CN101787084A - Method for preparing stichopus japonicus selenka glycosaminoglycans - Google Patents
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Abstract
The invention discloses a method for preparing stichopus japonicus selenka glycosaminoglycans, which comprises the following steps: (1) carrying out pretreatment on fresh sea cucumber; (2) carrying out alkaline hydrolysis on body wall of the sea cucumber; (3) carrying out dual-enzyme enzymatic hydrolysis on the body wall of the sea cucumber; (4) using trichloroacetic acid for removing proteins in dual-enzyme enzymatic hydrolysis solution after completing the enzymatic hydrolysis; (5) using ethanol for precipitating the stichopus japonicus selenka glycosaminoglycans; (6) drying for obtaining crude stichopus japonicus selenka glycosaminoglycans; (7) using potassium acetate for precipitating the stichopus japonicus selenka glycosaminoglycans; (8) decoloring the stichopus japonicus selenka glycosaminoglycans; (9) using the potassium acetate for precipitating the stichopus japonicus selenka glycosaminoglycans; and (10) drying for obtaining fine stichopus japonicus selenka glycosaminoglycans. The method can balance the relationship between the yield and the purity of the stichopus japonicus selenka glycosaminoglycans and improve the purity of the stichopus japonicus selenka glycosaminoglycans.
Description
Technical field
The present invention relates to a kind of preparation method of stichopus japonicus selenka glycosaminoglycans.Belong to the biotechnology natural active matter and extract the field.
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 that HBV infects, and the incidence of these regional HBV dependency hepatopathys such as primary hepatocarcinoma also is significantly higher than the low district of sending out that HBV such as middle part, America and south infect simultaneously.That chronic hepatitis B treatment mainly comprises is antiviral, immunomodulatory, anti-inflammatory protect the liver, anti-fibrosis and symptomatic treatment, wherein carries out effective antiviral therapy, but suppress the key that HBV DNA is treatment lastingly below PCR routine sensing range.Sulfated polysaccharide is no matter in vivo still external, all shown in various degree antiviral, antitumor and to immune regulating effect.Studies show that sulfated polysaccharide and other polyanionic materials can disturb the absorption and the intrusion of retrovirus and other viruses, some shows the inhibition activity to the reversed transcriptive enzyme of various retroviruss.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 bioactivity material provide a huge marine pharmaceutical resource storehouse for us.The special marine eco-environment and closely related property between the biologic chain make the marine organisms secondary metabolite have with terrestrial organism different chemical structure and the biological activity of differential high efficient, therefore the Application and Development to the marine organisms polysaccharide is significant.From the marine active polysaccharide, seek the active drug of treatment 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 made up of D-N-acetylamino galactosamine, D-glucuronic acid, L-Fucose and sulfate, and relative molecular mass is about 4-5 ten thousand dalton.Stichopus japonicus selenka glycosaminoglycans exists with ionic species in solution, has the ability with the ion or the interaction between component of lotus positive electricity, belongs to polyanion.Stichopus japonicus selenka glycosaminoglycans polyanion character and the not isomorphic map in solution have constituted the basis of its various biological effect.The extracting method of existing 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 glycosaminoglycan has significant plyability, big as molecular weight difference, degree is inconsistent, conformation is complicated, microstructure is inhomogeneous and the residue number change is big etc., especially on a specific protein glycan molecule, kind, the quantity of the glycosaminoglycan chains that it contains there are differences, and make existing stichopus japonicus selenka glycosaminoglycans preparation method be difficult to obtain the high stichopus japonicus selenka glycosaminoglycans of purity.
Summary of the invention
At the deficiency that prior art exists, technical problem to be solved by this invention is, a kind of preparation method of stichopus japonicus selenka glycosaminoglycans is provided, so that effectively prepare stichopus japonicus selenka glycosaminoglycans, and improves its purity, the relation of balance stichopus japonicus selenka glycosaminoglycans yield and purity.
