CN101100688A - Method for extracting and purifying dermatan sulfate in angler fish skin - Google Patents
Method for extracting and purifying dermatan sulfate in angler fish skin Download PDFInfo
- Publication number
- CN101100688A CN101100688A CNA2007100699590A CN200710069959A CN101100688A CN 101100688 A CN101100688 A CN 101100688A CN A2007100699590 A CNA2007100699590 A CN A2007100699590A CN 200710069959 A CN200710069959 A CN 200710069959A CN 101100688 A CN101100688 A CN 101100688A
- Authority
- CN
- China
- Prior art keywords
- add
- dermatan sulfate
- liquid
- water
- supernatant liquor
- 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
Landscapes
- Peptides Or Proteins (AREA)
Abstract
It is concerned with extraction and purification of sulfuric acid skin-lotion from waste skins of angler fish production. It is carried out by: extracting with compounded enzyme of pancreatin and papain, adding NaOH into extracting liquid to cut off glycopeptide bond while protecting glycosidic bond with NaPH4, removing protein by savage process, depositing sulfur acid skin lotion in alcohol, dissolving in water and centrifuging dissoluble substances, dialyzing to remove ions and micro-molecular substances, and frozen drying high purity sulfur acid skin lotion with ratio of sulfate radical to carboxylic radical = 1.07. The product is strong antithrombotic, with less side effect of hemorrhage, has disinfecting activity, and can be used in pharmaceutical field.
Description
Technical field
The invention belongs to biological pharmacy technical field, relate to the preparation of the biological medicine in animal body source, the extraction and purification method of dermatan sulfate in the Ju Ti Wei angler skin.
Background technology
Dermatan sulfate (DS) belongs to the mucopolysaccharide compound, it is topmost glycosaminoglycan in the animal skin, its molecule mainly is made up of the disaccharide chain that N-acetylgalactosamine 4-sulfuric acid and L-idose aldehyde constitute, and also has the disaccharide chain of D-glucuronic acid disaccharide chain, N-acetylgalactosamine 6-sulfuric acid and D-glucal formation etc. in addition.It has stronger anticoagulant active, transfers effects such as blood fat, anti-inflammatory, atherosclerosis in addition.It is strong that it is had an anti-bolt effect as medicine, and the characteristics of long half time are compared with heparin, and its outstanding advantage is there is not hemorrhage side effect, patient's better tolerance.Prove that through research of the experimental pharmacologies in nearly ten years and clinical trial dermatan sulfate is a non-heparin class antithrombotic agent safely and effectively.
For a long time, pigskin is the main raw material of preparation dermatan sulfate, also is the main source of commodity dermatan sulfate.Become the another approach of preparation dermatan sulfate in recent years with the raffinate behind pig, Roll mucous membrane or ox lung separation heparin, the Suleparoid.Yet because religious belief and people can't be accepted by everyone by the feedstock production dermatan sulfate with pig or ox the fear of mad cow disease and foot and mouth disease, security also will be under suspicion.
As far back as the sixties, people (Biochemical Prep 12,5-12,1968) such as Clifonelli J A and Roden L just once utilized pigskin to be the feedstock production dermatan sulfate.Specifically be to adopt the cetylpyridinium chloride that utilizes different concns after the papoid degraded to carry out combination to obtain the dermatan sulfate crude product with parsing, afterwards that crude product is water-soluble, with sodium hydroxide and Benedict precipitation, precipitation is dissolved in the acetate, the dialysis of in the sodium hydroxide and back, dialyzate is by the strong acid type cationic resin decopper(ing), and decopper(ing) liquid transfers to PH4-5 with acetate, is settled out dermatan sulfate with 2 times of volume ethanol at last.
1996, Voler (J Chromatogram B 685,27-34,1996) be raw material with the ox lung, with processing such as acetone, trichloromethane, methyl alcohol, papoid and Collagenase enzymolysis, ion exchange resin absorption, washing, wash-out, trichoroacetic acid(TCA) removes albumen, precipitation, centrifugal repeatedly, drying obtains exquisite mucopolysaccharide, mixes in the mucopolysaccharide solution again by adding the organic solvent of different volumes, as methyl alcohol, ethanol and acetone etc., different and they are separated according to the solvability of different mucopolysaccharides.
