulfate derived from shark bone in Example 10.
图3是实施例11中鲨鱼骨来源的低分子量硫酸软骨素的寡糖分布谱图。Fig. 3 is a distribution spectrum of oligosaccharides of low-molecular-weight chondroitin sulfate derived from shark bone in Example 11.
图4是实施例12中鲨鱼骨来源的低分子量硫酸软骨素的寡糖分布谱图。Fig. 4 is a distribution spectrum of oligosaccharides of low molecular weight chondroitin sulfate derived from shark bone in Example 12.
图5是实施例14中不同来源的低分子量硫酸软骨素的功效活性检测结果图。Figure 5 is a graph showing the efficacy and activity detection results of low molecular weight chondroitin sulfate from different sources in Example 14.
为便于本领域技术人员理解本发明内容，下面将结合具体实施例进一步描述本发明的技术方案，但以下内容不应以任何方式限制本发明权利要求书请求保护的范围。In order to facilitate those skilled in the art to understand the content of the present invention, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection claimed by the claims of the present invention in any way.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。其中硫酸软骨素裂解酶来源于本实验室保存的从海岸边、河边、农贸市场、屠宰厂和食堂处的土样、污水或淤泥中筛选、鉴定，并用大肠杆菌或枯草芽孢杆菌优化表达得到的，其最高酶活可达11976.5U/L，含有998个氨基酸，分子量113KDa，该硫酸软骨素裂解酶的氨基酸序列公开于本公司申请的另外一件发明专利中，其申请日：2019年4月3日，申请号：201910264385.5，公布日：2019年6月21日，公布号：CN109913437A，该专利申请的所有相关内容引入到本专利申请中。The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Among them, the chondroitin sulfate lyase is selected and identified from the soil samples, sewage or sludge at the coast, riverside, farmer’s market, slaughterhouse, and canteen preserved in our laboratory, and optimized and expressed by E. coli or Bacillus subtilis. The highest enzyme activity can reach 11976.5U/L, contains 998 amino acids, and has a molecular weight of 113KDa. The amino acid sequence of this chondroitin sulfate lyase is disclosed in another invention patent filed by our company. Its application date: April 2019 On the 3rd, application number: 201910264385.5, publication date: June 21, 2019, publication number: CN109913437A, all relevant contents of this patent application are incorporated into this patent application.
低分子量硫酸软骨素平均分子量的计算公式如下所示：The formula for calculating the average molecular weight of low molecular weight chondroitin sulfate is as follows:
其中，r
t＝r
U1+r
U2+r
U3+r
U4+r
U5。
Among them, r t =r U1 +r U2 +r U3 +r U4 +r U5 .
其中：r
u1：样品溶液中组份一(六糖和八糖)的峰响应值；M
W1是样品溶液中组份一的分子量；
Among them: ru1 : the peak response value of component one (hexasaccharide and octasaccharide) in the sample solution; M W1 is the molecular weight of component one in the sample solution;
r
u2：样品溶液中组份二(四糖)的峰响应值；M
W2是样品溶液中组份二的分子量；
r u2 : peak response value of component two (tetrasaccharide) in the sample solution; M W2 is the molecular weight of component two in the sample solution;
r
u3：样品溶液中组份三(四糖)的峰响应值；M
W3是样品溶液中组份三的分子量；
r u3 : peak response value of component three (tetrasaccharide) in the sample solution; M W3 is the molecular weight of component three in the sample solution;
r
u4：样品溶液中组份四(二糖)的峰响应值；M
W4是样品溶液中组份四的分子量；
r u4 : peak response value of component four (disaccharide) in the sample solution; M W4 is the molecular weight of component four in the sample solution;
r
u5：样品溶液中组份五(二糖)的峰响应值；M
W5是样品溶液中组份五的分子量；
r u5 : peak response value of component five (disaccharide) in the sample solution; M W5 is the molecular weight of component five in the sample solution;
r
t：样品溶液中组份一、组份二、组份三、组份四、组份五的峰响应值之和。
r t : The sum of the peak response values of component 1, component 2, component 3, component 4, and component 5 in the sample solution.
