CN1746191A - Extraction and preparation of comb scallop glycosaminoglycan - Google Patents
Extraction and preparation of comb scallop glycosaminoglycan Download PDFInfo
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- CN1746191A CN1746191A CN 200410035656 CN200410035656A CN1746191A CN 1746191 A CN1746191 A CN 1746191A CN 200410035656 CN200410035656 CN 200410035656 CN 200410035656 A CN200410035656 A CN 200410035656A CN 1746191 A CN1746191 A CN 1746191A
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Abstract
An extraction and production of merodont scallop mucopolysaccharide is disclosed. The process is carried out by weighing merodont scallop mucopolysaccharide visceral mass, adding into water, preparing uniform slurry, adding protease into uniform slurry, heating, reacting to obtain enzymolyzed liquid, centrifugalizing enzymolyzed liquid in centrifuge, separating out deposit, adding secondary alcohol solution into centrifugate, forming deposit, adding distilled water into separated deposit, preparing solution, heating, adding into active carbon, cooling, centrifugalizing, collecting supernatant, extraction filtering for supernatant to obtain purified liquid, super-filtering for purified liquid from filtering membrane to obtain filtrate with molecular weight 5k-100k, freezing and drying filtrate to obtain merodont scallop mucopolysaccharide. It achieves simple process, low cost and high purity. It can be used to prevent cardiovascular diseases.
Description
Technical field:
The present invention relates to a kind of extract from the chlamys farreri visceral mass that preparation has the vascular protection effect active substance---the method for chlamys farreri glycosaminoglycan (SS-GAG) belongs to the technological method to the further comprehensive processing and utilization of sea-food scallop.
Background technology:
Scallop culture mainly contains kinds such as chlamys farreri, bay scallop in China's development rapidly in recent years.The tankage of scallop gained after processing---scallop visceral mass (also claiming scallop body) major part is dropped, and causes significant wastage, and a large amount of scallop body resource letters are waited to develop.Contain multiple composition in the known scallop visceral mass, wherein glycosaminoglycan (GAG) is crucial active substance.According to domestic and foreign literature, contain multiple GAG in the sea mollusk, as the GAG in the animals such as stichopus japonicus, starfish, shark, Perna uiridis (Linnaeus), pteria martensii, have anti-freezing, lipopenicillinase, antitumor, anti-inflammatory, biological activity such as antiviral respectively.Caused the great attention of people to this family macromolecule active substance.Bibliographical information is arranged, from the clean limit of bay scallop, extract a kind of GAG of heparin sample, have anticoagulating active.Do not see both at home and abroad that for the method for from the chlamys farreri visceral mass, extracting the GAG that the vascular protection effect is arranged report is arranged.
Summary of the invention:
The objective of the invention is to provide a kind of material---method of chlamys farreri glycosaminoglycan that preparation has the protection vascular effect of from the chlamys farreri visceral mass, extracting, the physicochemical characteristics of the glycosaminoglycan of being extracted is: be the micro-yellow powder shape, odorless, tasteless, easy deliquescence, soluble in water; Be heparitin sample glycosaminoglycan, contained hexosamine is mainly galn, and hexuronic acid is based on iduronic acid; Its sulfate and carboxylic acid group's ratio is 1.69: 1; Designed preparation method has characteristics simple to operate, that reaction conditions is gentle, with low cost, yield and purity height, technology help fully utilizing.
The extraction preparation method of the chlamys farreri glycosaminoglycan that the present invention relates to is that the chlamys farreri visceral mass of culturing with the coastal waters is that raw material makes, and extracting and preparing technique is as follows:
With the aquatic foods product or the chlamys farreri visceral mass after freezing product and thawing weigh, add water and make homogenate, 8～32 ℃ of control room temperatures add proteolytic enzyme in homogenate, heated and stirred react 4～5 hours, must enzymolysis solution; Enzymolysis solution is centrifugal in whizzer, get centrifugate, discard throw out; Centrifugate is added the secondary ethanolic soln, form precipitation, separate, get throw out, reclaim supernatant liquor ethanol with whizzer; The throw out adding distil water is made into aqueous solution post-heating and adds gac stir, cooling is collected supernatant liquor after centrifugal, and it is heavy to discard, and forms sediment; To the supernatant liquor suction filtration, further remove the protein and the pigment of gac and absorption thereof, get clear liquid; With clear liquid filter membrane ultrafiltration, getting molecular weight is the filtrate of 5k～100k; With the filtrate lyophilize, promptly make the refining chlamys farreri glycosaminoglycan of yellow powder powder.
