CN103230617B - 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 and a kind of there is the premium properties such as good biocompatibility, biodegradable, adhesiveness, can quick-acting haemostatic powder, anti-inflammatory and antalgic, promotion wound compound hemorrhage.
Background technology
Hemorrhagely cause great danger to human life, seek the focus that hemorrhage is fast and effectively research both at home and abroad always.In all kinds of surgical operation or trauma care, wound surface is hemorrhage and be isolated from the outside and avoid infection, very large on the wound healing impact of patient.In war, according to statistics in latter 48 hours of wound in the cause of the death, hemorrhage is chief reason, accounts for 80% of all traumatic event.Wound severe loss of blood can make wounded's pain, shock, stupor even dead, and therefore hemostatic material needs fast effectively hemostasis, must have anti-inflammatory and antalgic, promote the effect of wound compound etc.
Traditional hemostatic material is first-aid kit, four-tailed bandage, tourniquet and binder etc. mainly, and this kind of material is not portable to be preserved with sterilization, and hemostasis is almost bad by mechanism effect.In recent years, people develop a class biological absorbable hemostatic material, wherein collagen-based hemostatic material gets more and more people's extensive concerning, collagen had both had good biocompatibility, biological degradability, hypotoxicity, poor antigen, again can the expression of active cell characteristic group, maintain the normal characteristic of cell and express, be conducive to the sticking of cell, grow, breed, break up, its anthemorrhagic performance is also comparatively outstanding, existing a large amount of bibliographical informations.
But the people such as Wei Hua preside over and bear " 15 " national high-tech research development plan (863 Program) project, develop " the medical bio skin graft " of high comprehensive performance, realize industrialization.This " medical bio skin graft " has good biocompatibility, and its anthemorrhagic performance is good.Liang Peihong etc. adopt pig tendon collagen to be polymerized by a certain percentage with chitosan, and glutaraldehyde cross-linking, composite sponge is made in lyophilization.Research shows that its bleeding stopping period is 2 minutes, can promote wound healing [(Liang Peihong, Ye Chunting, Li Siming, Deng. the animal experiment study [J] of compound Ι Collagen Type VI sponge wound hemostasis. the traumatology department's magazine, 2002,4 (5): 274-275)].If but above-mentioned material or similar collagen-based product do not use glutaraldehyde to be cross-linked, mechanical property cannot reach requirement, after crosslinked, not only material biocompatibility but also there will be problem; Use the inadequate or immune end peptide of collagen purity well not remove, cause certain immunogenicity, thus cause the wounded uncomfortable, and this series products can not alleviate the misery of the wounded, has been difficult to meet the requirement of people to 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 scholar studied with having done this class.Collagen, chitosan and Active Constituents in Rhubarb chrysophanol have been carried out organic composite by Zhu Chuhong etc., prepare one and there is polyfunctional the new pattern compress [(Zhu Chuhong such as quick-acting haemostatic powder, anti-inflammatory analgesic, Jiang Bo, Zeng Wen, Li Li, Mi Jianhong. a kind of Novel multifunctional hemostatic dressing. application publication number: CN 102258802A)].As can be seen here, hemostatic material in the past, remains the drawbacks such as long in bleeding stopping period, haemostatic effect is poor, and even indivedual hemostatic material also has certain immunogenicity.
In sum, prior art has the following disadvantages:
1. existing collagen-based hemorrhage mechanical property is not enough, and adhesiveness is poor, causes unnecessary trouble to operation technique;
2. existing collagen-based haemostatic performance is general, and function singleness, can not meet the requirement in market;
3. in existing collagen-based hemorrhage monomer material, type i collagen purity is inadequate, also can there is certain immunogenicity, is applied to clinically to cause imponderable injury to patient.
Summary of the invention
The object of the invention is to provide a kind of Collagen/chitosan micro-nano fiber composite hemostatic membrane material for the deficiencies in the prior art.Material is under hyperacoustic effect, medical bio skin graft is adopted to be that raw material extracts type i collagen, the type i collagen purity obtained 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 hemostatic membrane material.Use this material, can realize stopping blooding fast, effectively, also possess the advantages such as mechanical property is outstanding, good adhesion, anti-inflammatory analgesic, promotion wound healing.
