CN104208096A

CN104208096A - Insoluble polysaccharide compound with hemostatic function and preparation method thereof

Info

Publication number: CN104208096A
Application number: CN201310222368.8A
Authority: CN
Inventors: 范杰; 李云龙; 廖晓峰; 尚小强; 陈昊
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2013-06-04
Filing date: 2013-06-04
Publication date: 2014-12-17
Anticipated expiration: 2033-06-04
Also published as: CN104208096B

Abstract

The invention provides an insoluble polysaccharide compound with a hemostatic function. The compound includes insoluble polysaccharide and a blood component. The blood component is whole blood or plasma, and the insoluble polysaccharide is macromolecular polysaccharide insoluble in whole blood or plasma. According to the invention, by mixing the blood component with the insoluble polysaccharide, the blood component undergoes clotting reaction evenly and dispersedly on the surface of the insoluble polysaccharide, so that clotting factors generated in the clotting process can be adsorbed by the insoluble polysaccharide surface, and high clotting activity can be maintained, thereby obtaining a composite hemostatic material with an excellent clotting effect. The preparation method of the insoluble polysaccharide compound provided by the invention is simple, and is convenient and safe to use, thus being widely applicable to trauma and surgical hemostasis. The insoluble polysaccharide compound can be combined with other medical materials and medical instruments so as to be prepared into band aids, infusion bands, self-adhesive dressings, tablets and capsule core materials, operation packages and first-aid packets, etc. At the same time, the adsorption performance and other characteristics of the insoluble polysaccharide compound are utilized to adsorb antibiotics, painkillers and the like thereinto, thus achieving better curative effect.

Description

A kind of slightly solubility polysaccharides compound with hemostatic function and preparation method thereof

Technical field

The invention belongs to technical field of biomedical materials, be specifically related to a kind of slightly solubility polysaccharides compound with hemostatic function and preparation method thereof, and comprise the hemostatic material of this complex.

Background technology

Hemostasis is the committed step of emergency survival.Exploitation (3 minutes) just can control the hemostatic material of the emergency as aorta is bled so in a short period of time, can try to gain time precious to one for further medical treatment, greatly reduces the death that hemorrhage, wound etc. causes.

There is report slightly solubility polysaccharide being applied to hemostasis at present, wherein used more and mainly contained chitosan (CN202020720U, CN102526795A), cellulose (CN100348272C, CN1109756A) and starch (CN102178691B).Chitosan is the Natural polycations polysaccharide that chitin obtains after deacetylated; chitosan molecule have blood coagulation, antibacterial, promote the multiple effect such as wound healing, be a class good biocompatibility, easily biological-degradable, non-immunogenicity, non-irritating multifunctional macromolecule material.Be applied to the chitosan tourniquet HemCon Bandage that one of maximum hemostatic material of the market capacity of external haemorrhage is HemCon company of the U.S. at present, its main component is chitosan material.Chitosan promotes that the mechanism of blood coagulation is that activated blood platelet accelerates Coagulation test.Chitosan material has extremely strong viscosity simultaneously, and the surface that can stick to wound forms covering, stops wound bleeding, in wound, surgical hemostasis treatment, show very bright application prospect.But chitosan material is due to only activated blood platelet, and not obvious to the activation of blood coagulation passage, thus coagulating effectiveness is limited.Therefore research worker has to find the better chitosan-based coagulant material of exploitation coagulating effectiveness.It is the effective ways improving chitosan material coagulating effectiveness that thrombin is loaded to chitosan material surface.Such as thrombin is loaded to chitosan material surface by Chinese patent CN101053669A, CN102526795A report, enhances the coagulating effectiveness of material.But due to the effect of chitosan surface group and thrombin, cause the activity decrease of thrombin.Interaction force in addition due to chitosan and thrombin is more weak, and thus the adsorbance of thrombin is not high.Simultaneously due to thrombin expensive (thrombin of beef price about 30,000 yuan/gram), greatly limit it and load to the application of chitosan surface as hemostasis.

There is following defect in chitosan loaded thrombin hemostatic material in sum: 1) thrombin expensive (thrombin of beef price about 30,000 yuan/gram); 2) thrombin is loaded to the method on chitosan material surface, make both interaction forces weak, can not realize effective combination of thrombin and hemostatic material, the thrombin of load easily comes off, during for wound hemostasis, the thrombin come off may enter into blood vessel and cause thrombosis; 3) thrombin has larger loss of activity after loading to chitosan surface, and thus product is difficult to realize commercialization.

Compared with chitosan material, the biocompatibility of cellulose and starch material is better, and more weak to hematoblastic activation, haemostatic effect is relatively poorer.At present for cellulosic hemorrhage, mainly cellulose is modified or load thrombin, expensive due to load thrombin, and in loading process, the loss of thrombin vigor is comparatively large, is thus difficult to commercialization.And for the hemorrhage of starch, mainly utilize strong absorptive and the load thrombin of starch, be thus also faced with the loss of thrombin vigor and expensive problem.

In order to obtained haemostatic effect is good and can have the slightly solubility polysaccharide hemostatic material of Commercial Prospect, be necessary to solve in hemostatic material, effectively introduce thrombin technology and Cost Problems.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of slightly solubility polysaccharides compound with hemostatic function and preparation method thereof, and comprises the hemostatic material of this complex.

The present invention adopts following technical scheme:

Have a slightly solubility polysaccharides compound for hemostatic function, described complex comprises slightly solubility polysaccharide and blood constituent, and described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma.

In slightly solubility polysaccharides compound of the present invention, the inorganic ions (as calcium ion, magnesium ion etc.) existed in blood constituent causes Coagulation test to be excited, and original position generation Coagulation test generates the protein that Factor Xa, thrombin etc. promote blood coagulation, simultaneously, Fibrinogen in blood constituent is by Factor Xa, thrombins etc. promote that the protein transduction of blood coagulation changes into fibrin monomer, the fibrin monomer generated is at the surfaces cross-link of slightly solubility polysaccharide, raw webbed fibrin, slightly solubility polysaccharide surface is wrapped up by netted fibrin, according to the specific recognition effect of enzyme-to-substrate, the thrombins generated etc. promote that the fibrin that the protein of blood coagulation is wrapped in slightly solubility polysaccharide surface firmly adsorbs, and be evenly dispersed on fibrin, and can not be suppressed by inhibitor such as antithrombase in blood plasma, and keep higher activity.Which ensure that the efficient blood coagulation efficiency of slightly solubility polysaccharides compound of the present invention.And slightly solubility polysaccharides compound material of the present invention has the thrombin of activation and the characteristic of slightly solubility polysaccharide concurrently, can be widely used in wound and surgical hemostasis, have great advantage.