For solving the problems of the technologies described above, the technical solution used in the present invention is that a kind of preparation method of stichopus japonicus selenka glycosaminoglycans comprises the following steps: (1) bright stichopus japonicus pre-treatment; (2) alkaline hydrolysis stichopus japonicus body wall; (3) two enzyme enzymolysis stichopus japonicus body walls; (4) after enzymolysis finishes, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; (5) ethanol sedimentation stichopus japonicus selenka glycosaminoglycans; (6) dry stichopus japonicus selenka glycosaminoglycans raw sugar; (7) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans; (8) stichopus japonicus selenka glycosaminoglycans is decoloured; (9) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans; (10) dry stichopus japonicus selenka glycosaminoglycans refined sugar.
The preparation method of above-mentioned stichopus japonicus selenka glycosaminoglycans, it comprises following particular content:
(1) bright stichopus japonicus pre-treatment:
Use the distilled water flushing stichopus japonicus rapidly, remove the adherent foreign matter of stichopus japonicus body surface, dissect bright ginseng forward from the nearly anus of belly with microscler pocket knife, reject internal organ immediately, with distilled water flush away dirt, after weighing the stichopus japonicus body wall is placed clean large beaker rapidly, shred the stichopus japonicus body wall.Whole process should be rapidly and smooth, avoids the obvious self-dissolving of stichopus japonicus body wall as far as possible, otherwise can cause the loss of raw material.
(2) alkaline hydrolysis stichopus japonicus body wall: add 2 times of distilled waters to stichopus japonicus body wall weight, add the NaOH of 2mol/L then, the limit edged stirs, and avoids local superheating and alkali concn too high, and making NaOH solution final concentration is 0.5mo l/L, 4 ℃ of digested overnight.
(3) two enzyme enzymolysis of stichopus japonicus body wall:
A. trypsin digestion; Regulate pH value between the 8.2-9.0 with Glacial acetic acid.The trypsinase that adds 0.45% stichopus japonicus body wall quality, 52 ℃ of water-bath 5h fully stir in the water-bath process, and the NaOH solution of Dropwise 5 mmol/L is kept PH at 8.2-9.0; Then enzymolysis solution is cooled to below 30 ℃ termination reaction behind the maintenance 30mi n rapidly.
B. stomach en-enzymolysis:
Regulate pH value to 2.3 with 6mol/L HCl again, keep then adding the stomach en-of 0.40% stichopus japonicus body wall quality again between the pH value in 2 to 2.5, enzymolysis 4h in 50 ℃ water-bath, and fully stir at any time.Get enzymolysis solution 5ml in glass test tube before enzymolysis stops, the trichoroacetic acid(TCA) solution of Dropwise 5 % detects, and it is little when muddy that solution is, and illustrates that enzymolysis is more complete.Then enzymolysis solution is cooled to 30 ℃ rapidly, termination reaction behind the maintenance 30min.Collect supernatant liquor with the centrifugal 15min of 5000r/min then.
(4) after enzymolysis finished, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal, the addition of trichoroacetic acid(TCA) was 10% of an enzymolysis solution, progressively adds, and the limit edged stirs, and enzymolysis solution is become till the muddiness.12h is left standstill in solution refrigeration.Collect supernatant liquor with the centrifugal 25min of 5000r/min then.
(5) ethanol sedimentation stichopus japonicus selenka glycosaminoglycans:
The NaOH of Dropwise 5 mmol/L regulates the pH value to 6.8-7.2, and the centrifugal 10min of 7000r/min collects supernatant and adds dehydrated alcohol, and the limit edged stirs, and makes the ethanol final concentration be 55%, 4 ℃ and spends the night.
(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 acetone in the throw out are volatilized naturally, further dry during the throw out partial desiccation with low temperature vacuum drier, obtain rough stichopus japonicus selenka glycosaminoglycans.
(7) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans:
Crude polysaccharides fully was dissolved in distilled water by 1: 90, went precipitation with the centrifugal 15min of 3600r/min.Shift supernatant to new centrifuge tube, adding potassium acetate, to make its final concentration be 2mol/L, abundant mixing, 4 ℃ spend the night after, the centrifugal 10min of 7000r/min, collecting precipitation.
