CN102993323A - Method for extracting and purifying glycosaminoglycans (GAGs) from stichopus japonicus - Google Patents
Method for extracting and purifying glycosaminoglycans (GAGs) from stichopus japonicus Download PDFInfo
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- CN102993323A CN102993323A CN2012105849523A CN201210584952A CN102993323A CN 102993323 A CN102993323 A CN 102993323A CN 2012105849523 A CN2012105849523 A CN 2012105849523A CN 201210584952 A CN201210584952 A CN 201210584952A CN 102993323 A CN102993323 A CN 102993323A
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Abstract
The invention belongs to the field of extraction of biotechnology natural active materials and particularly relates to a method for extracting and purifying GAGs from stichopus japonicus. The method includes: step one, pre-processing fresh stichopus japonicus; step two, performing alkaline hydrolysis on the body wall of the stichopus japonicus; step three, performing two-enzyme enzymolysis on the body wall of the stichopus japonicus; step four, removing proteins in two-enzyme enzymatic hydrolysate through trichloroacetic acid after the enzymolysis; step five, precipitating the GAGs of the stichopus japonicus through ethanol; step six, drying GAGs raw sugar of the stichopus japonicus; step seven, purifying the GAGs of the stichopus japonicus by using a cellulose ion exchange column chromatography and a gel column sequentially; and step eight, drying GAGs refined sugar of the stichopus japonicus. According to the method, the relation between the yield and the purity of the GAGs of the stichopus japonicus can be balanced, and the purity of the GAGs of the stichopus japonicus can be improved.
Description
Technical field
The invention belongs to the biotechnology natural active matter and extract the field, be specifically related to a kind of also method of purifying glycosaminoglycan of from stichopus japonicus, extracting.
Background technology
Chronic hepatitis B is the health problem of a sternness facing of the whole world, China, South East Asia and Africa are the districts occurred frequently of HBV infection, and the incidence of these regional HBV related liver diseases of while such as primary hepatocarcinoma is also sent out the district apparently higher than hanging down of HBV infection such as middle part, America and south.That treatment of chronic mainly comprises is antiviral, immunomodulatory, anti-inflammatory protect the liver, anti-fibrosis and symptomatic treatment, wherein carries out effective antiviral therapy, but suppress lastingly the key that HBV DNA is treatment below PCR routine sensing range.Sulfated polysaccharide is no matter in vivo or external, all shown in various degree antiviral, antitumor and to immune regulating effect.Studies show that sulfated polysaccharide and other polyanionic materials can disturb absorption and the intrusion of retrovirus and other viruses, some shows the reverse transcription enzymeinhibition of various retroviruss active.The antiviral activity of sulfated polysaccharide at first comes from its polyanion characteristic, and the polyanion characteristic then mainly comes from the sulfate in its molecule.
The ocean is a huge natural product treasure-house, and abundant marine organisms and marine active substance provide a huge marine pharmaceutical resource storehouse for us.Therefore the special marine eco-environment and the Close relation between the biologic chain are significant to the Application and Development of marine polysaccharide so that the marine organisms secondary metabolite has the biological activity with terrestrial organism difference chemical structure and differential high efficient.From the marine active polysaccharide, seek the active drug for the treatment of chronic hepatitis B, caused the close attention of domestic and international the world of medicine.
