CN105524188A - Hyaluronan odd oligosaccharide monomer and preparation method thereof - Google Patents
Hyaluronan odd oligosaccharide monomer and preparation method thereof Download PDFInfo
- Publication number
- CN105524188A CN105524188A CN201510921459.XA CN201510921459A CN105524188A CN 105524188 A CN105524188 A CN 105524188A CN 201510921459 A CN201510921459 A CN 201510921459A CN 105524188 A CN105524188 A CN 105524188A
- Authority
- CN
- China
- Prior art keywords
- acid
- hyaluronic acid
- preparation
- oligosaccharide
- odd number
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K13/00—Sugars not otherwise provided for in this class
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides a hyaluronan odd oligosaccharide monomer and its preparation method. Polymerization degree of the hyaluronan odd oligosaccharide monomer is 3-23. The preparation method comprises the following steps: degrading hyaluronan with acid or hydrogen cation exchange resin, neutralizing with alkali, concentrating, precipitating with an organic solvent, centrifuging, concentrating a supernatant, and finally separating by gel column choromatography so as to obtain hyaluronan odd oligosaccharide monomers of different polymerization degrees. By one-time separation, high-purity hyaluronan odd oligosaccharide monomers with polymerization degree being 3-23 can be prepared. In addition, the product has advantages of novel structure, small molecular weight, good dissolvability and stable performance. The preparation technology is simple, low-cost and pollution-free, and is easy for industrialization. The hyaluronan odd oligosaccharide monomer obtained in the invention can be used in preparation of an oligosaccharide standard reagent or can be used as a bioactive substance or an intermediate to further prepare an oligosaccharide health product or an oligosaccharide drug. The product is fully utilized, and its function and activity are widely developed.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of hyaluronic acid odd number oligosaccharide monomer and preparation method thereof.
Background technology
Hyaluronic acid (Hyaluronicacid, HA) be a kind of polymeric acidic mucopolysaccharide without branched structure be made up of by β-(1 → 4) glycosidic link and β-(1 → 3) glycosidic link 2-Acetamido-2-deoxy-D-glucose (GlcNAc) and D-Glucose aldehydic acid (GlcA) disaccharides repeating unit, be distributed widely in the pod membrane of animal tissue cell's interstitial and some bacterium.Along with deepening continuously of the aspect researchs such as the domestic and international distribution to HA, chemical structure and physico-chemical property, find that HA has multiple biological function, as participated in lung and vascular disease and embryoplastic intracellular signaling, wound healings etc., make it have the extensively using value of uniqueness in fields such as food, medicine and makeup.Research also finds, the size that HA plays physiological action and its molecular weight is closely related, and LMW HA oligosaccharides promotes vasculogenesis, and HMW HA is inhibiting angiogenesis then.HA oligosaccharides as antagonist can with CD44 receptors bind, accelerate the cracking of CD44, thus reduce drug-resistant effect, the resistance of Tumor suppression.HA oligosaccharides, by promoting the expression of tumor suppressor gene (PTEN), suppresses the growth of PI3K/AKT signal transduction pathway and inhibition tumor cell.In addition, HA oligosaccharides can promote wound healing, inducing macrophage expresses multiple chemokine.
Research finds, the biomolecule action such as oligosaccharides and protein plays in the process of physiological function, and kind and its activity of the polymerization degree of oligosaccharides and reducing end or non-reducing end saccharide residue are closely related.Therefore, prepare novel structure, hyaluronic acid oligosaccharides that the polymerization degree is clear and definite, for the research of hyaluronic acid oligosaccharides structure activity relationship, find that the activity that hyaluronic acid oligosaccharides is new and the mode of action have great importance.