Slightly solubility polysaccharides compound of the present invention, can only be made up of slightly solubility polysaccharide and blood constituent, and described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma.

In addition, slightly solubility polysaccharides compound of the present invention can further include the additional inorganic ions that can promote to excite Coagulation test, namely described slightly solubility polysaccharides compound is made up of slightly solubility polysaccharide, blood constituent and additional inorganic ions, described additional inorganic ions can be selected from calcium ion, magnesium ion, strontium ion any one or multiple arbitrarily.By additional inorganic ions, the effect promoting Coagulation test further can be played.Preferably, the concentration of described additional inorganic ions in blood constituent is 2mM ~ 10mM.

As preferably, described slightly solubility polysaccharide is selected from natural macromolecule polysaccharide, the macromolecule polysaccharide of modification or their mixture.

Further, the macromolecule polysaccharide of described modification be selected from carboxy-modified macromolecule polysaccharide, amino modified macromolecule polysaccharide, sulfhydryl modified macromolecule polysaccharide any one or multiple arbitrarily.

Slightly solubility polysaccharide of the present invention can be natural in chemically treated macromolecule polysaccharide, described macromolecule polysaccharide can be selected from cellulose, lignin, starch, chitosan, agarose any one or multiple arbitrarily; Also can be the macromolecule polysaccharide through chemically treated modification, the macromolecule polysaccharide of described modification be selected from carboxy-modified macromolecule polysaccharide, amino modified macromolecule polysaccharide, sulfhydryl modified macromolecule polysaccharide any one or multiple arbitrarily, described macromolecule polysaccharide can be selected from cellulose, lignin, starch, chitosan, agarose any one or multiple arbitrarily; Slightly solubility polysaccharide of the present invention can also be natural in chemically treated macromolecule polysaccharide and the mixture through the macromolecule polysaccharide of chemically treated modification.

But slightly solubility polysaccharide of the present invention is not limited to this, every other base group modification, all the present invention is applicable to the slightly solubility polysaccharide material of blood coagulation unrestraint effect.

In slightly solubility polysaccharides compound of the present invention, described whole blood can be the whole blood of human or animal.Described blood plasma is pure blood plasma, or comprises the blood plasma of hemocyte, described hemocyte be selected from erythrocyte, leukocyte, macrophage, platelet any one or multiple arbitrarily.Described blood plasma can be animal blood slurry (such as the blood plasma of pig, rabbit, cattle, chicken, duck), also can be the blood plasma of people.Described blood plasma can be anticoagulate plasma or the blood plasma not adding anticoagulant, and described blood plasma can also be the blood plasma adding inorganic ions (such as calcium ion, magnesium ion, strontium ion).Various blood plasma is all applicable to the present invention.

In slightly solubility polysaccharides compound of the present invention, in described complex, slightly solubility polysaccharide mixes with arbitrary proportion with blood constituent, the difficult soluble polysaccharide of preferred mixed proportion: the mass ratio of blood constituent is 1:8 ~ 1:1.

The present invention also provides two kinds of preparation methoies with the slightly solubility polysaccharides compound of hemostatic function:

Method one comprises the steps:

1) mixed with blood constituent by slightly solubility polysaccharide, obtained slightly solubility polysaccharide-blood constituent composite, described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma; Described mixing temperature is 5 ~ 37 DEG C, and described incorporation time is 10 ~ 60min;

2) above-mentioned slightly solubility polysaccharide-blood constituent composite is dry, the obtained slightly solubility polysaccharides compound with hemostatic function.

In said method, described step 1) in slightly solubility polysaccharide mix with blood constituent, the inorganic ions (as calcium ion, magnesium ion etc.) existed in blood constituent causes Coagulation test to be excited, and original position generation Coagulation test generates the protein that Factor Xa, thrombin etc. promote blood coagulation, simultaneously, Fibrinogen in blood constituent is by Factor Xa, thrombins etc. promote that the protein transduction of blood coagulation changes into fibrin monomer, the fibrin monomer generated is at the surfaces cross-link of slightly solubility polysaccharide, raw webbed fibrin, slightly solubility polysaccharide surface is wrapped up by netted fibrin, according to the specific recognition effect of enzyme-to-substrate, the Factor Xa generated, thrombins etc. promote that the fibrin that the protein of blood coagulation is wrapped in slightly solubility polysaccharide surface firmly adsorbs, and be evenly dispersed on fibrin, and can not be suppressed by inhibitor such as antithrombase in blood plasma, and keep higher activity.

In said method, described step 1) in, the mixed proportion of slightly solubility polysaccharide and blood constituent is arbitrary proportion, the difficult soluble polysaccharide of preferred mixed proportion: the mass ratio of blood constituent is 1:8 ~ 1:1.

As preferably, in said method, described step 1) in, described whole blood can be the whole blood of human or animal; Described blood plasma is pure blood plasma, or comprises the blood plasma of hemocyte, described hemocyte be selected from erythrocyte, leukocyte, macrophage, platelet any one or multiple arbitrarily.Described blood plasma can be animal blood slurry (such as the blood plasma of pig, rabbit, cattle, chicken, duck), also can be the blood plasma of people.Described blood plasma can be anticoagulate plasma or the blood plasma not adding anticoagulant, and described blood plasma can also be the blood plasma adding inorganic ions (such as calcium ion, magnesium ion, strontium ion).Various blood plasma is all applicable to the present invention.

Preferred, described step 1) in, described blood plasma is the blood plasma adding calcium ion.

In said method, described step 1) in hybrid mode can be sound wave, stirring, vortex or described slightly solubility polysaccharide is mixed with blood constituent by modes such as syringe repeatedly aspirate.

Slightly solubility polysaccharide of the present invention can be natural in chemically treated macromolecule polysaccharide, described macromolecule polysaccharide can be selected from cellulose, lignin, starch, chitosan, agarose any one or multiple arbitrarily; Also can be the macromolecule polysaccharide through chemically treated modification, the macromolecule polysaccharide of described modification be selected from carboxy-modified macromolecule polysaccharide, amino modified macromolecule polysaccharide, sulfhydryl modified macromolecule polysaccharide any one or multiple arbitrarily, described macromolecule polysaccharide can be selected from cellulose, lignin, starch, chitosan, agarose any one or multiple arbitrarily.Slightly solubility polysaccharide of the present invention can also be natural in chemically treated macromolecule polysaccharide and the mixture through the macromolecule polysaccharide of chemically treated modification.

In said method, described step 2) in drying means can be vacuum drying or lyophilization, described vacuum drying temperature can be 20 DEG C ~ 38 DEG C, and the time is 0.5 ~ 5 h.

