CN104403026A - Method for separating heparan sulfate from animal lungs - Google Patents
Method for separating heparan sulfate from animal lungs Download PDFInfo
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- CN104403026A CN104403026A CN201410819670.6A CN201410819670A CN104403026A CN 104403026 A CN104403026 A CN 104403026A CN 201410819670 A CN201410819670 A CN 201410819670A CN 104403026 A CN104403026 A CN 104403026A
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Abstract
The invention discloses a method for separating heparan sulfate from animal lungs, belonging to the technical field of biological pharmacy. The method comprises the following steps: by using animal lungs as the raw material, carrying out composite enzyme degradation on an animal lung brine extract by using hydrolases, adding an oxidizer and activated carbon into the enzymolysis solution to perform oxidative adsorption decolorization, carrying out fractional precipitation on the oxidized solution by using acetone, dehydrating to obtain a heparan sulfate crude product, sequentially carrying out membrane separation and anion exchange chromatography on the crude product heparan sulfate water solution to separate the heparan sulfate from dermatan sulfate, chondroitin sulfate, heparin and other impurities, carrying out ultrafiltration concentration on the eluate through a 5000-7000Da ultrafiltration membrane, desalting by gel filtration chromatography, and freeze-drying to obtain the heparan sulfate refined product. The method has advantages of simple technological conditions, accessible raw materials and low cost, and is easy to implement; and the obtained heparan sulfate has high purity.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of method being separated Suleparoid from animal lungs.
Background technology
Suleparoid (Heparan sulfate, HS) belongs to mucopolysaccharide compounds, gains the name because of similar to heparin in its composition glycosyl and glycosidic link mode of connection.But based on the dissacharide units be connected by (1 → 4) with 2-N-kharophen 2-deoxidation alpha-D-glucose (comprising C6-O-sulfuric ester to replace) by β-D-Glucose aldehydic acid in its sugar chain.HS is closely connected with heparin but an independently class glycosaminoglycan (Casu, 2005).Compare HS degree with heparin lower, and degree of acetylation is higher.
Tumour be in the world today sickness rate the highest, to human health and the very harmful disease of life.According to statistics, in recent years in China, the number dying from tumour accounted for total toll ratio more than 25%, and some areas are more than 30%, and death toll and ratio increase just year by year.At present, there is no good curative drug, the exploitation of such medicine is remained to the focus of research in the world.Current research shows, HS is the new polyose medicament that Recent study finds to be played by the transfer of immunity moderation function and inhibition tumor cell antitumor action.At present, take HS as raw material, prepare its derivative by enzyme engineering or chemical method, be the focus of world research, obtain with it product innovation that pharmacological action is strong, side effect is little, curative effect is high.Thus, this product has broad application prospects.Its pharmacological action is strong, be its outstanding feature without obvious toxic side effect, the HS originated by ox lung or pig lung has very strong antitumor and antithrombotic acitivity.
China produces the mammiferous big countries such as ox, pig, sheep in the world, a large amount of ox lungs, pig lung and sheep lung etc. is had well not to utilize, with it for development of raw materials has the Suleparoid of high biological activity to have huge economic benefit and social benefit every year.The domestic report not yet having this product of exploitation, develops this product, can export bulk drug, be also developed as domestic new drug.
Summary of the invention
The object of this invention is to provide a kind of method being separated Suleparoid from animal lungs.