由鲨鱼骨来源的大分子硫酸软骨素酶解得到的低分子量硫酸软骨素中二糖(n＝0)、四糖(n＝1)、六糖(n＝2)、八糖(n＝3)的分子量有差异，其中n＝4的十糖和n＝5的十二糖含量非常少，因此在计算寡糖组合物的平均分子量时忽略不计，具体如下表1所示：Disaccharides (n=0), tetrasaccharides (n=1), hexasaccharides (n=2), octasaccharides (n=3) in low molecular weight chondroitin sulfate obtained by enzymatic hydrolysis of large molecular chondroitin sulfate derived from shark bone ) Has a difference in molecular weight, where the content of decasaccharides with n=4 and dodecasaccharides with n=5 are very small, so they are ignored when calculating the average molecular weight of the oligosaccharide composition, as shown in Table 1 below:
表1鲨鱼骨来源的低分子量硫酸软骨素的分子量分布Table 1 Molecular weight distribution of low molecular weight chondroitin sulfate derived from shark bone
| ## | 二糖(Da)Disaccharide (Da) | 四糖(Da)Tetrasaccharide (Da) | 六糖(Da)Hexaose (Da) | 八糖(Da)Octose (Da) |
| 11 | 379.1和459.1379.1 and 459.1 | 838.2和918.2838.2 and 918.2 | 1155.31155.3 | 1534.51534.5 |
实施例1 酶解反应Example 1 Enzymatic hydrolysis reaction
于5L玻璃烧杯中，加入纯化水2L，控制搅拌转速为400rpm，加入800g鲨鱼骨硫酸软骨素，待全部溶解后使用氢氧化钠溶液调pH至7.0，加入200U/L的硫酸软骨素裂解酶，保持体系30℃搅拌反应，反应6h检测平均分子量是否低于1000Da，若未反应完全，延长4h反应时间后继续中控。继续反应直至平均分子量低于1000Da判断为合格。In a 5L glass beaker, add 2L of purified water, control the stirring speed to 400rpm, add 800g of shark bone chondroitin sulfate, after all is dissolved, adjust the pH to 7.0 with sodium hydroxide solution, add 200U/L of chondroitin sulfate lyase, Keep the system at 30°C and stir the reaction. After 6 hours of reaction, check whether the average molecular weight is lower than 1000 Da. If the reaction is not complete, continue the central control after prolonging the reaction time for 4 hours. The reaction is continued until the average molecular weight is less than 1000 Da, and it is judged as qualified.
实施例2 酶解反应Example 2 Enzymatic hydrolysis reaction
于5L玻璃烧杯中，加入纯化水2L，控制搅拌转速为700rpm，加入400g鲨鱼骨硫酸软骨素，待全部溶解后使用氢氧化钠溶液调pH至6.5，加入300U/L的硫酸软骨素裂解酶，保持体系35℃搅拌反应，反应6h检测平均分子量是否低于1000Da，若未反应完全，延长4h反应时间后继续中控。继续反应直至水解液平均分子量低于1000Da判断为合格。In a 5L glass beaker, add 2L of purified water, control the stirring speed to 700rpm, add 400g of shark bone chondroitin sulfate, after all is dissolved, adjust the pH to 6.5 with sodium hydroxide solution, add 300U/L of chondroitin sulfate lyase, Keep the system at 35°C and stir the reaction, and check whether the average molecular weight is lower than 1000 Da after 6 hours of reaction. If the reaction is not complete, continue the central control after prolonging the reaction time for 4 hours. Continue the reaction until the average molecular weight of the hydrolyzate is less than 1000 Da, and it is judged as qualified.
实施例3 酶解反应Example 3 Enzymatic hydrolysis reaction
于5L玻璃烧杯中，加入纯化水2L，控制搅拌转速为100rpm，加入200g鲨鱼骨硫酸软骨素，待全部溶解后使用氢氧化钠溶液调pH至8.0，加入100U/L的硫酸软骨素裂解酶，保持体系25℃搅拌反应，反应6h检测平均分子量是否低于1000Da，若未反应完全，延长4h反应时间后继续中控。继续反应直至水解液平均分子量低于1000Da判断为合格。In a 5L glass beaker, add 2L of purified water, control the stirring speed to 100rpm, add 200g of shark bone chondroitin sulfate, after all is dissolved, adjust the pH to 8.0 with sodium hydroxide solution, add 100U/L of chondroitin sulfate lyase, Keep the system at 25°C and stir the reaction, and check whether the average molecular weight is lower than 1000 Da after 6 hours of reaction. If the reaction is not complete, continue the central control after prolonging the reaction time for 4 hours. Continue the reaction until the average molecular weight of the hydrolyzate is less than 1000 Da, and it is judged as qualified.
实施例4 酶解反应Example 4 Enzymatic hydrolysis reaction
于5L玻璃烧杯中，加入纯化水2L，控制搅拌转速为500rpm，加入1400g鲨鱼骨硫酸软骨素，待全部溶解后使用氢氧化钠溶液调pH至8.5，加入280U/L的硫酸软骨素裂解酶，保持体系28℃搅拌反应，反应6h检测平均分子量是否低于1000Da，若未反应完全，延长4h反应时间后继续中控。继续反应直至水解液平均分子量低于1000Da判断为合格。In a 5L glass beaker, add 2L of purified water, control the stirring speed to 500rpm, add 1400g shark bone chondroitin sulfate, adjust the pH to 8.5 with sodium hydroxide solution, add 280U/L chondroitin sulfate lyase, Keep the system at 28°C and stir the reaction. After 6 hours of reaction, check whether the average molecular weight is lower than 1000 Da. If the reaction is not complete, continue the central control after prolonging the reaction time for 4 hours. Continue the reaction until the average molecular weight of the hydrolyzate is less than 1000 Da, and it is judged as qualified.
