CN105801723B - A kind of method for quickly purifying of marine sulfate polysaccharide - Google Patents
A kind of method for quickly purifying of marine sulfate polysaccharide Download PDFInfo
- Publication number
- CN105801723B CN105801723B CN201610224470.5A CN201610224470A CN105801723B CN 105801723 B CN105801723 B CN 105801723B CN 201610224470 A CN201610224470 A CN 201610224470A CN 105801723 B CN105801723 B CN 105801723B
- Authority
- CN
- China
- Prior art keywords
- vacuum filtration
- ocean
- sulfuric acid
- thick many
- many candies
- 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.)
- Active
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
- C08B37/0069—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
- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Dermatology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a kind of method for quickly purifying of marine sulfate polysaccharide, ocean sulfuric acid Thick many candies are dissolved in phosphate-buffered salt by this method first, then pour into ion exchange resin filler, after after ocean, sulfuric acid Thick many candies are adsorbed in filler, with phosphoric acid buffer eluting salt, vacuum filtration；Low concentration sodium chloride solution is added, is eluted, vacuum filtration；High concentration sodium chloride solution is added, is eluted, vacuum filtration；Finally centrifuge, alcohol precipitation, dialysis freezes after one day, obtains marine sulfate polysaccharide.The present invention has following technical characterstic：Simplify purification step, save purification time, improve purification efficiency；Organic extraction solvent is not introduced, reduces cost, non-environmental-pollution；Method is stable, favorable reproducibility；Equipment is simple, and condition is easily controlled.
Description
Technical field
The present invention relates to the technical field of purification of marine sulfate polysaccharide, and QFF (Q are utilized more particularly to one kind
Sepharose Fast Flow anion-exchange, Sepharose anion exchanger resin) strong anion filler and decompression take out
Filter the method that device rapidly purifies sulfated polysaccharide.
Background technology
Sulfated polysaccharide is rich in the sulfuric acid mollusk of ocean, and structure often has height repetitive structure sequence, is easy to structure
Effect relation probe into and quality control.Wherein, sea cucumber is one of source of current main marine sulfate polysaccharide.Sea cucumber mainly has
Two kinds of sulfated polysaccharide：Sea cucumber chondroitin sulfate (Sea Cucumber Chondroitin Sufate, SC-CHS) and sea
It is cruel (sea Cucumber Fucan, SC-FUC) to join sulfated fucan.Wherein, sea cucumber chondroitin sulfate is a kind of band fucose
The acid mucopolysaccharide of side chain, the molecule being mainly made up of the acetylamino galactosamines of D mono-, the glucuronic acids of D mono- and the fucoses of L mono- are miscellaneous
Polysaccharide, it is the important component of sea cucumber polysaccharide.Sea cucumber chondroitin sulfate has reducing blood lipid and the physiologically active such as antitumor, clinically may be used
For a variety of diseases such as prevention of arterial atherosclerosis；Sea cucumber sulfated fucan is extremely that the straight chain being made up of L-fucose is more
Sugar.The sugar composition of the two is different, has the light base in part to occur Sulfation on sugar chain, the special construction of two kinds of sea cucumber polysaccharides,
It is peculiar for sea cucumber.Being rich in other sea urchin has fucoidan, and simple in construction, and repeatability is high.Marine sulfate polysaccharide
There is the spies such as the polytropy of the inhomogeneities of the inconsistency of degree, microstructure and molecular weight and residue quantity
Point, cause the complexity of marine sulfate polysaccharide purifying and structural analysis.
The extracting method of sulfated polysaccharide is usually to digest, and halogenated phosphates precipitation, is classified alcohol precipitation.The second of low concentration
Alcohol (<40%) chondroitin sulfate can be precipitated out, the ethanol of high concentration (>60%) fucoidan can be settled out
Come.And it is polysaccharide crude extract to be obtained by above method, the sulfated polysaccharide with compared with high bioactivity is obtained, also to be divided
From and purifying, the separation of polysaccharide and purifying mainly include two steps, first, non-polysaccharide component in separating extractive, such as miscellaneous egg
In vain, the impurity such as pigment, oligosaccharide and some micromolecular compounds, second, the polysaccharide very wide to molecular weight ranges is classified, is obtained
To the holosaccharide with certain molecular weight scope.