Method two comprises the steps:

1) by slightly solubility polysaccharide, blood constituent, inorganic ions mixing, obtained slightly solubility polysaccharide-blood constituent composite; Described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma; Described mixing temperature is 5 ~ 37 DEG C, and described incorporation time is 10 ~ 60min;

In said method, described step 1) in the object adding inorganic ions be promote to start blood coagulation passage, promote the generation of Coagulation test, described inorganic ions be selected from calcium ion, magnesium ion, strontium ion any one or multiple arbitrarily, described inorganic ions can from the soluble inorganic salt comprising above-mentioned ion, and the concentration of described additional inorganic ions in blood constituent is 2mM ~ 10mM.

In said method, described step 1) in slightly solubility polysaccharide, blood constituent, inorganic ions mixing, promote exciting of Coagulation test, original position generation Coagulation test generates the protein that Factor Xa, thrombin etc. promote blood coagulation, simultaneously, Fibrinogen in blood constituent is by Factor Xa, thrombins etc. promote that the protein transduction of blood coagulation changes into fibrin monomer, the fibrin monomer generated is at the surfaces cross-link of slightly solubility polysaccharide, raw webbed fibrin, slightly solubility polysaccharide surface is wrapped up by netted fibrin, according to the specific recognition effect of enzyme-to-substrate, the Factor Xa generated, thrombins etc. promote that the fibrin that the protein of blood coagulation is wrapped in slightly solubility polysaccharide surface firmly adsorbs, and be evenly dispersed on fibrin, and can not be suppressed by inhibitor such as antithrombase in blood plasma, and keep higher activity.

As preferably, in said method, described step 1) in, described whole blood can be the whole blood of human or animal.Described blood plasma is pure blood plasma, or comprises the blood plasma of hemocyte, described hemocyte be selected from erythrocyte, leukocyte, macrophage, platelet any one or multiple arbitrarily.Described blood plasma can be animal blood slurry (such as the blood plasma of pig, rabbit, cattle, chicken, duck), also can be the blood plasma of people.Described blood plasma can be anticoagulate plasma or the blood plasma not adding anticoagulant, and described blood plasma can also be the blood plasma adding inorganic ions (such as calcium ion, magnesium ion, strontium ion).Various blood plasma is all applicable to the present invention.

After the step of method one or method two completes, the obtained slightly solubility polysaccharides compound with hemostatic function can also be ground, sterilizing preserves, hemostatic material can be made more easily.

Described sterilizing adopts Co-60 ray sterilizing.

The present invention also provides a kind of material for stopping blooding, and described hemostatic material comprises slightly solubility polysaccharides compound of the present invention as effective ingredient, or also comprises other medically acceptable adjuvants or other drug.

The present invention also provides a kind of material for stopping blooding, it is characterized in that: described hemostatic material can be independent slightly solubility polysaccharides compound, also can be comprise slightly solubility polysaccharides compound and adjuvant, also can be comprise slightly solubility polysaccharides compound and other drug, can also be comprise slightly solubility polysaccharides compound, adjuvant and other drug, described adjuvant comprises Polyethylene Glycol, polyglycerol, calcium carbonate etc., and the preferred percetage by weight of described adjuvant is 0 ~ 80%; Described other drug can be the medicine with antiinflammatory, pain relieving, antibacterial functions, such as gentamycin, ofloxacin etc.

The dosage form of described hemostatic material can be ointment, paste, cream, hemostatic adhesive bandage, hemostatic gauze, hemostatic microsphere etc.

Tool of the present invention has the following advantages:

1, reaction condition is gentle, easy to operation, and cheaper starting materials is easy to get.The price of chitosan, cellulose, starch etc. and animal blood slurry is all very cheap.

2, pass through slightly solubility polysaccharide-blood plasma compound, the procoagulant Factor after Activated Coagulation maintains higher activity on the surface of material, makes slightly solubility polysaccharide-blood plasma composite have fabulous effect.

3, the slightly solubility polysaccharide-blood plasma complex of gained, maintains the characteristic of the good biocompatibility of slightly solubility polysaccharide own.Be conducive to being applied to various wound and surgical hemostasis.Utilize the absorption property etc. of material itself simultaneously, can adsorption antibacterial element, analgesic in wherein, compound medication.

The invention has the beneficial effects as follows: the material prepared by the present invention has a clear superiority in wound, surgical hemostasis treatment.Due to animal blood slurry very cheap (about 2 yuan/kg), by by animal blood slurry and macromolecule polysaccharide material mixing, the haemostatic effect and 10 of what the animal blood slurry of 1 yuan of price and macromolecule polysaccharide Material cladding obtained the have slightly solubility polysaccharides compound of hemostatic function, the effect that 000 yuan of thrombin of beef plays is suitable, greatly can reduce the cost of hemostatic material.Because blood plasma is in the surperficial blood coagulation of slightly solubility polysaccharides compound, after making the activation of coagulant, Factor Xa, thrombin etc. promote that the protein adsorption of blood coagulation is on the surface of slightly solubility polysaccharides compound.Due to this adsorption, make them can not be suppressed by antithrombase, thus keep very high activity on the surface of slightly solubility polysaccharides compound.And during separately by blood clot blood coagulation, because the Factor Xa, thrombin etc. that produce in blood clot forming process promote that the protein of blood coagulation is combined by inhibitor such as antithrombase soon, lose the activity of coagulant, thus, independent blood clot coagulant low effort.Macromolecule polysaccharide in slightly solubility polysaccharides compound can excite the platelet in blood constituent to promote Coagulation test, and the Factor Xa of surface active, thrombin etc. promote that the protein of blood coagulation can thrombin proenzyme in direct activation blood constituent, accelerate Coagulation test greatly, play the effect of quick-acting haemostatic powder.And slightly solubility polysaccharides compound maintains the good biocompatibility of macromolecule polysaccharide material, thus can not cause tissue reaction, also can not cause wound infection, use very safe, without serious toxic and side effects.Utilize the absorption property of macromolecule polysaccharide material simultaneously, can other drug is adsorbed in wherein as antiinflammatory, pain relieving, antimicrobial drug etc., obtain better curative effect.

Accompanying drawing explanation

Fig. 1 is the forming process schematic diagram of slightly solubility polysaccharides compound of the present invention.

Fig. 2 is the external coagulating effectiveness comparison diagram of chitosan of the present invention/blood plasma complex and natural blood coagulation, chitosan, blood clot, and error line is obtained by three groups of experimental datas.

Fig. 3 is the external coagulating effectiveness comparison diagram of chitosan of the present invention/blood plasma complex, chitosan/whole blood complex and natural blood coagulation, free thrombin, YUNNAN BAIYAO, chitosan/thrombin composite, and error line is obtained by three groups of experimental datas.