The dermatan sulfate purity height that above method obtains, but complex operation is difficult to use in suitability for industrialized production.Also there is not at present a kind of dermatan sulfate method of purification that is suitable for suitability for industrialized production.Rarer people extracts dermatan sulfate from fish-skin.
Summary of the invention
The purpose of this invention is to provide a kind of method that purifying obtains stay-in-grade high purity dermatan sulfate of Cong the angler skin, extracting, the unserviceable practical problems of fish-skin in the Xie Jue angler processing simultaneously, and the raw material of development potentiality is more arranged at the present situation exploitation that present most of dermatan sulfate derives from pig and ox.
Yi angler skin of the present invention is a raw material; main points are that the prozyme that adopts papoid and pancreatin to form extracts under suitable solid-liquid ratio, temperature and time; under the protection of sodium borohydride, add sodium hydroxide and remove glycopeptide; remove albumen by the savage method; pass through ethanol sedimentation again; dialysis deionizing and small molecules behind the resolution of precipitate, last lyophilize obtains the high purity dermatan sulfate.
The concrete operations step is as follows:
1. Xin Xian angler skin obtains the dry product raw material through the washing postlyophilization after tissue is smashed to pieces;
2. the weightmeasurement ratio by dry product raw material and water is 1: 20-1: 30, in the dry product raw material, add distilled water, add the prozyme that papoid and pancreatin are formed again, extracted 13-19 hour in 50-75 ℃ of heating in water bath, the cooling back is centrifugal, and obtaining supernatant liquor is the dermatan sulfate crude extract;
3. add solid sodium hydroxide in the crude extract, add sodium borohydride again, sodium hydroxide and sodium borohydride were respectively 0.5mol/L and 0.1mol/L at the final concentration of crude extract, in 30-50 ℃ of water-bath 20-30 hour, the centrifuging and taking supernatant liquor transfers the supernatant liquor pH value to 6.5-7.0 with acetic acid;
4. add volume and be the protein liquid that removes of supernatant liquor volume 1/3, should remove protein liquid and form and be by volume: propyl carbinol: trichloromethane 1: 2.5-1: 4.5, fully rock 15-30 minute after, the room temperature standing demix is removed lower floor's liquid, keeps supernatant liquid;
5. with supernatant liquid by the 4. repetitive operation 4 times of above step, obtain removing protein liquid;
6. remove protein liquid and add two volumes ethanol, left standstill collecting precipitation one day in 4 ℃;
7. will precipitate fully water-solublely, and centrifugally remove insoluble composition, it is 14000 dialysis tubing deionizing and small-molecule substance that the aqueous solution adopts molecular weight, obtains the dermatan sulfate of purifying after the lyophilize.
The add-on of the prozyme of above step in 2. is the 1.0-3.5% of dry product raw material weight, and the weight ratio of components of papoid and pancreatin is 1 in the prozyme: 1-1: 3.
Above step further describes as follows:
Xin Xian angler skin obtains the dry product raw material through the washing postlyophilization after tissue is smashed to pieces.The distilled water that adds the heavy 20-30 times of volume of dry product raw material, add the prozyme that accounts for the heavy 1-3.5% of raw material simultaneously, prozyme consists of papoid and pancreatin, its ratio 1 by weight: 1-1: 3 (w: w), extracted 13-19 hour in 50-75 ℃ of heating in water bath, the cooling back is centrifugal, and obtaining supernatant liquor is the dermatan sulfate crude extract.