Protein isolate matter method mainly has the chemistry side such as Sevag methods, trifluorotrichloroethane method, trichloroacetic acid method from polysaccharide
Method.Sevag methods are the most commonly used in the lab, but it is disadvantageous in that：Technological process is longer；Polysaccharide loss is more；Once
A small amount of protein can only be removed, generally requiring repeated multiple times could eliminate protein；Solvent uses volatile chloroform, consumption
Amount is big, is hazardous to the human body, and pollutes environment.The Deproteinated efficiency of trifluorotrichloroethane method is higher, but also because its is volatile, it is unsuitable
It is a large amount of to use.The sulfated polysaccharide extracted from sea mollusk, due to the enzymolysis process of early stage, in resulting acidic polysaccharose
The content of albumen is not high.
Contain a large amount of pigments in marine animal, this just brings very big difficulty to the purifying of marine sulfate polysaccharide, not only to dividing
The photometric degree of accuracy of light has a significant impact, but also has a strong impact on the quality of finished product.Existing method mainly uses activated carbon
Adsorption bleaching, this method decolorizing effect is general, and activated carbon adsorbs seriously to polysaccharide.H2O2Although decolorizing effect is pretty good, can drop
Polysaccharide is solved, so as to influence the bioactivity of polysaccharide, therefore to be considered carefully using its decolouring.Decolourize to purify for acidic polysaccharose,
It is most common method that cellulose anion, which exchanges column chromatography,.However, time-consuming for the method for chromatographic column, purifying rate it is low (<25%),
Can not meet the needs of largely producing.
The content of the invention
The technical problem to be solved in the present invention is in view of the shortcomings of the prior art, there is provided a kind of marine sulfate polysaccharide it is quick
Purification process.The present invention, which has, simplifies purification step, saves purification time, improves purification efficiency；Organic extraction solvent is not introduced,
Reduce cost, non-environmental-pollution；Method is stable, favorable reproducibility；The characteristics of equipment is simple, and condition is easily controlled.
In order to solve the above-mentioned technical problem, the present invention provides a kind of method for quickly purifying of marine sulfate polysaccharide, this method
Comprise the following steps：
(1), ocean sulfuric acid Thick many candies are dissolved in phosphate-buffered salt, obtain the aqueous solution of ocean sulfuric acid Thick many candies；
(2), the aqueous solution for the ocean sulfuric acid Thick many candies that step 1 obtains is poured into QFF fillers, ocean sulfuric acid Thick many candies with
The mass volume ratio of QFF fillers is 1/100-200 (g/ml)；After after ocean, sulfuric acid Thick many candies are adsorbed in QFF fillers, delayed with phosphoric acid
Eluting salt is rushed, is filtered by vacuum；
(3) add low concentration sodium chloride solution in the mixture after, being filtered by vacuum into step 2, elute, vacuum filtration；
(4) high concentration sodium chloride solution in the mixture after, being filtered by vacuum into step 3, elute, vacuum filtration；
(5) the holosaccharide liquid for collecting to obtain after, being filtered by vacuum in step 4 centrifuges, alcohol precipitation, and dialysis freezes after one day, obtains
Marine sulfate polysaccharide；
In the step 1 and step 2, the phosphate-buffered salt is the KH that concentration is 01M2PO4The aqueous solution, pH value is
6.3；In the step 3, the low concentration sodium chloride solution is the aqueous solution for the sodium chloride that concentration is 0-0.5M；The step 4
In, the high concentration sodium chloride solution is the aqueous solution for the sodium chloride that concentration is 1.5-4M；In the step 5, bag filter is used
Dialysis.
The present invention has the advantages that：Marine sulfate polysaccharide purification process such as Sevag methods, three of the invention and traditional
Trifluorotrichloroethane method, trichloroacetic acid method removing protein, activated carbon, H2O2Adsorption bleaching, ethanol precipitation method, quaternary salt deposit method,
Cellulose anion exchange column chromatography, gel filtration chromatography classification polysaccharide compare, only need a step can remove foreigh protein removing, depigmentation and
Polysaccharide is classified, organic solvent need not be added while simplifying operation, saves purification time, and do not interfere with the biology of sulfated polysaccharide
Activity, be advantageous to purify the quick preparation of marine sulfate polysaccharide.The method also has favorable reproducibility simultaneously, easy to operate, cost is low
The characteristics of.
Brief description of the drawings
Fig. 1 is that Superparamagnetic iron-oxide chondroitin sulfate is schemed through the GPC of quick elution method after purification；
Fig. 2 is that Superparamagnetic iron-oxide fucoidan is schemed through the GPC of quick elution method after purification.