Fig. 4 is different proportion of the present invention chitosan/blood plasma complex (mixing by the mass ratio of 1:0.5,1:1,1:2,1:4,1:8 respectively) and the external coagulating effectiveness comparison diagram of natural blood coagulation, chitosan, and error line is obtained by three groups of experimental datas.

Fig. 5 is the external coagulating effectiveness comparison diagram of cellulose of the present invention/blood plasma complex, cellulose/whole blood complex and natural blood coagulation, free thrombin, cellulose, cellulose/thrombin composite, and error line is obtained by three groups of experimental datas.

Fig. 6 is different proportion of the present invention starch/blood plasma complex (mixing by the mass ratio of 1:1,1:2,1:4 respectively) and the external coagulating effectiveness comparison diagram of natural blood coagulation, starch, and error line is obtained by three groups of experimental datas.

Fig. 7 is the external coagulating effectiveness comparison diagram of lignin of the present invention/blood plasma complex, agarose/blood plasma complex and natural blood coagulation, lignin, agarose, and error line is obtained by three groups of experimental datas.

Fig. 8 is the external coagulating effectiveness comparison diagram of amination of the present invention chitosan/blood plasma complex, Chitosan-Thiolated Polymers/blood plasma complex, carboxylated chitosan/blood plasma complex and natural blood coagulation, amination chitosan, Chitosan-Thiolated Polymers, carboxylated chitosan.Error line is obtained by three groups of experimental datas.

Detailed description of the invention

The invention provides a kind of slightly solubility polysaccharides compound with hemostatic function, described complex comprises slightly solubility polysaccharide and blood constituent, and described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma.

In the present invention, the forming process schematic diagram of slightly solubility polysaccharides compound as shown in Figure 1.In slightly solubility polysaccharides compound of the present invention, the inorganic ions (as calcium ion, magnesium ion etc.) existed in blood constituent causes Coagulation test to be excited, and original position generation Coagulation test generates the protein that Factor Xa, thrombin etc. promote blood coagulation, simultaneously, Fibrinogen in blood constituent is by Factor Xa, thrombins etc. promote that the protein transduction of blood coagulation changes into fibrin monomer, the fibrin monomer generated is at the surfaces cross-link of slightly solubility polysaccharide, raw webbed fibrin, slightly solubility polysaccharide surface is wrapped up by netted fibrin, according to the specific recognition effect of enzyme-to-substrate, the Factor Xa generated, thrombins etc. promote that the fibrin that the protein of blood coagulation is wrapped in slightly solubility polysaccharide surface firmly adsorbs, and be evenly dispersed on fibrin, and can not be suppressed by inhibitor such as antithrombase in blood plasma, and keep higher activity.Which ensure that the efficient blood coagulation efficiency of slightly solubility polysaccharides compound of the present invention.And slightly solubility polysaccharides compound material of the present invention has the thrombin of activation and the characteristic of slightly solubility polysaccharide concurrently, can be widely used in wound and surgical hemostasis, have great advantage.

In addition, slightly solubility polysaccharides compound of the present invention can also comprise the additional inorganic ions that can promote to excite Coagulation test, namely described slightly solubility polysaccharides compound is made up of slightly solubility polysaccharide, blood constituent and additional inorganic ions, described additional inorganic ions can be selected from calcium ion, magnesium ion, strontium ion any one or multiple arbitrarily.By additional inorganic ions, the effect promoting Coagulation test further can be played.Preferably, the concentration of described additional inorganic ions in blood constituent is 2mM ~ 10mM.

The present invention is further described below in conjunction with embodiment and accompanying drawing, for the whole blood of pig, blood plasma in embodiment and comparative example, preparation method and the coagulating effectiveness of slightly solubility polysaccharides compound of the present invention are described, with the whole blood of people or other animals, blood plasma for raw material prepare the method for slightly solubility polysaccharides compound, coagulating effectiveness rule all identical.Coagulating effectiveness is in clotting time, and clotting time is shorter, and coagulating effectiveness is better.

Comparative example 1

The mensuration of nature coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.During experiment in vitro, get the 0.2M CaCl of 40ul ₂solution joins in the centrifuge tube of 10ml, then adds the whole blood of 2ml constant temperature, and the time of whole blood coagulation is designated as nature clotting time.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, obtaining nature clotting time is 9.6min, and its coagulating effectiveness is shown in Fig. 2 ~ Fig. 8.

Comparative example 2

The mensuration of chitosan coagulating effectiveness: get 50mg chitosan (molecular weight 150,000 ~ 200,000) and join in the centrifuge tube of 10ml, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Then the 0.2M CaCl of 40ul is added ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of chitosan.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan is 4.1 min, and the coagulating effectiveness of chitosan is general as can be seen here, and its coagulating effectiveness is shown in Fig. 2 and Fig. 4.

Comparative example 3

The mensuration of blood clot coagulating effectiveness: 100ul blood plasma is placed in test tube, adds 10ul calcium ion, places and treats its blood coagulation half an hour.Then dry, blend; External clotting assay is carried out, by whole blood in advance 25 DEG C of constant temperature water bath half an hour at 25 DEG C.Add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate dry after the coagulating effectiveness of blood clot, time during whole blood coagulation is designated as the clotting time of blood clot.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining blood clot is 2.3 min, and its coagulating effectiveness is general, sees Fig. 2.

Embodiment 1

The preparation of chitosan/blood plasma complex: 50mg chitosan material (molecular weight 150,000 ~ 200,000) is mixed with 100ul anticoagulate plasma (mass ratio by 1:2), adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtain chitosan/blood plasma composite, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/blood plasma complex.

The mensuration of chitosan/blood plasma complex coagulating effectiveness: ground by chitosan/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/blood plasma complex is 1 min, and its coagulating effectiveness is better, sees Fig. 2.As can be seen here, chitosan with add calcium ion blood plasma compound after coagulating effectiveness greatly improve.

Obtained chitosan/blood plasma complex is shown in Fig. 2 with the external coagulating effectiveness comparison diagram of the natural blood coagulation of comparative example 1-3, chitosan, blood clot respectively, as shown in Figure 2, the coagulating effectiveness of chitosan/blood plasma complex is better than the coagulating effectiveness of chitosan and blood clot, is more better than the effect of nature blood coagulation.

Embodiment 2

The preparation of chitosan/whole blood complex: 50mg chitosan (molecular weight 150,000 ~ 200,000) is mixed with 100ul whole blood (mass ratio by 1:2), adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtain chitosan/whole blood composite, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/whole blood complex.