The preparation method of disclosed low-molecular-weight hyaluronic acid mainly contains enzymolysis process, Physical and chemical method at present.Wherein prepare low-molecular-weight hyaluronic acid with enzyme process maximum, Chinese patent application (publication No.: CN102690847A, CN101294179A, CN102876748A, CN103255187A, CN104178539A, CN101020724A) disclose enzyme process and prepare low-molecular-weight method.Chinese patent application (publication No.: CN104059166A, CN103993022A) discloses prepares the hyaluronic method of oligomerization from hyaluronic acid fermentation liquor.Chemical process prepares hyaluronic acid also has report, and Chinese patent application (publication No.: CN102174122A, publication number: CN1563108A) utilizes hyaluronic acid heat and sour unstable characteristic, prepares low-molecular-weight hyaluronic acid.Chinese patent application (publication No.: CN102850467A) discloses the method that low-molecular-weight hyaluronic acid is prepared in base catalysis in organic solvent.Chinese patent application (publication number: CN101429255A) discloses the method that acid catalysis in organic solvent prepares low-numerator sodium hyaluronate.The report that mechanical means prepares low-molecular-weight hyaluronic acid has, Chinese patent application (publication No.: CN101942037A) disclose mechanical milling method prepare molecular weight lower than 10,000 the method for low-molecular-weight hyaluronic acid.Chinese patent application (publication No.: CN102630231A) discloses and utilizes gac to decompose the method preparing low-molecular-weight hyaluronic acid.Low-molecular-weight hyaluronic acid prepared by these methods is mixture, and range of molecular weight distributions is wide, and structure is indefinite.Although Chinese patent application (publication No.: CN102304193A) discloses in acidic solution prepare the hyaluronic acid oligosaccharides method that the polymerization degree is 1-4, but hyaluronic acid oligosaccharide structure prepared by the method is indefinite, and the oligosaccharides polymerization degree is little, range of application is restricted.The Unidasa that Chinese patent application (publication No.: CN103484513A) utilizes leech to originate prepares the method for tetrose, six sugar, eight sugar and ten sugar, the hyaluronic acid oligosaccharides polymerization degree prepared by the method is even number, and its oligosaccharide structure sequence is unclear.Therefore, prepare specified molecular weight by simple process, mass-producing, the method for serial odd number hyaluronic acid oligosaccharide monomer that structure is clear and definite has not yet to see relevant report.
Summary of the invention
For Problems existing in current hyaluronic acid oligosaccharide monomer preparation, the object of this invention is to provide a kind of hyaluronic acid odd number oligosaccharide monomer and acid degradation preparation method thereof, it can make up above-mentioned deficiency of the prior art.The present invention is the new hyaluronic acid oligosaccharides solving the specific odd number polymerization degree of preparation, adopts diluted acid or solid phase acid hydrolysis, prepares serial hyaluronic acid oligosaccharides in conjunction with the flash liberation of low pressure gel chromatography, and product structure is clear and definite, inclusion-free remains, and easily realizes industrialization.
For achieving the above object, the present invention is achieved by the following technical solutions:
A kind of hyaluronic acid odd number oligosaccharide monomer, its general structure is as follows:
N=0 ~ 10 in wherein said structural formula, R=H or Na or K or NH
4, the reducing end of described hyaluronic acid odd number oligosaccharide monomer and non-reducing end are metal-salt or the ammonium salt of glucuronic acid or glucuronic acid.
Present invention also offers the preparation method of described hyaluronic acid odd number oligosaccharide monomer, it comprises the following steps:
(1) acid degradation: by molecular weight be the high molecular weight hyaluronic acid aqueous solution of 50 ~ 1000kDa under heated and stirred with diluted acid degraded or degrade with hydrogen type cation exchange resin, the volume ratio consumption of described diluted acid is 5 ~ 20 times of hyaluronic acid quality, the quality of described hydrogen type cation exchange resin is 1 ~ 5 times of hyaluronic acid quality, filter, neutralize with alkali after filtrate cooling, concentrated, add organic solvent deposit, centrifuged supernatant concentrating under reduced pressure obtains acid hydrolysis oligosaccharide mixture;
(2) gel column is separated: be separated by step (1) described acid hydrolysis oligosaccharide mixture low pressure gel chromatographic columns, take salts solution as eluent, Composition distribution on-line checkingi, collect according to elution peak, reduced vacuum concentrates postlyophilization respectively, obtains the hyaluronic acid odd number oligosaccharide monomer of different polymerization degree.