(8) stichopus japonicus selenka glycosaminoglycans decolouring:
Throw out fully is dissolved in the distilled water once more, regulates about pH to 9.0 with 2mol/L NaOH, 50 ℃ of insulations progressively add the decolouring that 30% hydrogen peroxide carries out polysaccharide and handle, and solution stops decolouring during near white.Solution is cooled to room temperature, leaves standstill 2h in 4 ℃ then.
The centrifugal 15min of 3600r/min goes precipitation, shifts supernatant liquor and be cooled to 0 ℃ to new centrifuge tube.Readjustment supernatant liquor pH is 2.0, and the centrifugal 15min of 3600r/min removes precipitation once more.
(9) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans:
Shift supernatant liquor to new centrifuge tube, adding potassium acetate, to make final concentration be 1.5mol/L, 4 ℃ of centrifugal 10min of back 7000r/min that spend the night, collecting precipitation.
(10) drying of stichopus japonicus selenka glycosaminoglycans refined sugar:
Throw out respectively washs 1 time with 80%, 95%, 100% ethanol respectively, washs respectively with dehydrated alcohol and acetone successively and dewaters 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.This polysaccharide odorless, tasteless has water absorbability, and is soluble in water, is soluble in hot water especially, and solution is viscosity, is insoluble to high concentration ethanol, acetone and other organic solvent.
The present invention has set up a kind of effective method for preparing stichopus japonicus selenka glycosaminoglycans, when alkaline process extracts, in conjunction with trypsinase and pepsic pair of collagenase treatment, promptly at first adopt alkaline purification stichopus japonicus tissue, use trypsinase and pepsin hydrolysis again, to strengthen the release of glycosaminoglycan in the stichopus japonicus body wall tissue.Be to improve the purity of stichopus japonicus selenka glycosaminoglycans, integrated application of the present invention the ethanol precipitation and the potassium acetate precipitator method, and use trichoroacetic acid(TCA) and remove protein.Method of the present invention can balance stichopus japonicus selenka glycosaminoglycans yield and the relation of purity, improves the purity of stichopus japonicus selenka glycosaminoglycans.
In the material involved in the present invention: fresh stichopus japonicus is available from market, Qingdao; Trypsinase, stomach en-are middle vast and boundless biotech firm product; Sodium hydroxide, dehydrated alcohol, acetone are analytical reagent, Laiyang, Shandong Fine Chemical Works product; Potassium acetate, trichoroacetic acid(TCA) are analytical reagent, those chemical reagent company limited products of Shanghai dust; The pH test paper is a Shandong rel health sterilization Science and Technology Ltd. product.
In the instrument involved in the present invention, desk-top refrigerated centrifuge is a German EPPnedorf company product; Freeze drier is SIM FD5508.
Embodiment
The present invention is described in more detail below in conjunction with specific embodiment.
Embodiment 1
A kind of preparation method of stichopus japonicus selenka glycosaminoglycans, it comprises following content:
(1) bright stichopus japonicus pre-treatment: use the distilled water flushing stichopus japonicus rapidly, remove the adherent foreign matter of stichopus japonicus body surface, dissect bright ginseng with microscler pocket knife forward from the nearly anus of belly, reject internal organ immediately, use distilled water flush away dirt more rapidly, after weighing the stichopus japonicus body wall is placed clean large beaker, shred the stichopus japonicus body wall then.Said process should be rapidly and smooth finishing, and avoids the obvious self-dissolving of stichopus japonicus body wall as far as possible.
(2) alkaline hydrolysis stichopus japonicus body wall: add 2 times of distilled waters to stichopus japonicus body wall weight, add the NaOH of 2mol/L then, the limit edged stirs, and avoids local superheating and alkali concn too high, and making NaOH solution final concentration is 0.5mol/L, 4 ℃ of digested overnight.