A kind of method being separated Suleparoid from animal lungs of the present invention, comprises the following steps:
1) saline water extraction: lungs are smashed to pieces, then add its weight 30-40 doubly 2% the NaCl aqueous solution (w/v), temperature controls at 90-95 DEG C, stir 5-6h, centrifugal, collect supernatant liquor and precipitation respectively, add in precipitation 80-120 doubly 2% the NaCl aqueous solution (w/v), repeat aforesaid operations 2-3 time;
2) enzymic hydrolysis: combining step 1) in supernatant liquor, adjustment pH is 9-11, and temperature is 40-50 DEG C, adds lytic enzyme, stir 20-24h, be then warming up to 90-95 DEG C, insulation 2-3h, centrifugal, discard precipitation, in supernatant liquor, add ethanol, precipitation 12-18h, obtains throw out;
3) oxidative decoloration: in step 2) gained throw out in add its weight 30-60 2%NaCl aqueous solution doubly, controlling lysate temperature is 40 DEG C-50 DEG C, and pH is 9-11, adds the KMnO of lysate volume 0.1%
4with 0.2% gac, stirring at room temperature 6-8h, centrifugal segregation precipitate, in supernatant liquor, add the alcohol settling 12-18h of 1 times of volume, obtain throw out;
4) acetone fractional precipitation: in step 3) gained throw out in add its weight 30-60 2%NaCl aqueous solution doubly, stirring and dissolving, add the acetone of 0.6-1.3 times of volume, precipitation 12-18h, centrifugal collecting precipitation, repeats aforesaid operations 3 times, the ethanol of 2-3 times of volume is added to throw out, abundant stirring, leave standstill to filter and remove ethanol, vacuum-drying obtains Suleparoid crude product;
5) ultrafiltration: add 80-120 times of volume purified water in Suleparoid crude product, stirring and dissolving, loop ultrafiltration 3-5h, collects trapped fluid;
6) ion-exchange chromatography: select strong anion exchange resin to 5) in trapped fluid carry out exchange adsorption, first carry out wash-out with the NaCl solution of the 0.5-1mol/L of 2-3 times of volume; Again with the NaCl solution wash-out of the 1.5mol/L of 2-3 times of volume, collect elutriant;
7) gel permeation chromatography: to 6) in elutriant carry out gel permeation chromatography, collect elutriant;
8) concentrated: to 7) in elutriant carry out the 1/20-1/30 that ultrafiltration is original volume, collect trapped fluid;
9) gel-filtration desalination: to 8) in elutriant carry out gel-filtration desalination, collect elutriant;
10) freeze-drying: by step 9) the elutriant freeze-drying of gained, obtain Suleparoid fine work.
Described step 2) in enzymic hydrolysis process, described lytic enzyme is the combination of the one or both in trypsinase, rnase II.
Described step 2) in enzymic hydrolysis process, tryptic consumption is the 0.5%-2.0% for lung weight, the amount adding ethanol be the 2-3 of supernatant volume doubly.
Described step 5) in, select molecular weight cut-off to be the ultra-filtration membrane of 5000-7000Da.
Described step 7) in, adopt Sephadex G-100 preparative chromatography post to carry out wash-out.
Described step 9) in adopt Sephadex G-10 chromatography column to carry out wash-out.
Molecular weight cut-off is selected to be the ultra-filtration membrane of 2000-4000Da.
Described animal lungs are Mammals lungs.
Compared with prior art, the invention has the advantages that: the present invention is separated the method for Suleparoid from animal lungs, processing condition are simple, are easy to realize, and raw material is easy to obtain, and cost is low, and gained Suleparoid purity is high.
