CN108070627A - A kind of preparation method of chondroitin sulfate D tetroses - Google Patents
A kind of preparation method of chondroitin sulfate D tetroses Download PDFInfo
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- CN108070627A CN108070627A CN201711216846.9A CN201711216846A CN108070627A CN 108070627 A CN108070627 A CN 108070627A CN 201711216846 A CN201711216846 A CN 201711216846A CN 108070627 A CN108070627 A CN 108070627A
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- chondroitin sulfate
- tetroses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
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- 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
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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Abstract
The present invention relates to a kind of preparation methods of chondroitin sulfate D tetroses, belong to technical field of biochemical industry.It is degraded using shark chondroitine as raw material using chondroitin sulfate A (CSA) C enzymes, through ethanol precipitation, anion-exchange chromatography separation, gel chromatography separation, is prepared chondroitin sulfate D tetroses.The beneficial effects of the invention are as follows：The chondroitin sulfate D tetroses of precise structure and molecular weight can be prepared, yield is larger, and the research for chondroitin sulfate oligosaccharide monomer provides possibility.
Description
Technical field
The present invention relates to a kind of preparation methods of chondroitin sulfate D tetroses, belong to technical field of biochemical industry.
Background technology
Chondroitin sulfate is a kind of mucopolysaccharide extracted from animal cartilage, is connected by acetylamino galactosamine and glucuronic acid
It connects, has use in every field such as medicine, health care, food, cosmetics.Chondroitin sulfate has been used for nerve at present
Bitterly, the clinical treatment of the diseases such as arthralgia, arthritis.The potential bioactivity of chondroitin sulfate includes anti-inflammatory, immunological regulation, god
Through nutrition and pre- preventing bone rarefaction etc..Yet with the permeability of its high molecular weight, the non-uniform feature of structure and cell membrane
Low factor, there are the problems such as bioavailability is low, curative effect is unstable for puritan filler chondroitin.
Low molecular chondroitin sulfate is increasingly valued by people, and United States Patent (USP) USP3405120 is it was demonstrated that molecular weight
In 3500-5300 dalton, chondroitin sulfate has most strong medicine in prevention atherosclerosis, rheumatic inflammation etc.
Reason activity.These low molecular chondroitin sulfates are still the mixture of different molecular weight.
Chinese patent《A kind of chondroitin sulfate disaccharides, tetrose, the preparation method of six sugar》(application number
201210159448.9) have been disclosed for the preparation method of different molecular weight oligosaccharides, including enzymolysis, molecular sieve, ion exchange and
Prepare the means such as electrophoresis.The yield of this method is smaller, and is limited to prepare electrophoresis yield, this technique can not realize amplification life
Production.
The separating and extracting process of chondroitin sulfate specific structure and molecular weight oligosaccharide is not yet reported that at present.
The content of the invention
In view of the above problems, the present invention provides a kind of preparation methods of chondroitin sulfate D tetroses.
The technical solution that the present invention solves above-mentioned technical problem is as follows：
A kind of preparation method of chondroitin sulfate D tetroses, includes the following steps：
(1) enzymic degradation of chondroitin sulfate D：Shark chondroitine 5g is dissolved in pH8.0 by the concentration of 5% (W/V) and delays
It rushes in solution, adds in 50IU chondroitin sulfate A (CSA) C enzymes, 37 DEG C of stirring enzymolysis 48h；After reaction, 5 times of 95% second of volume are added in
Alcohol precipitates, and freeze-drying obtains chondroitin sulfate oligosaccharide mixture；
Selection chondroitin sulfate A (CSA) C enzymes are for the non-sulphur of degradation of selectivity for the purpose for shark chondroitine of degrading
Aching and limp ossein D disaccharide components, and retain chondroitin sulfate D oligosaccharides as much as possible.
(2) anion exchange separation of chondroitin sulfate D oligosaccharide mixtures：The sample that step (1) is obtained is used
The NaCl solution of 0.15mol/L is dissolved into 0.1g/mL, chromatographic column 2.6 × 20cm, Q Sepharose Fast Flow it is strong it is cloudy from
Son exchanges filler, ultraviolet detection wavelength 232nm, sample size 10ml, the NaCl solution of mobile phase 0.15mol/L and 1.2mol/L,
Flow velocity 1.33mL/min, gradient elution collect obtained different component；
(3) the gel filtration desalination of chondroitin sulfate D oligosaccharides：The component that step (2) is collected into carries out G-25 glucans
Gel filtration desalination, it is concentrated freeze-dried after desalination；
(4) the gel filtration separation of chondroitin sulfate D oligosaccharides：The sample that step (3) freezes is dissolved into purified water
0.1g/mL, chromatographic column 1.6 × 100cm, Bio-Gel P6 polyacrylamide gel fillers, ultraviolet detection wavelength 232nm, flowing
Phase 0.2mol/LNaCl solution, flow velocity 0.4mL/min, sample size 2ml collect obtained different component；
(5) the gel filtration desalination of chondroitin sulfate D oligosaccharides：The component that step (4) is collected into carries out G-25 glucans
It is concentrated freeze-dried after gel filtration desalination, up to chondroitin sulfate D tetroses.