Embodiment
The present invention is further described below according to embodiment.
Embodiment 1：The fast purifying of Superparamagnetic iron-oxide chondroitin sulfate：
2g is completely dissolved in 50ml phosphoric acid buffers through digesting the Superparamagnetic iron-oxide chondroitin sulfate Thick many candies of 40% ethanol alcohol precipitation
In salt (pH6.3), the aqueous solution of sea cucumber sulfuric acid Thick many candies is obtained；200mlQFF fillers are filled in 500ml sand core funnels；
50ml Thick many candies solution is poured into sand core funnel, after polysaccharide is adsorbed in QFF, with phosphoric acid buffer eluting salt 400ml, connects vacuum
Pumping filter filters；Then mixture in sand core funnel adds 0.1M sodium chloride solutions into sand core funnel, elute 400ml, connect
Vavuum pump filters；Add 1.5M sodium chloride solutions in mixture after suction filtration in sand core funnel, connect vavuum pump suction filtration, co-elute
600ml；Obtained holosaccharide liquid centrifugation, alcohol precipitation are collected, dialysis freezes after one day.Holosaccharide 1.2g is obtained after dialysis, purifies
Yield more than 60%.The gel chromatography figure of Superparamagnetic iron-oxide chondroitin sulfate is as shown in figure 1, be single peak after purification, molecule
Measure for 7.3KDa, and compared with the Superparamagnetic iron-oxide chondroitin sulfate that Anion-adsorption chromatographic column traditional before obtains, purity
Unanimously, all it is single gel chromatography peak.
Embodiment 2：The fast purifying of Superparamagnetic iron-oxide fucoidan：
2g is completely dissolved in 50ml phosphoric acid through digesting the Superparamagnetic iron-oxide fucoidan Thick many candies of 60% ethanol alcohol precipitation
In buffer salt (pH6.3), the aqueous solution of sea cucumber sulfuric acid Thick many candies is obtained；400mlQFF fillers are filled in 500ml sand core funnels
In；50ml Thick many candies solution is poured into sand core funnel, after polysaccharide is adsorbed in QFF, with phosphoric acid buffer eluting salt 400ml, is connected
Vavuum pump filters；Then mixture in sand core funnel adds 0.5M sodium chloride solutions into sand core funnel, elute 400ml,
Connect vavuum pump suction filtration；Add 3.8M sodium chloride solutions in mixture after suction filtration in sand core funnel, connect vavuum pump suction filtration, wash altogether
De- 600ml；Obtained holosaccharide liquid centrifugation, alcohol precipitation are collected, dialysis freezes after one day.Holosaccharide 1.35g is obtained after dialysis, it is pure
Change yield more than 67.5%.The gel chromatography figure of Superparamagnetic iron-oxide fucoidan is as shown in Fig. 2 to be single after purification
Peak, molecular weight 32KDa, and the Superparamagnetic iron-oxide fucoidan obtained with Anion-adsorption chromatographic column traditional before
Compare, purity is consistent, is all single gel chromatography peak.
Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (3)
1. a kind of method for quickly purifying of marine sulfate polysaccharide, it is characterised in that this method comprises the following steps：
（1）, ocean sulfuric acid Thick many candies are dissolved in phosphate-buffered salt, obtain the aqueous solution of ocean sulfuric acid Thick many candies；
（2）, by step（1）The aqueous solution of obtained ocean sulfuric acid Thick many candies is poured into Sepharose anion exchanger resin filler,
The mass volume ratio of ocean sulfuric acid Thick many candies and filler is 1/20-1/400 g/ml；Treat that ocean sulfuric acid Thick many candies are adsorbed in agar
After sugared anion exchange resin filler, with phosphoric acid buffer eluting salt, vacuum filtration；
（3）, to step（2）Add 0-0.5M sodium chloride solutions in mixture after middle vacuum filtration, elute, vacuum filtration；
（4）, to step（3）Add 1.5-4M sodium chloride solutions in mixture after middle vacuum filtration, elute, vacuum filtration；
（5）, by step（4）Obtained holosaccharide liquid centrifugation, alcohol precipitation are collected after middle vacuum filtration, dialysis is freezed after one day, obtained extra large
Foreign sulfated polysaccharide.
2. the method for quickly purifying of marine sulfate polysaccharide according to claim 1, it is characterised in that the step（1）And step
Suddenly（2）In, the phosphate-buffered salt is the KH that concentration is 0.1M2PO4The aqueous solution, pH value 6.3.