Further: the mass volume ratio concentration that in described step (1), during acid degradation, high molecular weight hyaluronic acid is water-soluble is 5% ~ 20%.
Further: in described step (1), the temperature of acid degradation is 50 DEG C ~ 100 DEG C, reaction times 0.5h ~ 12h.
Further: in described step (1), diluted acid is at least one in hydrochloric acid, sulfuric acid, nitric acid, acetic acid, formic acid, oxalic acid, citric acid or trifluoroacetic acid, and the final concentration of described diluted acid is 0.01mol/L ~ 2.0mol/L.
Further: in described step (1), hydrogen type cation exchange resin is strongly acidic styrene type cation exchange resin.
Further: in described step (1), alkali is at least one in sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or ammonium hydroxide.
Further: in described step (1), organic solvent is methyl alcohol, ethanol, Virahol or acetone, and the volume ratio final concentration of organic solvent is 30% ~ 80%.
Further: described step (2) mesolow gel chromatography column packing is Superdex30, BioGelP10, BioGelP6 or BioGelP4.
Further: in described step (2), eluent is the aqueous solution of 0.05mol/L ~ 0.5mol/L bicarbonate of ammonia or ammonium acetate.
Compared with prior art, advantage of the present invention and positively effect are:
(1) the present invention adopts gel column chromatography separation to prepare hyaluronic acid oligosaccharide monomer, flash liberation can obtain the hyaluronic acid odd number oligosaccharide monomer of the polymerization degree 3 ~ 23, without the need to being separated further with ion exchange chromatography again, greatly simplify technological operation, improve efficiency prepared by oligosaccharide monomer.
(2) the present invention adopts bicarbonate of ammonia or ammonium acetate to be that hyaluronic acid odd number oligosaccharide monomer is prepared in the separation of elutriant gel column, obtain oligosaccharide monomer through concentrating under reduced pressure or lyophilize can remove the salt in moving phase repeatedly, overcome the problem of oligosaccharide monomer especially low polymerization degree oligosaccharide monomer desalination difficulty, improve the quality of product.
(3) the hyaluronic acid oligosaccharides polymerization degree that prepared by the present invention is odd number, fill up blank prepared by hyaluronic acid odd number oligosaccharides, and prepared oligosaccharides reducing end and non-reducing end are GlcA, the research for hyaluronic acid oligosaccharides structure activity relationship has obvious advantage.
(4) the hyaluronic acid odd number oligosaccharide monomer novel structure prepared of the present invention, stable in properties, can be used for preparing oligosaccharides standard reagent, or is prepared into oligosaccharides healthcare products or oligosaccharides medicine further as biologically active substance or intermediate.
(5) hyaluronic acid odd number oligosaccharide monomer provided by the invention has preparation technology simply, harmless, pollution-free, the feature of easy industrialization.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, other advantage and disadvantage of the present invention will become more clear.
Accompanying drawing explanation
Fig. 1 is the low pressure gel column chromatography separation graph that the present invention prepares hyaluronic acid odd number oligosaccharide monomer.
Fig. 2 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid trisaccharide.
Fig. 3 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid pentasaccharides.
Fig. 4 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid seven sugar.
Fig. 5 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid nine sugar.
Fig. 6 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid 11 sugar.
Fig. 7 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid ten trisaccharide.
Fig. 8 is the ESI/MS mass spectrum that the present invention prepares hyaluronic acid ten pentasaccharides.
Embodiment
Below in conjunction with drawings and Examples, technical scheme of the present invention is further described in detail.
Embodiment 1: hyaluronic acid odd number oligosaccharide monomer diluted acid degraded preparation
The diluted acid degraded preparation method of hyaluronic acid odd number oligosaccharide monomer described in the present embodiment comprises the following steps:
1, getting molecular weight is that 600kDa hyaluronic acid 75g adds heated and stirred in 1500mL distilled water and makes it dissolve completely, add sulfuric acid its final concentration is reached volume unit that in 0.01mol/L ~ 2mol/L(the present invention, diluted acid adds is mL, the unit of hyaluronic acid quality is g), 60 DEG C ~ 100 DEG C stirring in water bath hydrolysis 0.5h ~ 5h, after reaction terminates, reaction solution is cooled to room temperature, by sodium hydrate regulator solution pH value to neutral, add centrifugation after 3 times of volume ethanol, supernatant liquor reduced vacuum concentrates, and obtains hyaluronic acid oligosaccharide mixture after lyophilize.