Accompanying drawing explanation
Fig. 1 is the gel chromatography figure of the embodiment of the present invention 1,2 and 3 gained Suleparoid;
In figure, chromatographic band is respectively the HS of standard substance HS, the HS of ox lung, the HS of pig lung and sheep lung.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
From animal lungs, be separated the method for Suleparoid, comprise the following steps:
1) get pig lung 3Kg to smash to pieces, then add 120L 2%NaCl solution, 90 DEG C are stirred 5 hours, centrifugal, shift supernatant liquor and collecting precipitation, add the 2%NaCl purification of aqueous solutions of 300L, repeat aforesaid operations 2 times in precipitation;
2) combining step 1) in supernatant liquor, with NaOH solution adjustment pH be 9, at 40 DEG C, add Trypsin lytic enzyme 60g, stir 24 hours, then temperature is risen to 90 DEG C of insulations 3 hours, centrifugal, discard precipitation, in supernatant liquor, add ethanol, precipitate 18 hours, obtain throw out 626g;
3) in step 2) add the 2%NaCl aqueous solution of its weight 60 times in the throw out of gained, stirring and dissolving, obtains lysate; Control lysate temperature and be 40 DEG C DEG C, adjustment lysate pH is 9, adds the KMnO of lysate volume 0.1%
4with the gac of 0.2%, stirring at room temperature reacts 8 hours, and centrifugal segregation precipitates, and adds the long-pending alcohol settling of monoploid wherein 18 hours, obtains throw out;
4) in step 3) add the 2%NaCl aqueous solution of its weight 60 times in the throw out of gained, stirring and dissolving, obtains lysate, adds the acetone of 0.6 times of volume in lysate, and stirred at ambient temperature precipitates 18 hours, centrifugal collecting precipitation; In throw out, add the 2%NaCl aqueous solution of its weight 60 times of volumes, stirring and dissolving, obtains lysate, adds the acetone of 0.8 times of volume in lysate, and stirred at ambient temperature precipitates 18 hours, centrifugal collecting precipitation; The 2%NaCl aqueous solution of 60 times of volumes, stirring and dissolving, obtains lysate, adds the acetone of 1.2 times of volumes in lysate, and stirred at ambient temperature precipitates 18 hours, centrifugal collecting precipitation; The 2%NaCl aqueous solution of 60 times of volumes, stirring and dissolving, obtains lysate, in lysate, add the acetone of 1.3 times of volumes, stirred at ambient temperature precipitates 18 hours, centrifugal collecting precipitation, and in precipitation, add the ethanol of 99.9% of 2 times of volumes, remove ethanol, vacuum-drying obtains Suleparoid crude product;
5) to step 4) in Suleparoid crude product in add 100 times of volume purified water, stirring and dissolving, obtains lysate; Selective retention molecular weight is the ultra-filtration membrane ultrafiltration of 7000Da, loop ultrafiltration 5 hr collections trapped fluid;
6) select strong anion exchange resin to 5) in trapped fluid carry out exchange adsorption, first carry out wash-out with the NaCl aqueous solution of the 1mol/L of 2 times of volumes; Use the NaCl solution wash-out of the 1.5mol/L of 2 times of volumes again, and collect elutriant;
7) select Sephadex G-100 preparative chromatography post to 6) in elutriant carry out separation and purification, elution speed is 2ml/s, Fractional Collections elutriant;
8) select molecular weight be that the ultra-filtration membrane of 1000Da is to 7) in elutriant concentrate, collect trapped fluid;
9) select Sephadex G-10 chromatography column to 8) in elutriant carry out wash-out, Fractional Collections elutriant;
10) by elutriant freeze-drying, HS fine work is obtained; Its purity >=98%.
