CN108578791A - A kind of hirudin is modified the preparation method of anticoagulant material - Google Patents
A kind of hirudin is modified the preparation method of anticoagulant material Download PDFInfo
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- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
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- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
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- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/06—Use of macromolecular materials
- A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
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- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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Abstract
The present invention relates to technical field of biological medical material preparation, and in particular to a kind of hirudin is modified the preparation method of anticoagulant material.The present invention is by spider extracting solution, chitosan, Phellinus polysaccharide, glucose etc. is mixed to get induction liquid, induction liquid is poured into chitterlings, induction liquid weightening is sucked with eurysome golden thread leech, it squeezes out eurysome golden thread leech saliva and obtains purifying filtrate, by silk fibroin protein solution, gelatin solution, quadracycline, purify filtrate mixing, ultrafiltration obtains concentrate, finally hirudin is obtained by freeze-drying be modified anticoagulant material, contain multiple proteins and vitamin in gelatin solution, it can play the role of extra-nutrition to matrix, unsaturated fatty acid contained by fish scale, it can inhibit the oxidation of hemalbumin in blood, to avoid that blood coagulation effect occurs, hirudin energy anticoagulation, antithrombus formation, it can also inhibit the stimulation of the fibroblastic proliferation and fibrin ferment Human Umbilical Vein Endothelial Cells of thrombin induction, it has a extensive future.
Description
Technical field
The present invention relates to technical field of biological medical material preparation, and in particular to a kind of hirudin is modified anticoagulant material
Preparation method.
Background technology
Biomaterial is a kind of reparation can be used for animal organ and tissue and replacement, the Clinics and Practices of disease, and dynamic
Object bio-compatible, the material with property or function.Anticoagulant biomaterial is the important component of biomaterial, quilt
Be widely used in on human blood and the medical material that is in contact of tissue, such as hemodialysis system, extracorporeal circulation system, artificial
Heart valve, pacemaker, artificial blood vessel, intravascular stent, conduit etc..The anticoagulant material being in direct contact with blood is not only wanted
Ask with histocompatbility, inflammation will not be caused to bio-tissue, and require blood compatibility, can antithrombotic,
Blood coagulation phenomenon will not occur in material surface, it is also desirable to have elasticity similar with tissue, ductility and good resistance to tired
The biology such as labor is mechanical compatibility.
In order to improve the blood compatibility of medical material, home and abroad scholar filters out blood compatibility from current material
Preferable material, such as：Polyurethane, polyethylene, polyvinyl chloride, polystyrene, polyvinyl alcohol, polytetrafluoroethylene (PTFE), gathers poly(ether-urethane)
Amide etc..It, be from anti-freezing when the basis material to anticoagulant material carries out anticoagulation modification when preparing anticoagulant material
The firmness of blood agent, the bioactivity of anticoagulant and anticoagulant are comprehensive at several aspects such as the concentration of substrate material surface
It closes and considers.Currently, the main method for preparing anticoagulant material is by substrate material surface test tube of hepari, by being modified in basis material
Surface obtains polyoxy acetylene structure and by being modified basis material to obtain poly- Phosphorylcholine substance, however heparin is answered in clinic
It uses and will produce many adverse side effects, if medication can excessively cause hematostaxis and heparin-induced thrombocytopenia,
Prolonged application can generate temporary alopecia, osteoporosis and spontaneous fracture, to there is the Case treatment effect of Antithrombin deficiency
It is bad, in addition, the heparin structure of separate sources is widely different, causes its physiological activity unstable, detached from animal's liver
Heparin is it is also possible to induction and the relevant prion pollution of rabid ox disease etc..
In general, the above method is realized by physical absorption and chemical bonding.The former is easy, but the amount of anticoagulant
Change and be difficult to control, and the anticoagulation duration is not grown, easily dissolves and be lost in blood；Chemical conjugation methods prepare anticoagulant material
When material, usually in material surface chemical graft anticoagulant.Anticoagulant is bonded in by this method securely by covalent bond
Material surface changes the normal conformation of anticoagulant, cause the active part that there are anticoagulant functions to cross may be embedded or
It destroys, thus reduces its bioactivity so that anticoagulant effect reduces to some extent, and some even disappears completely, and loses
Application value clinically.In addition, in clinical application, due to factors such as hospital's particular surroundings, anticoagulant biomaterial is easy
Cause to infect.