2, get after described oligosaccharide mixture is dissolved in water and be separated with Superdex30 gel chromatographic columns, with 0.3mol/L ammonium acetate for elutriant, Composition distribution on-line checkingi, merges according to elution peak and collects, and concentrating under reduced pressure postlyophilization obtains different polymerization degree hyaluronic acid oligosaccharide monomer.
Sulfuric acid described in the present embodiment can use hydrochloric acid, nitric acid, acetic acid, formic acid, oxalic acid, citric acid or trifluoroacetic acid instead; Described sodium hydroxide can use potassium hydroxide, sodium carbonate, sodium bicarbonate or ammonium hydroxide instead; Described ethanol can use methyl alcohol, acetone or Virahol instead; Described Superdex30 gel filler can use BioGelP10 instead, BioGelP6 or BioGelP4; Described 0.3mol/L ammonium acetate eluant can use water or 0.05mol/L ~ 0.5mol/L bicarbonate of ammonia instead; The concentration of the acid of selecting in described method, temperature of reaction and time can affect molecular weight distribution and the product yield of product.
Embodiment 2: prepared by the acid degradation of hyaluronic acid odd number oligosaccharide monomer solid phase
Described in the present embodiment, the solid phase acid degradation preparation method of hyaluronic acid odd number oligosaccharide monomer comprises the following steps:
1, getting molecular weight is that 200kDa hyaluronic acid 200g adds heated and stirred in 2000mL distilled water and makes it dissolve completely, add polystyrene type strong cation-exchanging resin 400g, 50 DEG C ~ 90 DEG C water-bath 1h ~ 8h, filter, filtrate is extremely neutral by potassium hydroxide regulator solution pH value after being cooled to room temperature, add centrifugation after 4 times of volumes methanol, supernatant liquor reduced vacuum concentrates, after lyophilize hyaluronic acid oligosaccharide mixture.
2, get after described oligosaccharide mixture is dissolved in water and be separated with Bio-GelP6 gel chromatographic columns, with 0.1mol/L bicarbonate of ammonia for elutriant, Composition distribution on-line checkingi, merges according to elution peak and collects, and concentrating under reduced pressure postlyophilization obtains different polymerization degree hyaluronic acid oligosaccharide monomer.
Potassium hydroxide described in the present embodiment can use sodium hydroxide, sodium carbonate, sodium bicarbonate or ammonium hydroxide instead; Described methyl alcohol can use ethanol, acetone or Virahol instead; Described BioGelP6 gel filler can use Superdex30 instead, BioGelP10 or BioGelP4; Described elutriant 0.1mol/L bicarbonate of ammonia can use water or 0.05mol/L-0.5mol/L ammonium acetate instead; Molecular weight distribution and product yield that the amount of strong cation-exchanging resin, temperature of reaction and time can affect product is added in method.
As shown in Figure 1, adopt Superdex30 filler can be by the polymerization degree 3 ~ 23 all odd number hyaluronic acid oligosaccharides carry out good separation, BioGelP6, BioGelP10 or BioGelP4 can obtain similar separating resulting.
As shown in Figure 2, mass-to-charge ratio (m/z) 572.2 place is [M-H]
-peak, 285.6 places are [M-2H]
2-peak, molecular weight (Mr) is 573.2 dalton (Da) as calculated, for hyaluronic acid trisaccharide actual measurement molecular weight, consistent with its theoretical molecular 573.2Da, be made up of 2 GlcA and 1 2-Acetamido-2-deoxy-D-glucoses (GlcNAc), its structure sequence is GlcA-GlcNAc-GlcA.