Embodiment 2
From animal lungs, be separated the method for Suleparoid, comprise the following steps:
1) get ox lung 2.5Kg to smash to pieces, then add 100L 2%NaCl solution, 95 DEG C are stirred 6 hours, centrifugal, shift supernatant liquor and collecting precipitation.In precipitation, add the 2%NaCl solution of 200L, and repeat 2 times;
2) combining step 1) in supernatant liquor, adjusting pH by NaOH solution is 11,50 DEG C add Trypsin lytic enzyme 20g and rnase II 13g stir within 22 hours, then temperature is risen to 95 DEG C insulation 3 hours, centrifugal, discard precipitation, in supernatant liquor, add ethanol, precipitate 17 hours, obtain throw out 532g;
3) in step 2) add the 2%NaCl aqueous solution of its weight 50 times in the throw out of gained, stirring and dissolving, obtains lysate; Controlling lysate temperature is 50 DEG C, and adjustment lysate pH is 11, adds the KMnO of lysate volume 0.1%
4with the gac of 0.2%, stirring at room temperature reacts 6 hours, and centrifugal segregation precipitates, and adds the long-pending alcohol settling of monoploid wherein 12 hours, obtains throw out;
4) in step 3) add the 2%NaCl aqueous solution of its weight 30 times in the throw out of gained, stirring and dissolving, obtains lysate, adds the acetone of 0.6 times of volume in lysate, and stirred at ambient temperature precipitates 12 hours, centrifugal collecting precipitation; In throw out, add the 2%NaCl aqueous solution of its weight 30 times of volumes, stirring and dissolving, obtains lysate, adds the acetone of 0.8 times of volume in lysate, and stirred at ambient temperature precipitates 12 hours, centrifugal collecting precipitation; The 2%NaCl aqueous solution of 30 times of volumes, stirring and dissolving, obtains lysate, adds the acetone of 1.2 times of volumes in lysate, and stirred at ambient temperature precipitates 12 hours, centrifugal collecting precipitation; The 2%NaCl aqueous solution of 30 times of volumes, stirring and dissolving, obtain lysate, in lysate, add the acetone of 1.3 times of volumes, stirred at ambient temperature precipitates 12 hours, centrifugal collecting precipitation, and in precipitation, add the ethanol of 99.9% of 2 times of volumes, abundant stirring, leave standstill suction filtration and remove ethanol, vacuum-drying obtains Suleparoid crude product;
5) to step 4) in Suleparoid crude product in add 100 times of volume purified water, stirring and dissolving, obtains lysate; Selective retention molecular weight is the ultra-filtration membrane ultrafiltration of 5000Da, loop ultrafiltration 3 hr collections trapped fluid;
6) select strong anion exchange resin to 5) in trapped fluid carry out exchange adsorption, first carry out wash-out with the NaCl aqueous solution of the 0.5mol/L of 2.5 times of volumes; Use the NaCl solution wash-out of the 1.5mol/L of 2.5 times of volumes again, and collect elutriant;
7) select Sephadex G-100 preparative chromatography post to 6) in elutriant carry out separation and purification, elution speed is 2ml/s, Fractional Collections elutriant;
8) select molecular weight be that the ultra-filtration membrane of 1000Da is to 7) in elutriant concentrate, collect trapped fluid;
9) select Sephadex G-10 chromatography column to 8) in elutriant carry out desalination wash-out, Fractional Collections elutriant;
10) by elutriant freeze-drying, HS fine work is obtained; Its purity >=97%.
Embodiment 3
From animal lungs, be separated the method for Suleparoid, comprise the following steps:
1) get 2Kg to smash to pieces, then add 80L 2%NaCl solution, 92 DEG C are stirred 5 hours, centrifugal, shift supernatant liquor and collecting precipitation.In precipitation, add the 2%NaCl purification of aqueous solutions of 200L, and repeat 2 times;
2) combining step 1) in supernatant liquor, adjusting pH by NaOH solution is 10,45 DEG C add Trypsin lytic enzyme 36g and rnase II 24g stir within 24 hours, then temperature is risen to 92 DEG C insulation 3 hours, centrifugal, discard precipitation, in supernatant liquor, add ethanol, precipitate 15 hours, obtain throw out 626g;
3) in step 2) add the 2%NaCl aqueous solution of its weight 50 times in the throw out of gained, stirring and dissolving, obtains lysate; Controlling lysate temperature is 45 DEG C, and adjustment lysate pH is 10, adds the KMnO of lysate volume 0.1%
4with the gac of 0.2%, stirring at room temperature reacts 7 hours, and centrifugal segregation precipitates, and adds the long-pending alcohol settling of monoploid wherein 15 hours, obtains throw out;
