Active polypeptide repair material and preparation method thereof

Abstract

The invention discloses an active polypeptide repair material and a preparation method thereof. The collagen fiber membrane is a fiber membrane prepared by compounding type I collagen extracted from an animal source and type III collagen microspheres containing a placenta extract. The prepared active polypeptide repairing material acts on the skin surface intervened by intense pulsed light after wetting, water-soluble I-type collagen can be transdermally absorbed on a tiny wound surface, fibroblast migration and hyperplasia are stimulated, wound healing is accelerated, meanwhile, III-type collagen and placenta-carrying polypeptide act on the wound surface, and the wound surface can be effectively repaired. The skin elasticity is recovered, less wrinkles are formed, the skin is more elastic, and the effect of delaying senescence is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin repair, and particularly relates to an active polypeptide repair material and a preparation method thereof. Background Art

[0002] The skin is the organ with the largest mass and area in the animal body, and is also the largest peripheral sensor. It also contains main support systems such as blood, nerves and muscles, and has functions such as immune ability, emotional reactivity, ultraviolet perception ability and endocrine. However, with the increase of age, the skin will show visible changes (such as more wrinkles, deeper wrinkles, dryness and roughness, etc.). Skin aging will weaken its barrier function, make the skin dry and thin, increase the probability of skin diseases, and at the same time increase the risk of skin cancer. With the increase of the aging population, more and more people are seeking ways to maintain young and healthy skin. In recent years, optoelectronic technology has developed rapidly. By using its functions of blocking blood vessels and improving the microenvironment, it has a definite therapeutic effect on skin aging. At present, intense pulsed light is mostly used to treat skin aging, and satisfactory results can be achieved after multiple treatments.

[0003] Intense Pulsed Light (IPL) is a major progress in the field of optoelectronic therapy after laser technology. By using selective photothermal action, the output energy is selectively absorbed by melanin and hemoglobin. Therefore, phototherapy effects can be produced. Intense pulsed light produces reversible thermal damage to collagen and induces the contraction and remodeling of collagen fibers, resulting in the effect of photon rejuvenation. It can simultaneously treat skin beauty problems such as pigmentation, telangiectasia, inflammatory acne and hair removal.

[0004] The skin barrier refers to the physical and mechanical barrier effects produced by the epidermis, especially the stratum corneum, against external stimuli. The barrier function of the skin depends on keratinocytes rich in proteins and lamellar intercellular lipids. A good skin barrier helps to maintain the body's moisture, defend against microbial infections, and maintain electrolyte balance and metabolism. Intense pulsed light treatment will not cause visible skin damage, but it will increase the transepidermal water loss of the skin and also have an adverse impact on the skin barrier of patients, which is clinically manifested as flushing, sensitivity and other symptoms. Summary of the Invention

[0005] The purpose of the present invention is to provide an active polypeptide repair material and a preparation method thereof.

[0006] An active polypeptide repair material is a fiber membrane prepared by compounding type I collagen extracted from animal sources and type III collagen microspheres containing placental extract.

[0007] The animal is a pig, a cow, a sheep or a horse.

[0008] The preparation method of the active polypeptide repair material is carried out according to the following steps:

[0009] (1) Collagen extraction: Using animal-derived soft tissue as raw material, through terminal peptide removal treatment to remove immunogenicity, and through pyrogen removal and impurity protein removal treatment, purified type I and type III collagen are prepared;

[0010] (2) Placenta polypeptide preparation: Using animal placenta as raw material, through pre-washing treatment to remove immunogenicity, and enzyme extraction of placenta polypeptide;

[0011] (3) The purified type I collagen is prepared into a water-soluble type I collagen solution through acylation treatment;

[0012] (4) The purified type III collagen and placenta polypeptide are prepared into composite nanospheres, and then the water-soluble type I collagen solution is added and fully dispersed in purified water to prepare a solution with a concentration of 0.04 - 0.06%. The non-woven fabric is fully infiltrated therein to obtain the active polypeptide repair material.

[0013] The specific steps of the collagen extraction described in step (1) are as follows:

[0014] 1) Select fresh fetal animal skin, remove surface fat and minced meat, soak in pure water to remove blood, wash and cut into pieces, crush in a tissue crusher, and wash the crushed tissue with deionized water; Wash the crushed tissue with 0.05 - 0.15% Na2CO3 at 25°C with shaking for 20 - 28 h, with a material-liquid ratio of 1 g: 5 ml, and wash 3 - 5 times with deionized water; Finally, place it in an 8 - 12% NaCl solution and soak at 4°C for 20 - 28 h, with a material-liquid ratio of 1 g: 5 ml, centrifuge to collect the precipitate, and wash with deionized water;