The mensuration of chitosan/whole blood complex coagulating effectiveness: ground by chitosan/whole blood complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/whole blood complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/whole blood complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/whole blood complex is 1.1 min, and its coagulating effectiveness is better, sees Fig. 3.

Comparative example 4

The mensuration of free thrombin clot effect: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get thrombin (Sigma T4648 thrombin, manufacturer Sigma-Aldrich company) and be mixed with 1mg/ml solution, get 100ul and carry out external clotting assay.Then the 0.2M CaCl of 40ul is added ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/whole blood complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining thrombin is 0.67 min, and its coagulating effectiveness is fine, sees Fig. 3.

Comparative example 5

The preparation of chitosan/thrombin composite: with reference to the method for Chinese patent CN 102526795A, 5g chitosan is scattered in 30ml distilled water; Thrombin is dissolved in the phosphate buffer that pH is 7.0, obtains thrombin (Sigma T4648 thrombin, manufacturer Sigma-Aldrich company) the solution 10ml that mass concentration is 0.1%; Then by the two 3:1 mixing by volume, the part by weight of obtained chitosan and thrombin is the mixed solution 40mL of 500:1.Under the condition stirred, add 0.5g glycerol and 1g mannitol, and dropwise add the calcium chloride solution 50ul of mass concentration 5%, mix homogeneously, again under stirring, dropwise add volumetric concentration 1% glutaraldehyde solution 250ul to be cross-linked, in the mould of the then suitable shape of impouring, place 2 hours, after gel to be formed,-20 DEG C freezing 24 hours, then in-40 DEG C of vacuum lyophilizations 36 hours, reverse mould, grinds, encapsulates.Be fixed the chitosan/thrombin composite of thrombin.

The mensuration of chitosan/thrombin composite coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg this chitosan/thrombin composite, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/thrombin composite.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/thrombin composite is 2.2 min, contrast with the dissociate coagulating effectiveness of thrombin of comparative example 4, the coagulating effectiveness of the chitosan/thrombin composite of fixing thrombin reduces, illustrate that the fixing rear coagulating effectiveness of thrombin reduces, see Fig. 3.

Comparative example 6

The mensuration of YUNNAN BAIYAO coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get YUNNAN BAIYAO (batch number ZA12041) powder 0.1g, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of YUNNAN BAIYAO.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining YUNNAN BAIYAO is 4.2 min, and its coagulant effect is general, sees Fig. 3.

Chitosan/blood plasma complex that embodiment 1 is obtained, the natural blood coagulation of chitosan/whole blood complex that embodiment 2 is obtained and comparative example 1, the free thrombin of comparative example 4-6, YUNNAN BAIYAO, the coagulating effectiveness comparison diagram of chitosan/thrombin composite is shown in Fig. 3, as shown in Figure 3, chitosan/blood constituent complex (chitosan/blood plasma complex, chitosan/whole blood complex) coagulating effectiveness and free thrombin be more or less the same, and be greatly better than business-like hemostasia products, as YUNNAN BAIYAO, also chitosan/thrombin the composite securing thrombin is better than, because preparation method of the present invention is simple, raw material is cheap, thus prospect is very wide.

Embodiment 3

The preparation of the chitosan/blood plasma complex of different proportion: by 50mg chitosan (molecular weight 150,000 ~ 200,000) respectively with 25ul, 50ul, 100ul, 200ul, the mixing of 400ul anticoagulate plasma (presses 1:0.5 respectively, 1:1, 1:2, 1:4, the mass ratio mixing of 1:8), adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained chitosan/25ul blood plasma composite respectively, chitosan/50ul blood plasma composite, chitosan/100ul blood plasma composite, chitosan/200ul blood plasma composite, chitosan/400ul blood plasma composite, by above-mentioned blood plasma composite respectively at 30 DEG C of vacuum dryings, obtained chitosan/25ul blood plasma complex respectively, chitosan/50ul blood plasma complex, chitosan/100ul blood plasma complex, chitosan/200ul blood plasma complex, chitosan/400ul blood plasma complex.

The mensuration of the chitosan/blood plasma complex coagulating effectiveness of different proportion: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Above-mentioned blood plasma complex respectively gets 50mg respectively, adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its clotting time, time during whole blood coagulation is designated as the clotting time of chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, obtain the clotting time data of the chitosan/blood plasma complex of different proportion respectively in table 1.

The clotting time data of the chitosan/blood plasma complex of table 1 different proportion

Different proportion chitosan/blood plasma complex (presses 1:0.5 respectively, 1:1,1:2,1:4, the mass ratio mixing of 1:8) see Fig. 4 with the natural blood coagulation of comparative example 1-2, the external coagulating effectiveness comparison diagram of chitosan, the coagulating effectiveness of the visible different proportion chitosan/blood plasma complex of Fig. 4 is better than the coagulating effectiveness of nature blood coagulation, chitosan greatly, and the coagulating effectiveness of the chitosan of different proportion/blood plasma complex is all better.

Embodiment 4

The preparation of cellulose/blood plasma complex: 50mg cellulose (molecular weight 500,000 ~ 1,000,000) is mixed with 100ul anticoagulate plasma (mixing by the mass ratio of 1:2), then calcium ion is added, calcium ion concentration is made to be 5mM, 25 DEG C mix half an hour, obtain cellulose/blood plasma composite, by it at 30 DEG C of vacuum dryings, obtain cellulose/blood plasma complex.

The mensuration of cellulose/blood plasma complex coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get this complex of 50mg, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of cellulose/blood plasma complex, time during whole blood coagulation is designated as the clotting time of cellulose/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining cellulose/blood plasma complex is that 0.96min(is shown in Fig. 5), close to the coagulating effectiveness of free thrombin, fibres visible element/blood plasma complex has good blood coagulation facilitation.

Embodiment 5

The preparation of cellulose/whole blood complex: by 50mg cellulose (molecular weight 500,000 ~ 1,000,000) and 100ul whole blood mixed at room temperature (mixing by the mass ratio of 1:2), then calcium ion is added, calcium ion concentration is made to be 5mM, 25 DEG C mix half an hour, obtain cellulose/whole blood composite, by it at 30 DEG C of vacuum dryings, obtain cellulose/whole blood complex.

The mensuration of cellulose/whole blood complex coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get this complex of 50mg, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of cellulose/blood plasma complex, time during whole blood coagulation is designated as the clotting time of cellulose/whole blood complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining cellulose/whole blood complex is 1.3 min, sees Fig. 5, and fibres visible element/whole blood complex has larger blood coagulation facilitation.

