CN103230617A - Collagen/chitosan micro-nano fiber composite hemostatic membrane material and preparation method thereof - Google Patents
Collagen/chitosan micro-nano fiber composite hemostatic membrane material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a collagen/chitosan micro-nano fiber composite hemostatic membrane material and a preparation method thereof. The preparation method is characterized by comprising the following steps of: extracting a medical biological skin sheet serving as a raw material by an ultrasonic technology to prepare I type collagen; mixing hexafluoroisopropanol and acetic acid according to different volume ratios to prepare a spinning solvent; mixing the prepared I type collagen and chitosan in a certain mass ratio and adding the mixture into the spinning solvent; stirring in an ultrasonic cleaner at room temperature until the material is transparent to prepare electrostatic spinning mother liquid at the concentration of 4 to 10 percent; injecting the electrostatic spinning mother liquid into an electrostatic spinning machine and performing electrostatic spinning to obtain a collagen/chitosan micro-nano fiber composite membrane; soaking the collagen/chitosan micro-nano fiber composite membrane into natural biomacromolecules and Chinese herbal medicines sequentially; and freeze-drying to obtain the collagen/chitosan micro-nano fiber composite hemostatic membrane material. The collagen/chitosan micro-nano fiber composite hemostatic membrane material has excellent biocompatibility, biodegradability, adhesion property and the like, can quickly stop bleeding, resist inflammation, ease pain and promote wound healing, and can be applied to trauma hemostasis and repair and general civil hemostasis emergency treatment.
Description
Technical field
The present invention relates to a kind of premium properties such as excellent biological compatibility, biodegradable, adhesiveness that have, can quick-acting haemostatic powder, anti-inflammatory and antalgic, the compound hemorrhage of promotion wound.
Background technology
Hemorrhagely cause great danger to the human life, seek fast and effectively hemorrhage and be always the focus of research both at home and abroad.In all kinds of surgical operations or trauma care, wound surface is hemorrhage and be isolated from the outside and avoid infection, and is very big to patient's wound healing influence.In war, in back 48 hours of wound in the cause of the death, hemorrhage is chief reason, accounts for 80% of all wound accidents according to statistics.The wound severe loss of blood can make wounded's pain, shock, stupor even death, so hemostatic material needs fast effectively hemostasis, must have anti-inflammatory and antalgic, promote the effect that wound is compound etc.
Traditional hemostatic material mainly is first-aid kit, four-tailed bandage, tourniquet and binder etc., the not portable and sterilization preservation of this class material, and hemostasis is almost bad by the mechanism effect.In recent years, people develop the biological absorbable hemostatic material of a class, wherein collagen base hemostatic material gets more and more people's extensive concerning, collagen had both had excellent biological compatibility, biological degradability, hypotoxicity, poor antigen, expression that again can active cell characteristic group is kept the normal characteristic of cell and is expressed, and is conducive to sticking, grow, breed, breaking up of cell, its anthemorrhagic performance is also comparatively outstanding, existing a large amount of bibliographical information.