Stichopus japonicus selenka glycosaminoglycans (Stichopus Japomicus selenka GlycosaminoGlycans, SJ-GAG), be once called as Stichopus Japonicus Acidic Mucopolysaccharide or stichopus japonicus mucopolysaccharide, it is a kind of sulfated polysaccharide that from the stichopus japonicus body wall, extracts, system is comprised of D-N-acetylamino galactosamine, D-Glucose aldehydic acid, L-fucose and sulfate, relative molecular mass about 4 to 50,000 dalton.Stichopus japonicus selenka glycosaminoglycans exists with ionic species in solution, has the ability with ion or the interaction between component of positively charged, belongs to polyanion.Stichopus japonicus Tang amine glycan polyanion character and the basis that has consisted of its various biological effect in the difference at solution end.The extracting method of present stichopus japonicus selenka glycosaminoglycans mainly comprises alkaline lysis, enzyme digestion and ethanol precipitation.But above-mentioned preparation method can not obtain the high stichopus japonicus selenka glycosaminoglycans of purity, its major cause is that the chemical structure of Tang's amine glycan has significant plyability, such as the molecular weight difference method, degree is inconsistent, conception is complicated, microstructure is inhomogeneous and residue number change etc., especially on a specified protein glycan molecule, kind, the quantity of Tang's amine polysaccharide chains that it contains there are differences, so that the preparation method of existing stichopus japonicus selenka glycosaminoglycans is difficult to obtain the high stichopus japonicus selenka glycosaminoglycans of purity.
Summary of the invention
For solving above-mentioned problems of the prior art, the object of the present invention is to provide a kind of also method of purifying glycosaminoglycan of from stichopus japonicus, extracting, stichopus japonicus selenka glycosaminoglycans is used cellulose ion-exchange column chromatography method and gel column purifying successively, obtain the higher stichopus japonicus selenka glycosaminoglycans of purity.
Technical scheme of the present invention is: a kind of also method of purifying glycosaminoglycan of extracting from stichopus japonicus comprises the following steps: step 1: bright stichopus japonicus pre-treatment; Step 2: alkaline hydrolysis stichopus japonicus body wall; Step 3: two enzyme enzymolysis stichopus japonicus body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate stichopus japonicus selenka glycosaminoglycans with ethanol; Step 6: dry stichopus japonicus selenka glycosaminoglycans raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying stichopus japonicus selenka glycosaminoglycans; Step 8: dry stichopus japonicus selenka glycosaminoglycans refined sugar.
Optimize, the concrete steps of gel column purifying are in the described step 7: with the distilled water soaked overnight of 40g dextrane gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; 30mg glycosaminoglycan raw sugar with 10mL damping fluid dissolving upper props under 40～60 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
Optimize, described damping fluid adopts the NaAC of 0.5mol/L, and its pH is 6.0～6.5.
The g-100 of sephadex described in the present invention is dextrane gel, and the present invention does not have particular requirement to reagent, material and the instrument that adopts, and it is the commercially available prod, can buy by multiple commercial channel.
Beneficial effect of the present invention is: set up a kind of method that effectively prepares highly purified stichopus japonicus selenka glycosaminoglycans, when alkaline process extracts, in conjunction with trypsinase and pepsic pair of collagenase treatment, namely at first adopt alkaline purification stichopus japonicus tissue, use again trypsinase and pepsin hydrolysis, gather the release of glycosaminoglycan to strengthen stichopus japonicus body wall tissue.For improving the purity of glycosaminoglycan, the present invention uses cellulose ion-exchange column chromatography method and gel column purifying purifying stichopus japonicus selenka glycosaminoglycans raw sugar successively.Method of the present invention can the balance stichopus japonicus selenka glycosaminoglycans yield and the relation of purity, improved the purity of stichopus japonicus selenka glycosaminoglycans.
Embodiment
The present invention will be further described below in conjunction with specific embodiment.