The extraction preparation method of this chlamys farreri glycosaminoglycan, the proteolytic enzyme that adds in homogenate is neutral protease and trypsinase, and the neutral protease add-on is 15～20g/kg homogenate, and the trypsinase add-on is in 5～10g/kg homogenate, temperature of reaction is 50 ℃, and the time is 4～5 hours.
The extraction preparation method of this chlamys farreri glycosaminoglycan, when centrifugate was added 2 ethanolic solns, the ethanolic soln concentration of its adding was 90～95%, and its final concentration is 65～75%, and the reaction times is 12 hours.
The extraction preparation method of this chlamys farreri glycosaminoglycan, described when the throw out adding distil water is made into the aqueous solution, the concentration of aqueous solution that makes is 4～6%.
The extraction preparation method of this chlamys farreri glycosaminoglycan describedly adds gac to the aqueous solution, and its gac add-on is that gac weight and aqueous solution volume ratio are 1～2%, and its Heating temperature is 80 ℃.
This extraction preparation method, described with the clear liquid ultrafiltration, 100k and 5k filter membrane are successively used in its ultrafiltration, and final score amount is the filtrate of 5k～100k.
Preparation method of the present invention; one of its advantage is that this method has promoted the comprehensive utilization of chlamys farreri; remaining visceral mass is dropped as the waste material major part behind the scallop of the chlamys farreri processing in the past post; and this preparation method utilizes the glycosaminoglycan that waste extracts pharmaceutical use; it is with low cost, and the vascular protection effect that this chlamys farreri glycosaminoglycan has is to be found confirmation first.When extracting glycosaminoglycan, also can reclaim protein in a large number, increase its economic benefit and pharmaceutical use.
Two of present method advantage is that extracting method is simple, yield and purity height.This method adopts neutral protease and trypsinase to be hydrolyzed, and more a kind of proteolytic enzyme or pancreas homogenate hydrolysis are more abundant.This method directly adds proteolytic enzyme and is hydrolyzed in chlamys farreri visceral mass homogenate, make acetone powder compared with extracting the polysaccharide acetone degreasing that adds earlier commonly used in the document, and then it is simply many to carry out enzymolysis.
Embodiment:
Embodiment 1: takes by weighing fresh chlamys farreri visceral mass 2kg, under 20 ℃ of conditions of room temperature, adds water and make homogenate for 2 liters, get homogenate and add neutral protease 35g, and trypsinase 10g, electronic stirring got enzymolysis solution in 5 hours under 50 ℃ of conditions; Centrifugal about 40 minutes with 5000 rev/mins; Extracting centrifugal liquid discards throw out; Centrifugate is added 95% ethanol, and the ethanol final concentration is 70%, leaves standstill 12 hours after stirring, through 6000 rev/mins centrifugal 30 minutes, throw out, add one time 95% ethanolic soln washing again, 6000/ minute centrifugal 30 minutes, reclaim supernatant liquor (ethanol), collect the wet about 85g of throw out; Throw out is made into 5% solution 1700ml with distilled water, is heated to 80 ℃, add gac 17g, stirred 30 minutes; Supernatant liquor is collected in cooling back centrifugal (6000 rev/mins) 30 minutes, discards precipitation; Supernatant liquor is further removed the protein and the pigment of gac and absorption thereof through husky heart funnel suction filtration, gets clear liquid; Clear liquid with the ultrafiltration of 100k filter membrane, is got filtered solution, continue ultrafiltration with the 5k filter membrane again, must filter only liquid (molecular weight 5k～100k),, promptly get the refining chlamys farreri glycosaminoglycan 4.5g of yellow powder powder through lyophilize.
Embodiment 2: get the chlamys farreri visceral mass 2kg that has thawed, press embodiment 1 method and extract preparation chlamys farreri glycosaminoglycan, the final 4.0g chlamys farreri glycosaminoglycan that gets.