But people such as Wei Hua preside over and bear " 15 " national high-tech research development plans (863 Program) project, develop " the medical bio skin graft " of high comprehensive performance, have realized industrialization.This " medical bio skin graft " has excellent biological compatibility, and its anthemorrhagic performance is good.Liang Peihong etc. adopt pig tendon collagen and chitosan polymerization by a certain percentage, glutaraldehyde cross-linking, and composite sponge is made in lyophilization.Studies show that its bleeding stopping period is 2 minutes, can promote wound healing [(Li Siming is etc. the animal experiment study [J] of compound Ι Collagen Type VI sponge wound hemostasis for Liang Peihong, Ye Chunting. the traumatology department's magazine, 2002,4 (5): 274-275)].If but that above-mentioned material or similar collagen base product do not use glutaraldehyde to carry out is crosslinked, mechanical property can't reach requirement, crosslinked after, the biocompatibility of material but also can go wrong not only; Use the not enough or immunity end peptide of collagen purity well not remove, cause certain immunogenicity, thereby cause the wounded's discomfort, and this series products can not alleviate the wounded's misery, be difficult to satisfy people to the requirement of this series products.In order to solve the problem of above-mentioned material function singleness, the Chinese medicine that material and some can be possessed anti-inflammatory analgesic carries out compound, and a lot of scholars have done this class ground research.Zhu Chuhong etc. have carried out effective ingredient chrysophanol in collagen, chitosan and the Radix Et Rhizoma Rhei organic compound, a kind of polyfunctional new pattern compress [(Zhu Chuhong such as quick-acting haemostatic powder, anti-inflammatory analgesic that have have been prepared, Jiang Bo, Ceng Wen, Li Li, Mi Jianhong. a kind of Multifunction bleeding-stopping dressing. application publication number: CN 102258802A)].This shows that hemostatic material in the past remains in drawbacks such as bleeding stopping period length, haemostatic effect differences, even indivedual hemostatic material also has certain immunogenicity.
In sum, prior art has the following disadvantages:
1. existing collagen base hemorrhage mechanical property deficiency, adhesiveness is relatively poor, causes unnecessary trouble to operation technique;
2. existing collagen base hemorrhage anthemorrhagic performance is general, and function singleness can not satisfy the requirement in market;
3. type i collagen purity is not enough in the existing collagen base hemorrhage monomer material, also can have certain immunogenicity, is applied to clinically may cause imponderable injury to the patient.
Summary of the invention
The objective of the invention is provides a kind of collagen/chitosan micro-nano fiber composite haemostatic membrane material at the deficiencies in the prior art.Material is under action of ultrasonic waves, adopting the medical bio skin graft is that raw material extracts type i collagen, the type i collagen purity that obtains is higher, extraction ratio is also higher, antigenicity is lower, by electrostatic spinning, composite natral macromolecule and compound components of panax notoginseng, prepare collagen/chitosan micro-nano fiber composite haemostatic membrane material.Use this material, can realize fast, effectively stop blooding, also possess advantages such as mechanical property is outstanding, good adhesion, anti-inflammatory analgesic, promotion wound healing.
For realizing that the technical scheme that above-mentioned purpose of the present invention adopts is such, it is a kind of collagen/chitosan micro-nano fiber composite haemostatic membrane material, it can be fast, effectively hemostasis, anti-inflammatory analgesic and can promote wound healing, it is characterized in that the preparation method step is as follows:
(1) the I Collagen Type VI extracts: get a certain amount of medical bio skin graft, it is cut into the fritter of 0.5cm * 0.5cm, clean 3～5 times with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is that placing frequency is in the ultrasonic cleaner of 20～40kHz in 7.5 the Tris-NaCl buffer solution, effect 2～4h; Remove buffer, with distilled water with skin bit rinsing 2～5 times; The acetum that adds 0.5～1M of 30～50 times soaks 2～4h, under 4 ℃ of constant temperatures with refiner smash, homogenate, be transferred to then in the reactor, press the pepsin of 2% adding respective quality of tare weight; Under the ultrasound wave condition, 4 ℃ of constant temperature slowly stir enzymolysis 24～36h, per 3～5h effect, 1～2h; After reaction is finished, stop to stir, with the reactant liquor sucking filtration, regulate filtrate pH to 7.0～7.5, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 10～20h; With collagen solution centrifugal 10～30min under 10000～20000rpm, remove supernatant, precipitate is dissolved in 0.1～0.5M acetum, centrifugal 10～the 30min of 10000～20000rpm removes supernatant, dissolves repeatedly centrifugal 3～5 times, at last with the ultra-pure water washing precipitation, centrifugal, lyophilization, thus obtain high-purity I Collagen Type VI;