The technical scheme adopted for realizing above-mentioned purpose of the present invention is such, i.e. a kind of Collagen/chitosan micro-nano fiber composite hemostatic membrane material, it can stop blooding fast, effectively, anti-inflammatory analgesic and can promote wound healing, and it is characterized in that, preparation method step is as follows:
(1) NTx extracts: get a certain amount of medical bio skin graft, be cut into the fritter of 0.5cm × 0.5cm, with ultra-pure water cleaning 3 ~ 5 times, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is in the Tris-NaCl buffer solution of 7.5, is placed in ultrasonic cleaner that frequency is 20 ~ 40kHz, effect 2 ~ 4h; Remove buffer, with distilled water by skin bit rinsing 2 ~ 5 times; Add the acetum of the 0.5 ~ 1M of 30 ~ 50 times, soak 2 ~ 4h, under 4 DEG C of constant temperatures with refiner smash, homogenate, be then transferred in reactor, add the pepsin of respective quality by 2% of tare weight; Under Ultrasonic Conditions, 4 DEG C of Slow Isothermals stir enzymolysis 24 ~ 36h, every 3 ~ 5h effect 1 ~ 2h; After having reacted, stop stirring, by 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; By collagen solution centrifugal 10 ~ 30min under 10000 ~ 20000rpm, removing supernatant, by precipitate dissolves in 0.1 ~ 0.5M acetum, centrifugal 10 ~ the 30min of 10000 ~ 20000rpm, removing supernatant, dissolves centrifugal 3 ~ 5 times repeatedly, finally precipitate with milli-Q water, centrifugal, lyophilization, thus obtain high-purity I Collagen Type VI;
(2) electrostatic spinning prepares collagen/chitosan micro-nano fiber composite membrane: mixed with different volumes ratio with acetic acid by hexafluoroisopropanol, hexafluoroisopropanol content volume percentage ratio is 90% ~ 99%, above-mentioned NTx is mixed with a certain proportion of chitosan, be stirred to transparent under room temperature under ultrasound wave, be mixed with the mixed liquor of 4% ~ 10%, be electrostatic spinning mother solution, wherein chitosan content mass percent is 10% ~ 50%; Electrostatic spinning mother solution is injected electrostatic spinning machine and carries out electrostatic spinning, obtain collagen/chitosan micro-nano fiber composite membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: under Ultrasonic Conditions, Radix Notoginseng powder is immersed in 15 ~ 20h in 95% alcoholic solution of 10 ~ 20 times, be immersed in 5 ~ 10h in 95% alcoholic solution of 5 ~ 10 times again, merge twice alcohol extract after leaving standstill, stay and use it for anything else; Residue is dried, adds the flooding 15 ~ 20h of 10 ~ 15 times; Use 2 ~ 5 times of water retting 6 ~ 10h again, centrifugalize obtains filtrate, filtrate is concentrated, dry obtained thick dencichine powder;
(4) preparation of Collagen/chitosan micro-nano fiber composite hemostatic membrane material: under Ultrasonic Conditions, is soaked in one or more being selected from Polyethylene Glycol, alginate, hyaluronic acid, thrombin, fibrin or chondroitin sulfate by above-mentioned obtained collagen/chitosan micro-nano fiber composite membrane; And then impregnated in and adopt in the thick dencichine prepared of said method; 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, in step (1), use peptic activity of stomach for the purchase of 3000u/g, Sigma company; In step (2), hexafluoroisopropanol is analytical pure, and Beijing company of Hua Weirui section buys; In step (2), the chitosan of different deacetylation is prepared by the deacetylation of control chitin; The concentration of soak in step (4), soak time look the difference of material and different, and if sodium alginate is 2%, soak time is 10 ~ 20h etc., and soaking temperature is room temperature.