Embodiment 3: divide different time, different material amount to extract preparation chlamys farreri glycosaminoglycan by embodiment 1 method, concrete outcome sees Table 1.
| Sequence number | Time | Charging capacity (chlamys farreri visceral mass) (kg) | Chlamys farreri glycosaminoglycan output (g) | To raw material recovery rate (%) |
| 1 | 02.3.18 | 0.5 | 1.40 | 0.28 |
| 2 | 02.3.25 | 0.5 | 1.27 | 0.25 |
| 3 | 02.5.15 | 0.5 | 1.45 | 0.29 |
| 4 | 02.10.10 | 2 | 4.20 | 0.21 |
| 5 | 02.11.28 | 2 | 4.00 | 0.20 |
| 6 | 02.12.2 | 2 | 4.50 | 0.225 |
| 7 | 02.12.12 | 2 | 4.30 | 0.215 |
| 8 | 02.12.24 | 2 | 4.58 | 0.229 |
Table 1
The chlamys farreri glycosaminoglycan of extracting preparation has been carried out multinomial pharmacodynamic experiment; the result shows that extracting the chlamys farreri glycosaminoglycan that obtains with the inventive method has the obvious suppression effect to vascular smooth muscle cell proliferation; foam cell is formed with the obvious suppression effect, and damage has significant protective effect to human vascular endothelial.Following brief description is as the further proof to effect of the present invention.
(1) the chlamys farreri glycosaminoglycan is to the restraining effect of vascular smooth muscle cell proliferation
Divide a word with a hyphen at the end of a line and the hyperplasia of vascular smooth muscle cell (VSMCs) are the most basic pathological changes of atherosclerosis, also are the major causes of coronary artery moulding postoperative restenosis.This experimental observation the chlamys farreri glycosaminoglycan to the restraining effect of vascular smooth muscle cell proliferation and to the influence of the synthesis secretion function of vascular smooth muscle cell.
1. the chlamys farreri glycosaminoglycan is to the active influence of vascular smooth muscle cell proliferation
Get the thoracic aorta of rabbit and rat respectively, adopt collagenase digestion and tissue block adherent method to carry out that vascular smooth muscle cell is former is commissioned to train fosterly, treat cell attachment, merge and grow up to the cultivation of going down to posterity behind the fine and close individual layer, get 5-8 and experimentize for cell.Observational study the chlamys farreri glycosaminoglycan to the active effect of Prostatropin (bFGF) inductive vascular smooth muscle cell proliferation, the influence of expressing to the active effect of 20% foetal calf serum (FBS) inductive vascular smooth muscle cell proliferation, to the thrombocyte derivation somatomedin (PDGF) of vascular smooth muscle cell and proliferating cell nuclear antigen (PCNA), to the vascular smooth muscle cell proliferation effect of kinetics.Experimental result shows that the chlamys farreri glycosaminoglycan is at 50,100,200 μ gmL
-1Under the concentration, the vascular smooth muscle cell proliferation due to 20%FBS, the bFGF is all had obvious restraining effect, and the expression of PCNA, PDGF strengthens can reverse vascular smooth muscle cell proliferation the time.Thereby the propagation that S phase cell is prevented vascular smooth muscle cell can be synthesized, reduce to the chlamys farreri glycosaminoglycan by the DNA that suppresses the vascular smooth muscle cell S phase.
2. the chlamys farreri glycosaminoglycan is to the influence of the vascular smooth muscle cell anti-oxidant function of propagation
Set up Prostatropin (bFGF) inductive vascular smooth muscle cell proliferation model, adopt methods such as enzyme process and thiobarbituricacid method to measure the variation of activity, mda content and the Total antioxidant capacity of SOD superoxide-dismutase and Selenoperoxidase in the nutrient solution of respectively organizing cell, observe of the influence of different concns chlamys farreri glycosaminoglycan the anti-oxidant function of the vascular smooth muscle cell of propagation.Experimental result shows that chlamys farreri glycosaminoglycan (100ug/ml, 200ug/ml) can strengthen the Total antioxidant capacity of vascular smooth muscle cell and the activity of Selenoperoxidase, reduces the generation of superoxide mda, improves the anti-oxidant function of cell.