[0015] 2) According to the material-liquid ratio of 1 g: 10 ml, add 0.3 - 0.7 mol / L acetic acid to the crushed tissue, and then add pepsin accounting for 3 - 7% of the mass of the crushed tissue (pepsin activity 8000 - 12000 IU / g). After mixing evenly, carry out acidic enzymatic hydrolysis in a sealed container at 20 - 30 °C for 2 - 4 d; centrifuge the acidic enzymatic hydrolysate in a high-speed low-temperature centrifuge at 4 °C and 6000 - 8000 r / min for 10 - 20 min, aspirate the centrifuged supernatant, add it to a new container, adjust the pH value of the supernatant to 6.0 - 6.5 with 4 - 6 mol / L NaOH, add NaCl to a final concentration of 4.4 M, and perform salting out overnight; centrifuge, divide the precipitate into two parts, label them as A and B respectively. Take precipitate B and wash it with 4.4 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4 °C with shaking for 6 - 10 h; centrifuge, take the precipitate, extract it twice with 2.4 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4 °C, each time for 2 - 6 h; centrifuge, take the precipitate, extract it twice with 1.7 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4 °C, each time for 2 - 6 h; centrifuge to obtain the supernatant, add saturated sodium chloride for salting out with a volume ratio of 1:1, and let it stand at 4 °C overnight until no more precipitate precipitates. Label the precipitate as C;

[0016] 3) Put the precipitate A after salting out in step 2) into a dialysis container containing 0.4 - 0.6 mol / L acetic acid solution for dialysis, change the acetic acid solution every 10 - 14 h, and perform dialysis 2 - 4 times in total; then change the acetic acid solution in the dialysis container to distilled water for injection, change the distilled water for injection every 10 - 14 h, and perform dialysis 2 - 4 times in total; the product after dialysis is type I collagen;

[0017] 4) Extract the precipitate C after salting out in step 2) twice with 1 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4 °C, each time for 3 - 5 h. Centrifuge to obtain the supernatant, salt it out with 2 M NaCl to a final concentration of 1.5 M, and let it stand at 4 °C overnight until no more precipitate precipitates; put the precipitate into a dialysis container containing 0.4 - 0.6 mol / L acetic acid solution for dialysis, change the acetic acid solution every 10 - 14 h, and perform dialysis 2 - 4 times in total; then change the acetic acid solution in the dialysis container to distilled water for injection, change the distilled water for injection every 10 - 14 h, and perform dialysis 2 - 4 times in total; the product after dialysis is type III collagen.

[0018] The specific steps for preparing the placental polypeptide described in step (2) are as follows: Take 12 - 15 g of fresh animal placenta, add 40 - 60 ml of deionized water, and under ice bath conditions, use a tissue homogenizer to break it at 10000 - 14000 r / min until there are no obvious tissue fragments; the broken tissue homogenate is made up to 100 ml, the pH is adjusted to 6.3 - 6.7, add 0.4 - 0.6 g of papain (papain activity 2000 - 4000 IU / g), and keep it in a constant temperature water bath at 40 - 60 °C for 3 - 5 h; inactivate the enzyme solution at 90 °C for 8 - 12 min; centrifuge the inactivated enzyme solution at 5000 - 7000 r / min for 4 - 6 min, collect the supernatant, and freeze-dry it for 20 - 28 h to obtain the placental polypeptide.

[0019] The animal is a pig, cow, sheep, or horse.

[0020] The operation steps for the acylation of type I collagen described in step (3) are as follows: Dissolve 1.5 - 2.5 g of type I collagen in 0.4 - 0.6 M acetic acid solution to prepare a solution with a concentration of 4 - 6 mg / ml, adjust the pH of the solution with 0.5 - 1.5 M NaOH, continuously stir and add 0.5 - 0.7 g of succinic anhydride solution, continuously add 0.5 - 1.5 M sodium hydroxide solution during the reaction to keep the pH of the system between 7.0 - 7.5, the reaction time is 1.5 - 2.5 h, and the reaction temperature is 15 - 25 °C; after the reaction, dialyze with a 100 kd molecular weight dialysis bag for 2 - 4 d, change the water once every 10 - 14 h, after dialysis, place the dialyzed product at -40 °C for pre-freezing for 10 - 14 h, and then freeze-dry it in a -80 °C freeze-dryer for 20 - 28 h.

[0021] The succinic anhydride solution is prepared by dissolving succinic anhydride in acetone to form a 10% succinic anhydride acetone solution.

[0022] The operation steps for preparing the composite nanospheres described in step (4) are as follows: Disperse the placental polypeptide and type III collagen in physiological saline at a mass ratio of 1:(4 - 6) to prepare a mixed solution with a concentration of 0.04 - 0.06%, and prepare composite nanospheres in a low-temperature freeze spray dryer.

[0023] The mass ratio of the amount of type I collagen to the composite nanospheres described in step (4) is (8 - 12):1.

[0024] The beneficial effects of the present invention: The active polypeptide repair material prepared by the present invention, when wetted, acts on the skin surface after intense pulsed light intervention. At small wounds, it can transdermally absorb water-soluble type I collagen, stimulate the migration and proliferation of fibroblasts, accelerate wound healing. At the same time, type III collagen carrying placental polypeptide acts on the wound surface, restores skin elasticity, reduces wrinkle formation, makes the skin more elastic, and achieves the effect of delaying aging. Brief Description of the Drawings

[0025] Figure 1 It is a microscopic observation diagram of composite nanospheres. Detailed Embodiments

[0026] For ease of understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0027] Example 1

[0028] A method for preparing an active polypeptide repair material is carried out according to the following steps:

[0029] (1) Collagen extraction:

[0030] 1) Select fresh fetal cowhide, remove surface fat and minced meat, soak in pure water to remove blood, wash and cut into pieces, pulverize in a tissue pulverizer, and wash the pulverized tissue with deionized water; wash the pulverized tissue with 0.1% Na2CO3 at 25°C by shaking and soaking for 24 h, with a material-liquid ratio of 1 g: 5 ml, and wash 4 times with deionized water; finally place it in a 10% NaCl solution, soak at 4°C for 24 h, with a material-liquid ratio of 1 g: 5 ml, centrifuge to collect the precipitate, and wash with deionized water;

[0031] 2) According to the material-liquid ratio of 1 g: 10 ml, add 0.5 mol / L acetic acid to the pulverized tissue, and then add pepsin (pepsin activity 10000 IU / g) accounting for 5% of the mass of the pulverized tissue. After mixing evenly, carry out acid enzymatic hydrolysis in a sealed container at 25°C for 3 d; centrifuge the acid enzymatic hydrolysis solution in a high-speed low-temperature centrifuge at 4°C and 7000 r / min for 15 min, suck out the centrifuged supernatant, add it to a new container, adjust the pH value of the supernatant to 6.2 with 5 mol / L NaOH, add NaCl to a final concentration of 4.4 M, and carry out salting out overnight; centrifuge, divide the precipitate into two parts, respectively labeled as A and B, take precipitate B and wash it with 4.4 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C by shaking for 8 h; centrifuge, take the precipitate, extract it twice with 2.4 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C, each time for 4 h; centrifuge, take the precipitate, extract it twice with 1.7 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C, each time for 4 h; centrifuge to take the supernatant, add saturated sodium chloride with a volume ratio of 1:1 for salting out, and let it stand at 4°C overnight until no more precipitate precipitates, and label it as precipitate C;

[0032] 3) Put the precipitate A after salting out in step 2) into a dialysis container filled with 0.5 mol / L acetic acid solution for dialysis, change the acetic acid solution every 12 h, and perform dialysis 3 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 12 h, and perform dialysis 3 times in total; the product after dialysis is type I collagen;

[0033] 4) Extract the precipitate C after salting out in step 2) twice with 1M NaCl / 0.05M Tris-HCl at pH 7.5 at 4°C for 4 h each time, centrifuge to obtain the supernatant, salt out with 2M NaCl to a final concentration of 1.5M, and let it stand overnight at 4°C until no more precipitation occurs; put the precipitate into a dialysis container filled with 0.5 mol / L acetic acid solution for dialysis, change the acetic acid solution every 12 h, and perform dialysis 3 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 12 h, and perform dialysis 3 times in total; the product after dialysis is type III collagen;

[0034] (2) Preparation of placental polypeptide: Take 13.6 g of fresh bovine placenta, add 50 ml of deionized water, and under ice bath conditions, use a tissue homogenizer to break it at 12000 r / min until there are no obvious tissue fragments; make the volume of the broken tissue homogenate up to 100 ml, adjust the pH to 6.5, add 0.5 g of papain (papain activity 3000 IU / g), and keep it in a water bath at 50°C for 4 h; inactivate the enzyme solution at 90°C for 10 min; centrifuge the inactivated enzyme solution at 6000 r / min for 5 min, collect the supernatant, and freeze-dry it for 24 h to obtain placental polypeptide;

[0035] (3) Acylation of type I collagen: Dissolve 2 g of type I collagen in 0.5M acetic acid solution to prepare a solution with a concentration of 5 mg / ml, adjust the pH of the solution with 1M NaOH, continuously stir and add 0.6 g of succinic anhydride solution, continuously add 1M sodium hydroxide solution during the reaction to keep the system pH at 7.2, the reaction time is 2 h, and the reaction temperature is 20°C; after the reaction, dialyze with a 100 kd molecular weight dialysis bag for 3 d, change the water every 12 h, after dialysis, place the dialyzed product at -40°C for pre-freezing for 12 h, and then freeze-dry it in a -80°C freeze-dryer for 24 h; the succinic anhydride solution is prepared by dissolving succinic anhydride in acetone to form a 10% succinic anhydride acetone solution;

[0036] (4) Placenta polypeptide and type III collagen are fully dispersed in physiological saline at a mass ratio of 1:5 to prepare a mixed solution with a concentration of 0.05%. In a low-temperature freeze spray dryer, composite nano-microspheres are prepared. Then, water-soluble type I collagen solution is added and fully dispersed in purified water to prepare a solution with a concentration of 0.05%. The non-woven fabric is fully infiltrated therein to obtain an active polypeptide repair material; the mass ratio of the type I collagen to the composite nano-microspheres is 10:1.