实施例5 蛋白脱除Example 5 Protein removal
取2L实施例1中反应结束后的水解液，转移到离心机中，在4200rpm离心15min除去菌体，取上清，加入有机溶剂0.4L(二氯甲烷和异丙醇的体积比＝5:1)，脱除蛋白，在100rpm搅拌40min，在4200rpm继续离心15min，取出后倒出上层反应液。Take 2L of the hydrolyzed solution after the reaction in Example 1, transfer to a centrifuge, centrifuge at 4200rpm for 15min to remove the bacteria, take the supernatant, and add 0.4L of organic solvent (volume ratio of dichloromethane and isopropanol = 5: 1) Remove protein, stir at 100 rpm for 40 minutes, continue centrifugation at 4200 rpm for 15 minutes, and pour out the upper reaction solution after taking it out.
实施例6 蛋白脱除Example 6 Protein removal
取2.1L实施例2中反应结束后的水解液，转移到离心机中，在4200rpm离心15min除去菌体，取上清，加入有机溶剂0.7L(二氯甲烷和异丙醇的体积比＝4:1)，脱除蛋白，在500rpm搅拌10min，在3000rpm继续离心30min，取出后倒出上层反应液。Take 2.1L of the hydrolyzed solution after the reaction in Example 2 is transferred to a centrifuge, centrifuge at 4200rpm for 15min to remove the bacteria, take the supernatant, and add 0.7L of organic solvent (volume ratio of dichloromethane and isopropanol = 4 1), remove the protein, stir at 500 rpm for 10 min, continue centrifugation at 3000 rpm for 30 min, and pour out the upper reaction solution after taking it out.
实施例7 蛋白脱除Example 7 Protein removal
取2L实施例3中反应结束后的水解液，转移到离心机中，在4200rpm离心15min除去菌体，取上清，加入有机溶剂1L(二氯甲烷和异丙醇的体积比＝3:1)，脱除蛋白，在300rpm搅拌30min，在5000rpm继续离心10min，取出后倒出上层反应液。Take 2L of the hydrolysate after the reaction in Example 3, transfer to a centrifuge, centrifuge at 4200rpm for 15min to remove the bacteria, take the supernatant, and add 1L of organic solvent (volume ratio of dichloromethane and isopropanol=3:1 ), remove the protein, stir at 300 rpm for 30 min, continue centrifugation at 5000 rpm for 10 min, and pour out the upper reaction solution after taking it out.
实施例8 蛋白脱除Example 8 Protein removal
取2.3L实施例4中反应结束后的水解液，通过超滤系统，在低温条件下，采用截留分子量5-8万的膜包进行超滤，使蛋白去除得到超滤后反应液。Take 2.3L of the hydrolyzed liquid after the reaction in Example 4, pass through an ultrafiltration system, under low temperature conditions, use a membrane package with a molecular weight cut-off of 50,000 to 80,000 to perform ultrafiltration to remove the protein to obtain a reaction solution after ultrafiltration.
实施例9 醇沉干燥Example 9 Alcohol precipitation and drying
将2L实施例5得到的上层反应液经0.22um囊式滤芯过滤除菌至洁净区中，然后滴加到20L无水乙醇中，搅拌0.5h，静置2h，固体完全沉淀，抽去上清，离心过滤收集固体，固体于真空干燥箱中45℃真空干燥24h，至干燥失重不超过10％，得到650g低分子量硫酸软骨素产品，收率为81.3％(即650g低分子量硫酸软骨素与800g鲨鱼骨硫酸软骨素原料的比值)，用考马斯亮蓝法检测蛋白含量为0.3％，用液质联用仪测定其分子量分布如图1所示，出峰时 间为5.879min的组分一主要为六糖和八糖的组合物，含量为9.51％；出峰时间为8.632min组分二为四糖，含量为33.83％；出峰时间为8.966min的组分三也为四糖，含量为5.07％；出峰时间为9.846min的组分四为二糖，含量为47.08％；出峰时间为10.786min的组分五也为二糖，含量为2.03％；出峰时间为12.506min的组分六为盐峰；因此包括二糖、四糖、六糖和八糖的低分子量硫酸软骨素总含量为97.52％，主要以二糖和四糖为主，其中二糖和四糖含量之和为88.01％；由鲨鱼骨酶解得到的低分子量硫酸软骨素的平均分子量为704.3～741.2Da，具体计算过程如下：2L of the upper reaction liquid obtained in Example 5 was filtered through a 0.22um capsule filter and sterilized into a clean area, and then added dropwise to 20L of absolute ethanol, stirred for 0.5h, and allowed to stand for 2h. The solid was completely precipitated, and the supernatant was removed. , The solid was collected by centrifugal filtration, and the solid was dried in a vacuum drying oven at 45°C for 24 hours until the loss on drying did not exceed 10% to obtain 650g of low-molecular-weight chondroitin sulfate product with a yield of 81.3% (ie 650g of low-molecular-weight chondroitin sulfate and 800g The ratio of shark bone chondroitin sulfate raw materials), the protein content was determined by Coomassie Brilliant Blue method to be 0.3%, and the molecular weight distribution measured by liquid mass spectrometry is shown in Figure 1. The component with peak time of 5.879min is mainly The composition of hexasaccharide and octasaccharide, the content is 9.51%; the peak time is 8.632min, the second component is tetrasaccharide, the content is 33.83%; the peak time is 8.966min, the component three is also the tetrasaccharide, the content is 5.07 %; component four with a peak time of 9.846min is a disaccharide with a content of 47.08%; component five with a peak time of 10.786min is also a disaccharide with a content of 2.03%; the component with a peak time of 12.506min Six is a salt peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 97.52%, mainly disaccharides and tetrasaccharides, and the sum of the content of disaccharides and tetrasaccharides is 88.01%; the average molecular weight of low molecular weight chondroitin sulfate obtained from shark bone enzymatic hydrolysis is 704.3～741.2Da, the specific calculation process is as follows:
假定组分一中全部为六糖，则Assuming that all hexasaccharides in component one, then
假定组分一中全部为八糖，则Assuming that all octasaccharides in component one, then
实施例10 醇沉干燥Example 10 Alcohol precipitation and drying
将2L实施例6得到的上层反应液经0.22um囊式滤芯过滤除菌至洁净区中，然后滴加到16L无水乙醇中，搅拌0.5h，静置2h，固体完全沉淀，抽去上清，离心过滤收集固体，固体于真空干燥箱中50℃真空干燥24h，至干燥失重不超过10％，得到320g低分子量硫酸软骨素产品，收率为80.0％(即320g低分子量硫酸软骨素与400g鲨鱼骨硫酸软骨素原料的比值)，用考马斯亮蓝法检测蛋白含量为0.4％，用液质联用仪测定其分子量分布如图2所示，出峰时间为5.878min的组分一为六糖和八糖，含量为9.50％；出峰时间为8.625min的组分二为四糖，含量为33.90％；出峰时间为8.958min的组分三也为四糖，含量为5.08％；出峰时间为9.838min的组分四为二糖，含量为46.86％；出峰时间为10.785min的组分五也为二糖，含量为2.13％；出峰时间为12.505min的组分六为盐峰；因此包括二糖、四糖、六糖和八糖的低分子量硫酸软骨素总含量为97.47％，主要以二糖和四糖为主，其中二糖和四糖含量之和为87.97％；由鲨鱼骨酶解得到的低分子量硫酸软骨素的平均分子量为704.7～741.6Da，具体计算过程如下：2L of the upper reaction liquid obtained in Example 6 was filtered through a 0.22um capsule filter and sterilized into a clean area, and then added dropwise to 16L of absolute ethanol, stirred for 0.5h, and allowed to stand for 2h. The solid was completely precipitated, and the supernatant was removed. , Centrifugal filtration to collect the solids, the solids were dried in a vacuum drying oven at 50°C for 24 hours until the drying weight loss did not exceed 10% to obtain 320g of low molecular weight chondroitin sulfate product with a yield of 80.0% (that is, 320g of low molecular weight chondroitin sulfate and 400g Shark bone chondroitin sulfate raw material ratio), the protein content was determined by Coomassie Brilliant Blue method to be 0.4%, and the molecular weight distribution measured by liquid mass spectrometry is shown in Figure 2. The peak time of 5.878min component one is six Sugars and octasaccharides, the content is 9.50%; the second component with a peak time of 8.625min is tetrasaccharides with a content of 33.90%; the component three with a peak time of 8.958min also contains tetrasaccharides with a content of 5.08%; The component four with a peak time of 9.838min is a disaccharide with a content of 46.86%; the component five with a peak time of 10.785min is also a disaccharide with a content of 2.13%; the component six with a peak time of 12.505min is a salt Peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 97.47%, mainly disaccharides and tetrasaccharides, of which the sum of disaccharides and tetrasaccharides content is 87.97%; The average molecular weight of low-molecular-weight chondroitin sulfate obtained from shark bone enzymatic hydrolysis is 704.7～741.6Da. The specific calculation process is as follows:
假定组分一中全部为六糖，则Assuming that all hexasaccharides in component one, then
假定组分一中全部为八糖，则Assuming that all octasaccharides in component one, then
实施例11 醇沉干燥Example 11 Alcohol precipitation and drying
将2L实施例7得到的上层反应液经0.22um囊式滤芯过滤除菌至洁净区中，然后滴加到24L无水乙醇中，搅拌0.5h，静置2h，固体完全沉淀，抽去上清，离心过滤收集固体，固体于真空干燥箱中40℃真空干燥24h，至干燥失重不超过10％，得到150g低分子量硫酸软骨素产品，收率为75.0％(即150g低分子量硫酸软骨素与200g鲨鱼骨硫酸软骨素原料的比值)，用考马斯亮蓝法检测蛋白含量为0.4％，用液质联用仪测定其分子量分布如图3所示，出峰时间为5.879min的组分一为六糖和八糖，含量为8.62％；出峰时间为8.632min的组分二为四糖，含量为30.79％；出峰时间为8.966min的组分三也为四糖，含量为4.41％；出峰时间为9.872min的组分四为二糖，含量为52.49％；出峰时间为10.786min的组分五也为二糖，含量为2.02％；出峰时间为12.512min的组分六为盐峰；因此包括二糖、四糖、六糖和八糖的低分子量硫酸软骨素总含量为98.32％，主要以二糖和四糖为主，其中二糖和四糖含量之和为89.70％；由鲨鱼骨酶解得到的低分子量硫酸软骨素的平均分子量为679.2～712.5Da，具体计算过程如下：2L of the upper reaction liquid obtained in Example 7 was filtered and sterilized by a 0.22um capsule filter into a clean area, and then added dropwise to 24L of absolute ethanol, stirred for 0.5h, and allowed to stand for 2h. The solid was completely precipitated, and the supernatant was removed. , Centrifugal filtration to collect the solids, the solids were dried in a vacuum drying oven at 40°C for 24 hours, until the drying weight loss did not exceed 10%, to obtain 150g of low molecular weight chondroitin sulfate product with a yield of 75.0% (that