(2) electrostatic spinning prepares collagen/chitosan micro-nano composite fiber membrane: with hexafluoroisopropanol and acetic acid with the different volumes mixed, hexafluoroisopropanol content volume percentage ratio is 90%～99%, above-mentioned I Collagen Type VI is mixed with a certain proportion of chitosan, under ultrasound wave, be stirred to transparent under the room temperature, be mixed with 4%～10% mixed liquor, be the electrostatic spinning mother solution, wherein chitosan content mass percent is 10%～50%; The electrostatic spinning mother solution is injected electrostatic spinning machine carry out electrostatic spinning, obtain collagen/chitosan micro-nano composite fiber membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: under the ultrasound wave condition, Radix Notoginseng powder is immersed in 15～20h in 10～20 times 95% alcoholic solution, be immersed in 5～10h in 5～10 times 95% alcoholic solution again, leave standstill the back and merge twice alcohol extract, stay to use it for anything else; Residue is dried, add flooding 15～20h of 10～15 times; 2～5 times of water retting 6～10h of reuse, centrifugalize obtains filtrate, and filtrate is concentrated, and drying makes thick dencichine powder;
(4) preparation of collagen/chitosan micro-nano fiber composite haemostatic membrane material: under the ultrasound wave condition, the above-mentioned collagen/chitosan micro-nano composite fiber membrane that makes is soaked in is selected from Polyethylene Glycol, alginate, hyaluronic acid, thrombin, fibrin or the chondroitin sulfate one or more; And then impregnated in the thick dencichine that adopts method for preparing; Finally by lyophilization, dosage is 6～30KGy/h
60The gamma-rays sterilization that Co produces, formed package is finished product.
In above-mentioned preparation method, use peptic activity of stomach to be 3000u/g in the step (1), Sigma company buys; Hexafluoroisopropanol is analytical pure in the step (2), and company of Beijing Hua Weirui section buys; The chitosan of different deacetylations is to prepare by the deacetylation of controlling chitin in the step (2); The concentration of soak in the step (4), difference that soak time is looked material and difference is 2% as sodium alginate, soak time is 10～20h etc., soaking temperature is room temperature.
Performance parameter index determining method such as following table that collagen/chitosan micro-nano fiber composite haemostatic membrane material described in claim 1 has:
The anthemorrhagic performance assay method of the collagen/chitosan micro-nano fiber composite haemostatic membrane material described in claim is as follows:
Getting internal diameter is the glass cleaning test tube of 8mm, adds above-mentioned material 0.1g, adds calcium chloride 0.1ml again; Shake all the back and in test tube, add blood 0.5ml, jog, be mixed, clock immediately, in 37 ℃ of water baths, observe, every tilt gently test tube 1 time of 30s, observe and have or not blood coagulation.From add blood begin to clock inclination test tube clot freeze solidly on the pipe end when not flowing till, namely clotting time repeats six times.
The present invention has following advantage:
(1) performance is complementary and collaborative between the monomer of composite: material monomer is combined with each other, can brings into play the advantage of material monomer itself, composite can produce again that material monomer itself does not possess or than the monomer combination property that promotes to some extent of performance before.As electrostatic spinning is prepared collagen/chitosan micro-nano composite fiber membrane and natural macromolecular material, compound components of panax notoginseng is compound, composite is possessing the excellent biological compatibility of collagen itself, good biodegradability, is promoting wound healing etc., both had the outstanding anthemorrhagic performance of chitosan, antibiotic/bacteriostasis property, had hemostasis, the anti-inflammatory analgetic effect of Radix Notoginseng again concurrently.The aspects such as ability of the material mechanical performance after compound, anthemorrhagic performance, healing of wound all increase, and become a kind of hemorrhage of high comprehensive performance;
(2) the collagen/chitosan micro-nano composite fiber membrane for preparing of process using electrostatic spinning technique is not adding the mechanical property that strengthened collagen under the situation of cross-linking agent, anthemorrhagic performance etc.;
(3) extraction of type i collagen is to be raw material with the medical bio skin graft in the composite, under the ultrasound wave condition, adopts sour enzyme combined techniques to extract to obtain.Gained collagen purity, extraction ratio are higher, and immunogenicity is lower;
(4) in whole process of preparation, all used ultrasound wave, given full play to effects such as hyperacoustic cavitation effect, mechanical effect, heat effect, the preparation of material has been produced great influence;
(5) the collagen base micro-nano fiber composite membrane material high comprehensive performance for preparing, diverse in function is carried, uses, preserves all very convenient.