The beneficial effects of the invention are as follows：Using shark chondroitine as raw material, specific has selected chondroitin sulfate A (CSA) C
Enzyme is degraded, and through ethanol precipitation, anion exchange separation, gel separation, the sulphur of precise structure and molecular weight is prepared
Aching and limp ossein D tetroses, the research for chondroitin sulfate oligosaccharide monomer provide possibility.
The used separation equipment scale and Chinese patent of the present invention《A kind of chondroitin sulfate disaccharides, tetrose, six sugar
Preparation method》(application number 201210159448.9) quite, but the single chondroitin sulfate D tetroses of every batch of output may be up to
100mg, and Chinese patent《A kind of chondroitin sulfate disaccharides, tetrose, the preparation method of six sugar》Inventory ability 40mg, yield is very
It is small.
So technique described in the invention is designed for specific structure chondroitin sulfate D oligosaccharides, this technique step
It suddenly can be specially to the chondroitin sulfate of high sulphation, as chondroitin sulfate D or chondroitin sulfate E oligosaccharides progress high efficiency are pure
Change, this programme can not according to it is existing it is theoretical speculate obtain, but drawn after combination product characteristic test large-scale purification material.
Based on the above technical solutions, the present invention can also be improved as follows.
Further, in step (2), the anion exchange separation of chondroitin sulfate D oligosaccharide mixtures last is collected
A elution fraction.
Advantageous effect using above-mentioned further scheme is：The negative charge density highest of chondroitin sulfate D oligosaccharides, therefore
It finally is eluted out in anion exchange.
Further, in step (4), the last one elution group after chondroitin sulfate D oligosaccharides gel filtration separation is collected
Point.
Advantageous effect using above-mentioned further scheme is：The charge density highest of chondroitin sulfate D oligosaccharides, so in the presence of
In anion exchange in the component of last elution, and its molecular weight is minimum, therefore in gel filtration separation equally most
After be eluted out.
Description of the drawings
The QFF anion-exchange chromatography figures of Fig. 1 chondroitin sulfate oligosaccharides；
The P6 gel chromatography figures of Fig. 2 chondroitin sulfate oligosaccharides；
The enzymolysis chromatograms of Fig. 3 chondroitin sulfate D tetroses；
The mass spectrogram of Fig. 4 chondroitin sulfate D tetroses.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
Embodiment one
(1) shark chondroitine 5g is dissolved in by the concentration of 5% (W/V) in pH8.0 buffer solutions, adds in 50IU sulfuric acid
Chondroitin A C enzymes, 37 DEG C of stirring enzymolysis 48h；After reaction, 5 times of 95% ethanol precipitations of volume are added in, freeze-drying obtains sulphur
Aching and limp ossein oligosaccharide mixture.
(2) sample that step (1) obtains is dissolved into 0.1g/mL, chromatographic column specification with the NaCl solution of 0.15mol/L
2.6 × 20cm, Q Sepharose Fast Flow strong anions exchange filler, ultraviolet detection wavelength 232nm, sample size 10ml,
The NaCl solution of mobile phase 0.15mol/L and 1.2mol/L, flow velocity 1.33mL/min, gradient elution collect elution volume 1350-
At 1550ml, i.e. the last one wave crest.See Fig. 1.
(3) it is concentrated freeze-dried after the component progress G-25 dextran gel filtration desalinations being collected into step (2).
(4) sample that step (3) freezes is dissolved into 0.1g/mL, chromatographic column 1.6 × 100cm, Bio-Gel with purified water
P6 polyacrylamide gels, ultraviolet detection wavelength 232nm, mobile phase 0.2mol/LNaCl solution, flow velocity 0.4mL/min, sample introduction
2ml is measured, is collected at elution volume 210-230ml, i.e. the last one wave crest.See Fig. 2.
(5) it is concentrated freeze-dried after the component progress G-25 dextran gel filtration desalinations being collected into step (4).
Product Validation：The detection of chondroitin sulfate D tetroses
Using the preliminary purity for judging product of Chinese Pharmacopoeia enzymolysis liquid phase process and and disaccharides structure, the results show sulfuric acid
Bis- sugared contents of chondroitin D are 95%.See Fig. 3.
Confirm that its molecular weight is 1078 by Mass Spectrometer Method, it is consistent with chondroitin sulfate D tetrose calculated values, it was demonstrated that production
Product are chondroitin sulfate D tetroses.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modifications, equivalent replacements and improvements are made should all be included in the protection scope of the present invention.