Add solid NaOH in the crude extract and remove glycopeptide, add NaPH simultaneously
4The protection glycosidic link makes NaOH and NaPH
4Final concentration is respectively 0.5mol/L and 0.1mol/L, mixes, and in 30-50 ℃ of water-bath 20-30 hour, the centrifuging and taking supernatant liquor transferred pH value to 6.5-7.0 with acetic acid.Supernatant liquor adopts the operation of savage method to remove albumen 5 times, leaves standstill collecting precipitation one day for 4 ℃ except that liquid behind the albumen adds two volumes ethanol.
To precipitate water-solublely, if having that insolubles is centrifugal to be removed, it is 14000 dialysis tubing deionizing and small-molecule substance that the aqueous solution adopts molecular weight, obtains the high purity dermatan sulfate after the lyophilize.
The dermatan sulfate product that present method makes after measured, sulfate and carboxylic acid group's ratio are 1.07, contain hydroxyl, carboxyl, kharophen, sulfate etc. through Infrared spectroscopy, through the gel liquid chromatography be accredited as unimodal and appearance time identical with the dermatan sulfate standard substance, be accredited as single band through cellulose acetate membrane electrophoresis, confirm that product is the high purity dermatan sulfate.
In sum, Ke Yi angler skin of the present invention is that feedstock production obtains the high purity dermatan sulfate, and method is reasonable, is fit to suitability for industrialized production.
Embodiment
Embodiment one
(1), raw material Wei angler skin dry product 10g.Add papoid 0.125g, pancreatin 0.125g, distilled water 300mL, in water-bath 70-75 ℃ of extraction 17-19 hour, the cooling back was centrifugal, obtains supernatant liquor 225mL.
(2), add sodium hydroxide 4.500g in the supernatant liquor, sodium borohydride 0.851g stirs and makes solid dissolving, NaOH and NaPH after measured
4Final concentration in supernatant liquor is respectively 0.5mol/L and 0.1mol/L, handles 24 hours for water-bath 45-50 ℃, and is centrifugal, and supernatant liquor transfers pH value to neutral with acetic acid, and liquid is 203mL altogether.
(3), (trichloromethane: (v: v)), shake well 20-25 minute, standing demix in separating funnel discarded lower floor's liquid to propyl carbinol=2: 1 after the layering except that protein liquid to add 68mL in the liquid.
(4), supernatant liquid repeating step (3) operation is 4 times.Obtain supernatant liquor 68mL.
(5), in the supernatant liquor that obtains, add 136mL ethanol, 4 ℃ left standstill one day, collecting precipitation, use the 50mL dissolved in distilled water, the centrifuging and taking supernatant liquor is loaded on molecular weight and is in 14000 the dialysis tubing the tap water dialysis two days of flowing, distill water dialysis one day, dialysis back liquid freezing drying gets high purity dermatan sulfate 0.154g.
Embodiment two
(1) raw material Wei angler skin dry product 10g.Add papoid 0.100g, pancreatin 0.250g, distilled water 300mL, in water-bath 51-56 ℃ of extraction 13-15 hour, the cooling back was centrifugal, obtains supernatant liquor 228mL.
(2) add sodium hydroxide 4.560g in the supernatant liquor, sodium borohydride 0.862g stirs and makes the solid dissolving, handles 30 hours for water-bath 30-35 ℃, and centrifugal, to neutral, liquid is 210mL altogether with the acetic acid adjust pH for supernatant liquor.
(3) (trichloromethane: (v: v)), shake well 15-20 minute, standing demix in separating funnel discarded lower floor's liquid to propyl carbinol=3: 1 after the layering except that protein liquid to add 70mL in the liquid.
(4) supernatant liquid repeating step (3) operation is 4 times.Obtain supernatant liquor 70mL.
(5) supernatant liquor adds 140mL ethanol, 4 ℃ left standstill one day, collecting precipitation, use the 50mL dissolved in distilled water, the centrifuging and taking supernatant liquor is loaded on molecular weight and is in 14000 the dialysis tubing the tap water dialysis two days of flowing, distill water dialysis one day, dialysis back liquid freezing drying gets high purity dermatan sulfate 0.161g.