Description of drawings
Fig. 1. the SEM stereoscan photograph of collagen/chitosan micro-nano composite fiber membrane.
The specific embodiment
Below by implementing that the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention and further specifies; and can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make nonessential improvement and adjustment according to the content of foregoing invention.
Embodiment 1
(1) type i collagen extracts: the medical bio skin graft of getting 10 weight portions, it is cut into the fritter of 0.5cm * 0.5cm, clean 5 times with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is that placing frequency is in the ultrasonic cleaner of 20kHz in 7.5 the Tris-NaCl buffer solution, effect 2h; Remove buffer, with distilled water with twice of skin bit rinsing; The acetum that adds 30 times 0.5M soaks 2h, under 4 ℃ of constant temperatures with refiner smash, homogenate, be transferred to then in the reactor, add the pepsin of 0.2 weight portion; Be under the ultrasound wave condition of 30kHz in frequency, 4 ℃ of enzymolysis 36h under the slow stirring conditions down, every 3h effect 1h; After reaction is finished, stop to stir, with the reactant liquor sucking filtration, regulate filtrate pH to 7.0, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 10h; With collagen solution centrifugal 30min under 10000rpm, remove supernatant, precipitate is dissolved in the 0.5M acetum the centrifugal 30min of 10000rpm, remove supernatant, dissolve repeatedly centrifugal 5 times, at last with the ultra-pure water washing precipitation, centrifugal, lyophilization, thus obtain the high-purity type i collagen;
(2) electrostatic spinning prepares collagen/chitosan micro-nano composite fiber membrane: the hexafluoroisopropanol of 49 parts by volume is mixed with the acetic acid of 1 parts by volume, stir; Be during 80% chitosan is dissolved in the deacetylation of the type i collagen of 4.5 weight portions and 0.5 weight portion, be mixed with the mixed solution of 10% collagen/chitosan, under frequency is the ultrasound wave condition of 20kHz, be stirred to transparently under the room temperature, make the electrostatic spinning mother solution; The electrostatic spinning mother solution is injected electrostatic spinning machine, under the condition of voltage 18kv, spinning speed 0.03mm/min, receiving range 10cm, carry out electrostatic spinning, obtain collagen/chitosan micro-nano composite fiber membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: be under the ultrasound wave condition of 30kHz in frequency, the Radix Notoginseng powder of 10 weight portions is immersed in 20h in 95% alcoholic solution of 200 parts by volume, be immersed in 10h in 95% alcoholic solution of 100 parts by volume again, leave standstill the back and merge twice alcohol extract, stay to use it for anything else; Residue is dried, add the ultra-pure water lixiviate 20h of 150 parts by volume; The ultra-pure water dipping 10h of reuse 50 parts by volume, centrifugalize obtains filtrate, and filtrate is concentrated, and drying makes thick dencichine powder;
(4) collagen/chitosan micro-nano fiber composite haemostatic membrane material: be under the ultrasound wave condition of 20kHz in frequency, the above-mentioned collagen base micro-nano composite fiber membrane that makes is soaked in 2% sodium alginate and the 0.1M calcium chloride; And then impregnated in 8h in the thick dencichine that adopts method for preparing; Finally by lyophilization, dosage is 15KGy/h
60The gamma-rays sterilization that Co produces, formed package is finished product.