The performance parameter index determining method that Collagen/chitosan micro-nano fiber composite hemostatic membrane material as described in claim 1 has is as following table:
The anthemorrhagic performance assay method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material is as described in the accompanying claims as follows:
Get the glass cleaning test tube that internal diameter is 8mm, add above-mentioned material 0.1g, then add calcium chloride 0.1ml; Shake in all backward test tube and add blood 0.5ml, jog, to be mixed, clock immediately, observe in 37 DEG C of water baths, every 30s inclination test tube 1 time gently, observe with or without blood coagulation.The inclination test tube clot that clocks from adding blood freezes solidly on when not flowing at the bottom of pipe, i.e. clotting time, repeats six times.
The present invention has following advantage:
(1) between the monomer of composite, performance complement is with collaborative: be combined with each other by material monomer, can play the advantage of material monomer itself, composite can produce again the combination property that material monomer itself does not possess or promotes to some extent than performance before monomer.As collagen/chitosan micro-nano fiber composite membrane and natural macromolecular material, compound components of panax notoginseng compound prepared by electrostatic spinning, composite possesses the good biocompatibility, good biodegradability, promotion wound healing etc. of collagen itself, both there is the outstanding anthemorrhagic performance of chitosan, antibacterial/bacteriostasis property, had again the hemostasis of Radix Notoginseng, anti-inflammatory analgetic effect concurrently.The aspects such as the 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) technique adopts the collagen/chitosan micro-nano fiber composite membrane that electrostatic spinning technique prepares, and enhances the mechanical property of collagen, anthemorrhagic performance etc. when not adding cross-linking agent;
(3) in composite, the extraction of type i collagen is with medical bio skin graft for raw material, under Ultrasonic Conditions, adopts acid-enzyme binding-method extraction to obtain.Gained collagen purity, extraction ratio are higher, and immunogenicity is lower;
(4) in whole preparation process, all employ ultrasound wave, given full play to the effects such as hyperacoustic cavitation effect, mechanical effect, heat effect, great impact is created on the preparation of material;
(5) the collagen-based micro-nano fiber composite film material high comprehensive performance prepared, diverse in function, carry, use, preserve all very convenient.
Accompanying drawing explanation
Fig. 1. the SEM stereoscan photograph of collagen/chitosan micro-nano fiber composite membrane.
Detailed description of the invention
Below by enforcement, the present invention is specifically described; what be necessary to herein means out is that the present embodiment is only further described for the present invention; and limiting the scope of the invention can not be interpreted as, 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 getting 10 weight portions, be cut into the fritter of 0.5cm × 0.5cm, 5 times are cleaned with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is in the Tris-NaCl buffer solution of 7.5, is placed in ultrasonic cleaner that frequency is 20kHz, effect 2h; Remove buffer, with distilled water by skin bit rinsing twice; Add the acetum of the 0.5M of 30 times, soak 2h, under 4 DEG C of constant temperatures with refiner smash, homogenate, be then transferred in reactor, add the pepsin of 0.2 weight portion; Be under the Ultrasonic Conditions of 30kHz in frequency, enzymolysis 36h under slow stirring condition, every 3h effect 1h at 4 DEG C; After having reacted, stop stirring, by reactant liquor sucking filtration, regulate filtrate pH to 7.0, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 10h; By collagen solution centrifugal 30min under 10000rpm, removing supernatant, by precipitate dissolves in 0.5M acetum, the centrifugal 30min of 10000rpm, removing supernatant, repeatedly dissolves centrifugal 5 times, finally precipitates with milli-Q water, centrifugal, lyophilization, thus obtain high-purity I-type collagen;
(2) electrostatic spinning prepares collagen/chitosan micro-nano fiber composite membrane: mixed by the acetic acid of the hexafluoroisopropanol of 49 parts by volume with 1 parts by volume, stir; During the chitosan being 80% by the deacetylation of the type i collagen of 4.5 weight portions and 0.5 weight portion is dissolved in, be mixed with the mixed solution of the collagen/chitosan of 10%, be stirred to transparent under the Ultrasonic Conditions of 20kHz in frequency under room temperature, make electrostatic spinning mother solution; Electrostatic spinning mother solution is injected electrostatic spinning machine, under the condition of voltage 18kv, spinning speed 0.03mm/min, receiving range 10cm, carries out electrostatic spinning, obtain collagen/chitosan micro-nano fiber composite membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: be under the Ultrasonic Conditions 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, merge twice alcohol extract after leaving standstill, stay and use it for anything else; Residue is dried, adds the ultra-pure water lixiviate 20h of 150 parts by volume; Again with the ultra-pure water dipping 10h of 50 parts by volume, centrifugalize obtains filtrate, filtrate is concentrated, dry obtained thick dencichine powder;
(4) Collagen/chitosan micro-nano fiber composite hemostatic membrane material: be under the Ultrasonic Conditions of 20kHz in frequency, is soaked in 2% sodium alginate and 0.1M calcium chloride by above-mentioned obtained collagen-based micro-nano fiber composite membrane; And then impregnated in and adopt 8h in the thick dencichine prepared of said method; Finally by lyophilization, dosage is 15KGy/h
60the gamma-rays sterilization that Co produces, formed package, is finished product.