Therefore, it is necessary to anticoagulant biomaterials to have both good anticoagulation function and anti-microbial property.
Invention content
Present invention mainly solves the technical issues of, for based on heparin, and heparin is in clinic in current anticoagulant material
Using above will produce many adverse side effects, as medication can excessively cause hematostaxis and heparin-induced decrease of platelet
Disease, due to factors such as hospital's particular surroundings, causes anticoagulant biomaterial to easily cause the defect of infection in clinical application,
Provide a kind of preparation method of hirudin modification anticoagulant material.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is：
A kind of hirudin is modified the preparation method of anticoagulant material, it is characterised in that specifically preparation process is：
（1）100~150g live bodies spider is taken to be homogenized 2~3 times with colloid mill, it is isolated by gained slurries screen filtration
Filtrate is spider extracting solution, the spider extracting solution that takes 120~150mL above-mentioned, 60~80g chitosans, 4~5g Phellinus polysaccharide and
12~15g glucose mixes, then is diluted to 4~5L with distilled water, and stirring and dissolving obtains induction liquid；
（2）Above-mentioned induction liquid is poured into the chitterlings after exenterating, one section of chitterlings is filled into induction liquid, is tightened with line
Intestinal tube both ends make its expansion, obtain the intestinal tube of the liquid containing induction, and the dispersion of 400~500g eurysome golden thread leech is placed in glass guide channel, will
The liquid intestinal tube containing induction is laid in glass guide channel, is taken out after allowing eurysome golden thread leech freely to suck enteral induction liquid；
（3）Manual compression goes out eurysome golden thread leech saliva, obtains natural hirudin crude product, by natural hirudin crude product sieve mistake
Filter, isolated filtrate is put into beaker, then beaker is placed in water bath with thermostatic control and is heated, and is removed with screen filtration after cooling
Filter residue, isolated purifying filtrate；
（4）20~25g fibroin albumens dried are scattered in 200~300mL sodium hydroxide solutions, heat temperature raising is kept the temperature
To dispersion liquid, continues dropwise addition 90~100mL acetums after being cooled to room temperature into dispersion liquid, obtain silk fibroin protein solution；
（5）It takes 40~50g grass carp dry fish scales to be added in the beaker equipped with 400~450mL aseptic deionized waters, beaker water-bath is added
Heat, heating, then 4~5g trypsase is added in beaker, heat preservation enzymolysis, filtering removal filtrate obtains enzymolysis fish scale, will digest
Fish scale is washed with deionized 3~5 times, then the enzymolysis fish scale after washing is mixed with deionized water, and heating water bath heating is gone out
Enzyme, cooling, heat preservation are concentrated to give gelatin solution；
（6）200~220mL silk fibroin protein solutions, 80~100mL gelatin solutions, 2~3g quadracyclines and 400~500mL is pure
Change filtrate mixing, carry out ultrafiltration with ultrafiltration membrane, obtain concentrate, concentrate is placed in freeze drier, freeze-drying obtains water
Leech element is modified anticoagulant material.
Step（1）The mesh size specification used is 100 mesh, and induced fluid product is 4~5L after distilled water dilution.
Step（2）The chitterlings are one section every 40~50cm points, and eurysome golden thread leech passes through Nature enemy 10~15
It, it is 30~50min that eurysome golden thread leech, which sucks the induction liquid time, sucked eurysome golden thread leech weightening after induction liquid reach 1000~
It is taken out when 1200g.
Step（3）The mesh size specification is 200 mesh, and water bath with thermostatic control temperature is 45~50 DEG C, heating time 40
~45min, sieve used in cooled and filtered are 180 mesh.
Step（4）The mass fraction of the sodium hydroxide solution is 5%, and temperature is 40~45 DEG C after heat temperature raising, heat preservation
Time is 2~3h, and the mass fraction of acetum is 2%.
Step（5）Temperature is 35~40 DEG C after the beaker warming-in-water, and heat preservation enzymolysis time is 4~5h, digests fish
Squama and deionized water mixing quality ratio are that temperature is 90~95 DEG C after 1 ︰ 10, then heating water bath heating, the enzyme deactivation time is 10~
15min, temperature is 55~60 DEG C after cooling, and heat preservation concentration time is 6~7h.