3. the method for quickly purifying of marine sulfate polysaccharide according to claim 1, it is characterised in that the step（5）In, make
Dialysed with bag filter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610224470.5A CN105801723B (en) | 2016-04-12 | 2016-04-12 | A kind of method for quickly purifying of marine sulfate polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610224470.5A CN105801723B (en) | 2016-04-12 | 2016-04-12 | A kind of method for quickly purifying of marine sulfate polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105801723A CN105801723A (en) | 2016-07-27 |
| CN105801723B true CN105801723B (en) | 2018-02-09 |
Family
ID=56459283
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610224470.5A Active CN105801723B (en) | 2016-04-12 | 2016-04-12 | A kind of method for quickly purifying of marine sulfate polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105801723B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113912745A (en) * | 2021-08-12 | 2022-01-11 | 浙江大学 | Separation and purification method of glycosaminoglycan and preparation method of sulfated oligosaccharide |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555054A (en) * | 2009-05-19 | 2009-10-14 | 山东齐东方投资有限公司 | Method for extracting microbial polysaccharide flocculant by membrane concentration and pneumatic drying method |
| CN102838692A (en) * | 2011-06-24 | 2012-12-26 | 谭科 | Extraction method for heparin sodium |
| CN103992415B (en) * | 2014-05-06 | 2016-01-20 | 广州医科大学附属第一医院 | The method of the extraction purification of an authentic gas soup polysaccharide |
| CN104031158B (en) * | 2014-06-10 | 2016-08-17 | 浙江宇翔生物科技有限公司 | A kind of technique extracting polysaccharide sulfate from Thallus Laminariae (Thallus Eckloniae) |
-
2016
- 2016-04-12 CN CN201610224470.5A patent/CN105801723B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105801723A (en) | 2016-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102775523B (en) | Process for preparing high-purity low-molecular heparin sodium | |
| CN105294874B (en) | A kind of method for efficiently separating Black Ganoderma immunocompetence polysaccharide | |
| CN105175566B (en) | Polyamide column and macroporous resin column connection post method remove the method for protein and pigment in Radix Panacis Quinquefolii polysaccharide extract | |
| CN105801723B (en) | A kind of method for quickly purifying of marine sulfate polysaccharide | |
| CN101698683B (en) | Method for extracting polysaccharides from ovidutus ranae | |
| CN112679564B (en) | Method for separating and purifying specific compound arginine glycoside AF of ginseng | |
| JP2023542661A (en) | Method for separating and purifying p-coumaroylated anthocyanin | |
| CN106967186B (en) | A method of extracting heparin-like compound from air bladder | |
| CN110256597B (en) | Method for reducing heavy metal residues in ganoderma lucidum polysaccharide by membrane method | |
| CN108727328A (en) | A kind of high efficiency extraction and purification process of blueberry anthocyanin | |
| CN101089017A (en) | Process of separating and purifying melittin | |
| CN108822164A (en) | The preparation process of high quality monosialotetrahexose ganglioside sodium | |
| CN105055540B (en) | The technique that macroporous absorbent resin isolates and purifies polygonum flaccidum flavones | |
| CN104984739B (en) | A kind of preparation method and applications of gelatin affinity chromatography medium | |
| CN1253446C (en) | Method for preparing high content proanthocyanidin | |
| CN113440547B (en) | Method for separating and purifying Japanese thistle herb total glycosides by adopting macroporous resin series dynamic axial compression column | |
| CN109400743B (en) | Method for extracting sulfated galactan from sea grapes | |
| CN114057907A (en) | Method for extracting, separating and purifying red ginseng polysaccharide | |
| CN113912745A (en) | Separation and purification method of glycosaminoglycan and preparation method of sulfated oligosaccharide | |
| CN102617727B (en) | Thymalfasin compound and novel preparation method thereof | |
| CN107098985B (en) | A method for extracting acidic polysaccharides from residue of radix Ginseng after ethanol extraction | |
| CN106046066B (en) | A method of purifying prepares high-purity xylobiose | |
| CN116675784B (en) | Oyster glycosaminoglycan with alpha-glucosidase inhibition effect and preparation method thereof | |
| CN105037451B (en) | A kind of method of hyperfiltration treatment flavomycoin zymotic fluid | |
| CN103130916A (en) | Novel technology for extracting high purity heparin sodium from intestinal mucosa |
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 |