Major parameter performance such as the following table of the collagen/chitosan micro-nano fiber composite haemostatic membrane material that makes through said method:
Embodiment 2
(1) type i collagen extracts: the medical bio skin graft of getting 10 weight portions, it is cut into the fritter of 0.5cm * 0.5cm, clean 5 times with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is that placing frequency is in the ultrasonic cleaner of 30kHz in 7.5 the Tris-NaCl buffer solution, effect 2h; Remove buffer, with distilled water with twice of skin bit rinsing; The acetum that adds 40 times 0.5M soaks 2h, under 4 ℃ of constant temperatures with refiner smash, homogenate, being transferred to then in the reactor, adding the pepsin of 0.2 weight portion, is under the ultrasound wave condition of 30kHz in frequency, 4 ℃ are slowly stirred enzymolysis 24h, every 3h effect 1h down.After reaction is finished, stop to stir, with the reactant liquor sucking filtration, regulate filtrate pH to 7.5, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 15h; With collagen solution centrifugal 10min under 20000rpm, remove supernatant, precipitate is dissolved in the 0.5M acetum the centrifugal 10min of 20000rpm, remove supernatant, dissolve repeatedly centrifugal 5 times, at last with the ultra-pure water washing precipitation, centrifugal, lyophilization, thus obtain the high-purity type i collagen;
(2) electrostatic spinning prepares collagen/chitosan micro-nano composite fiber membrane: the hexafluoroisopropanol of 45 parts by volume is mixed with the acetic acid of 5 parts by volume, stir; Be during 80% chitosan is dissolved in the deacetylation of the type i collagen of 2 weight portions and 1 weight portion, be mixed with the mixed solution of 6% collagen/chitosan, under frequency is the ultrasound wave condition of 20kHz, be stirred to transparently under the room temperature, make the electrostatic spinning mother solution; The electrostatic spinning mother solution is injected electrostatic spinning machine, under the condition of voltage 20kv, spinning speed 0.01mm/min, receiving range 5cm, carry out electrostatic spinning, obtain collagen/chitosan micro-nano composite fiber membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: be under the ultrasound wave condition of 30kHz in frequency, the Radix Notoginseng powder of 10 weight portions is immersed in 15h in 95% alcoholic solution of 150 parts by volume, be immersed in 6h in 95% alcoholic solution of 100 parts by volume again, leave standstill the back and merge twice alcohol extract, stay to use it for anything else; Residue is dried, add the ultra-pure water lixiviate 20h of 200 parts by volume; The ultra-pure water dipping 15h of reuse 150 parts by volume, centrifugalize obtains filtrate, and filtrate is concentrated, and drying makes thick dencichine powder;
(4) preparation of collagen/chitosan micro-nano fiber composite haemostatic membrane material: be under the ultrasound wave condition of 25kHz in frequency, the above-mentioned collagen base micro-nano composite fiber membrane that makes be soaked in 8h in the mixed liquor of 2% sodium alginate, 4% Polyethylene Glycol and 0.1M calcium chloride; And then impregnated in 6h in the thick dencichine that adopts method for preparing; Finally by lyophilization, dosage is 20KGy/h
60The gamma-rays sterilization that Co produces, formed package is finished product.