3, the chlamys farreri glycosaminoglycan is to the vascular smooth muscle cell Nitric Oxide Synthase (NOS) of propagation and the influence of nitrogen protoxide (NO) content
Set up bFGF inductive rabbit vascular smooth muscle cell proliferation model.Adopt nitrate reductase method to measure the content of cell intracellular nitric oxide (NO), with colorimetric method for determining cell Nitric Oxide Synthase (NOS) vigor.Experimental result shows that the chlamys farreri glycosaminoglycan can improve content and the NOS activity of NO in the vascular smooth muscle cell.
4, the chlamys farreri glycosaminoglycan is to the influence of platelet-derived growth factor A chain (PDGF-A) mRNA expression in the vascular smooth muscle cell of propagation
Detect the expression of PDGF-A mRNA in the vascular smooth muscle cell with in situ hybridization, use DNAStar software design P of Rats DGF-A specificity cDNA oligonucleotide probe, synthetic and by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd through 5 ' end digoxigenin labeled, combine because of it can specificity take place with PDGF-A mRNA in the cytoplasm, can detect PDGF-A mRNA and its relative content in the sample by the digoxin detection kit.The result shows that the PDGF-A chain mRNA positive in the vascular smooth muscle cell after the SS-GAG of 100 μ g/ml and 200 μ g/ml is hatched is transcribed and weakened, and positive cell quantity reduces.
Above-mentioned result of study shows, the chlamys farreri glycosaminoglycan can by suppress cell DNA synthetic, reduce S phase cell, suppress in the vascular smooth muscle cell PDGF-A chain gene and transcribe and suppress vascular smooth muscle cell and transform to synthetic phenotype by shrinking phenotype, thereby the inhibition vascular smooth muscle cell proliferation; The generation that the chlamys farreri glycosaminoglycan also can be by reducing lipid peroxide simultaneously or superoxide is discharged increase, thus the antioxygenation of vascular smooth muscle cell is strengthened; In addition, the chlamys farreri glycosaminoglycan can improve total NOS vigor, the synthetic and release of increase NO of vascular smooth muscle cell; More than effect all can help suppressing atherosclerotic formation, and the protection vascular smooth muscle cell is avoided oxidative damage.
(2) the chlamys farreri glycosaminoglycan is to the restraining effect of foam cell formation
Foam cell is the main cytopathy in the early stage pathological change fat line of atherosclerosis (AS).The early stage monocyte of AS at first adheres to blood vessel endothelium, the subcutaneous scavenger cell that is divided in migrating to then, and the latter mainly engulfs a large amount of ox-LDL by scavenger receptor (SR) and forms foam cell, and then formation of deposits AS patch.Vascular endothelial growth factor (VEGF) is that a kind of cell mitogen of high degree of specificity is former, has the effect that promotes that cell fission propagation and blood vessel make up.In AS patch tissue, vegf expression obviously increases, and plays an important role in the AS early-stage development.This experiment is a material with the U937 human monocyte, foundation is adopted the influence to foam formation and vegf expression of enzyme process and enzyme linked immunological (ELISA) method research chlamys farreri glycosaminoglycan by Ox LDL (LDL) inductive monocyte source foam cell model.Experimental result shows that the chlamys farreri glycosaminoglycan can suppress the formation of foam cell, and intracellular total cholesterol, free cholesterol and cholesteryl ester are reduced, thereby and reverses the effect that the high expression level of VEGF in foam cell brought into play anti-AS.
(2) the chlamys farreri glycosaminoglycan is to the provide protection of human vascular endothelial damage
Atherosclerosis is the pathologic basis of cardiovascular and cerebrovascular diseases, and injury of vascular endothelial cells and dysfunction thereof are initiating, the key links that atherosclerosis takes place.It is to prevent and treat atherosclerotic key that the protection vascular endothelial cell is avoided damage.This experiment adopts Human umbilical vein endothelial cells (HUVEC) strain (CRL-2480) of vitro culture, studied SS-GAG to the vascular endothelial cell oxidative damage after the influence of proliferation activity and synthesis secretion function.