is, 150g of low molecular weight chondroitin sulfate and 200g Shark bone chondroitin sulfate raw material ratio), the protein content was determined by the Coomassie Brilliant Blue method to be 0.4%, and the molecular weight distribution measured by liquid mass spectrometry is shown in Figure 3. The peak time of 5.879min component one is six The content of sugar and octasaccharide is 8.62%; the second component with a peak time of 8.632min is tetrasaccharide, the content is 30.79%; the component three with a peak time of 8.966min is also a tetrasaccharide, the content is 4.41%; The component four with a peak time of 9.872min is a disaccharide with a content of 52.49%; the component five with a peak time of 10.786min is also a disaccharide with a content of 2.02%; the component six with a peak time of 12.512min is a salt Peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 98.32%, mainly disaccharides and tetrasaccharides, of which the sum of disaccharides and tetrasaccharides content is 89.70%; The average molecular weight of low-molecular-weight chondroitin sulfate obtained from shark bone enzymatic hydrolysis is 679.2～712.5Da. The specific calculation process is as follows:
假定组分一中全部为六糖，则Assuming that all hexasaccharides in component one, then
假定组分一中全部为八糖，则Assuming that all octasaccharides in component one, then
实施例12 喷雾干燥Example 12 Spray drying
将2L实施例8得到的反应液经0.22um囊式滤芯过滤除菌后进行喷雾干燥，喷雾干燥参数为：进风温度为120℃，出风温度为60℃，流速为100rpm。得到1200g低分子量硫酸软骨素产品，收率为85.7％(即1200g低分子量硫酸软骨素与1400g鲨鱼骨硫酸软骨素原料的比值)，用考马斯亮蓝法检测蛋白含量为0.5％，用液质联用仪测定其分子量分布如图4所示，出峰时间为5.876min的组分一为六糖和八糖，含量为8.50％；出峰时间为8.636min的组分二为四糖，含量为30.59％；出峰时间为8.963min的组分三也为四糖，含量为4.43％；出峰时间为9.870min的组分四为二糖，含量为52.69％；出峰时间为10.783min的组分五也为二糖，含量为2.14％；出峰时间为12.510min的组分六为盐峰；因此包括二糖、四糖、六糖和八糖的低分子量硫酸软骨素总含量为98.35％，主要以二糖和四糖为主，其中二糖和四糖含量之和为89.85％；由鲨鱼骨酶解得到的低分子量硫酸软骨素的平均分子量为677.4～710.2Da，具体计算过程如下：2L of the reaction solution obtained in Example 8 was filtered and sterilized by a 0.22um capsule filter and spray-dried. The spray-drying parameters were as follows: the inlet air temperature was 120°C, the outlet air temperature was 60°C, and the flow rate was 100 rpm. Obtained 1200g low molecular weight chondroitin sulfate product with a yield of 85.7% (that is, the ratio of 1200g low molecular weight chondroitin sulfate to 1400g shark bone chondroitin sulfate raw material). The protein content was determined to be 0.5% by the Coomassie brilliant blue method. The molecular weight distribution measured by the instrument is shown in Figure 4. The first component with a peak time of 5.876 min is hexasaccharides and octasaccharides, and the content is 8.50%; the second component with a peak time of 8.636 min is tetrasaccharides, and the content is 30.59%; component three with a peak time of 8.963min is also a tetrasaccharide with a content of 4.43%; a component four with a peak time of 9.870min is a disaccharide with a content of 52.69%; the group with a peak time of 10.783min Fraction five is also disaccharide with a content of 2.14%; component six with peak time of 12.510min is salt peak; therefore, the total content of low molecular weight chondroitin sulfate including disaccharides, tetrasaccharides, hexasaccharides and octasaccharides is 98.35% , Mainly disaccharides and tetrasaccharides, the sum of the content of disaccharides and tetrasaccharides is 89.85%; the average molecular weight of low molecular weight chondroitin sulfate obtained from shark bone enzymatic hydrolysis is 677.4～710.2Da, the specific calculation process is as follows:
假定组分一中全部为六糖，则Assuming that all hexasaccharides in component one, then
假定组分一中全部为八糖，则Assuming that all octasaccharides in component one, then
实施例13Example 13
分别使用牛软骨、猪软骨、鸡软骨以及鸡软骨和鲨鱼骨的混骨按照实施例1的酶解反应、实施例5的蛋白脱除工艺以及实施例9的醇沉干燥工艺，得到另外四种不同来源的低分子量硫酸软骨素。Using bovine cartilage, porcine cartilage, chicken cartilage, and a mixture of chicken cartilage and shark bone, respectively, according to the enzymatic hydrolysis reaction of Example 1, the protein removal process of Example 5, and the alcohol precipitation drying process of Example 9 were obtained. Low molecular weight chondroitin sulfate from different sources.