Major parameter performance such as the following table of the collagen/chitosan micro-nano fiber composite haemostatic membrane material that makes through said method:
Embodiment 3
(1) type i collagen extracts: the medical bio skin graft of getting 10 weight portions, it is cut into the fritter of 0.5cm * 0.5cm, clean 5 times with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is that placing frequency is in the ultrasonic cleaner of 30kHz in 7.5 the Tris-NaCl buffer solution, effect 2h; The buffer that inclines, with distilled water with twice of skin bit rinsing; The acetum that adds 50 times 0.5M soaks 2h, under 4 ℃ of constant temperatures with refiner smash, homogenate, be transferred to then in the reactor, add the pepsin of 0.2 weight portion; Be under the ultrasound wave condition of 20kHz in frequency, 4 ℃ are slowly stirred enzymolysis 36h, every 3h effect 1h down; After reaction is finished, stop to stir, with the reactant liquor sucking filtration, regulate filtrate pH to 7.3, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 20h.With collagen solution centrifugal 20min under 15000rpm, remove supernatant, precipitate is dissolved in the 0.5M acetum the centrifugal 20min of 15000rpm, remove supernatant, dissolve repeatedly centrifugal 5 times, at last with the ultra-pure water washing precipitation, centrifugal, lyophilization, thus obtain the high-purity type i collagen;
(2) electrostatic spinning prepares collagen/chitosan micro-nano composite fiber membrane: the hexafluoroisopropanol of 48 parts by volume is mixed with the acetic acid of 2 parts by volume, stir; Be during 70% chitosan is dissolved in the deacetylation of the type i collagen of 2.5 weight portions and 0.5 weight portion, be mixed with the mixed solution of 6% collagen/chitosan, under the ultrasound wave condition, be stirred to transparently under the room temperature, make the electrostatic spinning mother solution; The electrostatic spinning mother solution is injected electrostatic spinning machine, under the condition of voltage 19kv, spinning speed 0.05mm/min, receiving range 10cm, carry out electrostatic spinning, obtain collagen/chitosan micro-nano composite fiber membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: be under the ultrasound wave condition of 25kHz in frequency, the Radix Notoginseng powder of 10 weight portions is immersed in 15h in 95% alcoholic solution of 200 parts by volume, be immersed in 10h in 95% alcoholic solution of 100 parts by volume again, leave standstill the back and merge twice alcohol extract, stay to use it for anything else; Residue is dried, add the ultra-pure water lixiviate 15h of 150 parts by volume; The ultra-pure water dipping 10h of reuse 100 parts by volume, centrifugalize obtains filtrate, and filtrate is concentrated, and drying makes thick dencichine powder;
(4) preparation of collagen/chitosan micro-nano fiber composite haemostatic membrane material: be under the ultrasound wave condition of 25kHz in frequency, the above-mentioned collagen base micro-nano composite fiber membrane that makes be soaked in 8h in 40% alcohol mixed solution of 3% hyaluronic acid and 1% chondroitin sulfate; And then impregnated in 8h in the thick dencichine that adopts method for preparing; Finally by lyophilization, dosage is 30KGy/h
60The gamma-rays sterilization that Co produces, formed package is finished product.
Major parameter performance such as the following table of the collagen/chitosan micro-nano fiber composite haemostatic membrane material that makes through said method:
?。
Claims (8)
1. collagen/chitosan micro-nano fiber composite haemostatic membrane material is characterized in that described compound haemostatic membrane material possesses following physicochemical property:
(1) sodium chloride content :≤3% (m/m);
(2) content of beary metal :≤10 μ g/g (m/m);
(3) hydroxyproline content: the 8%(m ∕ m that is not less than total protein content);
(4) cytotoxicity: cell-cytotoxic reaction is not more than 1 grade.