Step（6）The ultrafiltration retaining molecular weight is 7000~8000D, and the ultrafiltration time is 8~10h, freeze-drying
Temperature is -40~-20 DEG C, and sublimation drying is 3~4h.
The beneficial effects of the invention are as follows：
（1）The present invention crosses sieve with spider defibrination and obtains spider extracting solution, by spider extracting solution, chitosan, Phellinus polysaccharide, grape
Sugared mixed diluting dissolves to obtain induction liquid, and induction liquid is poured into chitterlings, intestinal tube both ends are tightened with line, obtains the liquid containing induction
Intestinal tube freely sucks induction liquid weightening with eurysome golden thread leech, then manual compression goes out eurysome golden thread leech saliva, obtains natural hirudin
Crude product obtains purifying filtrate by heating sieving, gelatin solution is concentrated to give with heat preservation after the dry fish scale enzymolysis of grass carp, by fibroin egg
White solution, gelatin solution, quadracycline, purifying filtrate mixing, carry out ultrafiltration with ultrafiltration membrane, obtain concentrate, finally through supercooling
Jelly is dried to obtain hirudin and is modified anticoagulant material, and suppression is added using the extracting solution of spider as induction liquid in the present invention wherein
The glucide of bacterium anti-inflammatory and enhancing immunity of organism makes anticoagulant material have certain anti-infection ability, made bright of fish scale
Contain multiple proteins and vitamin in glue, extra-nutrition, and a variety of insatiable hungers contained by fish scale can be played the role of to matrix
And aliphatic acid, the oxidation of hemalbumin in blood can be inhibited, to avoid that blood coagulation effect occurs；
（2）Hirudin energy anticoagulation, antithrombus formation and the coagulation factor for preventing catalyzed by thrombin extracted in the present invention
The further blood stasis phenomenon such as activation and platelet response, in addition, it can also inhibit fibroblastic proliferation of thrombin induction
With the stimulation of fibrin ferment Human Umbilical Vein Endothelial Cells, compared with heparin, not only dosage is few for it, will not cause bleeding, also not dependent on endogenous
Property confactor, and heparin then has the danger to cause bleeding, antithrombase is past in the pathogenic process of disseminated intravascular coagulation
Toward can reduce, the effect of this will limit heparin, preferable effect is had using hirudin, passing through spider extracting solution and anti-inflammatory enhances
Immune carbohydrate is compound to obtain induction liquid, which can specifically bind after being sucked by leech with hirudin, form functionalization
Anticoagulant material, fibroin albumen and gelatin solution can form protein microsphere, can be used as the load of anticoagulation material in blood
Body has multiple hydroxyls that can be combined with polypeptide type hirudin on the ring of biocidal property quadracycline, improves the negative of hirudin on carrier
Carrying capacity has a extensive future.
Specific implementation mode
It takes 100~150g live bodies spider to be homogenized 2~3 times with colloid mill, 100 mesh screens of gained slurries is filtered, detach
Obtained filtrate is spider extracting solution, the spider extracting solution that takes 120~150mL above-mentioned, 60~80g chitosans, 4~5g Phellinus
Polysaccharide and the mixing of 12~15g glucose, then it is diluted to 4~5L with distilled water, stirring and dissolving obtains induction liquid；By above-mentioned induction liquid
It pours into the chitterlings after exenterating, fills induction liquid by being one section every 40~50cm, tightening intestinal tube both ends with line makes
It is expanded, and obtains the intestinal tube of the liquid containing induction, and 400~500g eurysome golden thread leech dispersion in hungry 10~15 days is placed in glass guide channel
In, induction liquid intestinal tube will be contained and be laid in glass guide channel, eurysome golden thread leech is allowed freely to suck enteral induction 30~50min of liquid,
Until eurysome golden thread leech integrally increases weight to 1000~1200g；Manual compression goes out eurysome golden thread leech saliva, and it is thick to obtain natural hirudin
200 mesh screens of natural hirudin crude product are filtered, isolated filtrate are put into beaker, then beaker is placed in 45 by product