1, the chlamys farreri glycosaminoglycan is to the influence of the vascular inner skin cell proliferating activity of oxidative damage
By morphological observation, cell counting, mtt assay carried out the influence of the cell-proliferation activity damage that the chlamys farreri glycosaminoglycan causes the Fenton system respectively, the influence of cell-proliferation activity damage that poly-lysine (PL) is caused, to the influence of the vascular endothelial cell form of OX-LDL damage and proliferation activity and to H
2O
2The vascular inner skin cell proliferating activity of damage influence four experiments.Comprehensive above-mentioned experimental result as seen, the chlamys farreri glycosaminoglycan can obviously be improved the inhibition of the vascular inner skin cell proliferating activity that four kinds of oxidative damages cause, proliferation activity to normal vascular endothelial cell also has promoter action, illustrate that the chlamys farreri glycosaminoglycan has the ability of anti-vascular endothelial cell oxidative damage, the mechanism of action may with its inhibition active oxygen (H
2O
2) formation or directly to remove established active oxygen relevant, thereby antagonism H
2O
2Endotheliocyte is protected in the peroxide injury effect of vascular endothelial cell, understood that furtherly the chlamys farreri glycosaminoglycan is in the vital role of preventing and treating aspect the atherosclerosis.
2, the chlamys farreri glycosaminoglycan discharges the influence of serum lactic dehydrogenase (LDH) to the endotheliocyte of Fenton system damage
Set up Fenton system endothelial cell damage model, observe of the influence of chlamys farreri glycosaminoglycan lactic dehydrogenase enzyme activity in the culture fluid of endothelial cell.The result shows the control group through the damage of Fenton system, the LDH vigor obviously increases, illustrate that oxyradical has direct toxic action to endotheliocyte, and the chlamys farreri glycosaminoglycan can reduce the LDH vigor of damage control group, prompting chlamys farreri glycosaminoglycan has anti-oxidant function, can suppress oxyradical cell membrane and mitochondrial damage, alleviate the direct toxic action of lipid peroxide and meta-bolites thereof endotheliocyte.This also is one of mechanism of chlamys farreri glycosaminoglycan study of anti-atherogenic effect.
3, the chlamys farreri glycosaminoglycan is to the vascular endothelial cell intracellular nitric oxide of OX-LDL damage and the influence of nitric oxide synthetase
Set up vascular endothelial cell OX-LDL damage model, adopt nitrate reductase method to measure the content of NO in the endotheliocyte; The utilization chemical colorimetry, the activity of NOS in the mensuration cell.Experimental result shows, but OX-LDL coup injury vascular endothelial cell suppresses the activity of its endothelial cell type NO synthetic enzyme (eNOS), suppresses the synthetic of NO directly or indirectly and discharges.The chlamys farreri glycosaminoglycan can alleviate the above-mentioned effect of OX-LDL, and what further prove the chlamys farreri glycosaminoglycan has provide protection to vascular endothelial cell.
4, the chlamys farreri glycosaminoglycan is to the vascular endothelial cell oxidase system of OX-LDL damage and the influence of oxidation products
Set up vascular endothelial cell OX-LDL damage model, adopt xanthine oxidase, measure the activity of SOD in the cell; Use the thiobarbituricacid development process, measure MDA content in the vascular endothelial cell.Experimental result shows that the vascular endothelial cell oxidative damage that OX-LDL causes is suppressed its SOD activity, and the generation of the representative meta-bolites MDA of lipid peroxide obviously increases; SOD activity in the chlamys farreri glycosaminoglycan protection group endotheliocyte of three kinds of dosage (50 μ g/ml, 100 μ g/ml, 200 μ g/ml) is recovered gradually with the increase of the concentration of chlamys farreri glycosaminoglycan, and MDA content also reduces gradually simultaneously.Therefore can think that the chlamys farreri glycosaminoglycan has provide protection to the vascular endothelial cell lipid peroxidation injury that OX-LDL causes, thereby prove that further the chlamys farreri glycosaminoglycan is relevant with its antioxygenation to Vascular Endothelial mechanism.
5, the chlamys farreri glycosaminoglycan is to prostacyclin I in the vascular endothelial cell of OX-LDL damage
2And thromboxane A
2Influence
Set up vascular endothelial cell OX-LDL damage model, adopt the prostacyclin I of measured by radioimmunoassay vascular endothelial cell
2Metabolite content and thromboxane A
2Metabolite content.This experimental result shows that OX-LDL can suppress prostacyclin I
2Synthetic, stimulate vascular endothelial cell secretion thromboxane A
2, break prostacyclin I
2-thromboxane A
2Balance, thereby promoted atherosclerotic the development.The chlamys farreri glycosaminoglycan can reverse The above results by different approaches, promptly by rising prostacyclin I
2Level reduces thromboxane A
2Level is kept the equilibrium state of the two, thereby stops the formation of atherosclerosis (AS), and this may be one of important mechanism of chlamys farreri glycosaminoglycan study of anti-atherogenic effect.