(3) two enzyme enzymolysis of stichopus japonicus body wall:
A. trypsin digestion: regulate pH value to 8.2 with Glacial acetic acid.The trypsinase that adds 0.45% stichopus japonicus body wall quality, 52 ℃ of water-bath 5h fully stir the NaOH solution of Dropwise 5 mmol/L down, keep PH 8.2; Then enzymolysis solution is cooled to 30 ℃ rapidly, termination reaction behind the maintenance 30min.
B. stomach en-enzymolysis: regulate pH value to 2.3 with 6mol/L HCl again, keep then adding the stomach en-of 0.40% stichopus japonicus body wall quality again between the pH value in 2 to 2.5, enzymolysis 4h in 50 ℃ water-bath, and fully stirring at any time.Get enzymolysis solution 5ml in glass test tube before enzymolysis stops, the trichoroacetic acid(TCA) solution of Dropwise 5 % detects, and it is little when muddy that solution is, and illustrates that enzymolysis is more complete.Then enzymolysis solution is cooled to 30 ℃ rapidly, termination reaction behind the maintenance 30min.Collect supernatant liquor with the centrifugal 15min of 5000r/min then.
(4) after enzymolysis finishes, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; The addition of trichoroacetic acid(TCA) is 10% of an 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 cold compartment of refrigerator.Collect supernatant liquor with the centrifugal 25min of 5000r/min then.
(5) ethanol sedimentation stichopus japonicus selenka glycosaminoglycans: the NaOH of Dropwise 5 mmol/L regulates pH value to 6.8 in above-mentioned supernatant liquor, and the centrifugal 10min of 7000r/min collects supernatant and adds dehydrated alcohol, and the limit edged stirs, and makes the ethanol final concentration be 55%, 4 ℃ and spends the night.
(6) dry stichopus japonicus selenka glycosaminoglycans raw sugar: with the centrifugal 10min of above-mentioned solution 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 acetone in the throw out are volatilized naturally, further dry during the throw out partial desiccation with low temperature vacuum drier, obtain rough stichopus japonicus selenka glycosaminoglycans.
(7) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans: Crude polysaccharides fully is dissolved in distilled water by 1: 90 ratio, goes to precipitate with the centrifugal 15min of 3600r/min.Shift supernatant to new centrifuge tube, adding potassium acetate, to make its final concentration be 2mol/L, abundant mixing, 4 ℃ spend the night after, the centrifugal 10min of 7000r/min, collecting precipitation.
(8) stichopus japonicus selenka glycosaminoglycans decolouring: throw out fully is dissolved in the distilled water once more, regulates pH to 9.0 with 2mol/LNaOH, 50 ℃ of insulations progressively add the decolouring that 30% hydrogen peroxide carries out polysaccharide and handle, and solution stops decolouring during near white.Solution is cooled to room temperature, places 4 ℃ of refrigerators to leave standstill 2h then.The centrifugal 15min of 3600r/min goes precipitation, shifts supernatant liquor and be cooled to 0 ℃ to new centrifuge tube.Readjustment supernatant liquor pH is 2.0, and the centrifugal 15min of 3600r/min removes precipitation once more.
(9) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans: shift supernatant liquor to new centrifuge tube, adding potassium acetate, to make final concentration be 1.5mol/L, and 4 ℃ are spent the night.The centrifugal 10min of 7000r/min, collecting precipitation.
(10) drying of stichopus japonicus selenka glycosaminoglycans refined sugar: throw out is respectively washed 1 time with 80%, 95%, 100% ethanol respectively, 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 dry unformed powder that obtains white is refining polysaccharide.
This polysaccharide odorless, tasteless has water absorbability, and is soluble in water, is soluble in hot water especially, and solution is viscosity, is insoluble to high concentration ethanol, acetone and other organic solvent.