Comparative example 7

The mensuration of cellulose coagulating effectiveness: get 50mg cellulose (molecular weight 500,000 ~ 1,000,000) and join in the centrifuge tube of 10ml, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as cellulosic clotting time.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, obtaining cellulosic clotting time is 8.1 min, and cellulosic coagulating effectiveness is poor as can be seen here, and its coagulating effectiveness is shown in Fig. 5.

Comparative example 8

The preparation of cellulose/thrombin composite: with reference to the method for Chinese patent CN 102526795A, 5g cellulose is scattered in 30ml distilled water; Thrombin is dissolved in the phosphate buffer that pH is 7.0, obtains thrombin (Sigma T4648 thrombin, manufacturer Sigma-Aldrich company) the solution 10ml that mass concentration is 0.1%; Then by the two 3: 1 mixing by volume, the part by weight of obtained cellulose and thrombin is the mixed solution 40mL of 500: 1.Under the condition stirred, add 0.5g glycerol and 1g mannitol, and dropwise add the calcium chloride solution 50ul of mass concentration 5%, mix homogeneously, again under stirring, dropwise add volumetric concentration 1% glutaraldehyde solution 250ul to be cross-linked, in the mould of the then suitable shape of impouring, place 2 hours, after gel to be formed,-20 DEG C freezing 24 hours, then in-40 DEG C of vacuum lyophilizations 36 hours, reverse mould, grinds, encapsulates.Be fixed the cellulose/thrombin composite of thrombin.

The mensuration of cellulose/thrombin composite coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg this cellulose/thrombin composite, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of the cellulose/thrombin composite of fixing thrombin, time during whole blood coagulation is designated as the clotting time of cellulose/blood plasma composite.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining cellulose/blood plasma composite is 2.6 min, contrast with the dissociate coagulating effectiveness of thrombin of comparative example 4, the coagulating effectiveness of the cellulose/thrombin composite of fixing thrombin reduces, illustrate that the fixing rear coagulating effectiveness of thrombin reduces, see Fig. 5.

Obtained cellulose/blood plasma complex, the natural blood coagulation of cellulose/whole blood complex and comparative example 1, the free thrombin of comparative example 4, the cellulose of comparative example 7, the external coagulating effectiveness comparison diagram that comparative example 8 fixes the cellulose/thrombin composite of thrombin is shown in Fig. 5, Fig. 5 is visible, cellulose/blood constituent complex (cellulose/blood plasma complex, cellulose/whole blood complex) coagulating effectiveness suitable with the coagulating effectiveness of free thrombin, greatly be better than nature blood coagulation, cellulosic coagulating effectiveness, also the coagulating effectiveness of the cellulose/thrombin composite of fixing thrombin is better than, the coagulating effectiveness of the cellulose/thrombin composite of fixing thrombin, lower than free thrombin, illustrates that the fixing rear coagulating effectiveness of thrombin reduces.

Comparative example 9

The mensuration of starch coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Getting 50mg starch (molecular weight 50,000 ~ 200,000) joins in the centrifuge tube of 10ml, then adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of starch.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining starch is 7.2 min, and the coagulating effectiveness of starch is general as can be seen here, and its coagulating effectiveness is shown in Fig. 6.

Embodiment 6

The preparation of the starch/blood plasma complex of different proportion: by 50mg starch (molecular weight 50,000 ~ 200,000) respectively with 50ul, 100ul, 200ul anticoagulate plasma mixed at room temperature (presses 1:1 respectively, 1:2, the mass ratio mixing of 1:4), adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained starch/50ul blood plasma composite respectively, starch/100ul blood plasma composite, starch/200ul blood plasma composite, by above-mentioned blood plasma composite respectively at 30 DEG C of vacuum dryings, obtained starch/50ul blood plasma complex respectively, starch/100ul blood plasma complex, starch/200ul blood plasma complex.

The mensuration of the starch/blood plasma complex coagulating effectiveness of different proportion: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Above-mentioned blood plasma complex respectively gets 50mg respectively, adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of starch/blood plasma complex, time during whole blood coagulation is designated as the clotting time of starch/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining the starch/blood plasma complex of different proportion is respectively 1.4min, 1.1min, 0.9min.See Fig. 6.

Different proportion starch/blood plasma complex (presses 1:1 respectively, 1:2, the mass ratio mixing of 1:4) see Fig. 6 with the natural blood coagulation of comparative example 1, the external coagulating effectiveness comparison diagram of comparative example 9 starch, the coagulating effectiveness of the visible different proportion starch/blood plasma complex of Fig. 6 is better than the coagulating effectiveness of nature blood coagulation, starch greatly, and the coagulating effectiveness of the starch of different proportion/blood plasma complex is all better.

Comparative example 10

The mensuration of lignin coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Getting 50mg lignin (molecular weight 50,000 ~ 200,000) joins in the centrifuge tube of 10ml, then adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of lignin.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining lignin is 10.1 min, and the coagulating effectiveness of lignin is poor as can be seen here, and its coagulating effectiveness is shown in Fig. 7.

Embodiment 7

The preparation of lignin/blood plasma complex: the ratio magnetic force of 50mg lignin (molecular weight 50,000 ~ 200,000) and 100ul anticoagulate plasma 1:2 is in mass ratio mixed, adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained lignin/blood plasma composite, by it at 30 DEG C of vacuum dryings, obtain lignin/blood plasma complex.

The mensuration of lignin/blood plasma complex coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg this lignin/blood plasma complex, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of lignin/blood plasma complex, time during whole blood coagulation is designated as the clotting time of lignin/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining lignin/blood plasma complex is 1.3min, and its coagulating effectiveness is better, and its coagulating effectiveness is shown in Fig. 7.

Comparative example 11

The mensuration of agarose coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Getting 50mg agarose (molecular weight 20,000 ~ 100,000) joins in the centrifuge tube of 10ml, then adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of agarose.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining agarose is 12.1min, and agarose does not have blood coagulation enhancing effect as can be seen here, and its coagulating effectiveness is shown in Fig. 7.

Embodiment 8

The preparation of agarose/blood plasma complex: the ratio magnetic force of 50mg agarose (molecular weight 20,000 ~ 100,000) and 100ul anticoagulate plasma 1:2 is in mass ratio mixed, adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained agarose/blood plasma composite, by it at 30 DEG C of vacuum dryings, obtain agarose/blood plasma complex.

The mensuration of agarose/blood plasma complex coagulating effectiveness: get 50mg this agarose/blood plasma complex, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of agarose/blood plasma complex, time during whole blood coagulation is designated as the clotting time of agarose/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining agarose/blood plasma complex is 1.5 min, and its coagulating effectiveness is shown in Fig. 7.