实施例14 功效活性检测Example 14 Efficacy and activity detection
采用细胞活性测定CCK法，考察不同来源的低分子量硫酸软骨素和大分子硫酸软骨素，对损伤的软骨细胞的修复作用，其功效活性检测结果如图5所示，结果表明由鲨鱼骨或鸡骨中的一种或其混合骨酶解得到的低分子量硫酸软骨素相对鲨鱼骨来源的大分子硫酸软骨素(硫酸软骨素多糖)，在50～100μg/mL浓度内对1mM双氧水损伤的软骨细胞均有更明显的修复作用，修复能力在14％～23％之间，可用于治疗关节损伤。其中鲨鱼源的低分子量硫酸软骨素修复效果优于鸡软骨、猪软骨、牛软骨和混骨来源的低分子量硫酸软骨素。在50μg/mL浓度时，由鲨鱼骨、鸡软骨、猪软骨、牛软骨和混骨来源酶解得到的低分子量硫酸软骨素均比鲨鱼骨来源的大分子硫酸软骨素，对1mM双氧水损伤的软骨细胞有更明显的修复作用。The CCK method for cell viability determination was used to investigate the repair effects of low molecular weight chondroitin sulfate and macromolecular chondroitin sulfate from different sources on damaged chondrocytes. The results of the efficacy and activity test are shown in Figure 5, and the results show that it is made from shark bone or chicken. The low-molecular-weight chondroitin sulfate obtained by enzymatic hydrolysis of one kind of bone or its mixed bone is relative to the macromolecular chondroitin sulfate (chondroitin sulfate polysaccharide) derived from shark bone, which can damage chondrocytes damaged by 1mM hydrogen peroxide at a concentration of 50-100μg/mL Both have a more obvious repairing effect, the repairing ability is between 14% and 23%, and it can be used to treat joint injuries. Among them, shark-derived low-molecular-weight chondroitin sulfate has a better repair effect than chicken cartilage, porcine cartilage, bovine cartilage, and mixed bone-derived low-molecular-weight chondroitin sulfate. At a concentration of 50μg/mL, the low-molecular-weight chondroitin sulfate derived from shark bone, chicken cartilage, porcine cartilage, bovine cartilage and mixed bone-derived enzymatic hydrolysis is better than shark bone-derived macromolecular chondroitin sulfate, and has better effects on cartilage damaged by 1mM hydrogen peroxide. Cells have a more obvious repair effect.
Claims (14)
- 一种低分子量硫酸软骨素，其特征在于：所述低分子量硫酸软骨素的平均分子量低于1000道尔顿，其分子量分布范围窄，主要以硫酸软骨素二糖和硫酸软骨素四糖为主，硫酸软骨素二糖含量占比为43～60％，硫酸软骨素四糖含量占比为30～45％，硫酸软骨素二糖和硫酸软骨素四糖含量之和大于87％；所述低分子量硫酸软骨素的结构通式如下式I所示：A low-molecular-weight chondroitin sulfate, characterized in that: the average molecular weight of the low-molecular-weight chondroitin sulfate is less than 1000 Daltons, and its molecular weight distribution range is narrow, mainly composed of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide , The content of chondroitin sulfate disaccharide is 43-60%, the content of chondroitin sulfate tetrasaccharide is 30-45%, and the sum of the content of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is greater than 87%; The general structural formula of molecular weight chondroitin sulfate is shown in the following formula I:
式I：n＝0～5，R 1,R 2,R 3＝-H或-SO 3Na。 Formula I: n=0 to 5, R 1 , R 2 , R 3 =-H or -SO 3 Na. - 根据权利要求1所述的低分子量硫酸软骨素，其特征在于：所述低分子量硫酸软骨素的平均分子量为590～830Da，进一步优选为677～742Da。The low-molecular-weight chondroitin sulfate of claim 1, wherein the average molecular weight of the low-molecular-weight chondroitin sulfate is 590-830 Da, more preferably 677-742 Da.
- 根据权利要求1所述的低分子量硫酸软骨素，其特征在于：所述低分子量硫酸软骨素中硫酸软骨素二糖含量占比为48～55％，硫酸软骨素四糖含量占比为35～40％。The low-molecular-weight chondroitin sulfate of claim 1, wherein the content of chondroitin sulfate disaccharides in the low-molecular-weight chondroitin sulfate accounts for 48-55%, and the content of chondroitin sulfate tetrasaccharides accounts for 35-5%. 40%.