Embodiment 1
The method of extracting glycosaminoglycan from stichopus japonicus of present embodiment comprises the following steps: step 1: bright stichopus japonicus pre-treatment; Step 2: alkaline hydrolysis stichopus japonicus body wall; Step 3: two enzyme enzymolysis stichopus japonicus body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate stichopus japonicus selenka glycosaminoglycans with ethanol; Step 6: dry stichopus japonicus selenka glycosaminoglycans raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying stichopus japonicus selenka glycosaminoglycans; Step 8: dry stichopus japonicus selenka glycosaminoglycans refined sugar.The concrete grammar of each step is:
Step 1: bright stichopus japonicus pre-treatment; Use rapidly the distilled water flushing stichopus japonicus, removes the foreign matter that the stichopus japonicus body surface adheres to, with microscler pocket knife from belly only anus dissect forward aquatic foods and join, reject immediately internal organ, with distilled water flush away dirt, after the weighing stichopus japonicus body wall is placed clean large beaker rapidly, shred the stichopus japonicus body wall;
Step 2: alkaline hydrolysis stichopus japonicus body wall: add the distilled water of 2 times of weight in the stichopus japonicus body wall, then add the NaOH of 2mol/L, the limit edged stirs, and making NaOH solution final concentration is 0.5mol/L, 4 ℃ of digested overnight;
Step 3: two enzyme enzymolysis of stichopus japonicus body wall:
Trypsin digestion: reconcile pH value to 8.2 with Glacial acetic acid, add the trypsinase of stichopus japonicus body wall quality 0.45%, 52 ℃ of water-bath 5h fully stir in the water-bath process, and drip the NaOH solution of 5 mmol/L, keep pH 8.2; Then enzymolysis solution is cooled to rapidly below 30 ℃ termination reaction behind the maintenance 30min;
Stomach en-enzymolysis: reconcile between the pH value to 2 with 6mol/L HCl, add again the stomach en-of stichopus japonicus body wall quality 0.40%, in 50 ℃ water-bath, continue enzymolysis 4h, fully stir in the water-bath process; Enzymolysis is got in enzymolysis solution 5mL and the glass test tube before stopping, and the trichoroacetic acid(TCA) solution of dropping 5% detects, and when solution is little muddiness, enzymolysis solution is cooled to rapidly below 30 ℃, keeps the 30min enzymolysis reaction; Then collect supernatant with the centrifugal 15min of 5000r/min;
Step 4: after enzymolysis is complete, process two enzyme enzymolysis supernatant with trichoroacetic acid(TCA), the addition of trichoroacetic acid(TCA) is 10% of enzymolysis solution, progressively adds, and the limit edged stirs, and enzymolysis solution is become till the muddiness; Solution is kept at leaves standstill 12h in the refrigerating chamber, then collect supernatant with the centrifugal 25min of 5000r/min;
Step 5: ethanol precipitation stichopus japonicus selenka glycosaminoglycans: the NaOH solution that drips 5mmol/L is reconciled the pH value to 6.8 of supernatant liquor, and the centrifugal 10min of 7000r/min collects supernatant and adds dehydrated alcohol, and the limit edged stirs, and making the ethanol final concentration is 55%, 4 ℃ of precipitation of spending the night;
Step 6: dry stichopus japonicus selenka glycosaminoglycans raw sugar: the centrifugal 10min of 7000r/min, collecting precipitation, the washing with alcohol with 95% 3 times is washed respectively with dehydrated alcohol and acetone successively and is dewatered 1 time; Ethanol and the acetone of precipitating species are volatilized naturally, further dry with low temperature vacuum drier during the throw out partial desiccation, obtain rough stichopus japonicus selenka glycosaminoglycans;
Step 7: at first with 50g diethylaminoethyl cellulose damping fluid soaked overnight, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; With 10mL damping fluid dissolving upper props under 40 ℃, with each 150mL gradient elution of NaCl of damping fluid and 1.5mol/L, flow velocity employing 8mL/h is with the collection of 3mL/ pipe with 30mg glycosaminoglycan raw sugar; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
Then with the distilled water soaked overnight of 40g sephadex g-100 gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; The above-mentioned enriched material of 30mg with 10mL damping fluid dissolving upper props under 40 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated, obtains the stichopus japonicus selenka glycosaminoglycans refined sugar.Described damping fluid adopts the NaAC of 0.5mol/L, and its pH is 6.0.
Step 8: dry stichopus japonicus selenka glycosaminoglycans refined sugar:
The stichopus japonicus selenka glycosaminoglycans refined sugar is used respectively 80%, 95%, 100% washing with alcohol 1 time, wash respectively with dehydrated alcohol and acetone successively again and dewater 1 time; Ethanol and acetone in the throw out are volatilized naturally, and during the throw out partial desiccation, further dry with low temperature vacuum drier, the unformed powder of the white dried that obtains is refining polysaccharide.