Embodiment 2
The preparation method of the stichopus japonicus selenka glycosaminoglycans of present embodiment, the difference of itself and embodiment 1 is following two portions, all the other contents are identical:
The first, during a. trypsin digestion in two enzyme enzymolysis process of step (3) stichopus japonicus body wall, regulate pH value to 9.0 with Glacial acetic acid, the trypsinase that adds 0.45% stichopus japonicus body wall quality then, 52 ℃ of water-bath 5h fully stir the NaOH solution of Dropwise 5 mmol/L down, keep PH 9.0;
The second, when step (5) ethanol sedimentation stichopus japonicus selenka glycosaminoglycans, the NaOH of Dropwise 5 mmol/L regulates pH value to 7.2 in supernatant liquor.
Embodiment 3
The preparation method of the stichopus japonicus selenka glycosaminoglycans of present embodiment, the difference of itself and embodiment 1 is following two portions, all the other contents are identical:
The first, during a. trypsin digestion in two enzyme enzymolysis process of step (3) stichopus japonicus body wall, regulate pH value to 8.5 with Glacial acetic acid, the trypsinase that adds 0.45% stichopus japonicus body wall quality then, 52 ℃ of water-bath 5h fully stir the NaOH solution of Dropwise 5 mmo l/L down, keep PH 8.5;
The second, when step (5) ethanol sedimentation stichopus japonicus selenka glycosaminoglycans, the NaOH of Dropwise 5 mmol/L regulates pH value to 7.0 in supernatant liquor.
The preparation method of the stichopus japonicus selenka glycosaminoglycans of being implemented in the various embodiments described above all can balance stichopus japonicus selenka glycosaminoglycans yield and the relation of purity, improves the purity of stichopus japonicus selenka glycosaminoglycans.
Claims (2)
1. the preparation method of a stichopus japonicus selenka glycosaminoglycans comprises the following steps: (1) bright stichopus japonicus pre-treatment; (2) alkaline hydrolysis stichopus japonicus body wall; (3) two enzyme enzymolysis stichopus japonicus body walls; (4) after enzymolysis finishes, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; (5) use the ethanol sedimentation stichopus japonicus selenka glycosaminoglycans; (6) dry stichopus japonicus selenka glycosaminoglycans raw sugar; (7) precipitate stichopus japonicus selenka glycosaminoglycans with potassium acetate; (8) stichopus japonicus selenka glycosaminoglycans decolouring; (9) precipitate stichopus japonicus selenka glycosaminoglycans with potassium acetate; (10) dry stichopus japonicus selenka glycosaminoglycans refined sugar.
2. the preparation method of stichopus japonicus selenka glycosaminoglycans according to claim 1, it comprises following content:
(1) bright stichopus japonicus pre-treatment: use the distilled water flushing stichopus japonicus rapidly, remove the adherent foreign matter of stichopus japonicus body surface, dissect bright ginseng forward from the nearly anus of belly with microscler pocket knife, reject internal organ immediately, with distilled water flush away dirt, after weighing the stichopus japonicus body wall is placed clean large beaker rapidly, shred the stichopus japonicus body wall;
(2) alkaline hydrolysis stichopus japonicus body wall: add the distilled water of 2 times of weight in the stichopus japonicus body wall, add the NaOH of 2mol/L then, the limit edged stirs, and making NaOH solution final concentration is 0.5mol/L, 4 ℃ of digested overnight;
(3) two enzyme enzymolysis of stichopus japonicus body wall:
A. trypsin digestion: regulate pH value to 8.2-9.0 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 the NaOH solution of Dropwise 5 mmol/L are kept PH at 8.2-9.0; Then enzymolysis solution is cooled to below 30 ℃ termination reaction behind the maintenance 30min rapidly;
B. stomach en-enzymolysis:
Regulate the pH value between 2-2.5 with 6mol/L HCl again, add the stomach en-of stichopus japonicus body wall quality 0.40% again, in 50 ℃ water-bath, continue enzymolysis 4h, fully stir in the water-bath process; Enzymolysis is got enzymolysis solution 5ml in glass test tube before stopping, and the trichoroacetic acid(TCA) solution of Dropwise 5 % detects, and when solution is little muddiness, enzymolysis solution is cooled to rapidly below 30 ℃, keeps the 30min enzymolysis reaction; Collect supernatant liquor with the centrifugal 15min of 5000r/min then;
(4) after enzymolysis finishes, handle two enzyme enzymolysis supernatant liquors with trichoroacetic acid(TCA), the addition of trichoroacetic acid(TCA) is 10% of an 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, collect supernatant liquor with the centrifugal 25min of 5000r/min then;
(5) ethanol sedimentation stichopus japonicus selenka glycosaminoglycans: the NaOH solution of Dropwise 5 mmol/L is regulated the pH value of supernatant liquor to 6.8-7.2, 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;
(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 acetone in the throw out are volatilized naturally, further dry during the throw out partial desiccation with low temperature vacuum drier, obtain rough stichopus japonicus selenka glycosaminoglycans;
(7) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans: Crude polysaccharides fully is dissolved in distilled water by 1: 90 mass ratio, goes to precipitate with the centrifugal 15min of 3600r/min; Shift supernatant to new centrifuge tube, adding potassium acetate to its final concentration is 2mol/L, abundant mixing, 4 ℃ of precipitations of spending the night, the centrifugal 10min of 7000r/min then, collecting precipitation;
(8) stichopus japonicus selenka glycosaminoglycans decolouring: throw out fully is dissolved in the distilled water once more, regulates pH to 9.0 with 2mol/LNaOH, 50 ℃ of insulations progressively add the decolouring that 30% hydrogen peroxide carries out polysaccharide and handle, and solution stops decolouring during near white; Solution is cooled to room temperature, leaves standstill 2h in 4 ℃ then; The centrifugal 15min of 3600r/min goes precipitation, shifts supernatant liquor and be cooled to 0 ℃ to new centrifuge tube, and readjustment supernatant liquor pH is 2.0, and the centrifugal 15min of 3600r/min removes small amount of precipitate once more;
(9) potassium acetate precipitation stichopus japonicus selenka glycosaminoglycans:
Shift supernatant liquor to new centrifuge tube, adding potassium acetate to its final concentration is 1.5mol/L, 4 ℃ of precipitations of spending the night, the centrifugal 10min of 7000r/min then, collecting precipitation;
(10) dry stichopus japonicus selenka glycosaminoglycans:
Throw out is used 80%, 95%, 100% washing with alcohol 1 time respectively, 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.
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| CN102372788A (en) * | 2010-08-25 | 2012-03-14 | 天津科技大学 | Mactra veneriformis glycosaminoglycan extraction method |
| CN102372788B (en) * | 2010-08-25 | 2013-09-18 | 天津科技大学 | Mactra veneriformis glycosaminoglycan extraction method |
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| CN102993327A (en) * | 2012-12-30 | 2013-03-27 | 青岛市市立医院 | Method for preparing black holothurian glycosaminoglycan |
| CN102993324A (en) * | 2012-12-30 | 2013-03-27 | 青岛市市立医院 | Method for extracting holothuria leucospilota glycosaminoglycan |
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| CN103044564A (en) * | 2012-12-30 | 2013-04-17 | 青岛市市立医院 | Preparation method for holothuria leucospilota glycosaminoglycans |
| CN103087207A (en) * | 2012-12-30 | 2013-05-08 | 青岛市市立医院 | Method for extracting holothuria leucospilota glycosaminoglycans |
| CN102993323A (en) * | 2012-12-30 | 2013-03-27 | 青岛市市立医院 | Method for extracting and purifying glycosaminoglycans (GAGs) from stichopus japonicus |
| CN111011567A (en) * | 2019-11-28 | 2020-04-17 | 程红 | Tabletting candy with memory improving function and preparation method thereof |
| CN111925461A (en) * | 2020-08-31 | 2020-11-13 | 四川大学 | Fibrocartilage glycosaminoglycan and extraction method thereof |
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