Embodiment three
(1) raw material Wei angler skin dry product 10g.Add papoid 0.050g, pancreatin 0.100g, distilled water 260mL extracted 17 hours for 65 ℃ in water-bath, and the cooling back is centrifugal, obtains supernatant liquor 200mL.
(2) add in the liquid 67mL remove protein liquid (trichloromethane: propyl carbinol=4.5: 1 (v: v)), shake well 30 minutes, standing demix in separating funnel discards lower floor's liquid after the layering.
(3) supernatant liquid repeating step (2) operation is 4 times.Obtain supernatant liquor 67mL.
(4) supernatant liquor adds 134mL ethanol, 4 ℃ left standstill one day, collecting precipitation, use the 50mL dissolved in distilled water, the centrifuging and taking supernatant liquor is loaded on molecular weight and is in 14000 the dialysis tubing the tap water dialysis two days of flowing, distill water dialysis one day, dialysis back liquid freezing drying gets high purity dermatan sulfate 0.147g.
The embodiment that the present invention exemplifies is a preferred embodiments.Need to prove that in data area disclosed in this invention, the dermatan sulfate purity that this programme extracted can reach more than 95%.
Claims (2)
1. the extraction and purification method of dermatan sulfate in the angler skin is characterized in that following steps:
1. Xin Xian angler skin obtains the dry product raw material through the washing postlyophilization after tissue is smashed to pieces;
2. the weightmeasurement ratio by dry product raw material and water is 1: 20-1: 30, in the dry product raw material, add distilled water, add the prozyme that papoid and pancreatin are formed again, extracted 13-19 hour in 50-75 ℃ of heating in water bath, the cooling back is centrifugal, and obtaining supernatant liquor is the dermatan sulfate crude extract;
3. add solid sodium hydroxide in the crude extract, add sodium borohydride again, sodium hydroxide and sodium borohydride were respectively 0.5mol/L and 0.1mol/L at the final concentration of crude extract, in 30-50 ℃ of water-bath 20-30 hour, the centrifuging and taking supernatant liquor transfers the supernatant liquor pH value to 6.5-7.0 with acetic acid;
4. add volume and be the protein liquid that removes of supernatant liquor volume 1/3, should remove protein liquid and form and be by volume: propyl carbinol: trichloromethane 1: 2.5-1: 4.5, fully rock 15-30 minute after, the room temperature standing demix is removed lower floor's liquid, keeps supernatant liquid;
5. with supernatant liquid by the 4. repetitive operation 4 times of above step, obtain removing protein liquid;
6. remove protein liquid and add two volumes ethanol, left standstill collecting precipitation one day in 4 ℃;
7. will precipitate fully water-solublely, and centrifugally remove insoluble composition, it is 14000 dialysis tubing deionizing and small-molecule substance that the aqueous solution adopts molecular weight, obtains the dermatan sulfate of purifying after the lyophilize.