4) in step 3) add the 2%NaCl aqueous solution of its weight 50 times in the throw out of gained, stirring and dissolving, obtains lysate, adds the acetone of 0.6 times of volume in lysate, and stirred at ambient temperature precipitates 15 hours, centrifugal collecting precipitation; In throw out, add the 2%NaCl aqueous solution of its weight 50 times of volumes, stirring and dissolving, obtains lysate, adds the acetone of 0.8 times of volume in lysate, and stirred at ambient temperature precipitates 15 hours, centrifugal collecting precipitation; The 2%NaCl aqueous solution of 50 times of volumes, stirring and dissolving, obtains lysate, adds the acetone of 1.2 times of volumes in lysate, and stirred at ambient temperature precipitates 15 hours, centrifugal collecting precipitation; The 2%NaCl aqueous solution of 50 times of volumes, stirring and dissolving, obtains lysate, in lysate, add the acetone of 1.3 times of volumes, stirred at ambient temperature precipitates 15 hours, centrifugal collecting precipitation, and in precipitation, add the ethanol of 99.9% of 2 times of volumes, remove ethanol, vacuum-drying obtains Suleparoid crude product;
5) to step 4) in Suleparoid crude product in add 110 times of volume purified water, stirring and dissolving, obtains lysate; Selective retention molecular weight is the ultra-filtration membrane ultrafiltration of 6000Da, loop ultrafiltration 5 hr collections trapped fluid;
6) select strong anion exchange resin to 5) in trapped fluid carry out exchange adsorption, first carry out wash-out with the NaCl aqueous solution of the 0.7mol/L of 3 times of volumes; Use the NaCl solution wash-out of the 1.5mol/L of 3 times of volumes again, and collect elutriant;
7) select Sephadex G-100 preparative chromatography post to 6) in elutriant carry out separation and purification, elution speed is 2ml/s, Fractional Collections elutriant;
8) select molecular weight be that the ultra-filtration membrane of 1000Da is to 7) in elutriant concentrate, collect trapped fluid;
9) select Sephadex G-10 chromatography column to 8) in elutriant carry out wash-out, Fractional Collections elutriant;
10) by elutriant freeze-drying, HS fine work is obtained; Its purity >=99%.
Can be learnt by Fig. 1, adopt HS fine work prepared by present method, impurity is few, and purity is high.
Claims (8)
1. from animal lungs, be separated a method for Suleparoid, it is characterized in that, comprise the following steps:
1) saline water extraction: lungs are smashed to pieces, then add its weight 30-40 doubly 2% the NaCl aqueous solution (w/v), temperature controls at 90-95 DEG C, stir 5-6h, centrifugal, collect supernatant liquor and precipitation respectively, add in precipitation 80-120 doubly 2% the NaCl aqueous solution (w/v), repeat aforesaid operations 2-3 time;
2) enzymic hydrolysis: combining step 1) in supernatant liquor, adjustment pH is 9-11, and temperature is 40-50 DEG C, adds lytic enzyme, stir 20-24h, be then warming up to 90-95 DEG C, insulation 2-3h, centrifugal, discard precipitation, in supernatant liquor, add ethanol, precipitation 12-18h, obtains throw out;
3) oxidative decoloration: in step 2) gained throw out in add its weight 30-60 2%NaCl aqueous solution doubly, controlling lysate temperature is 40 DEG C-50 DEG C, and pH is 9-11, adds the KMnO of lysate volume 0.1%
4with 0.2% gac, stirring at room temperature 6-8h, centrifugal segregation precipitate, in supernatant liquor, add the alcohol settling 12-18h of 1 times of volume, obtain throw out;
4) acetone fractional precipitation: in step 3) gained throw out in add its weight 30-60 2%NaCl aqueous solution doubly, stirring and dissolving, add the acetone of 0.6-1.3 times of volume, precipitation 12-18h, centrifugal collecting precipitation, repeats aforesaid operations 3 times, the ethanol of 2-3 times of volume is added to throw out, abundant stirring, leave standstill to filter and remove ethanol, vacuum-drying obtains Suleparoid crude product;
5) ultrafiltration: add 80-120 times of volume purified water in Suleparoid crude product, stirring and dissolving, loop ultrafiltration 3-5h, collects trapped fluid;
6) ion-exchange chromatography: select strong anion exchange resin to 5) in trapped fluid carry out exchange adsorption, first carry out wash-out with the NaCl solution of the 0.5-1mol/L of 2-3 times of volume; Again with the NaCl solution wash-out of the 1.5mol/L of 2-3 times of volume, collect elutriant;
7) gel permeation chromatography: to 6) in elutriant carry out gel permeation chromatography, collect elutriant;
8) concentrated: to 7) in elutriant carry out the 1/20-1/30 that ultrafiltration is original volume, collect trapped fluid;
9) gel-filtration desalination: to 8) in elutriant carry out gel-filtration desalination, collect elutriant;
10) freeze-drying: by step 9) the elutriant freeze-drying of gained, obtain Suleparoid fine work.