40~45min is heated in~50 DEG C of water bath with thermostatic control, is filtered with 180 mesh screens after cooling and is removed filter residue, isolated purifying filter
Liquid；20~25g fibroin albumens dried are scattered in the sodium hydroxide solution that 200~300mL mass fractions are 5%, heating rises
Temperature keeps the temperature 2~3h, obtains dispersion liquid, continue dropwise addition 90~100mL mass after being cooled to room temperature into dispersion liquid to 40~45 DEG C
The acetum that score is 2%, obtains silk fibroin protein solution；Taking the addition of 40~50g grass carp dry fish scales to be equipped with, 400~450mL is sterile to be gone
In the beaker of ionized water, by beaker heating water bath, 35~40 DEG C are warming up to, then 4~5g trypsase is added in beaker, heat preservation
4~5h of enzymolysis, filtering removal filtrate, obtains enzymolysis fish scale, and enzymolysis fish scale is washed with deionized 3~5 times, then will be after washing
Enzymolysis fish scale and deionized water be that 1 ︰ 10 is mixed in mass ratio, heating water bath is warming up to 90~95 DEG C, 10~15min of enzyme deactivation,
55~60 DEG C are cooled to, heat preservation 6~7h of concentration obtains gelatin solution；200~220mL silk fibroin protein solutions, 80~100mL is bright
Glue, 2~3g quadracyclines and 400~500mL purifying filtrate mixing, with the ultrafiltration that molecule interception is 7000~8000D
Film carries out 8~10h of ultrafiltration, obtains concentrate, concentrate is placed in freeze drier, be freeze-dried under the conditions of -40~-20 DEG C
3~4h obtains hirudin and is modified anticoagulant material.
It takes 100g live bodies spider to be homogenized 2 times with colloid mill, 100 mesh screens of gained slurries is filtered, isolated filter
Liquid is spider extracting solution, spider extracting solution, 60g chitosans, 4g Phellinus polysaccharide and the mixing of 12g glucose for taking 120mL above-mentioned,
It is diluted to 4L with distilled water again, stirring and dissolving obtains induction liquid；Above-mentioned induction liquid is poured into the chitterlings after exenterating,
Induction liquid is filled by being one section every 40cm, tightening intestinal tube both ends with line makes its expansion, obtains the intestinal tube of the liquid containing induction, will be hungry
It starves the dispersion of 10 days 400g eurysome golden thread leech to be placed in glass guide channel, induction liquid intestinal tube will be contained and be laid in glass guide channel, allow expanded letter gold thread
Leech freely sucks enteral induction liquid 30min, until eurysome golden thread leech integrally increases weight to 1000g；Manual compression goes out expanded letter gold
Line leech saliva, obtains natural hirudin crude product, and 200 mesh screens of natural hirudin crude product are filtered, by isolated filtrate
It is put into beaker, then beaker is placed in 45 DEG C of water bath with thermostatic control and heats 40min, filter removal with 180 mesh screens after cooling and filter
Slag, isolated purifying filtrate；The 20g fibroin albumens dried are scattered in the sodium hydroxide solution that 200mL mass fractions are 5%
In, 40 DEG C are heated to, 2h is kept the temperature, obtains dispersion liquid, continues dropwise addition 90mL mass point after being cooled to room temperature into dispersion liquid
Number is 2% acetum, obtains silk fibroin protein solution；Take 40g grass carp dry fish scales that the burning equipped with 400mL aseptic deionized waters is added
In cup, by beaker heating water bath, 35 DEG C are warming up to, then 4g trypsase is added in beaker, heat preservation enzymolysis 4h, filtering removal filter
Liquid obtains enzymolysis fish scale, enzymolysis fish scale is washed with deionized 3 times, then the enzymolysis fish scale after washing is pressed with deionized water
Mass ratio mixes for 1 ︰ 10, and heating water bath is warming up to 90 DEG C, enzyme deactivation 10min, is cooled to 55 DEG C, and heat preservation concentration 6h obtains gelatin
Liquid；200mL silk fibroin protein solutions, 80mL gelatin solutions, 2g quadracyclines and 400mL purifying filtrate are mixed, retained with molecule
The ultrafiltration membrane that amount is 7000D carries out ultrafiltration 8h, obtains concentrate, concentrate is placed in freeze drier, cold under the conditions of -20 DEG C
Dry 3h is lyophilized, obtains hirudin and is modified anticoagulant material.