[0037] Example 2

[0038] A preparation method of an active polypeptide repair material is carried out according to the following steps:

[0039] (1) Collagen extraction:

[0040] 1) Select fresh fetal pig skin, remove the surface fat and minced meat, soak in pure water to remove blood, wash and cut into pieces, pulverize in a tissue pulverizer, and wash the pulverized tissue with deionized water; wash the pulverized tissue with 0.08% Na2CO3 at 25°C by shaking and soaking for 20 h, with a material-liquid ratio of 1 g:5 ml, and wash 3 times with deionized water; finally, place it in an 8% NaCl solution and soak at 4°C for 20 h, with a material-liquid ratio of 1 g:5 ml, centrifuge to collect the precipitate, and wash it with deionized water;

[0041] 2) According to the material-liquid ratio of 1 g:10 ml, add 0.4 mol / L acetic acid to the pulverized tissue, and then add pepsin (pepsin activity 9000 IU / g) accounting for 4% of the mass of the pulverized tissue. After mixing evenly, carry out acidic enzymatic hydrolysis in a sealed container at 22°C for 2 d; centrifuge the acidic enzymatic hydrolysis solution in a high-speed low-temperature centrifuge at 4°C and 6000 r / min for 10 min, suck the centrifuged supernatant into a new container, adjust the pH value of the supernatant to 6.0 with 4 mol / L NaOH, add NaCl to a final concentration of 4.4 M, and carry out salting out overnight; centrifuge, divide the precipitate into two parts, and label them as A and B respectively. Take precipitate B and wash it with 4.4 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4°C by shaking for 6 h; centrifuge, take the precipitate, extract it twice with 2.4 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4°C, 3 h each time; centrifuge, take the precipitate, extract it twice with 1.7 M NaCl / 0.05 M Tris-HCl with pH 7.5 at 4°C, 3 h each time; centrifuge to take the supernatant, add saturated sodium chloride with a volume ratio of 1:1 for salting out, and let it stand at 4°C overnight until no more precipitate precipitates, and label the precipitate as C;

[0042] 3) The precipitate A after salting out in step 2) is placed in a dialysis container containing 0.4 mol / L acetic acid solution for dialysis, and the acetic acid solution is replaced every 10 h for a total of 2 times; then the acetic acid solution in the dialysis container is replaced with injection water, and the injection water is replaced every 10 h for a total of 2 times; the product after dialysis is type I collagen;

[0043] 4) The precipitate C after salting out in step 2) is extracted with 1M NaCl / 0.05M Tris-HCl at pH 7.5 at 4°C for 2 times, 3 h each time. After centrifugation, the supernatant is taken, and salted out with 2M NaCl to a final concentration of 1.5M, and left standing at 4°C overnight until no more precipitate precipitates; the precipitate is placed in a dialysis container containing 0.4 mol / L acetic acid solution for dialysis, and the acetic acid solution is replaced every 10 h for a total of 2 times; then the acetic acid solution in the dialysis container is replaced with injection water, and the injection water is replaced every 10 h for a total of 2 times; the product after dialysis is type III collagen;

[0044] (2) Preparation of placental polypeptide: Take 12 g of fresh pig placenta, add 40 ml of deionized water, and use a tissue homogenizer to break it at 10000 r / min in an ice bath until there are no obvious tissue fragments; the broken tissue homogenate is made up to 100 ml, the pH is adjusted to 6.3, 0.4 g of papain (papain activity 2500 IU / g) is added, and it is kept in a water bath at 40°C for 3 h; the enzyme solution is inactivated at 90°C for 8 min; the inactivated enzyme solution is centrifuged at 5000 r / min for 4 min, the supernatant is collected, and freeze-dried for 20 h to obtain placental polypeptide;

[0045] (3) Acylation of type I collagen: Dissolve 1.5 g of type I collagen in 0.4M acetic acid solution to prepare a solution with a concentration of 4 mg / ml. Adjust the pH of the solution with 0.5M NaOH, continuously stir and add 0.5 g of succinic anhydride solution. During the reaction, continuously add 0.5M sodium hydroxide solution to keep the pH of the system at 7.0. The reaction time is 1.5 h, and the reaction temperature is 18°C; after the reaction, dialysis is carried out with a 100 kd molecular weight dialysis bag for 2 d, and the water is changed every 10 h. After dialysis, the dialyzed product is pre-frozen at -40°C for 10 h, and then freeze-dried at -80°C in a freeze dryer for 20 h; the succinic anhydride solution is succinic anhydride dissolved in acetone to prepare a 10% succinic anhydride acetone solution;

[0046] (4) Placenta polypeptide and type III collagen are fully dispersed in physiological saline according to a mass ratio of 1:4 to prepare a mixed solution with a concentration of 0.04%. In a low-temperature freeze spray dryer, composite nano-microspheres are prepared. Then, a water-soluble type I collagen solution is added and fully dispersed in purified water to prepare a solution with a concentration of 0.04%. The non-woven fabric is fully soaked in it to obtain an active polypeptide repair material; the mass ratio of the type I collagen to the composite nano-microspheres is 8:1.

[0047] Example 3

[0048] A preparation method of an active polypeptide repair material is carried out according to the following steps:

[0049] (1) Collagen extraction:

[0050] 1) Select fresh fetal lambskin, remove surface fat and minced meat, soak in pure water to remove blood, wash and cut into pieces, pulverize in a tissue pulverizer, and wash the pulverized tissue with deionized water; wash the pulverized tissue with 0.12% Na2CO3 at 25°C by shaking and soaking for 28 h, with a material-liquid ratio of 1 g:5 ml, and wash 5 times with deionized water; finally, place it in an 8-12% NaCl solution and soak at 4°C for 28 h, with a material-liquid ratio of 1 g:5 ml, centrifuge to collect the precipitate, and wash it with deionized water;