The major parameter performance of the Collagen/chitosan micro-nano fiber composite hemostatic membrane material obtained through said method is as following table:
embodiment 2
(1) type i collagen extracts: the medical bio skin graft getting 10 weight portions, be cut into the fritter of 0.5cm × 0.5cm, 5 times are cleaned with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is in the Tris-NaCl buffer solution of 7.5, is placed in ultrasonic cleaner that frequency is 30kHz, effect 2h; Remove buffer, with distilled water by skin bit rinsing twice; Add the acetum of the 0.5M of 40 times, soak 2h, under 4 DEG C of constant temperatures with refiner smash, homogenate, then being transferred in reactor, adding the pepsin of 0.2 weight portion, is under the Ultrasonic Conditions of 30kHz in frequency, slowly enzymolysis 24h is stirred, every 3h effect 1h at 4 DEG C.After having reacted, stop stirring, by reactant liquor sucking filtration, regulate filtrate pH to 7.5, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 15h; By collagen solution centrifugal 10min under 20000rpm, removing supernatant, by precipitate dissolves in 0.5M acetum, the centrifugal 10min of 20000rpm, removing supernatant, repeatedly dissolves centrifugal 5 times, finally precipitates with milli-Q water, centrifugal, lyophilization, thus obtain high-purity I-type collagen;
(2) electrostatic spinning prepares collagen/chitosan micro-nano fiber composite membrane: mixed by the acetic acid of the hexafluoroisopropanol of 45 parts by volume with 5 parts by volume, stir; During the chitosan being 80% by the deacetylation of the type i collagen of 2 weight portions and 1 weight portion is dissolved in, be mixed with the mixed solution of the collagen/chitosan of 6%, be stirred to transparent under the Ultrasonic Conditions of 20kHz in frequency under room temperature, make electrostatic spinning mother solution; Electrostatic spinning mother solution is injected electrostatic spinning machine, under the condition of voltage 20kv, spinning speed 0.01mm/min, receiving range 5cm, carries out electrostatic spinning, obtain collagen/chitosan micro-nano fiber composite membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: be under the Ultrasonic Conditions 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, merge twice alcohol extract after leaving standstill, stay and use it for anything else; Residue is dried, adds the ultra-pure water lixiviate 20h of 200 parts by volume; Again with the ultra-pure water dipping 15h of 150 parts by volume, centrifugalize obtains filtrate, filtrate is concentrated, dry obtained thick dencichine powder;
(4) preparation of Collagen/chitosan micro-nano fiber composite hemostatic membrane material: be under the Ultrasonic Conditions of 25kHz in frequency, is soaked in 8h in the mixed liquor of 2% sodium alginate, 4% Polyethylene Glycol and 0.1M calcium chloride by above-mentioned obtained collagen-based micro-nano fiber composite membrane; And then impregnated in and adopt 6h in the thick dencichine prepared of said method; Finally by lyophilization, dosage is 20KGy/h
60the gamma-rays sterilization that Co produces, formed package, is finished product.