Embodiment 2
The method of extracting glycosaminoglycan from stichopus japonicus of present embodiment comprises the following steps: step 1: bright stichopus japonicus pre-treatment; Step 2: alkaline hydrolysis stichopus japonicus body wall; Step 3: two enzyme enzymolysis stichopus japonicus body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate stichopus japonicus selenka glycosaminoglycans with ethanol; Step 6: dry stichopus japonicus selenka glycosaminoglycans raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying stichopus japonicus selenka glycosaminoglycans; Step 8: dry stichopus japonicus selenka glycosaminoglycans refined sugar.The concrete grammar of each step is:
Step 1: bright stichopus japonicus pre-treatment; Use rapidly the distilled water flushing stichopus japonicus, removes the foreign matter that the stichopus japonicus body surface adheres to, with microscler pocket knife from belly only anus dissect forward aquatic foods and join, reject immediately internal organ, with distilled water flush away dirt, after the weighing stichopus japonicus body wall is placed clean large beaker rapidly, shred the stichopus japonicus body wall;
Step 2: alkaline hydrolysis stichopus japonicus body wall: add the distilled water of 2 times of weight in the stichopus japonicus body wall, then add the NaOH of 2mol/L, the limit edged stirs, and making NaOH solution final concentration is 0.5mol/L, 4 ℃ of digested overnight;
Step 3: two enzyme enzymolysis of stichopus japonicus body wall:
Trypsin digestion: reconcile pH value to 9.0 with Glacial acetic acid, add the trypsinase of stichopus japonicus body wall quality 0.45%, 52 ℃ of water-bath 5h fully stir in the water-bath process, and drip the NaOH solution of 5 mmol/L, keep pH 9.0; Then enzymolysis solution is cooled to rapidly below 30 ℃ termination reaction behind the maintenance 30min;
Stomach en-enzymolysis: reconcile between the pH value to 2.5 with 6mol/L HCl, add again the stomach en-of stichopus japonicus body wall quality 0.40%, in 50 ℃ water-bath, continue enzymolysis 4h, fully stir in the water-bath process; Enzymolysis is got in enzymolysis solution 5mL and the glass test tube before stopping, and the trichoroacetic acid(TCA) solution of dropping 5% detects, and when solution is little muddiness, enzymolysis solution is cooled to rapidly below 30 ℃, keeps the 30min enzymolysis reaction; Then collect supernatant with the centrifugal 15min of 5000r/min;
Step 4: after enzymolysis is complete, process two enzyme enzymolysis supernatant with trichoroacetic acid(TCA), the addition of trichoroacetic acid(TCA) is 10% of enzymolysis solution, progressively adds, and the limit edged stirs, and enzymolysis solution is become till the muddiness; Solution is kept at leaves standstill 12h in the refrigerating chamber, then collect supernatant with the centrifugal 25min of 5000r/min;
Step 5: ethanol precipitation stichopus japonicus selenka glycosaminoglycans: the NaOH solution that drips 5mmol/L is reconciled the pH value to 7.2 of supernatant liquor, and the centrifugal 10min of 7000r/min collects supernatant and adds dehydrated alcohol, and the limit edged stirs, and making the ethanol final concentration is 55%, 4 ℃ of precipitation of spending the night;
Step 6: dry stichopus japonicus selenka glycosaminoglycans raw sugar: the centrifugal 10min of 7000r/min, collecting precipitation, the washing with alcohol with 95% 3 times is washed respectively with dehydrated alcohol and acetone successively and is dewatered 1 time; Ethanol and the acetone of precipitating species are volatilized naturally, further dry with low temperature vacuum drier during the throw out partial desiccation, obtain rough stichopus japonicus selenka glycosaminoglycans;
Step 7: at first with 50g diethylaminoethyl cellulose damping fluid soaked overnight, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; With 10mL damping fluid dissolving upper props under 60 ℃, with each 150mL gradient elution of NaCl of damping fluid and 1.5mol/L, flow velocity employing 8mL/h is with the collection of 3mL/ pipe with 30mg glycosaminoglycan raw sugar; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
Then with the distilled water soaked overnight of 40g sephadex g-100 gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; The above-mentioned enriched material of 30mg with 10mL damping fluid dissolving upper props under 60 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated, obtains the stichopus japonicus selenka glycosaminoglycans refined sugar.Described damping fluid adopts the NaAC of 0.5mol/L, and its pH is 6.5.