[0051] 2) According to a material-liquid ratio of 1 g:10 ml, add 0.6 mol / L acetic acid to the pulverized tissue, and then add pepsin (pepsin activity 11000 IU / g) accounting for 6% of the mass of the pulverized tissue. After mixing evenly, carry out acid enzymatic hydrolysis in a sealed container at 28°C for 4 d; centrifuge the acid enzymatic hydrolysate in a high-speed low-temperature centrifuge at 4°C and 8000 r / min for 20 min, suck out the centrifuged supernatant, add it to a new container, adjust the pH value of the supernatant to 6.5 with 6 mol / L NaOH, add NaCl to a final concentration of 4.4 M, and carry out salting out overnight; centrifuge, divide the precipitate into two parts, respectively labeled as A and B. Take precipitate B and wash it by shaking at 4°C for 10 h with 4.4 M NaCl / 0.05 M Tris-HCl with pH 7.5; centrifuge, take the precipitate, extract it twice at 4°C with 2.4 M NaCl / 0.05 M Tris-HCl with pH 7.5, 6 h each time; centrifuge, take the precipitate, extract it twice at 4°C with 1.7 M NaCl / 0.05 M Tris-HCl with pH 7.5, 6 h each time; centrifuge to obtain the supernatant, add saturated sodium chloride with a volume ratio of 1:1 for salting out, and let it stand at 4°C overnight until no more precipitate precipitates, and label the precipitate as C;

[0052] 3) Place the precipitate A after salting out in step 2) into a dialysis container filled with 0.6 mol / L acetic acid solution for dialysis, change the acetic acid solution every 14 h, and perform dialysis 4 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 14 h, and perform dialysis 4 times in total; the product after dialysis is type I collagen;

[0053] 4) Extract the precipitate C after salting out in step 2) twice with 1 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C for 5 h each time, centrifuge to obtain the supernatant, salt out with 2 M NaCl to a final concentration of 1.5 M, and let it stand overnight at 4°C until no more precipitation occurs; place the precipitate into a dialysis container filled with 0.6 mol / L acetic acid solution for dialysis, change the acetic acid solution every 14 h, and perform dialysis 4 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 14 h, and perform dialysis 4 times in total; the product after dialysis is type III collagen;

[0054] (2) Preparation of placental polypeptide: Take 15 g of fresh sheep placenta, add 60 ml of deionized water, and under ice bath conditions, use a tissue homogenizer to break it at 14000 r / min until there are no obvious tissue fragments; make the volume of the broken tissue homogenate up to 100 ml, adjust the pH to 6.7, add 0.6 g of papain (papain activity 2500 IU / g), and keep it in a constant temperature water bath at 60°C for 5 h; inactivate the enzyme solution at 90°C for 12 min; centrifuge the inactivated enzyme solution at 7000 r / min for 6 min, collect the supernatant, and freeze-dry it for 28 h to obtain placental polypeptide;

[0055] (3) Acylation of type I collagen: Dissolve 2.5 g of type I collagen in 0.6 M acetic acid solution to prepare a solution with a concentration of 6 mg / ml, adjust the pH of the solution with 1.5 M NaOH, continuously stir and add 0.7 g of succinic anhydride solution, continuously add 1.5 M sodium hydroxide solution during the reaction to keep the system pH at 7.5, the reaction time is 2.5 h, and the reaction temperature is 25°C; after the reaction, dialyze with a 100 kd molecular weight dialysis bag for 4 d, change the water every 14 h, after dialysis, place the dialyzed product at -40°C for pre-freezing for 14 h, and then freeze-dry it in a -80°C freeze dryer for 28 h; the succinic anhydride solution is prepared by dissolving succinic anhydride in acetone to form a 10% succinic anhydride acetone solution;

[0056] 4) The placental polypeptide and type III collagen are fully dispersed in physiological saline at a mass ratio of 1:6 to prepare a mixed solution with a concentration of 0.06%. The mixed solution is then processed in a low-temperature freeze spray dryer to prepare composite nano microspheres. Subsequently, a water-soluble type I collagen solution is added and fully dispersed in purified water to prepare a solution with a concentration of 0.06%. The non-woven fabric is fully soaked in this solution to obtain an active polypeptide repair material; the mass ratio of the type I collagen to the composite nano microspheres is 12:1.

[0057] Comparative Example 1:

[0058] A preparation method of a repair material is carried out according to the following steps:

[0059] (1) Collagen extraction:

[0060] 1) Select fresh fetal cowhide, remove the surface fat and minced meat, soak it in pure water to remove blood, wash and cut it into pieces, then crush it in a tissue crusher. Wash the crushed tissue with deionized water; wash the crushed tissue with 0.1% Na2CO3 at 25°C by shaking and soaking for 24 h, with a material-liquid ratio of 1 g:5 ml, and wash it 4 times with deionized water; finally, place it in a 10% NaCl solution and soak it at 4°C for 24 h, with a material-liquid ratio of 1 g:5 ml, centrifuge to collect the precipitate, and wash it with deionized water;