The major parameter performance of the Collagen/chitosan micro-nano fiber composite hemostatic membrane material obtained through said method is as following table:
embodiment 3
(1) type i collagen extracts: the medical bio skin graft getting 10 weight portions, be cut into the fritter of 0.5cm × 0.5cm, 5 times are cleaned with ultra-pure water, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is in the Tris-NaCl buffer solution of 7.5, is placed in ultrasonic cleaner that frequency is 30kHz, effect 2h; Incline buffer, with distilled water by skin bit rinsing twice; Add the acetum of the 0.5M of 50 times, soak 2h, under 4 DEG C of constant temperatures with refiner smash, homogenate, be then transferred in reactor, add the pepsin of 0.2 weight portion; Be under the Ultrasonic Conditions of 20kHz in frequency, slowly stir enzymolysis 36h at 4 DEG C, every 3h effect 1h; After having reacted, stop stirring, by reactant liquor sucking filtration, regulate filtrate pH to 7.3, adding ultimate density is 1.5mol/L ammonium sulfate powder, leaves standstill 20h.By collagen solution centrifugal 20min under 15000rpm, removing supernatant, by precipitate dissolves in 0.5M acetum, the centrifugal 20min of 15000rpm, removing supernatant, repeatedly dissolves centrifugal 5 times, finally precipitates with milli-Q water, centrifugal, lyophilization, thus obtain high-purity I-type collagen;
(2) electrostatic spinning prepares collagen/chitosan micro-nano fiber composite membrane: mixed by the acetic acid of the hexafluoroisopropanol of 48 parts by volume with 2 parts by volume, stir; During the chitosan being 70% by the deacetylation of the type i collagen of 2.5 weight portions and 0.5 weight portion is dissolved in, is mixed with the mixed solution of the collagen/chitosan of 6%, is stirred to transparent under room temperature under Ultrasonic Conditions, make electrostatic spinning mother solution; Electrostatic spinning mother solution is injected electrostatic spinning machine, under the condition of voltage 19kv, spinning speed 0.05mm/min, receiving range 10cm, carries out electrostatic spinning, obtain collagen/chitosan micro-nano fiber composite membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: be under the Ultrasonic Conditions 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, merge twice alcohol extract after leaving standstill, stay and use it for anything else; Residue is dried, adds the ultra-pure water lixiviate 15h of 150 parts by volume; Again with the ultra-pure water dipping 10h of 100 parts by volume, centrifugalize obtains filtrate, filtrate is concentrated, dry obtained thick dencichine powder;
(4) preparation of Collagen/chitosan micro-nano fiber composite hemostatic membrane material: be under the Ultrasonic Conditions of 25kHz in frequency, is soaked in 8h in 40% alcohol mixed solution of 3% hyaluronic acid and 1% chondroitin sulfate by above-mentioned obtained collagen-based micro-nano fiber composite membrane; And then impregnated in and adopt 8h in the thick dencichine prepared of said method; Finally by lyophilization, dosage is 30KGy/h
60the gamma-rays sterilization that Co produces, formed package, is finished product.