Step 8: dry stichopus japonicus selenka glycosaminoglycans refined sugar:
The stichopus japonicus selenka glycosaminoglycans refined sugar is used respectively 80%, 95%, 100% washing with alcohol 1 time, wash respectively with dehydrated alcohol and acetone successively again and dewater 1 time; Ethanol and acetone in the throw out are volatilized naturally, and during the throw out partial desiccation, further dry with low temperature vacuum drier, the unformed powder of the white dried that obtains is refining polysaccharide.
The method of extracting glycosaminoglycan from stichopus japonicus of implementing in the embodiment of the invention all can balance stichopus japonicus selenka glycosaminoglycans yield and the relation of purity, the purity of raising stichopus japonicus selenka glycosaminoglycans.
Claims (3)
1. one kind is extracted the also method of purifying glycosaminoglycan from stichopus japonicus, it is characterized in that: comprise the following steps: step 1: bright stichopus japonicus pre-treatment; Step 2: alkaline hydrolysis stichopus japonicus body wall; Step 3: two enzyme enzymolysis stichopus japonicus body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate stichopus japonicus selenka glycosaminoglycans with ethanol; Step 6: dry stichopus japonicus selenka glycosaminoglycans raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying stichopus japonicus selenka glycosaminoglycans; Step 8: dry stichopus japonicus selenka glycosaminoglycans refined sugar.
2. a kind of from stichopus japonicus, the extraction and the method for purifying glycosaminoglycan according to claim 1, it is characterized in that: the concrete steps of gel column purifying are in the described step 7: with the distilled water soaked overnight of 40g dextrane gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; 30mg glycosaminoglycan raw sugar with 10mL damping fluid dissolving upper props under 40～60 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
3. a kind of the extraction from stichopus japonicus and the method for purifying glycosaminoglycan according to claim 2, it is characterized in that: described damping fluid adopts the NaAC of 0.5mol/L, and its pH is 6.0～6.5.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104435359A (en) * | 2014-12-09 | 2015-03-25 | 山东好当家海洋发展股份有限公司 | Liver-protecting stichopus japonicus traditional Chinese medicine oral solution and preparation method thereof |
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| CN101724086A (en) * | 2009-11-25 | 2010-06-09 | 深圳海王药业有限公司 | Oligomerization pineapple ginseng glycosaminoglycan and preparation method thereof |
| CN101787084A (en) * | 2010-02-12 | 2010-07-28 | 青岛市市立医院 | Method for preparing stichopus japonicus selenka glycosaminoglycans |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724086A (en) * | 2009-11-25 | 2010-06-09 | 深圳海王药业有限公司 | Oligomerization pineapple ginseng glycosaminoglycan and preparation method thereof |
| CN101787084A (en) * | 2010-02-12 | 2010-07-28 | 青岛市市立医院 | Method for preparing stichopus japonicus selenka glycosaminoglycans |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104435359A (en) * | 2014-12-09 | 2015-03-25 | 山东好当家海洋发展股份有限公司 | Liver-protecting stichopus japonicus traditional Chinese medicine oral solution and preparation method thereof |
| CN104435359B (en) * | 2014-12-09 | 2019-03-08 | 山东好当家海洋发展股份有限公司 | A kind of protect liver stichopus japonicus traditional Chinese medicine oral liquid and preparation method thereof |
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Application publication date: 20130327 |