[0061] 2) According to the material-liquid ratio of 1 g:10 ml, add 0.5 mol / L acetic acid to the crushed tissue, and then add pepsin (pepsin activity 10000 IU / g) accounting for 5% of the mass of the crushed tissue. After mixing evenly, carry out acidic enzymatic hydrolysis in a sealed container at 25°C for 3 d; centrifuge the acidic enzymatic hydrolysis solution in a high-speed low-temperature centrifuge at 4°C and 7000 r / min for 15 min, suck out the centrifuged supernatant, add it to a new container, adjust the pH value of the supernatant to 6.2 with 5 mol / L NaOH, add NaCl to a final concentration of 4.4 M, and carry out salting out overnight; centrifuge, divide the precipitate into two parts, respectively labeled as A and B. Take precipitate B and wash it by shaking at 4°C for 8 h with 4.4 M NaCl / 0.05 M Tris-HCl with pH 7.5; centrifuge, take the precipitate, extract it twice at 4°C with 2.4 M NaCl / 0.05 M Tris-HCl with pH 7.5, each time for 4 h; centrifuge, take the precipitate, extract it twice at 4°C with 1.7 M NaCl / 0.05 M Tris-HCl with pH 7.5, each time for 4 h; centrifuge to obtain the supernatant, add saturated sodium chloride with a volume ratio of 1:1 for salting out, and let it stand at 4°C overnight until no more precipitate precipitates. Label the precipitate as C;

[0062] 3) Put the precipitate A after salting out in step 2) into a dialysis container filled with 0.5 mol / L acetic acid solution for dialysis. Replace the acetic acid solution every 12 h, and perform dialysis 3 times in total; then replace the acetic acid solution in the dialysis container with injection water, and replace the injection water every 12 h, and perform dialysis 3 times in total; the product after dialysis is type I collagen;

[0063] 4) Extract the precipitate C after salting out in step 2) with 1 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4 °C for 2 times, each time for 4 h. Centrifuge to obtain the supernatant, and salt out with 2 M NaCl to a final concentration of 1.5 M, and let it stand at 4 °C overnight until no more precipitate precipitates; put the precipitate into a dialysis container filled with 0.5 mol / L acetic acid solution for dialysis. Replace the acetic acid solution every 12 h, and perform dialysis 3 times in total; then replace the acetic acid solution in the dialysis container with injection water, and replace the injection water every 12 h, and perform dialysis 3 times in total; the product after dialysis is type III collagen;

[0064] (2) Acylation of type I collagen: Dissolve 2 g of type I collagen in 0.5 M acetic acid solution to prepare a solution with a concentration of 5 mg / ml. Adjust the pH of the solution with 1 M NaOH, continuously stir and add 0.6 g of succinic anhydride solution. During the reaction, continuously add 1 M sodium hydroxide solution to maintain the pH of the system at 7.2. The reaction time is 2 h, and the reaction temperature is 20 °C; after the reaction, dialyze with a 100 kd molecular weight dialysis bag for 3 d, change the water every 12 h. After dialysis, place the dialyzed product at -40 °C for pre-freezing for 12 h, and then freeze-dry it in a -80 °C freeze dryer for 24 h; the succinic anhydride solution is a 10% succinic anhydride acetone solution prepared by dissolving succinic anhydride in acetone;

[0065] 3) Disperse type III collagen fully in physiological saline to prepare a solution with a concentration of 0.05%, and prepare nano-microspheres in a low-temperature freeze spray dryer. Then add a water-soluble type I collagen solution and disperse it fully in purified water to prepare a solution with a concentration of 0.05%. Immerse the non-woven fabric in it to obtain a repair material; the mass ratio of the dosage of type I collagen to nano-microspheres is 10:1.

[0066] Comparative Example 2:

[0067] A preparation method of an active polypeptide repair material is carried out according to the following steps:

[0068] (1) Collagen extraction:

[0069] 1) Select fresh fetal bovine skin, remove the surface fat and minced meat, soak it in pure water to remove blood, wash it, cut it into pieces, pulverize it in a tissue pulverizer, and wash the pulverized tissue with deionized water; wash the pulverized tissue with 0.1% Na2CO3 by shaking and soaking at 25 °C for 24 h, with a solid-liquid ratio of 1 g: 5 ml, and wash it 4 times with deionized water; finally, place it in a 10% NaCl solution and soak it at 4 °C for 24 h, with a solid-liquid ratio of 1 g: 5 ml, centrifuge to collect the precipitate, and wash it with deionized water;

[0070] 2) According to the solid-liquid ratio of 1 g: 10 ml, add 0.5 mol / L acetic acid to the pulverized tissue, and then add pepsin (pepsin activity 10000 IU / g) accounting for 5% of the mass of the pulverized tissue. After mixing evenly, carry out acid enzymatic hydrolysis in a sealed container at 25 °C for 3 d; centrifuge the acid enzymatic hydrolysate in a high-speed low-temperature centrifuge at 4 °C and 7000 r / min for 15 min, suck out the centrifuged supernatant, add it to a new container, adjust the pH value of the supernatant to 6.2 with 5 mol / L NaOH, add NaCl to a final concentration of 4.4 M, and carry out salting out overnight; centrifuge and take the precipitate for standby;

[0071] 3) Put the precipitate after salting out in step 2) into a dialysis container filled with 0.5 mol / L acetic acid solution for dialysis, change the acetic acid solution every 12 h, and carry out dialysis 3 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 12 h, and carry out dialysis 3 times in total; the product after dialysis is type I collagen;