- 一种权利要求1～3任一项所述的低分子量硫酸软骨素的制备方法，其特征在于：将大分子硫酸软骨素原料经过硫酸软骨素裂解酶的酶解得到平均分子量可稳定地控制在低于1000道尔顿的低分子量硫酸软骨素产物，其分子量分布范围窄，产物主要以硫酸软骨素二糖和硫酸软骨素四糖为主，硫酸软骨素二糖含量占比为43～60％，硫酸软骨素四糖含量占比为30～45％，硫酸软骨素二糖和硫酸软骨素四糖含量之和大于87％；所述低分子量硫酸软骨素的结构通式如下式I所示：A method for preparing low molecular weight chondroitin sulfate according to any one of claims 1 to 3, characterized in that: the macromolecular chondroitin sulfate raw material is enzymatically hydrolyzed by chondroitin sulfate lyase to obtain an average molecular weight that can be stably controlled at Low molecular weight chondroitin sulfate products below 1000 Daltons have a narrow molecular weight distribution range. The products are mainly chondroitin sulfate disaccharides and chondroitin sulfate tetrasaccharides, and the content of chondroitin sulfate disaccharides accounts for 43-60% , The content of chondroitin sulfate tetrasaccharide is 30-45%, and the sum of the content of chondroitin sulfate disaccharide and chondroitin sulfate tetrasaccharide is greater than 87%; the general structural formula of the low molecular weight chondroitin sulfate is shown in the following formula I:
式I：n＝0～5；R 1,R 2,R 3＝-H或-SO 3Na。 Formula I: n=0 to 5; R 1 , R 2 , R 3 =-H or -SO 3 Na. - 根据权利要求4所述的低分子量硫酸软骨素的制备方法，其特征在于：所述硫酸软骨素裂解酶从海岸边、河边、农贸市场、屠宰厂和食堂处的土样、污水或淤泥中筛选、鉴定，并用大肠杆菌或枯草芽孢杆菌优化表达得到。The method for preparing low-molecular-weight chondroitin sulfate according to claim 4, wherein the chondroitin sulfate lyase is obtained from soil samples, sewage or silt at coasts, rivers, farmers markets, slaughterhouses and canteens. Screening, identification, and optimized expression using Escherichia coli or Bacillus subtilis.
- 根据权利要求4所述的低分子量硫酸软骨素的制备方法，其特征在于：所述大分子硫酸软骨素原料来源于陆生和海洋动物的软骨组织，包括鸡软骨，猪软骨，牛软骨或鲨鱼骨中的一种或几种混合。The method for preparing low-molecular-weight chondroitin sulfate according to claim 4, wherein the macromolecular chondroitin sulfate raw materials are derived from cartilage tissues of terrestrial and marine animals, including chicken cartilage, porcine cartilage, bovine cartilage or shark One or several mixtures in bone.
- 根据权利要求6所述的低分子量硫酸软骨素的制备方法，其特征在于：所述大分子硫酸软骨素原料来源于鲨鱼骨。The method for preparing low molecular weight chondroitin sulfate according to claim 6, wherein the macromolecular chondroitin sulfate raw material is derived from shark bone.
- 根据权利要求4所述的低分子量硫酸软骨素的制备方法，其特征在于：酶解反应的操作条件为，所述硫酸软骨素裂解酶相对每升发酵液的添加量为100～300U/L，大分子硫酸软骨素原料的浓度为100～700g/L，酶解时间为6～10h，酶解温度为25～35℃，搅拌转速为100～700rpm，酶解pH为6.5～8.5。The method for preparing low-molecular-weight chondroitin sulfate according to claim 4, wherein the operating conditions of the enzymatic hydrolysis reaction are that the added amount of the chondroitin sulfate lyase per liter of fermentation broth is 100-300 U/L, The concentration of the macromolecular chondroitin sulfate raw material is 100-700g/L, the enzymatic hydrolysis time is 6-10h, the enzymatic hydrolysis temperature is 25-35°C, the stirring speed is 100-700rpm, and the enzymatic hydrolysis pH is 6.5-8.5.
- 根据权利要求4所述的低分子量硫酸软骨素的制备方法，其特征在于：酶解反应后的水解液通过混合溶剂脱除蛋白，水解液与混合溶剂的体积比为2～5:1，混合溶剂中二氯甲烷和异丙醇的体积比为3～5:1，100～500rpm搅拌10～40min，3000～5000rpm离心10～30min，取上层反应液。The method for preparing low-molecular-weight chondroitin sulfate according to claim 4, wherein the hydrolyzed solution after enzymatic hydrolysis is deproteinized by a mixed solvent, and the volume ratio of the hydrolyzed solution to the mixed solvent is 2-5:1, and the mixture The volume ratio of dichloromethane and isopropanol in the solvent is 3 to 5:1, stirring at 100 to 500 rpm for 10 to 40 minutes, centrifuging at 3000 to 5000 rpm for 10 to 30 minutes, and taking the upper reaction solution.