It takes 125g live bodies spider to be homogenized 2 times with colloid mill, 100 mesh screens of gained slurries is filtered, isolated filter
Liquid is spider extracting solution, and spider extracting solution, 70g chitosans, 4.5g Phellinus polysaccharide and the 14g glucose for taking 135mL above-mentioned are mixed
It closes, then 4.5L is diluted to distilled water, stirring and dissolving obtains induction liquid；It is small that above-mentioned induction liquid is poured into the pig after exenterating
Enteral fills induction liquid by being one section every 45cm, and tightening intestinal tube both ends with line makes its expansion, obtains the intestinal tube of the liquid containing induction,
Hungry 13 days 450g eurysome golden thread leech dispersion is placed in glass guide channel, induction liquid intestinal tube will be contained and be laid in glass guide channel, allows width
Body gold thread leech freely sucks enteral induction liquid 40min, until eurysome golden thread leech integrally increases weight to 1100g；Manual compression goes out
Eurysome golden thread leech saliva obtains natural hirudin crude product, and 200 mesh screens of natural hirudin crude product are filtered, will be isolated
Filtrate be put into beaker, then beaker is placed in 47 DEG C of water bath with thermostatic control and heats 43min, it is cooling after with the filtering of 180 mesh screens
Remove filter residue, isolated purifying filtrate；The 22g fibroin albumens dried are scattered in the hydroxide that 250mL mass fractions are 5%
In sodium solution, 43 DEG C are heated to, 2.5h is kept the temperature, obtains dispersion liquid, continue to be added dropwise into dispersion liquid after being cooled to room temperature
The acetum that 95mL mass fractions are 2%, obtains silk fibroin protein solution；Taking the addition of 45g grass carp dry fish scales to be equipped with, 425mL is sterile to be gone
In the beaker of ionized water, by beaker heating water bath, 37 DEG C are warming up to, then 4.5g trypsase is added in beaker, heat preservation enzymolysis
4.5h, filtering removal filtrate, obtains enzymolysis fish scale, enzymolysis fish scale is washed with deionized 4 times, then by the enzymolysis fish after washing
Squama is that 1 ︰ 10 is mixed in mass ratio with deionized water, and heating water bath is warming up to 93 DEG C, enzyme deactivation 13min, is cooled to 57 DEG C, heat preservation is dense
Contracting 6.5h, obtains gelatin solution；210mL silk fibroin protein solutions, 90mL gelatin solutions, 2.5g quadracyclines and 450mL are purified and filtered
Liquid mixes, and the ultrafiltration membrane for being 7500D with molecule interception carries out ultrafiltration 9h, obtains concentrate, and concentrate is placed in freeze drier
In, it is freeze-dried 3.5h under the conditions of -30 DEG C, hirudin is obtained and is modified anticoagulant material.
It takes 150g live bodies spider to be homogenized 3 times with colloid mill, 100 mesh screens of gained slurries is filtered, isolated filter
Liquid is spider extracting solution, spider extracting solution, 80g chitosans, 5g Phellinus polysaccharide and the mixing of 15g glucose for taking 150mL above-mentioned,
It is diluted to 5L with distilled water again, stirring and dissolving obtains induction liquid；Above-mentioned induction liquid is poured into the chitterlings after exenterating,
Induction liquid is filled by being one section every 50cm, tightening intestinal tube both ends with line makes its expansion, obtains the intestinal tube of the liquid containing induction, will be hungry
It starves the dispersion of 15 days 500g eurysome golden thread leech to be placed in glass guide channel, induction liquid intestinal tube will be contained and be laid in glass guide channel, allow expanded letter gold thread
Leech freely sucks enteral induction liquid 50min, until eurysome golden thread leech integrally increases weight to 1200g；Manual compression goes out expanded letter gold
Line leech saliva, obtains natural hirudin crude product, and 200 mesh screens of natural hirudin crude product are filtered, by isolated filtrate
It is put into beaker, then beaker is placed in 50 DEG C of water bath with thermostatic control and heats 45min, filter removal with 180 mesh screens after cooling and filter
Slag, isolated purifying filtrate；The 25g fibroin albumens dried are scattered in the sodium hydroxide solution that 300mL mass fractions are 5%
In, 45 DEG C are heated to, 3h is kept the temperature, obtains dispersion liquid, continues dropwise addition 100mL mass point after being cooled to room temperature into dispersion liquid
Number is 2% acetum, obtains silk fibroin protein solution；It takes 50g grass carp dry fish scales to be added and 400~450mL aseptic deionized waters is housed
Beaker in, by beaker heating water bath, be warming up to 40 DEG C, then 5g trypsase is added in beaker, heat preservation enzymolysis 5h is filtered off
Except filtrate, enzymolysis fish scale is obtained, enzymolysis fish scale is washed with deionized 5 times, then by the enzymolysis fish scale and deionization after washing
Water is that 1 ︰ 10 is mixed in mass ratio, and heating water bath is warming up to 95 DEG C, enzyme deactivation 15min, is cooled to 60 DEG C, and heat preservation concentration 7h is obtained
Gelatin solution；220mL silk fibroin protein solutions, 100mL gelatin solutions, 3g quadracyclines and 500mL purifying filtrate are mixed, molecule is used
The ultrafiltration membrane that interception is 8000D carries out ultrafiltration 10h, obtains concentrate, concentrate is placed in freeze drier, in -40 DEG C of items
It is freeze-dried 4h under part, hirudin is obtained and is modified anticoagulant material.