[0072] (2) Preparation of placental polypeptide: Take 13.6 g of fresh bovine placenta, add 50 ml of deionized water, and use a tissue homogenizer to break it at 12000 r / min under ice bath conditions until there are no obvious tissue fragments; make the broken tissue homogenate up to 100 ml, adjust the pH to 6.5, add 0.5 g of papain (papain activity 3000 IU / g), and carry out a constant temperature water bath at 50 °C for 4 h; inactivate the enzymatic hydrolysate at 90 °C for 10 min; centrifuge the inactivated enzymatic hydrolysate at 6000 r / min for 5 min, collect the supernatant, and freeze-dry it for 24 h to obtain placental polypeptide;

[0073] (3) Acylation of type I collagen: 2 g of type I collagen was dissolved in 0.5 M acetic acid solution to prepare a solution with a concentration of 5 mg / ml. The solution pH was adjusted with 1 M NaOH, and 0.6 g of succinic anhydride solution was added while stirring continuously. During the reaction, 1 M sodium hydroxide solution was continuously added to maintain the system pH at 7.2. The reaction time was 2 h and the reaction temperature was 20°C. After the reaction was completed, the solution was dialyzed with a 100 kd molecular weight dialysis bag for 3 days, and the water was changed once every 12 h. After the dialysis was completed, the dialyzed product was pre-frozen at -40°C for 12 h and then frozen in a -80°C freeze dryer for 24 h. The succinic anhydride solution was prepared by dissolving succinic anhydride in acetone to prepare a 10% succinic anhydride acetone solution.

[0074] 4) The placental polypeptide is fully dispersed in physiological saline to prepare a mixed solution with a concentration of 0.05%, and nano-microspheres are prepared in a low-temperature freeze spray dryer. Then, a water-soluble type I collagen solution is added and fully dispersed in purified water to prepare a solution with a concentration of 0.05%, and the non-woven fabric is fully infiltrated therein to prepare an active polypeptide repair material; the mass ratio of the type I collagen to the nano-microspheres is 10:1.

[0075] Experimental example:

[0076] (1) Take the composite nanospheres prepared in Example 1 and observe them under a microscope. Figure 1 As shown, the composite microspheres are round and uniform.

[0077] (2) Establishment of a mouse skin aging model: 8-week-old clean-grade mice were adaptively fed under normal conditions for 1 week and allowed to drink water freely; D-gal was intraperitoneally injected at 500 mg / kg / d, and the weight of the mice was measured every week to adjust the injection dose. The experiment was divided into a control group: collagen fiber membrane; an experimental group: the repair materials prepared in Examples 1-3 and Comparative Examples 1-2. The back epidermal hair was removed before injection every week, and the two groups of membrane materials were placed on the left and right sides of the back and fixed, and wet compressed for 20 minutes. After 8 consecutive weeks of treatment, recovery feeding was carried out for 3 days without any treatment, and normal feeding and drinking water were performed. After the end of the recovery feeding, the mice's eyeballs were removed for blood collection and the mice were killed by cervical dislocation. The back skin tissue was collected for the detection of the gene expression of skin aging-related proteins MMP-1a and MMP-3. The experimental results were statistically analyzed using SPSS24.0 software, and the quantitative data results were used. ±(mean ± standard deviation), the Kolmogorov-Smirnov test was used for data normality test, and for data that met the normal distribution, the t-test was used to compare the mean differences between the two groups, with P<0.05 as the difference being statistically significant. The test results are shown in Table 1:

[0078] Table 1

[0079]

[0080] Note: * represents P < 0.05 compared with the group of Example 1, and ** represents P < 0.01.

[0081] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An active polypeptide repair material, characterized in that, It is a fiber membrane prepared by compounding type I collagen extracted from animal sources and type III collagen microspheres containing placental extract.

2. The active polypeptide repair material according to claim 1, wherein The animals are pigs, cows, sheep or horses.

3. The preparation method of the active polypeptide repair material according to claim 1, characterized in that, The following steps are carried out: (1) Collagen extraction: Using animal-derived soft tissues as raw materials, through terminal peptide removal treatment to remove immunogenicity, and through pyrogen removal and impurity protein removal treatment to prepare purified type I and type III collagen; (2) Placenta polypeptide preparation: Using animal placenta as raw materials, through pre-washing treatment to remove immunogenicity, and enzymatically extracting placenta polypeptide; (3) Acylating the purified type I collagen to prepare a water-soluble type I collagen solution; (4) Preparing composite nano-microspheres from the purified type III collagen and placenta polypeptide, then adding the water-soluble type I collagen solution and fully dispersing it in purified water to prepare a solution with a concentration of 0.04 - 0.06%, and fully soaking non-woven fabric in it to obtain an active polypeptide repair material.