2. the method being separated Suleparoid from animal lungs according to claim 1, is characterized in that, described step 2) in enzymic hydrolysis process, described lytic enzyme is the combination of the one or both in trypsinase, rnase II.
3. the method being separated Suleparoid from animal lungs according to claim 1 and 2, it is characterized in that, described step 2) in enzymic hydrolysis process, tryptic consumption is the 0.5%-2.0% for lung weight, the amount adding ethanol be the 2-3 of supernatant volume doubly.
4. the method being separated Suleparoid from animal lungs according to claim 1, is characterized in that, described step 5) in, select molecular weight cut-off to be the ultra-filtration membrane of 5000-7000Da.
5. the method being separated Suleparoid from animal lungs according to claim 1, is characterized in that, described step 7) in, adopt Sephadex G-100 preparative chromatography post to carry out wash-out.
6. the method being separated Suleparoid from animal lungs according to claim 1, is characterized in that, described step 9) in adopt Sephadex G-10 chromatography column to carry out wash-out.
7. the method being separated Suleparoid from animal lungs according to claim 1, is characterized in that, selects molecular weight cut-off to be the ultra-filtration membrane of 2000-4000Da.
8. the method being separated Suleparoid from animal lungs according to claim 1, is characterized in that, described animal lungs are Mammals lungs.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037584A (en) * | 2015-07-02 | 2015-11-11 | 杭州惠顺生物科技有限公司 | Method for separating heparitin from heparin by-product waste protein |
| CN105504098A (en) * | 2016-01-08 | 2016-04-20 | 东营天东制药有限公司 | Technology for extracting heparin sulfate from duodena |
| CN108424468A (en) * | 2018-02-06 | 2018-08-21 | 浦江县昂宝生物技术有限公司 | A kind of preparation process of abalone polysaccharide |
| CN113896813A (en) * | 2021-11-26 | 2022-01-07 | 重庆望业药物研究有限公司 | Preparation method of high-purity heparan sulfate |
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| CN1817911A (en) * | 2006-03-08 | 2006-08-16 | 中国海洋大学 | Heparitin sulfate from rat tissue and production thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105037584A (en) * | 2015-07-02 | 2015-11-11 | 杭州惠顺生物科技有限公司 | Method for separating heparitin from heparin by-product waste protein |
| CN105504098A (en) * | 2016-01-08 | 2016-04-20 | 东营天东制药有限公司 | Technology for extracting heparin sulfate from duodena |
| CN108424468A (en) * | 2018-02-06 | 2018-08-21 | 浦江县昂宝生物技术有限公司 | A kind of preparation process of abalone polysaccharide |
| CN113896813A (en) * | 2021-11-26 | 2022-01-07 | 重庆望业药物研究有限公司 | Preparation method of high-purity heparan sulfate |
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Effective date of registration: 20170209 Address after: Zhang Huang industrial park 272300 Shandong city of Jining province Yutai County Applicant after: Shandong Chenzhong Biological Pharmaceutical Co., Ltd. Applicant after: Wuhan Xinuo Han Pu Biological Technology Development Co Ltd Address before: Zhang Huang industrial park 272300 Shandong city of Jining province Yutai County Applicant before: Shandong Chenzhong Biological Pharmaceutical Co., Ltd. |
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Application publication date: 20150311 |