- 根据权利要求4所述的低分子量硫酸软骨素的制备方法，其特征在于：酶解反应后的水解液通过超滤的方式脱除蛋白得到反应液。The method for preparing low-molecular-weight chondroitin sulfate according to claim 4, wherein the hydrolysate after the enzymatic hydrolysis reaction is subjected to ultrafiltration to remove proteins to obtain a reaction solution.
- 根据权利要求9所述的低分子量硫酸软骨素的制备方法，其特征在于：将脱除蛋白后的上层反应液，经过0.22um囊式滤芯过滤除菌，然后将反应液加入到8～12倍体积量的无水乙醇中，醇沉，真空干燥。The preparation method of low-molecular-weight chondroitin sulfate according to claim 9, characterized in that: the upper reaction solution after deproteinization is filtered through a 0.22um capsule filter to sterilize, and then the reaction solution is added to 8-12 times The volume of anhydrous ethanol, alcohol precipitation, vacuum drying.
- 据权利要求10所述的低分子量硫酸软骨素的制备方法，其特征在于：将脱除蛋白后的反应液，经过0.22um囊式滤芯过滤除菌后进行喷雾干燥。The method for preparing low-molecular-weight chondroitin sulfate according to claim 10, characterized in that the deproteinized reaction solution is filtered and sterilized by a 0.22um capsule filter and then spray-dried.
- 根据权利要求1所述的低分子量硫酸软骨素，其特征在于：由鲨鱼骨和鸡软骨中的一种或其混合骨酶解得到的低分子量硫酸软骨素相对鲨鱼骨的大分子硫酸软骨素，在50～100μg/mL浓度内对1mM双氧水损伤的软骨细胞有更明显的修复作用，修复率在14％～23％之间。The low-molecular-weight chondroitin sulfate of claim 1, wherein the low-molecular-weight chondroitin sulfate obtained by enzymatic hydrolysis of one of shark bone and chicken cartilage or its mixed bone is relative to the macromolecular chondroitin sulfate of shark bone. At a concentration of 50-100μg/mL, it has a more obvious repair effect on chondrocytes damaged by 1mM hydrogen peroxide, and the repair rate is between 14% and 23%.
- 根据权利要求1～3任一项所述的低分子量硫酸软骨素，其特征在于：所述低分子量硫酸软骨素具有在制备医药品、化妆品、保健品和食品领域方面的应用。The low-molecular-weight chondroitin sulfate according to any one of claims 1 to 3, wherein the low-molecular-weight chondroitin sulfate has applications in the preparation of pharmaceuticals, cosmetics, health products, and food.
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| CN201980013421.9A CN111741963A (en) | 2019-11-01 | 2019-11-01 | Low-molecular-weight chondroitin sulfate and preparation method thereof |
| PCT/CN2019/115120 WO2021081999A1 (en) | 2019-11-01 | 2019-11-01 | Low-molecular-weight chondroitin sulfate and preparation method therefor |
| EP20882136.3A EP4053290A4 (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, composition containing same, and preparation method therefor and use thereof |
| KR1020227018141A KR20220091552A (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, composition containing same, and method for preparing same and use thereof |
| AU2020374934A AU2020374934A1 (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, composition containing same, and preparation method therefor and use thereof |
| CN202080076790.5A CN114846147A (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, compositions comprising the same, methods of making the same, and uses thereof |
| PCT/CN2020/126003 WO2021083384A1 (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, composition containing same, and preparation method therefor and use thereof |
| BR112022008397A BR112022008397A8 (en) | 2019-11-01 | 2020-11-02 | LOW MOLECULAR WEIGHT CHONDROITIN SULFATE, COMPOSITION, PREPARATION METHOD AND USE |
| JP2022525450A JP2023500294A (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, composition, method of preparation and use thereof |
| CA3159355A CA3159355A1 (en) | 2019-11-01 | 2020-11-02 | Low molecular weight chondroitin sulfate, composition, preparation method and use thereof |
| US17/335,837 US11572421B2 (en) | 2019-11-01 | 2021-06-01 | Low molecular weight chondroitin sulfate, composition, preparation method and use thereof |
| US18/092,051 US20230250199A1 (en) | 2019-11-01 | 2022-12-30 | Low molecular weight chondroitin sulfate, composition, preparation method and use thereof |
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| CN113603732B (en) * | 2021-08-25 | 2024-02-13 | 江南大学 | Non-animal chondroitin sulfate oligosaccharide and preparation method thereof |
| CN114533654B (en) * | 2021-12-29 | 2023-11-21 | 南京乐韬生物科技有限公司 | Low-molecular chondroitin sulfate composite hydrogel and preparation method and application thereof |
| CN114480182B (en) * | 2022-01-10 | 2023-10-31 | 中国农业大学 | Arthrobacter pseudolaris PL-410 and application thereof in production of chondroitin lyase |
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