6, the chlamys farreri glycosaminoglycan is to vascular endothelial cell vaso-active substance endothelin (ET) and the influence of Angiotensin II (AII) excretory
Set up vascular endothelial cell Fenton system model of oxidative, adopt radioimmunoassay method, utilize homogeneous phase competition principle directly to measure the content of ET and AII in the culture fluid of endothelial cell.This experiment finds that the oxyradical that the Fenton system is produced can make endotheliocyte secretion endothelin increase, and the chlamys farreri glycosaminoglycan can obviously suppress the secretion of endotheliocyte endothelin, and high density chlamys farreri glycosaminoglycan group endothelin secretory volume is starkly lower than low dose group.
This experiment confirms that also after oxyradical caused endothelial cell damage, the secretion of Angiotensin II also obviously increased.According to domestic and foreign literature, Angiotensin II has proinflammatory effect, can promote vascular endothelial cell generation adhesion molecule and stimulate smooth muscle cell to produce interleukin-6, participates in the formation of AS.Angiotensin can also make monocyte chemotactic factor (MCP-1) raise, promote monocyte with the endotheliocyte adhesion and to vessel invasion, Angiotensin II can activate smooth muscle cell (SMC) phosphatidyl inositol kinase, causes activating SMC mitotic division, promotes the smooth muscle cell apoptosis.Angiotensin II causes development of atherosclerosis by above mechanism.This research prompting, chlamys farreri glycosaminoglycan suppress vascular endothelial cell secretion Angiotensin II, so, may be by the generation and the development of above mechanism retardance AS pathology.
7, the chlamys farreri glycosaminoglycan is to the influence of vascular endothelial cell adhesion molecule-1 (VCAM-1) expression of oxidative damage initiation
Set up vascular endothelial cell Fenton system model of oxidative, adopt enzyme-linked immunoassay method, both double antibodies sandwich ELLSA method detected VCAM-1.This experimental result shows; the expression of the endotheliocyte VCAM-1 of the chlamys farreri glycosaminoglycan protection of adding various dose is starkly lower than the damage control group; and presenting with chlamys farreri glycosaminoglycan dosage increases; VCAM-1 expresses the trend that reduces successively; show that the chlamys farreri glycosaminoglycan can suppress vascular endothelial cell adhesion molecule-1 and express; vascular endothelial cell damage is had protective effect, and its effect is strengthened with dosage.
8, the chlamys farreri glycosaminoglycan suppresses the research that Thr6 PDGF BB B chain (PDGF-BB) is expressed
Set up vascular endothelial cell Fenton system model of oxidative, adopt double antibodies sandwich ELISA method to measure the expression amount of PDGF-BB.Discover endotheliocyte under the oxyradical that the Fenton system is produced stimulates, PDGF-BB expresses and obviously increases, and the chlamys farreri glycosaminoglycan can suppress the expression of PDGF-BB.Known Thr6 PDGF BB (PDGF) promotes one of most important factor of smooth muscle proliferation when being atherosclerosis, PDGF plays a crucial role in AS generating process, vessel wall inflammation and reparation.Originally studies show that the chlamys farreri glycosaminoglycan suppresses the generation of vascular endothelial cell PDGF, thereby weakened the effect of the mitogenesis of PDGF, short smooth muscle proliferation and conversion, therefore help weakening and blocking the generation and the development of AS focus, this may be one of important mechanism of its study of anti-atherogenic effect.
Comprehensive above-mentioned experimental result shows; the chlamys farreri glycosaminoglycan has good protective action to vascular endothelial cell damage; illustrate the chlamys farreri glycosaminoglycan study of anti-atherogenic effect may with its protection endotheliocyte, the activity etc. that the secretion that suppresses vascular endothelial cell impaired back endothelin and Angiotensin II increases, reverses expression enhancing, anti peroxidation of lipid, increase endothelial type nitric oxide synthase and the prostaglandin synthetase of VCAM-1, PDGF-BB has substantial connection.This control for atherosclerosis and coronary heart disease provides new theory and experimental basis.