Comparative example 12

The mensuration of carboxylated chitosan coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Getting the carboxylated chitosan of 50mg (molecular weight 150,000 ~ 200,000) joins in the centrifuge tube of 10ml, then adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of carboxylated chitosan.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining carboxylated chitosan is 3.3 min, and carboxylated chitosan has stronger blood coagulation enhancing effect as can be seen here, and its coagulating effectiveness is shown in Fig. 8.

Embodiment 9

The preparation of carboxylated chitosan/blood plasma complex: the ratio magnetic force of carboxylated for 50mg chitosan (molecular weight 150,000 ~ 200,000) and 100ul anticoagulate plasma 1:2 is in mass ratio mixed, adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained carboxylated chitosan/blood plasma composite, by it at 30 DEG C of vacuum dryings, obtain carboxylated chitosan/blood plasma complex.

The mensuration of carboxylated chitosan/blood plasma complex coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get this carboxylated chitosan/blood plasma complex of 50mg, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of carboxylated chitosan/blood plasma complex, time during whole blood coagulation is designated as the clotting time of carboxylated chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining carboxylated chitosan/blood plasma complex is 0.89min, and its coagulating effectiveness is fine, sees Fig. 8.

Comparative example 13

The mensuration of amination chitosan coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Getting 50mg amination chitosan (molecular weight 150,000 ~ 200,000) joins in the centrifuge tube of 10ml, then adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of amination chitosan.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining amination chitosan is 5.9 min, and amination chitosan blood coagulation enhancing effect is more weak as can be seen here, and its coagulating effectiveness is shown in Fig. 8.

Embodiment 10

The preparation of amination chitosan/blood plasma complex: the ratio magnetic force of 50mg amination chitosan (molecular weight 150,000 ~ 200,000) and 100ul anticoagulate plasma 1:2 is in mass ratio mixed, adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained amination chitosan/blood plasma composite, by it at 30 DEG C of vacuum dryings, obtain amination chitosan/blood plasma complex.

The mensuration of amination chitosan/blood plasma complex coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get this amination chitosan/blood plasma complex of 50mg, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of amination chitosan/blood plasma complex, time during whole blood coagulation is designated as the clotting time of amination chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining amination chitosan/blood plasma complex is 1.4 min, and its coagulating effectiveness is better, and its coagulating effectiveness is shown in Fig. 8.

Comparative example 14

The preparation of Chitosan-Thiolated Polymers: take 0.8072 Chitosan-Thiolated Polymers, adds 8ml thioglycolic acid, 4ml acetic anhydride, and after stirring, add two concentrated sulphuric acids, be put in constant temperature oscillator the 3h that vibrates, placement is spent the night.Be washed till neutrality with water or ammonia, put into the oven dry of 50 DEG C, vacuum drying oven, obtain yellowish-brown gelatin-like solid, put in ground brown bottle and save backup.

The mensuration of Chitosan-Thiolated Polymers coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Getting 50mg Chitosan-Thiolated Polymers (molecular weight 150,000 ~ 200,000) joins in the centrifuge tube of 10ml, then adds the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, time during whole blood coagulation is designated as the clotting time of Chitosan-Thiolated Polymers.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining Chitosan-Thiolated Polymers is 4.2 min, and Chitosan-Thiolated Polymers has stronger blood coagulation enhancing effect as can be seen here, and its coagulating effectiveness is shown in Fig. 8.

Embodiment 11

The preparation of Chitosan-Thiolated Polymers/blood plasma complex: the ratio magnetic force of 50mg Chitosan-Thiolated Polymers (molecular weight 150,000 ~ 200,000) and 100ul anticoagulate plasma 1:2 is in mass ratio mixed, adding calcium ion makes its concentration be 5mM, 25 DEG C mix half an hour, obtained Chitosan-Thiolated Polymers/blood plasma composite, by it at 30 DEG C of vacuum dryings, obtain Chitosan-Thiolated Polymers/blood plasma complex.

The mensuration of Chitosan-Thiolated Polymers/blood plasma complex coagulating effectiveness: carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg this Chitosan-Thiolated Polymers/blood plasma complex, add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate the clotting time of Chitosan-Thiolated Polymers/blood plasma complex, time during whole blood coagulation is designated as the clotting time of Chitosan-Thiolated Polymers/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining Chitosan-Thiolated Polymers/blood plasma complex is 1.1 min, and its coagulating effectiveness is better, and its coagulating effectiveness is shown in Fig. 8.

Embodiment 12

The preparation of chitosan/blood plasma complex: 50mg chitosan material (molecular weight 150,000 ~ 200,000) is mixed with 100ul anticoagulate plasma (mass ratio by 1:2), adding magnesium ion makes its concentration be 5mM, 30 DEG C of mixing 15min, obtain chitosan/blood plasma composite, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/blood plasma complex.

The mensuration of chitosan/blood plasma complex coagulating effectiveness: ground by chitosan/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/blood plasma complex is 1.8 min, and its coagulating effectiveness is better, in table 2.As can be seen here, chitosan with add magnesium ion blood plasma compound after coagulating effectiveness greatly improve.

Embodiment 13

The preparation of chitosan/blood plasma complex: 50mg chitosan material (molecular weight 150,000 ~ 200,000) is mixed with 100ul anticoagulate plasma (mass ratio by 1:2), adding strontium ion makes its concentration be 8 mM, 20 DEG C of mixing 30min, obtain chitosan/blood plasma composite, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/blood plasma complex.

The mensuration of chitosan/blood plasma complex coagulating effectiveness: ground by chitosan/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/blood plasma complex is 1.3 min, and its coagulating effectiveness is better, in table 2.As can be seen here, chitosan with add strontium ion blood plasma compound after coagulating effectiveness greatly improve.

Embodiment 14

The preparation of chitosan/blood plasma complex: 50mg chitosan material (molecular weight 150,000 ~ 200,000) is mixed with the blood plasma that 100ul now gets (mass ratio by 1:2), 35 DEG C of mixing 15min, obtain chitosan/blood plasma composite, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/blood plasma complex.

The mensuration of chitosan/blood plasma complex coagulating effectiveness: ground by chitosan/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/blood plasma complex is 1.1 min, and its coagulating effectiveness is better, in table 2.As can be seen here, after chitosan and blood plasma compound, coagulating effectiveness improves greatly.

Embodiment 15

The preparation of chitosan/whole blood complex: 50mg chitosan material (molecular weight 150,000 ~ 200,000) is mixed with the whole blood that 100ul now gets (mass ratio by 1:2), 35 DEG C of mixing 15min, obtain chitosan/whole blood composite, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/whole blood complex.