2. according to the method for the extraction and purification of dermatan sulfate in the claim 1 Suo Shu angler skin, the add-on that it is characterized by the prozyme of step in 2. is the 1.0-3.5% of dry product raw material weight, and the weight ratio of components of papoid and pancreatin is 1 in the prozyme: 1-1: 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100699590A CN100572547C (en) | 2007-07-09 | 2007-07-09 | The extracting and purifying method of dermatan sulfate in the angler skin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100699590A CN100572547C (en) | 2007-07-09 | 2007-07-09 | The extracting and purifying method of dermatan sulfate in the angler skin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101100688A true CN101100688A (en) | 2008-01-09 |
| CN100572547C CN100572547C (en) | 2009-12-23 |
Family
ID=39035136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2007100699590A Expired - Fee Related CN100572547C (en) | 2007-07-09 | 2007-07-09 | The extracting and purifying method of dermatan sulfate in the angler skin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100572547C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676619A (en) * | 2011-12-20 | 2012-09-19 | 浙江省海洋开发研究院 | Comprehensive utilization process for marine fish skins |
| CN103849670A (en) * | 2013-10-22 | 2014-06-11 | 中国海洋大学 | Method of preparing high F-value collagen peptide by hydrolyzing anglerfish fishskins |
| CN105199015A (en) * | 2015-11-03 | 2015-12-30 | 山东美佳集团有限公司 | Method for extracting heparinoids from turbot skin |
-
2007
- 2007-07-09 CN CNB2007100699590A patent/CN100572547C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676619A (en) * | 2011-12-20 | 2012-09-19 | 浙江省海洋开发研究院 | Comprehensive utilization process for marine fish skins |
| CN102676619B (en) * | 2011-12-20 | 2013-12-25 | 浙江省海洋开发研究院 | Comprehensive utilization process for marine fish skins |
| CN103849670A (en) * | 2013-10-22 | 2014-06-11 | 中国海洋大学 | Method of preparing high F-value collagen peptide by hydrolyzing anglerfish fishskins |
| CN103849670B (en) * | 2013-10-22 | 2015-12-02 | 中国海洋大学 | A kind of Shui Xie angler leather is for the method for high F value collagen peptide |
| CN105199015A (en) * | 2015-11-03 | 2015-12-30 | 山东美佳集团有限公司 | Method for extracting heparinoids from turbot skin |
| CN105199015B (en) * | 2015-11-03 | 2017-12-22 | 山东美佳集团有限公司 | A kind of method that heparan is extracted in the fish-skin from turbot |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100572547C (en) | 2009-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | Structural characterization and in vitro antioxidant activities of chondroitin sulfate purified from Andrias davidianus cartilage | |
| US8399430B2 (en) | Alkylated semi synthetic glycosaminoglycosan ethers, and methods for making and using thereof | |
| Liu et al. | A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity | |
| JPH11504018A (en) | Production and use of sulfated oligosaccharides | |
| CN101885782B (en) | Method for purifying dermatan sulfate from heparin byproduct | |
| JP6555431B2 (en) | Moisturizing topical agent | |
| Ragab et al. | Anticoagulation, fibrinolytic and the cytotoxic activities of sulfated hemicellulose extracted from rice straw and husk | |
| KR910006811B1 (en) | Process for preparing high purity dermatan sulfate | |
| JP2012503683A (en) | Oninoyaga polysaccharide sulfate derivative, its production method and its use | |
| KR102082276B1 (en) | High purity heparin and production method therefor | |
| CN110776578B (en) | Low-molecular sea cucumber glycosaminoglycan and application thereof | |
| CN100572547C (en) | The extracting and purifying method of dermatan sulfate in the angler skin | |
| CN102477103A (en) | Platycodon grandiflorum polysaccharide, and degradation product, preparation method and application thereof | |
| US5948405A (en) | Fucans with low molecular weight having anticoagulant, antithrombinic and antithromobic activity | |
| CN102936291B (en) | Method for preparing selenylation poly mannuronic acid and application of selenylation poly mannuronic acid | |
| US5268366A (en) | Polysaccharide composition or polysaccharide having heparinoid activity, process for producing the same, and anticoagulant containing the same as active ingredient | |
| CN1155621C (en) | Extraction refining method for aloe-polysaccharide | |
| US2959583A (en) | Method of purifying sulfated polysaccharides | |
| CN104119457A (en) | Heparin sodium and dermatan sulfate combined production process | |
| KR20140044826A (en) | Shark-like chondroitin sulphate and process for the preparation thereof | |
| CN116284468A (en) | Tremella aurantialba acidic polysaccharide, preparation method thereof and application thereof in improving ulcerative colitis | |
| AU686161B2 (en) | Remitting agent for nephrotic syndrome and hepatopathy symptoms | |
| CN103788231B (en) | A kind of method of preparing high-purity sulfuric acid Chondroitin A from rabbit ear cartilage | |
| CN104725532B (en) | A kind of method of chondroitin sulfate and dermatan sulfate content in accurate quantification control heparin/heparan | |
| CN113248630A (en) | Fucoidin composition, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20091223 Termination date: 20100709 |