The anticoagulant material that comparative example is produced with Shanghai City company is as a comparison case
The anticoagulant material being modified in anticoagulant material and comparative example to hirudin produced by the present invention is detected, testing result
As shown in table 1：
Mechanics Performance Testing device therefor is electronic universal material testing machine（Test condition：Tensile speed is 500mm/min, is surveyed
Testing bar size is 115mm × 6mm × 2mm）.
Hemolysis rate is tested using YBB0003-2003 standards.
Antibiotic rate is tested using QB/T2591-2003 standards.
The measurement of clotting index
It extracts Rabbit Heart new blood and homemade sodium citrate anticoagulant is added（The volume ratio of blood and anticoagulation instrument agent
It is 9：1）For use.
The hirudin of the present invention is modified anticoagulant material and comparative example anticoagulant material is cut into 1cm2Sample, lie in
In beaker, it is put into 37 DEG C of heating 5min of water-bath and is added dropwise immediately after 0.1mL rabbit new bloods are added drop-wise on sample
0.2mol/LCaCl2Solution 0.02mL starts coagulation process, and 20mL deionized waters are added after 5min, are vibrated under 37 DEG C of constant temperature
5min then takes out solution and surveys its OD with ultraviolet specrophotometer（415nm）Value.
OD after 0.1mL blood is diluted to 20mL with deionized water（415nm）Value is assumed to 100 and makes reference value, and BCI=
100×OD（415nm）/100.BCI is lower, and blood coagulation was imitated better.
The measurement of bleeding stopping period by the hirudin of the present invention be modified anticoagulant material and comparative example anticoagulant material be cut into 2 ×
2cm2Fritter, it is spare after weighing.The identical rabbit of 12 sizes is divided into four groups, every group 3, will be distinguished on 4 kinds of hemostatic materials
Weighed medical cotton ball is covered, cotton is taken away after hemostasis by compression 10s, observes the haemostatic effect of a variety of materials.
Before the measurement hemostasis of amount of bleeding, accurate weighing cotton balls quality m1；After the completion of hemostasis, accurate weighing sucks the cotton of blood
Ball quality m2；Calculate amount of bleeding m=m2-m1。
1 performance measurement result of table
From the data in table 1, it can be seen that hirudin produced by the present invention is modified anticoagulant material, there is excellent mechanical performance, anticoagulant property
Can and anti-microbial property and comprehensive performance it is preferable, hence it is evident that be better than comparative example.Therefore, there is wide prospect of the application.