The mensuration of chitosan/whole blood complex coagulating effectiveness: ground by chitosan/whole blood complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/whole blood complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/whole blood complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/blood plasma complex is 1.3 min, and its coagulating effectiveness is better, in table 2.As can be seen here, after chitosan and whole blood compound, coagulating effectiveness improves greatly.

Embodiment 16

The preparation of chitosan/starch/blood plasma complex: 25mg chitosan material (molecular weight 150,000 ~ 200,000) 25mg starch (molecular weight 100,000) is mixed with 100ul anticoagulate plasma (mass ratio by 1:2), adding calcium ion makes its concentration be 5mM, 15 DEG C of mixing 50min, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/starch/blood plasma complex.

The mensuration of chitosan/starch/blood plasma complex coagulating effectiveness: ground by chitosan/starch/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/starch/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/starch/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/starch/blood plasma complex is 1.0min, and its coagulating effectiveness is better, in table 2.As can be seen here, chitosan/starch mixture with add calcium ion blood plasma compound after coagulating effectiveness greatly improve.

Embodiment 17

The preparation of Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex: 25mg Chitosan-Thiolated Polymers (molecular weight 150,000 ~ 200,000) the carboxylated chitosan of 25mg (molecular weight 150,000 ~ 200,000) is mixed with 100ul anticoagulate plasma (mass ratio by 1:2), adding calcium ion makes its concentration be 5mM, 37 DEG C of mixing 10min, by it at 30 DEG C of vacuum drying 5h, obtain Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex.

The mensuration of Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex coagulating effectiveness: ground by Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex is 1 min, and its coagulating effectiveness is better, in table 2.As can be seen here, after the blood plasma compound of Chitosan-Thiolated Polymers/carboxylated chitosan compound and interpolation calcium ion, coagulating effectiveness improves greatly.

Embodiment 18

The preparation of chitosan/carboxylated chitosan/blood plasma complex: 25mg chitosan (molecular weight 150,000 ~ 200,000) the carboxylated chitosan of 25mg (molecular weight 150,000 ~ 200,000) is mixed with 100ul anticoagulate plasma (mass ratio by 1:2), adding calcium ion makes its concentration be 5mM, 5 DEG C of mixing 60min, by it at 30 DEG C of vacuum drying 5h, obtain chitosan/carboxylated chitosan/blood plasma complex.

The mensuration of chitosan/carboxylated chitosan/blood plasma complex coagulating effectiveness: ground by chitosan/carboxylated chitosan/blood plasma complex, carry out external clotting assay at 25 DEG C, by whole blood in advance 25 DEG C of constant temperature water bath half an hour.Get 50mg chitosan/carboxylated chitosan/blood plasma complex, then add the 0.2M CaCl of 40ul ₂, finally add the whole blood of 2ml constant temperature, investigate its coagulating effectiveness, time during whole blood coagulation is designated as the clotting time of chitosan/carboxylated chitosan/blood plasma complex.By the step of said determination clotting time in triplicate, get the meansigma methods of three records, the clotting time obtaining chitosan/carboxylated chitosan/blood plasma complex is 0.9min, and its coagulating effectiveness is better, in table 2.As can be seen here, after the blood plasma compound of chitosan/carboxylated chitosan compound and interpolation calcium ion, coagulating effectiveness improves greatly.

The clotting time data of table 2 slightly solubility polysaccharides compound

Complex	Clotting time/min
		Chitosan/whole blood complex (interpolation magnesium ion)	1.8
Chitosan/whole blood complex (interpolation strontium ion)	1.3
		Chitosan and blood plasma complex (not adding inorganic ions)	1.1
Chitosan/whole blood complex (not adding inorganic ions)	1.3
		Chitosan/starch/blood plasma complex	1.0
Chitosan-Thiolated Polymers/carboxylated chitosan/blood plasma complex	1.0
		Chitosan/carboxylated chitosan/blood plasma complex	0.9

Above-described embodiment is only not used in for illustration of the present invention and limits the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.

Claims

1. one kind has the slightly solubility polysaccharides compound of hemostatic function, it is characterized in that: described complex comprises slightly solubility polysaccharide and blood constituent, described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma.

2. slightly solubility polysaccharides compound according to claim 1, is characterized in that: also comprise inorganic ions further in described slightly solubility polysaccharides compound.

3. slightly solubility polysaccharides compound according to claim 2, is characterized in that: described inorganic ions be selected from calcium ion, magnesium ion, strontium ion any one or multiple arbitrarily.

4. the slightly solubility polysaccharides compound according to any one of claim 1-3, is characterized in that: described slightly solubility polysaccharide is selected from natural macromolecule polysaccharide, the macromolecule polysaccharide of modification or their mixture.

5. slightly solubility polysaccharides compound according to claim 4, is characterized in that: the macromolecule polysaccharide of described modification be selected from carboxy-modified macromolecule polysaccharide, amino modified macromolecule polysaccharide, sulfhydryl modified macromolecule polysaccharide any one or multiple arbitrarily.

6. the slightly solubility polysaccharides compound according to claim 4 or 5, is characterized in that: described macromolecule polysaccharide be selected from cellulose, lignin, starch, chitosan, agarose any one or multiple arbitrarily.

7. the slightly solubility polysaccharides compound according to any one of claim 1-3, it is characterized in that: in described complex, slightly solubility polysaccharide mixes with arbitrary proportion with blood constituent, the difficult soluble polysaccharide of preferred mixed proportion: the mass ratio of blood constituent is 1:8 ~ 1:1.

8. the preparation method with the slightly solubility polysaccharides compound of hemostatic function according to claim 1, is characterized in that comprising the steps:

9. the preparation method with the slightly solubility polysaccharides compound of hemostatic function according to claim 2, is characterized in that comprising the steps:

1) by slightly solubility polysaccharide, blood constituent, inorganic ions mixing, obtained slightly solubility polysaccharide-blood constituent composite, described blood constituent is whole blood or blood plasma, and described slightly solubility polysaccharide is the macromolecule polysaccharide being insoluble to whole blood or blood plasma; Described mixing temperature is 5 ~ 37 DEG C, and described incorporation time is 10 ~ 60min;

10. preparation method according to claim 9, is characterized in that: described inorganic ions be selected from calcium ion, magnesium ion, strontium ion any one or multiple arbitrarily.

11. 1 kinds, for the material stopped blooding, is characterized in that: described hemostatic material comprises slightly solubility polysaccharides compound described in any one of claim 1-7 as effective ingredient, or also comprises medically acceptable adjuvant or other drug.

12. hemostatic materials according to claim 11, the dosage form of described hemostatic material is selected from ointment, paste, cream, hemostatic adhesive bandage, hemostatic gauze, hemostatic microsphere.