Claims (3)
1. a kind of preparation method of chondroitin sulfate D tetroses, which is characterized in that comprise the following steps：
(1) enzymic degradation of chondroitin sulfate D：It is molten to be dissolved in pH8.0 bufferings by the concentration of 5% (W/V) by shark chondroitine 5g
In liquid, 50IU chondroitin sulfate A (CSA) C enzymes, 37 DEG C of stirring enzymolysis 48h are added in；After reaction, 5 times of 95% ethyl alcohol of volume are added in sink
It forms sediment, freeze-drying obtains chondroitin sulfate oligosaccharide mixture；
(2) anion exchange separation of chondroitin sulfate D oligosaccharide mixtures：The sample that step (1) is obtained is with 0.15mol/L's
NaCl solution is dissolved into 0.1g/mL, and the exchange of chromatographic column specification 2.6 × 20cm, Q Sepharose Fast Flow strong anions is filled out
Material, ultraviolet detection wavelength 232nm, sample size 10ml, the NaCl solution of mobile phase 0.15mol/L and 1.2mol/L, flow velocity
1.33mL/min, gradient elution collect obtained different component；
(3) the gel filtration desalination of chondroitin sulfate D oligosaccharides：The component that step (2) is collected into carries out G-25 sephadexes
Desalination is filtered, it is concentrated freeze-dried after desalination；
(4) the gel filtration separation of chondroitin sulfate D oligosaccharides：The sample that step (3) freezes is dissolved into 0.1g/ with purified water
ML, chromatographic column 1.6 × 100cm, Bio-Gel P6 polyacrylamide gel fillers, ultraviolet detection wavelength 232nm, mobile phase
0.2mol/LNaCl solution, flow velocity 0.4mL/min, sample size 2ml collect obtained different component；
(5) the gel filtration desalination of chondroitin sulfate D oligosaccharides：The component that step (4) is collected into carries out G-25 sephadexes
It is concentrated freeze-dried after filtering desalination, up to chondroitin sulfate D tetroses.
2. a kind of preparation method of chondroitin sulfate D tetroses according to claim 1, it is characterised in that：In step (2)
In, the last one elution fraction of the anion exchange separation of collection chondroitin sulfate D oligosaccharide mixtures.
3. a kind of preparation method of chondroitin sulfate D tetroses according to claim 1 or 2, it is characterised in that：In step
(4) in, the last one elution fraction after chondroitin sulfate D oligosaccharides gel filtration separation is collected.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711216846.9A CN108070627A (en) | 2017-11-28 | 2017-11-28 | A kind of preparation method of chondroitin sulfate D tetroses |
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| CN201711216846.9A CN108070627A (en) | 2017-11-28 | 2017-11-28 | A kind of preparation method of chondroitin sulfate D tetroses |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575329A (en) * | 2020-05-26 | 2020-08-25 | 山东大学 | Preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property |
| CN112195211A (en) * | 2020-12-07 | 2021-01-08 | 滨州医学院 | Preparation process of chondroitin sulfate with wide molecular weight |
| WO2021081999A1 (en) * | 2019-11-01 | 2021-05-06 | 南京汉欣医药科技有限公司 | Low-molecular-weight chondroitin sulfate and preparation method therefor |
| WO2021083384A1 (en) | 2019-11-01 | 2021-05-06 | 南京信朗企业管理咨询有限公司 | Low molecular weight chondroitin sulfate, composition containing same, and preparation method therefor and use thereof |
-
2017
- 2017-11-28 CN CN201711216846.9A patent/CN108070627A/en not_active Withdrawn
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021081999A1 (en) * | 2019-11-01 | 2021-05-06 | 南京汉欣医药科技有限公司 | Low-molecular-weight chondroitin sulfate and preparation method therefor |
| WO2021083384A1 (en) | 2019-11-01 | 2021-05-06 | 南京信朗企业管理咨询有限公司 | Low molecular weight chondroitin sulfate, composition containing same, and preparation method therefor and use thereof |
| US11572421B2 (en) | 2019-11-01 | 2023-02-07 | Nanjing Hanxin Pharmaceutical Technology Co., Ltd. | Low molecular weight chondroitin sulfate, composition, preparation method and use thereof |
| CN111575329A (en) * | 2020-05-26 | 2020-08-25 | 山东大学 | Preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property |
| CN111575329B (en) * | 2020-05-26 | 2021-12-07 | 山东大学 | Preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property |
| CN112195211A (en) * | 2020-12-07 | 2021-01-08 | 滨州医学院 | Preparation process of chondroitin sulfate with wide molecular weight |
| CN112195211B (en) * | 2020-12-07 | 2021-04-16 | 滨州医学院 | Preparation process of chondroitin sulfate with wide molecular weight |
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