The major parameter performance of the Collagen/chitosan micro-nano fiber composite hemostatic membrane material obtained through said method is as following table:
。
Claims (8)
1. a preparation method for Collagen/chitosan micro-nano fiber composite hemostatic membrane material, is characterized in that the method comprises the following steps:
(1) NTx extracts: get a certain amount of medical bio skin graft, be cut into the fritter of 0.5cm × 0.5cm, with ultra-pure water cleaning 3 ~ 5 times, after be immersed in 0.05mol/L Tris, 1mol/L NaCl, pH is in the Tris-NaCl buffer solution of 7.5, is placed in ultrasonic cleaner that frequency is 20 ~ 40kHz, effect 2 ~ 4h; Remove buffer, with distilled water by skin bit rinsing 2 ~ 5 times; Add the acetum of the 0.5 ~ 1M of 30 ~ 50 times, soak 2 ~ 4h, under 4 DEG C of constant temperatures with refiner smash, homogenate, be then transferred in reactor, add the pepsin of respective quality by 2% of tare weight; Under Ultrasonic Conditions, 4 DEG C of Slow Isothermals stir enzymolysis 24 ~ 36h, every 3 ~ 5h effect 1 ~ 2h; After having reacted, stop stirring, by 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; By collagen solution centrifugal 10 ~ 30min under 10000 ~ 20000rpm, removing supernatant, by precipitate dissolves in 0.1 ~ 0.5M acetum, centrifugal 10 ~ the 30min of 10000 ~ 20000rpm, removing supernatant, dissolves centrifugal 3 ~ 5 times repeatedly, finally precipitate with milli-Q water, centrifugal, lyophilization, thus obtain high-purity I Collagen Type VI;
(2) electrostatic spinning prepares collagen/chitosan micro-nano fiber composite membrane: mixed with different volumes ratio with acetic acid by hexafluoroisopropanol, hexafluoroisopropanol content volume percentage ratio is 90% ~ 99%, above-mentioned NTx is mixed with a certain proportion of chitosan, be stirred to transparent under room temperature under ultrasound wave, be mixed with the mixed liquor of 4% ~ 10%, be electrostatic spinning mother solution, wherein chitosan content mass percent is 10% ~ 50%; Electrostatic spinning mother solution is injected electrostatic spinning machine and carries out electrostatic spinning, obtain collagen/chitosan micro-nano fiber composite membrane;
(3) extraction of the effective ingredient dencichine of Radix Notoginseng powder: under Ultrasonic Conditions, Radix Notoginseng powder is immersed in 15 ~ 20h in 95% alcoholic solution of 10 ~ 20 times, be immersed in 5 ~ 10h in 95% alcoholic solution of 5 ~ 10 times again, merge twice alcohol extract after leaving standstill, stay and use it for anything else; Residue is dried, adds the flooding 15 ~ 20h of 10 ~ 15 times; Use 2 ~ 5 times of water retting 6 ~ 10h again, centrifugalize obtains filtrate, filtrate is concentrated, dry obtained thick dencichine powder;
(4) preparation of Collagen/chitosan micro-nano fiber composite hemostatic membrane material: under Ultrasonic Conditions, is soaked in one or more being selected from Polyethylene Glycol, alginate, hyaluronic acid, thrombin, fibrin or chondroitin sulfate by above-mentioned obtained collagen/chitosan micro-nano fiber composite membrane; And then impregnated in and adopt in the thick dencichine prepared of said method; Finally by lyophilization, dosage is 6 ~ 30KGy/h
60the gamma-rays sterilization that Co produces, formed package, is finished product.
2. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, is characterized in that, the ultrasonic frequency of described enforcement is 20 ~ 40kHz, and power is 50 ~ 120w.
3. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, is characterized in that, described NTx purity >=95%, molecular weight is about 300,000.
4. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, is characterized in that, described electrospinning conditions is: voltage 15 ~ 25kv, liquid inventory are 0.01 ~ 0.3mm/min, receiving range is 5 ~ 25cm; The apparent viscosity of electrostatic spinning mother solution is 10 ~ 120mPa.s, and measuring temperature is 20 DEG C, and rotating speed is 32r/min.
5. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, it is characterized in that, described collagen/chitosan micro-nano fiber composite membrane is after obtaining, to be immersed in phosphate buffer 2 days, remove the hexafluoroisopropanol remained in film, period changes a buffer every 4h.
6. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, is characterized in that, described deacetylating degree of chitosan is 60% ~ 80%.
7. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, it is characterized in that, it is for raw material with medical bio skin graft that described NTx extracts, under hyperacoustic condition, employing acid-enzyme binding-method extracts NTx, the NTx obtained like this is purer, and extraction ratio is higher.
8. the preparation method of Collagen/chitosan micro-nano fiber composite hemostatic membrane material according to claim 1, is characterized in that, described compound hemostatic 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: 8% (m/m) being not less than total protein content;
(4) cytotoxicity: cell-cytotoxic reaction is not more than 1 grade.
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