As shown in Figure 3, mass-to-charge ratio 951.3 place is [M-H]
-peak, 475.2 places are [M-2H]
2-peak, 316.4 places are [M-3H]
3-peak, Mr is 952.3Da as calculated, and be hyaluronic acid pentasaccharides actual measurement molecular weight, consistent with its theoretical molecular 952.3Da, be made up of 3 GlcA and 2 GlcNAc, its structure sequence is GlcA-GlcNAc-GlcA-GlcNAc-GlcA.
As shown in Figure 4, mass-to-charge ratio 664.7 place is [M-2H]
2-peak, 442.8 places are [M-3H]
3-peak, 331.9 places are [M-4H]
4-peak, Mr is 1331.4Da as calculated, and be hyaluronic acid seven sugar actual measurement molecular weight, consistent with its theoretical molecular 1331.4Da, be made up of 4 GlcA and 3 GlcNAc, its structure sequence is GlcA-GlcNAc-[GlcA-GlcNAc]
2-GlcA.
As shown in Figure 5, mass-to-charge ratio 569.2 place is [M-3H]
3-peak, 426.6 places are [M-4H]
4-peak, 341.5 places are [M-5H]
5-peak, Mr is 1710.6Da as calculated, and be hyaluronic acid nine sugar actual measurement molecular weight, conform to its theoretical molecular 1710.5Da, be made up of 5 GlcA and 4 GlcNAc, its structure sequence is GlcA-GlcNAc-[GlcA-GlcNAc]
3-GlcA.
As shown in Figure 6, mass-to-charge ratio 695.6 place is [M-3H]
3-peak, 521.4 places are [M-4H]
4-peak, 416.9 places are [M-5H]
5-peak, Mr is 2089.8Da as calculated, and be hyaluronic acid 11 sugar actual measurement molecular weight, conform to its theoretical molecular 2089.6Da, be made up of 6 GlcA and 5 GlcNAc, its structure sequence is GlcA-GlcNAc-[GlcA-GlcNAc]
4-GlcA.
As shown in Figure 7, mass-to-charge ratio 822.0 place is [M-3H]
3-peak, 616.2 places are [M-4H]
4-peak, 493.0 places are [M-5H]
5-peak, Mr is 2469.0Da as calculated, and be hyaluronic acid ten trisaccharide actual measurement molecular weight, conform to its theoretical molecular 2468.7Da, be made up of 7 GlcA and 6 GlcNAc, its structure sequence is GlcA-GlcNAc-[GlcA-GlcNAc]
5-GlcA.
As shown in Figure 8, mass-to-charge ratio 948.7 place is [M-3H]
3-peak, 711.2 places are [M-4H]
4-peak, 568.8 places are [M-5H]
5-peak, 473.8 places are [M-6H]
6-peak, Mr is 2848.8Da as calculated, and be hyaluronic acid ten pentasaccharides actual measurement molecular weight, conform to its theoretical molecular 2847.8Da, be made up of 8 GlcA and 7 GlcNAc, its structure sequence is GlcA-GlcNAc-[GlcA-GlcNAc]
6-GlcA.
The present invention utilizes acid degradation high molecular weight hyaluronic acid and attached gel pillar layer separation obtains serial hyaluronic acid odd number oligosaccharide monomer, and this preparation method's technique is simple, environmental protection, and cost is low, and easy industrialization is produced.
In hyaluronic acid odd number oligosaccharide monomer molecule prepared by the present invention, reducing end and non-reducing end are saturated glucuronic acid (GlcA), and namely all oligosaccharide monomers all have [GlcA-GlcNAc]
nthe repeating structure feature of-GlcA, its structure and the standby even number hyaluronic acid oligosaccharide structure ([GlcA-GlcNAc] of Unidasa legal system
n) or hyaluronic acid even number oligosaccharide structure (the △ GlcA-[GlcNAc-GlcA] for preparing of chondroitinase
n-GlcNAc, wherein △ GlcA is unsaturated glucuronic acid) obviously different, the oligosaccharide compound of these novel structures, not only enrich the diversity of oligosaccharide structure, and be of great significance for carrying out the tool such as the generation of different types of structure hyaluronic acid oligosaccharides and disease, the relation of development.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. a hyaluronic acid odd number oligosaccharide monomer, is characterized in that its general structure is as follows:
N=0 ~ 10 in wherein said structural formula, R=H or Na or K or NH
4, the reducing end of described hyaluronic acid odd number oligosaccharide monomer and non-reducing end are metal-salt or the ammonium salt of glucuronic acid or glucuronic acid.
2. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 1, it is characterized in that, it comprises the following steps:
(1) acid degradation: by molecular weight be the high molecular weight hyaluronic acid aqueous solution of 50 ~ 1000kDa under heated and stirred with diluted acid degraded or degrade with hydrogen type cation exchange resin, the volume ratio consumption of described diluted acid is 5 ~ 20 times of hyaluronic acid quality, the quality of described hydrogen type cation exchange resin is 1 ~ 5 times of hyaluronic acid quality, filter, neutralize with alkali after filtrate cooling, concentrated, add organic solvent deposit, centrifuged supernatant concentrating under reduced pressure obtains acid hydrolysis oligosaccharide mixture;
(2) gel column is separated: be separated by step (1) described acid hydrolysis oligosaccharide mixture low pressure gel chromatographic columns, take salts solution as eluent, Composition distribution on-line checkingi, collect according to elution peak, reduced vacuum concentrates postlyophilization respectively, obtains the hyaluronic acid odd number oligosaccharide monomer of different polymerization degree.
3. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: the mass volume ratio concentration that in described step (1), during acid degradation, high molecular weight hyaluronic acid is water-soluble is 5% ~ 20%.
4. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: in described step (1), the temperature of acid degradation is 50 DEG C ~ 100 DEG C, reaction times 0.5h ~ 12h.
5. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, it is characterized in that: in described step (1), diluted acid is at least one in hydrochloric acid, sulfuric acid, nitric acid, acetic acid, formic acid, oxalic acid, citric acid or trifluoroacetic acid, and the final concentration of described diluted acid is 0.01mol/L ~ 2.0mol/L.
6. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: in described step (1), hydrogen type cation exchange resin is strongly acidic styrene type cation exchange resin.
7. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: in described step (1), alkali is at least one in sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or ammonium hydroxide.
8. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: in described step (1), organic solvent is methyl alcohol, ethanol, Virahol or acetone, and the volume ratio final concentration of organic solvent is 30% ~ 80%.
9. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: described step (2) mesolow gel chromatography column packing is Superdex30, BioGelP10, BioGelP6 or BioGelP4.
10. the preparation method of hyaluronic acid odd number oligosaccharide monomer according to claim 2, is characterized in that: in described step (2), eluent is the aqueous solution of 0.05mol/L ~ 0.5mol/L bicarbonate of ammonia or ammonium acetate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510921459.XA CN105524188B (en) | 2015-12-14 | 2015-12-14 | A kind of hyaluronic acid odd number oligosaccharide monomer and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510921459.XA CN105524188B (en) | 2015-12-14 | 2015-12-14 | A kind of hyaluronic acid odd number oligosaccharide monomer and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105524188A true CN105524188A (en) | 2016-04-27 |
| CN105524188B CN105524188B (en) | 2018-04-03 |
Family
ID=55766742
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510921459.XA Active CN105524188B (en) | 2015-12-14 | 2015-12-14 | A kind of hyaluronic acid odd number oligosaccharide monomer and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105524188B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820271A (en) * | 2016-05-25 | 2016-08-03 | 苏州景卓生物技术有限公司 | Preparation and purification method of scale micromolecular sodium hyaluronate |