4. The preparation method of the active polypeptide repair material according to claim 3, characterized in that The specific steps of the collagen extraction in step (1) are as follows: 1) Select fresh fetal animal skin, remove surface fat and minced meat, soak in purified water to remove blood, wash and cut into pieces, crush in a tissue crusher, and wash the crushed tissue with deionized water; Wash the crushed tissue with 0.05 - 0.15% Na2CO3 at 25°C with shaking and soaking for 20 - 28 h, with a material-liquid ratio of 1 g:5 ml, and wash with deionized water 3 - 5 times; Finally, place it in an 8 - 12% NaCl solution and soak at 4°C for 20 - 28 h, with a material-liquid ratio of 1 g:5 ml, centrifuge to collect the precipitate, and wash with deionized water; 2) According to a material-liquid ratio of 1 g:10 ml, add 0.3 - 0.7 mol / L acetic acid to the crushed tissue, and then add pepsin accounting for 3 - 7% of the mass of the crushed tissue (pepsin activity 8000 - 12000 IU / g), mix evenly, and carry out acidic enzymatic hydrolysis in a sealed container at 20 - 30°C for 2 - 4 d; Centrifuge the acidic enzymatic hydrolysis solution in a high-speed and low-temperature centrifuge at 4°C and 6000 - 8000 r / min for 10 - 20 min, suck out the centrifuged supernatant, add it to a new container, adjust the pH value of the supernatant to 6.0 - 6.5 with 4 - 6 mol / L NaOH, add saturated NaCl to a final concentration of 4.4 M, and carry out salting out overnight; Centrifuge, divide the precipitate into two parts, respectively labeled as A and B, take precipitate B and wash it with 4.4 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C with shaking for 6 - 10 h; Centrifuge, take the precipitate, and extract it twice with 2.4 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C, each time for 2 - 6 h; Centrifuge, take the precipitate, and extract it twice with 1.7 M NaCl / 0.05 M Tris-HCl at pH 7.5 at 4°C, each time for 2 - 6 h; Centrifuge to obtain the supernatant, add saturated sodium chloride with a volume ratio of 1:1 for salting out, and let it stand at 4°C overnight until no more precipitate precipitates. This precipitate is labeled as C; 3) Put the precipitate A after salting out in step 2) into a dialysis container containing 0.4 - 0.6 mol / L acetic acid solution for dialysis, change the acetic acid solution every 10 - 14 h, and dialyze 2 - 4 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 10 - 14 h, and dialyze 2 - 4 times in total; the product after dialysis is type I collagen; 4) Extract the precipitate C after salting out in step 2) with 1M NaCl / 0.05M Tris-HCl at pH 7.5 at 4°C for 2 times, 3 - 5 h each time, centrifuge to obtain the supernatant, salt out with 2M NaCl to a final concentration of 1.5M, and let it stand overnight at 4°C until no more precipitate precipitates; put the precipitate into a dialysis container containing 0.4 - 0.6 mol / L acetic acid solution for dialysis, change the acetic acid solution every 10 - 14 h, and dialyze 2 - 4 times in total; then change the acetic acid solution in the dialysis container to injection water, change the injection water every 10 - 14 h, and dialyze 2 - 4 times in total; the product after dialysis is type III collagen.

5. The preparation method of the active polypeptide repair material according to claim 3, wherein The specific steps for preparing the placental polypeptide in step (2) are as follows: Take 12 - 15 g of fresh animal placenta, add 40 - 60 ml of deionized water, and under ice bath conditions, use a tissue homogenizer to break it at 10000 - 14000 r / min until there are no obvious tissue fragments; make the volume of the broken tissue homogenate up to 100 ml, adjust the pH to 6.3 - 6.7, add 0.4 - 0.6 g of papain (papain activity 2000 - 4000 IU / g), and keep it in a constant temperature water bath at 40 - 60°C for 3 - 5 h; inactivate the enzyme solution at 90°C for 8 - 12 min; centrifuge the inactivated enzyme solution at 5000 - 7000 r / min for 4 - 6 min, collect the supernatant, and freeze-dry it for 20 - 28 h to obtain the placental polypeptide.

6. The preparation method of the active polypeptide repair material according to claims 4-5, characterized in that, The animal is a pig, a cow, a sheep or a horse.

7. The preparation method of the active polypeptide repair material according to claim 3, wherein, The acylation operation steps of the type I collagen in step (3) are as follows: Dissolve 1.5 - 2.5 g of type I collagen in 0.4 - 0.6 M acetic acid solution to prepare a solution with a concentration of 4 - 6 mg / ml, adjust the pH of the solution with 0.5 - 1.5 M NaOH, continuously stir and add 0.5 - 0.7 g of succinic anhydride solution, continuously add 0.5 - 1.5 M sodium hydroxide solution during the reaction to keep the system pH between 7.0 - 7.5, the reaction time is 1.5 - 2.5 h, and the reaction temperature is 15 - 25°C; after the reaction, dialyze with a 100 kd molecular weight dialysis bag for 2 - 4 d, change the water once every 10 - 14 h, after dialysis, place the dialyzed product at -40°C for pre-freezing for 10 - 14 h, and then freeze-dry it with a -80°C freeze dryer for 20 - 28 h.

8. The preparation method of the active polypeptide repair material according to claim 7, wherein The succinic anhydride solution is prepared by dissolving succinic anhydride in acetone to form a 10% succinic anhydride acetone solution.

9. The preparation method of the active polypeptide repair material according to claim 3, wherein, The operation steps for preparing the composite nanospheres described in step (4) are as follows: Placenta polypeptide and type III collagen are fully dispersed in physiological saline at a mass ratio of 1:(4 - 6) to prepare a mixed solution with a concentration of 0.04 - 0.06%, and then the composite nanospheres are prepared in a low-temperature freeze spray dryer.

10. The preparation method of the active polypeptide repair material according to claim 3, characterized in that, The mass ratio of the dosage of type I collagen to the composite nanospheres described in step (4) is (8 - 12):1.

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