Claims (7)
1. a kind of hirudin is modified the preparation method of anticoagulant material, it is characterised in that specifically preparation process is：
（1）100~150g live bodies spider is taken to be homogenized 2~3 times with colloid mill, it is isolated by gained slurries screen filtration
Filtrate is spider extracting solution, the spider extracting solution that takes 120~150mL above-mentioned, 60~80g chitosans, 4~5g Phellinus polysaccharide and
12~15g glucose mixes, then is diluted to 4~5L with distilled water, and stirring and dissolving obtains induction liquid；
（2）Above-mentioned induction liquid is poured into the chitterlings after exenterating, one section of chitterlings is filled into induction liquid, is tightened with line
Intestinal tube both ends make its expansion, obtain the intestinal tube of the liquid containing induction, and the dispersion of 400~500g eurysome golden thread leech is placed in glass guide channel, will
The liquid intestinal tube containing induction is laid in glass guide channel, is taken out after allowing eurysome golden thread leech freely to suck enteral induction liquid；
（3）Manual compression goes out eurysome golden thread leech saliva, obtains natural hirudin crude product, by natural hirudin crude product sieve mistake
Filter, isolated filtrate is put into beaker, then beaker is placed in water bath with thermostatic control and is heated, and is removed with screen filtration after cooling
Filter residue, isolated purifying filtrate；
（4）20~25g fibroin albumens dried are scattered in 200~300mL sodium hydroxide solutions, heat temperature raising is kept the temperature
To dispersion liquid, continues dropwise addition 90~100mL acetums after being cooled to room temperature into dispersion liquid, obtain silk fibroin protein solution；
（5）It takes 40~50g grass carp dry fish scales to be added in the beaker equipped with 400~450mL aseptic deionized waters, beaker water-bath is added
Heat, heating, then 4~5g trypsase is added in beaker, heat preservation enzymolysis, filtering removal filtrate obtains enzymolysis fish scale, will digest
Fish scale is washed with deionized 3~5 times, then the enzymolysis fish scale after washing is mixed with deionized water, and heating water bath heating is gone out
Enzyme, cooling, heat preservation are concentrated to give gelatin solution；
（6）200~220mL silk fibroin protein solutions, 80~100mL gelatin solutions, 2~3g quadracyclines and 400~500mL is pure
Change filtrate mixing, carry out ultrafiltration with ultrafiltration membrane, obtain concentrate, concentrate is placed in freeze drier, freeze-drying obtains water
Leech element is modified anticoagulant material.
2. a kind of hirudin according to claim 1 is modified the preparation method of anticoagulant material, it is characterised in that：Step
（1）The mesh size specification used is 100 mesh, and induced fluid product is 4~5L after distilled water dilution.
3. a kind of hirudin according to claim 1 is modified the preparation method of anticoagulant material, it is characterised in that：Step
（2）The chitterlings are one section every 40~50cm points, and eurysome golden thread leech passes through Nature enemy 10~15 days, eurysome golden thread leech
It is 30~50min to suck the induction liquid time, has sucked and has been taken out when eurysome golden thread leech weightening reaches 1000~1200g after inducing liquid.
4. a kind of hirudin according to claim 1 is modified the preparation method of anticoagulant material, it is characterised in that：Step
（3）The mesh size specification is 200 mesh, and water bath with thermostatic control temperature is 45~50 DEG C, and heating time is 40~45min, cooling
It is 180 mesh to filter sieve used afterwards.
5. a kind of hirudin according to claim 1 is modified the preparation method of anticoagulant material, it is characterised in that：Step
（4）The mass fraction of the sodium hydroxide solution is 5%, and temperature is 40~45 DEG C after heat temperature raising, and soaking time is 2~3h,
The mass fraction of acetum is 2%.
6. a kind of hirudin according to claim 1 is modified the preparation method of anticoagulant material, it is characterised in that：Step
（5）Temperature is 35~40 DEG C after the beaker warming-in-water, and heat preservation enzymolysis time is 4~5h, digests fish scale and deionized water
Mixing quality ratio is that temperature is 90~95 DEG C after 1 ︰ 10, then heating water bath heating, and the enzyme deactivation time is 10~15min, warm after cooling
Degree is 55~60 DEG C, and heat preservation concentration time is 6~7h.
7. a kind of hirudin according to claim 1 is modified the preparation method of anticoagulant material, it is characterised in that：Step
（6）The ultrafiltration retaining molecular weight be 7000~8000D, the ultrafiltration time be 8~10h, freeze-drying temperature be -40~-
20 DEG C, sublimation drying is 3~4h.
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Cited By (2)
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| CN110623275A (en) * | 2019-09-18 | 2019-12-31 | 周维海 | Composite amino acid gilt-edged leech polypeptide food with anticoagulant and antithrombotic effects and preparation method thereof |
| CN116987181A (en) * | 2023-09-27 | 2023-11-03 | 北京元延医药科技股份有限公司 | High biological activity natural hirudin and method for preparing same in high yield |
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