| CN106518934A (en) * | 2016-11-21 | 2017-03-22 | 苏州大学 | Unsaturated hyaluronic acid odd oligosaccharide and preparation method thereof |
| CN110698522A (en) * | 2019-09-19 | 2020-01-17 | 中国海洋大学 | Odd-numbered chondroitin oligosaccharide monomer and preparation method and application thereof |
| WO2020034953A1 (en) * | 2018-08-14 | 2020-02-20 | 江南大学 | Method for preparing odd-numbered hyaluronic acid oligosaccharides by double enzyme hydrolysis |
| CN113735984A (en) * | 2021-08-17 | 2021-12-03 | 苏州大学卫生与环境技术研究所 | Preparation process of carrageenan odd oligosaccharide |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563108A (en) * | 2004-04-13 | 2005-01-12 | 阮春学 | Method for preparing transparent calcium hyaIuronate in low molecular weight |
| CN101293934A (en) * | 2008-05-26 | 2008-10-29 | 江南大学 | Method for preparing small molecular weight hyaluronic acid with hydrogen phosphide and ascorbic acid oxidative degradation method |
| CN103436640A (en) * | 2013-07-12 | 2013-12-11 | 泰山医学院 | Preparation method for galactooligosaccharide composed of [-->5)beta-D-Galf(1-->] glycosyl |
| CN103484513A (en) * | 2013-10-22 | 2014-01-01 | 江南大学 | Method for preparing small-molecule oligomeric hyaluronic acid through enzyme method |
| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
| CN104059166A (en) * | 2013-03-21 | 2014-09-24 | 上海昊海生物科技股份有限公司 | Method for preparing oligomeric hyaluronic acid by hyaluronic acid broth |
| CN104388499A (en) * | 2014-11-14 | 2015-03-04 | 山东大学 | Method for preparing hyaluronan oligosaccharide chain modified flavonoid compound carrying glucuronic acid on terminal |
| CN104610467A (en) * | 2015-01-27 | 2015-05-13 | 江南大学 | Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide |
-
2015
- 2015-12-14 CN CN201510921459.XA patent/CN105524188B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563108A (en) * | 2004-04-13 | 2005-01-12 | 阮春学 | Method for preparing transparent calcium hyaIuronate in low molecular weight |
| CN101293934A (en) * | 2008-05-26 | 2008-10-29 | 江南大学 | Method for preparing small molecular weight hyaluronic acid with hydrogen phosphide and ascorbic acid oxidative degradation method |
| CN104059166A (en) * | 2013-03-21 | 2014-09-24 | 上海昊海生物科技股份有限公司 | Method for preparing oligomeric hyaluronic acid by hyaluronic acid broth |
| CN103436640A (en) * | 2013-07-12 | 2013-12-11 | 泰山医学院 | Preparation method for galactooligosaccharide composed of [-->5)beta-D-Galf(1-->] glycosyl |
| CN103484513A (en) * | 2013-10-22 | 2014-01-01 | 江南大学 | Method for preparing small-molecule oligomeric hyaluronic acid through enzyme method |
| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
| CN104388499A (en) * | 2014-11-14 | 2015-03-04 | 山东大学 | Method for preparing hyaluronan oligosaccharide chain modified flavonoid compound carrying glucuronic acid on terminal |
| CN104610467A (en) * | 2015-01-27 | 2015-05-13 | 江南大学 | Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide |
Non-Patent Citations (6)
| Title |
|---|
| CHARLES D. BLUNDELL，等: "Enzymatic and chemical methods for the generation of pure hyaluronan oligosaccharides with both odd and even numbers of monosaccharide units", 《ANALYTICAL BIOCHEMISTRY》 * |
| CHERNG-KANG PERNG等: "In Vivo Angiogenesis Effect of Porous Collagen Scaffold with Hyaluronic Acid Oligosaccharides", 《JOURNAL OF SURGICAL RESEARCH》 * |
| PETER NEBINGER，等: "HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF EVEN-AND ODD-NUMBERED HYALURONATE OLIGOSACCHARIDES", 《JOURNAL OF CHROMATOGRAPHY》 * |
| YUKO INOUE，等: "PREPARATION, BY CHEMICAL DEGRADATION OF HYALURONIC ACID, OF A SERIES OF EVEN- AND ODD-NUMBERED OLIGOSAC-CHARIDES HAVING A 2-ACETAMIDO-2-DEOXY-D-GLUCOSE AND A D-GLUCURONIC ACID RESIDUE, RESPECTIVELY, AT THE REDUCING END", 《CARBOHYDRATE RESEARCH》 * |
| 吴成玲，等: "亲水相互作用色谱-高分辨质谱分析不同糖胺聚糖寡糖", 《分析化学》 * |