2. collagen/chitosan micro-nano fiber composite haemostatic membrane preparation methods as claimed in claim 1 is characterized in that, comprises the steps:
(1) the I Collagen Type VI extracts: get a certain amount of medical bio skin graft, it is cut into the fritter of 0.5cm * 0.5cm, clean 3～5 times with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is that placing frequency is in the ultrasonic cleaner of 20～40kHz in 7.5 the Tris-NaCl buffer solution, effect 2～4h; Remove buffer, with distilled water with skin bit rinsing 2～5 times; The acetum that adds 0.5～1M of 30～50 times soaks 2～4h, under 4 ℃ of constant temperatures with refiner smash, homogenate, be transferred to then in the reactor, press the pepsin of 2% adding respective quality of tare weight; Under the ultrasound wave condition, 4 ℃ of constant temperature slowly stir enzymolysis 24～36h, per 3～5h effect, 1～2h; After reaction is finished, stop to stir, with the reactant liquor sucking filtration, regulate filtrate pH to 7.0～7.5, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 10～20h; With collagen solution centrifugal 10～30min under 10000～20000rpm, remove supernatant, precipitate is dissolved in 0.1～0.5M acetum, centrifugal 10～the 30min of 10000～20000rpm removes supernatant, dissolves repeatedly centrifugal 3～5 times, at last with the ultra-pure water washing precipitation, centrifugal, lyophilization, thus obtain high-purity I Collagen Type VI;
(2) electrostatic spinning prepares collagen/chitosan micro-nano composite fiber membrane: with hexafluoroisopropanol and acetic acid with the different volumes mixed, hexafluoroisopropanol content volume percentage ratio is 90%～99%, above-mentioned I Collagen Type VI is mixed with a certain proportion of chitosan, under ultrasound wave, be stirred to transparent under the room temperature, be mixed with 4%～10% mixed liquor, be the electrostatic spinning mother solution, wherein chitosan content mass percent is 10%～50%; The electrostatic spinning mother solution is injected electrostatic spinning machine carry out electrostatic spinning, obtain collagen/chitosan micro-nano composite fiber membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: under the ultrasound wave condition, Radix Notoginseng powder is immersed in 15～20h in 10～20 times 95% alcoholic solution, be immersed in 5～10h in 5～10 times 95% alcoholic solution again, leave standstill the back and merge twice alcohol extract, stay to use it for anything else; Residue is dried, add flooding 15～20h of 10～15 times; 2～5 times of water retting 6～10h of reuse, centrifugalize obtains filtrate, and filtrate is concentrated, and drying makes thick dencichine powder;
(4) preparation of collagen/chitosan micro-nano fiber composite haemostatic membrane material: under the ultrasound wave condition, the above-mentioned collagen/chitosan micro-nano composite fiber membrane that makes is soaked in is selected from Polyethylene Glycol, alginate, hyaluronic acid, thrombin, fibrin or the chondroitin sulfate one or more; And then impregnated in the thick dencichine that adopts method for preparing; Finally by lyophilization, dosage is 6～30KGy/h
60The gamma-rays sterilization that Co produces, formed package is finished product.
3. the ultrasonic frequency of implementing described in the claim 2 is 20～40 kHz, and power is 50～120w.
4. the described collagen/chitosan micro-nano of claim 2 fiber composite haemostatic membrane preparation methods is characterized in that, described I Collagen Type VI purity 〉=95%, and molecular weight is about 300,000.
5. the preparation method of claims 2 described collagen/chitosan micro-nano composite fiber membranes is characterized in that, described electrostatic spinning condition is: voltage 15～25kv, liquid inventory are that 0.01～0.3mm/min, receiving range are 5～25cm; The apparent viscosity of electrostatic spinning mother solution is 10～120mPa
.S, measuring temperature is 20 ℃, rotating speed is 32r/min.
6. the described collagen/chitosan micro-nano of claim 2 fiber composite haemostatic membrane preparation methods, it is characterized in that, described collagen/chitosan micro-nano composite fiber membrane is after making, be immersed in the phosphate buffer 2 days, remove the hexafluoroisopropanol that remains in the film, period interval 4h changes buffer one time.
7. the described collagen/chitosan micro-nano of claim 2 fiber composite haemostatic membrane preparation methods is characterized in that, described deacetylating degree of chitosan is 60%～80%.
8. the described collagen/chitosan micro-nano of claim 2 fiber composite haemostatic membrane preparation methods, it is characterized in that, it is to be raw material with the medical bio skin graft that described I Collagen Type VI extracts, under hyperacoustic condition, adopt sour enzyme combined techniques to extract the I Collagen Type VI, the I Collagen Type VI that obtains like this is purer, and extraction ratio is higher.
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