Claims (6)
1, the extraction preparation method of chlamys farreri glycosaminoglycan, it is characterized in that its extracting and preparing technique is as follows: with the aquatic foods product or the chlamys farreri visceral mass after freezing product and thawing weigh, add water and make homogenate, 8～32 ℃ of control room temperatures, in homogenate, add proteolytic enzyme, heated and stirred was reacted 4～5 hours, got enzymolysis solution; Enzymolysis solution is centrifugal in whizzer, get centrifugate, discard throw out; Centrifugate is added the secondary ethanolic soln, form precipitation, separate, get throw out, reclaim supernatant liquor ethanol with whizzer; The throw out adding distil water is made into aqueous solution post-heating and adds the gac stirring, and cooling is after centrifugal, and the collection supernatant liquor discards precipitation; To the supernatant liquor suction filtration, further remove the protein and the pigment of gac and absorption thereof, get clear liquid; With clear liquid filter membrane ultrafiltration, getting molecular weight is the filtrate of 5k～100k; With the filtrate lyophilize, promptly make the refining chlamys farreri glycosaminoglycan of yellow powder powder.
2, the extraction preparation method of chlamys farreri glycosaminoglycan according to claim 1, it is characterized in that the proteolytic enzyme that adds is neutral protease and trypsinase in homogenate, the neutral protease add-on is 15～20g/kg homogenate, the trypsinase add-on is in 5～10g/kg homogenate, and temperature of reaction is 50 ℃.
3, the extraction preparation method of chlamys farreri glycosaminoglycan according to claim 1, when it is characterized in that centrifugate added 2 ethanolic solns, the ethanolic soln concentration of its adding is 90～95%, and its final concentration is 65～75%, and the reaction times is 12 hours.
4, the extraction preparation method of chlamys farreri glycosaminoglycan according to claim 1, when it is characterized in that the throw out adding distil water is made into the aqueous solution, the concentration of aqueous solution that makes is 4～6%.
5, the extraction preparation method of chlamys farreri glycosaminoglycan according to claim 1 is characterized in that the aqueous solution is added gac to be stirred, and its gac add-on is that gac weight and aqueous solution volume ratio are 1-2%, and its Heating temperature is 80 ℃.
6, the extraction preparation method of chlamys farreri glycosaminoglycan according to claim 1 is characterized in that with the clear liquid ultrafiltration 100k and 5k filter membrane are successively used in its ultrafiltration, and final score amount is the filtrate of 5k～100k.
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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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| CN103127162A (en) * | 2011-12-03 | 2013-06-05 | 青岛大学 | Application of scallop glycosaminoglycan in preparing dementia resistant medicine |
| CN103127162B (en) * | 2011-12-03 | 2015-02-04 | 青岛大学 | Application of scallop glycosaminoglycan in preparing dementia resistant medicine |
| CN109053922A (en) * | 2018-07-20 | 2018-12-21 | 大连工业大学 | Scallop Viscus Polysaccharide, extracting method and its application and pharmaceutical composition |
| CN109053922B (en) * | 2018-07-20 | 2020-12-22 | 大连工业大学 | Patinopecten yessoensis visceral polysaccharide, extraction method and application thereof, and pharmaceutical composition |
| CN111320707A (en) * | 2020-03-31 | 2020-06-23 | 大连工业大学 | Patinopecten yessoensis skirt polysaccharide and extraction method and application thereof |
| CN111320707B (en) * | 2020-03-31 | 2021-09-24 | 大连工业大学 | Patinopecten yessoensis skirt polysaccharide and extraction method and application thereof |
| CN113481270A (en) * | 2021-06-08 | 2021-10-08 | 中国科学院海洋研究所 | Method for extracting functional glycopeptide from scallop skirt |
| CN113481270B (en) * | 2021-06-08 | 2023-07-04 | 中国科学院海洋研究所 | Method for extracting glycopeptide from scallop skirt |
| CN113481271A (en) * | 2021-06-22 | 2021-10-08 | 中国科学院南海海洋研究所 | Marine biological active peptide capable of effectively relieving skin sunburn and preparation method and application thereof |
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