| 张真庆，等: "寡糖的生物活性及海洋性寡糖的潜在应用价值", 《中国海洋药物》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105820271A (en) * | 2016-05-25 | 2016-08-03 | 苏州景卓生物技术有限公司 | Preparation and purification method of scale micromolecular sodium hyaluronate |
| CN106518934A (en) * | 2016-11-21 | 2017-03-22 | 苏州大学 | Unsaturated hyaluronic acid odd oligosaccharide and preparation method thereof |
| CN106518934B (en) * | 2016-11-21 | 2019-05-03 | 苏州大学 | Preparation method of unsaturated hyaluronic acid odd oligosaccharide |
| WO2020034953A1 (en) * | 2018-08-14 | 2020-02-20 | 江南大学 | Method for preparing odd-numbered hyaluronic acid oligosaccharides by double enzyme hydrolysis |
| CN110698522A (en) * | 2019-09-19 | 2020-01-17 | 中国海洋大学 | Odd-numbered chondroitin oligosaccharide monomer and preparation method and application thereof |
| CN113735984A (en) * | 2021-08-17 | 2021-12-03 | 苏州大学卫生与环境技术研究所 | Preparation process of carrageenan odd oligosaccharide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105524188B (en) | 2018-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105524188A (en) | Hyaluronan odd oligosaccharide monomer and preparation method thereof | |
| CN102336840B (en) | Triple-helical Tremellan, preparation method and application thereof | |
| US20210155720A1 (en) | Method for Preparing Hyaluronan Odd-numbered Oligosaccharides by Double Enzyme Hydrolysis | |
| Wende et al. | Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods | |
| CN103059163B (en) | Method for preparing alginate oligosaccharide monomers by using microwave radiation | |
| CN108530561B (en) | Method for extracting high-purity heparan sulfate from heparin production waste | |
| CN107739417A (en) | A kind of preparation method of Cationized hyaluronic acid | |
| CN106349407A (en) | Low-molecular-weight fucosylated chondroitin sulfate, preparation method thereof and application of low-molecular-weight fucosylated chondroitin sulfate to preparation of medicine for resisting Trousseau syndrome | |
| CN104498564A (en) | Low molecular weight chondroitin sulfate preparation method | |
| CN106432538A (en) | Method for preparing chitin oligosaccharides, chitooligosaccharides and chitosan oligosaccharides | |
| CN102807630B (en) | Low-molecular-weight chitosan and glucosamine co-production technology | |
| CN113603732B (en) | Non-animal chondroitin sulfate oligosaccharide and preparation method thereof | |
| CN102020724A (en) | Method for extracting sodium hyaluronate from fermentation liquor containing hyaluronic acid | |
| RU2670767C1 (en) | Method for producing low molecular weight heparin | |
| CN101422415A (en) | Moisture-absorption humectant and preparation method thereof | |
| CN107987179B (en) | Application of low-sulfated fucan in preparation of immunopotentiator | |
| CN110698522B (en) | Odd-numbered chondroitin oligosaccharide monomer and preparation method and application thereof | |
| CN102993244A (en) | Oligorhamnose monomer and preparation method thereof | |
| CN107011456B (en) | A kind of green algae polysaccharide and preparation method thereof | |
| CN100999537A (en) | Even agar gel oligo sugar alcohol monomer and preparation process thereof | |
| CN101279990A (en) | Iota-carrageenan even oligosacchride monomer and preparation thereof | |
| CN103864942B (en) | Middle molecular weight hydroxyethyl starch and method of purification thereof | |
| CN104877046A (en) | Preparation method of 3-site substituted benzoyl-beta-cyclodextrin | |
| CN103436640A (en) | Preparation method for galactooligosaccharide composed of [-->5)beta-D-Galf(1-->] glycosyl | |
| Saravanan et al. | Partial sequencing, structural characterization, and anticoagulant activity of heparan sulfate and sulfated chitosan from selected Indian marine mollusks |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |