Transdermal absorption nano sheep placenta collagen, and preparation method and application thereof

Abstract

The application relates to the technical field of biomaterials, in particular to transdermal absorption nanometer sheep placental collagen as well as a preparation method and application thereof. The preparation method comprises the following steps: raw material pretreatment, decellularization treatment, de-terminus peptide, purification, collagen self-assembly, and nanometer collagen preparation; in the collagen self-assembly step, collagen protein solution, transdermal peptide and EDC are crosslinked at 4 DEG C for 24 hours, and residual EDC is dialyzed away by using a dialysis bag; the obtained transdermal peptide-collagen crosslinking product is mixed with a collagen protein solution with a weight percentage of 5%, pH is adjusted to 7 by using PBS to obtain a collagen protein mixture; the collagen protein mixture is placed at 37 DEG C for 30 minutes, the collagen protein mixture is self-assembled to form a jelly-like gel. By means of the performance of collagen protein self-assembly, after being embedded with the transdermal peptide, hydrophilic nanometer collagen protein can effectively penetrate into the skin by using high-pressure nanometer technology in the self-assembled collagen protein network.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomaterials, and particularly relates to a transdermal absorption nano sheep placenta collagen and a preparation method and application thereof. BACKGROUND

[0002] Collagen, as an important biological macromolecule, is widely present in the skin, bones, tendons and other tissues of animals. Due to its good biocompatibility, biodegradability and biological activity, it has shown a broad application prospect in many fields such as biomedical, cosmetics, food and the like. The sheep placenta, as a kind of natural biomaterial, is rich in collagen, especially type III and type IV collagen, and has high nutritional value and medicinal value.

[0003] At present, the traditional animal collagen extraction is mainly derived from the skin or tendon. The tissue cells in these parts are relatively few, and the cell impurities are easy to handle during extraction. However, the sheep placenta contains a large amount of placental cell tissues and rich cell secretions and other metabolites, which makes the traditional preparation method of sheep placenta collagen have many disadvantages. The traditional preparation process of sheep placenta collagen is relatively rough, and is easily contaminated by a large amount of tissue cell fragments and cell lysates, thereby resulting in low collagen purity, high cost and poor biological activity. In addition, the traditional animal collagen has a fibrous macromolecular structure, which cannot be effectively absorbed by the skin when applied to the surface of the skin.

[0004] Therefore, it is of great practical significance to develop a preparation method of nano sheep placenta collagen with high efficiency, low cost, high purity and high biological activity. SUMMARY

[0005] The application provides a transdermal absorption nano sheep placenta collagen and a preparation method and application thereof. By means of the self-assembly performance of collagen, the hydrophilic nano collagen can effectively penetrate into the skin by using the high-pressure nanometer technology in the self-assembled collagen network after being chimeric with a transdermal peptide.

[0006] In a first aspect, the application provides a preparation method of a transdermal absorption nano sheep placenta collagen, which adopts the following technical scheme:

[0007] The preparation method of the transdermal absorption nano sheep placenta collagen specifically comprises the following steps: raw material pretreatment, decellularization treatment, de-endothelial peptide, purification, collagen self-assembly and preparation of nano collagen.

[0008] In the step of collagen self-assembly, the collagen protein solution is crosslinked with a transdermal peptide and EDC at 4 DEG C for 24 hours to obtain a transdermal peptide-collagen protein crosslinking product, and the residual EDC is removed by using a dialysis bag.

[0009] The transdermal peptide-collagen crosslinking product is mixed with a collagen solution with a weight percentage of 5%, the pH is adjusted to 7 with PBS to obtain a collagen mixture; the collagen mixture is placed at 37℃ for 30 min, and the collagen mixture is self-assembled to form a jelly-like gel.

[0010] Optionally, the transdermal peptide is added in an amount of 0.1-0.5%.

[0011] In a specific embodiment, the transdermal peptide is added in an amount of 0.01%, 0.05%, 0.1%, 0.25%, or 0.5%, respectively.

[0012] In some specific embodiments, the transdermal peptide is added in an amount of 0.01-0.05%, 0.01-0.1%, 0.01-0.25%, 0.05-0.1%, 0.05-0.25%, 0.05-0.5%, 0.1-0.25%, 0.1-0.5%, or 0.25-0.5%, respectively.

[0013] Optionally, the weight ratio of the transdermal peptide-collagen crosslinking product to the collagen solution with a weight percentage of 5% is 1:(10-20).

[0014] In a specific embodiment, the weight ratio of the transdermal peptide-collagen crosslinking product to the collagen solution with a weight percentage of 5% is 1:1, 1:10, or 1:20, respectively.

[0015] In some specific embodiments, the weight ratio of the transdermal peptide-collagen crosslinking product to the collagen solution with a weight percentage of 5% is 1:(1-10) or 1:(10-20).

[0016] Optionally, the preparation of the nanocollagen comprises the following steps: the gel is slowly stirred using a stirrer at a stirring speed of 200-500 rpm, and after uniform dispersion without obvious particles, further nanofication is performed using a pressure nanosyringe, the extrusion pressure is 50-100 bar, and the extrusion cycle number is 1-3 times. The extruder filter membrane uses a 50 nm and 100 nm carbonate membrane to obtain a transdermal absorption nanosheep placenta collagen.

[0017] Optionally, the extrusion pressure is 70-80 bar.

[0018] In a specific embodiment, the extrusion pressure is 50, 70, 80, or 100 bar, respectively.

[0019] In some specific embodiments, the extrusion pressure is 50-70, 50-80, 70-80, 70-100, or 80-100 bar.

[0020] Optionally, the number of cycles is 2-3 cycles, respectively.

[0021] In one embodiment, the number of cycles is 1 cycle, 2 cycles, or 3 cycles, respectively.

[0022] In some embodiments, the number of cycles is 1-2 cycles or 2-3 cycles.

[0023] Optionally, the particle size of the filter membrane of the extruder is 50 nm.

[0024] Optionally, the particle size of the filter membrane of the extruder is 100 nm.

[0025] Optionally, the decellularization step is: adding sheep placenta tissue powder into PBS buffer solution containing 0.1-1% neutral protease, with pH value of 6.5-7.5; the temperature of the enzymatic reaction is 4-8℃; static enzymolysis; the time of the enzymatic reaction is 24-48h.

[0026] After the enzymatic reaction is completed, EDTA-containing PBS termination solution is added in a volume ratio of 1:1 to end the enzymolysis; the enzymolysis solution is centrifuged at a speed of 2000-3000 rpm for 20-30 min to obtain a concentrated decellularized extracellular matrix supernatant.

[0027] Optionally, the step of removing the terminal peptide is: adding the decellularized extracellular matrix supernatant into a citric acid buffer solution containing 0.05-0.1% pepsin, with pH value of 2.5; the temperature of the enzymatic reaction is 4-8℃; continuously stirring to accelerate the enzymolysis, and the time of the enzymatic reaction is 12-24h.

[0028] After the enzymatic reaction is completed, the enzymolysis solution is centrifuged at a speed of 8000-10000 rpm for 20-30 min to obtain a supernatant.

[0029] In one embodiment, a preparation method of transdermal absorption nano sheep placenta collagen, the preparation method specifically comprises the following steps:

[0030] (1) Raw material pretreatment

[0031] Fresh, postpartum sheep placenta tissue material is selected to ensure its reliable source and no disease contamination. The sheep placenta tissue material is subjected to strict quality testing, including appearance inspection, microbial testing, etc. The material is washed with pure water for 2-3 times, with each washing time of 5-10 min. to remove impurities and possible contaminants.

[0032] The tissue material is soaked in 10% saline solution for 24-72 hours to allow the tissue fluid to fully leach out and effectively remove the fishy smell of the tissue material. The tissue material is then soaked in normal saline solution for 10-15 minutes, repeated 3-5 times to fully wash out the salt in the tissue material.

[0033] The treated tissue material is placed in a freeze dryer for freeze drying. The dried sheep placenta tissue material is placed in a pulverizer for pulverization to obtain sheep placenta tissue powder.

[0034] (2) Decellularization treatment

[0035] The sheep placenta tissue powder is added to a PBS buffer solution (pH 6.5-7.5) containing 0.1-1% neutral protease. The enzymatic reaction temperature is 4-8°C. The enzymatic reaction is carried out for 24-48 hours.

[0036] After the enzymatic reaction is completed, a PBS termination solution containing EDTA is added at a volume ratio of 1:1 to terminate the enzymatic reaction, and the enzymatic solution is centrifuged to remove the detached cells.

[0037] The separated tissue cells are removed by centrifugation to obtain a thick decellularized extracellular matrix supernatant.

[0038] (3) Removal of telopeptide

[0039] To remove the telopeptide of collagen and reduce immunogenicity, the decellularized extracellular matrix supernatant obtained above is added to a citric acid buffer solution (pH 2.5) containing 0.05-0.1% pepsin. The enzymatic reaction temperature is 4-8°C. The enzymatic reaction is carried out for 12-24 hours with continuous stirring to accelerate the enzymatic reaction.

[0040] After the enzymatic reaction is completed, the enzymatic solution is centrifuged to remove the thick mucous at the bottom that has not been completely enzymatically cut.

[0041] The unenzymatically cut impurities and cell fragments are removed by centrifugation to obtain a supernatant.

[0042] (4) Purification

[0043] The supernatant obtained above is filtered using a polyacrylamide filter to further remove particulate impurities. The collagen is then precipitated using 15% sodium chloride, and the collagen precipitate is washed 3 times with 3-5% high-salt solution. The collagen precipitate is then dialyzed overnight in a 30KD dialysis bag to remove excess salt, small molecular impurities, and enzymes, etc., to obtain a collagen solution.

[0044] The polyacrylamide filter membrane has a particle size of 0.45 um.

[0045] The purity of the collagen solution is detected by a UV spectrophotometer at 230 nm and 280 nm, and the purity of the collagen is calculated. The purity of the collagen solution can reach more than 90%.

[0046] (5) Collagen self-assembly

[0047] The concentration of the collagen solution is adjusted to 1%, and the transdermal peptide is added at a weight percentage of 0.01-0.5% and mixed, and then EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) is added at a weight percentage of 1%, and cross-linked at 4°C for 24 h to obtain a transdermal peptide-collagen cross-linking product. The residual EDC is dialyzed using a 30K dialysis bag.

[0048] The transdermal peptide-collagen cross-linking product is quantified, mixed with a collagen solution at a weight percentage of 5% at a weight ratio of 1:(1-20), and the pH of the mixture is adjusted to 7 using 5xPBS to obtain a collagen mixture. The collagen mixture is placed at 37°C for 30 min, and at this time the collagen mixture will self-assemble to form a jelly-like gel.

[0049] (6) Preparation of nano-collagen

[0050] The gel is slowly stirred using a stirrer at a stirring speed of 200-500 rpm, and after uniform dispersion without obvious particles, further nanofication is performed using a pressure nanometer extruder at an extrusion pressure of 50-100 bar and an extrusion cycle number of 1-3 times. The extruder filter membrane uses a 50 nm and 100 nm carbonate membrane to obtain a transdermal absorption nano-ovine placenta collagen.

[0051] Specifically, the preparation flowchart of the nano-ovine placenta collagen is as shown in Figure 2 After the transdermal peptide and the placenta collagen protein are cross-linked, a transdermal peptide-collagen cross-linking product is formed; after mixing with the collagen solution, a jelly-like gel (hydrogel) including the transdermal peptide-collagen cross-linking product and the collagen is formed, and after treatment by the extruder filter membrane (nanofilter membrane), a transdermal absorption nano-ovine placenta collagen in the form of "transdermal peptide-collagen cross-linking product wrapped collagen" is formed.

[0052] In a second aspect, the application provides a transdermal absorption nano-ovine placenta collagen prepared by the above preparation method.

[0053] In a third aspect, the application provides a use of the above transdermal absorption nano-ovine placenta collagen in the preparation of a composition for moisturizing and / or anti-wrinkle.

[0054] Optionally, the composition is a cosmetic or a food.

[0055] Optionally, the composition is a health product.

[0056] In summary, the present application includes at least one of the following beneficial technical effects:

[0057] 1. The present application uses nanotechnology to prepare transdermal absorption nanometer sheep placenta collagen with uniform particle size and high biological activity, which significantly improves the thermal stability and transdermal absorption efficiency of placenta collagen.

[0058] 2. In the preparation method of the present application, the steps of decellularization and depeptization are separated, the yield is higher, and pure collagen can be effectively obtained, avoiding the pollution of cell fragments.

[0059] 3. The preparation method of the present application is simple, low in cost and easy to operate, and is suitable for large-scale production.

[0060] 4. The transdermal absorption nanometer sheep placenta collagen prepared by the present application has good biocompatibility, biodegradability and biological activity, and has wide application prospects in the fields of biomedicine, cosmetics and food.

[0061] 5. The present application realizes the high-value utilization of sheep placenta, reduces resource waste and environmental pollution, and has important economic and social significance. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 Circular dichroism spectrum of the transdermal absorption nanometer sheep placenta collagen prepared by the present application.

[0063] Figure 2 Preparation flow chart of the nanometer sheep placenta collagen in step (5) and step (6) of the preparation method of the present application. DETAILED DESCRIPTION

[0064] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are for the purpose of describing specific embodiments only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term belongs.

[0065] It should be noted that the terms "first", "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "multiple" is two or more.

[0066] The endpoints of the ranges and any values disclosed in this application are not limited to the precise values recited as the exact dimensions are not critical to the application. Any numeric range recited is intended to include all values from the lower value to the upper value, inclusive of both values, and to also disclose all individual values or sub-ranges stemming from or between the recited values in the range. Any reference to claim combinations in the claims or detailed description is not meant, and shall not be interpreted to imply that a combination of claims is, or must be, claimed in any particular application.

[0067] In the present application, the term "comprising" or "including" is an open term, i.e. including the stated features but not excluding others.

[0068] The present application provides a preparation method of transdermally absorbable nanometer sheep placenta collagen. The preparation method specifically comprises the following steps:

[0069] (1) Raw material pretreatment

[0070] Fresh postpartum sheep placenta tissue material is selected to ensure its reliable source and no disease pollution. The sheep placenta tissue material is subjected to strict quality testing, including appearance inspection, microbial testing, etc. The material is washed with pure water for 2-3 times, and each washing time is 5-10 min. to remove impurities and possible pollutants.

[0071] The tissue material is soaked in 10% saline for 24-72 h to make the tissue fluid fully exude and effectively remove the fishy smell of the tissue material. Then the tissue material is soaked in physiological saline for 10-15 min repeatedly for 3-5 times to fully wash out the salt in the tissue material.

[0072] The above treated tissue material is placed in a freeze dryer for freeze drying. The dried sheep placenta tissue material is placed in a pulverizer for pulverization to obtain sheep placenta tissue powder.

[0073] (2) Decellularization treatment

[0074] The sheep placenta tissue powder is added to a PBS buffer solution (pH value is 6.5-7.5) containing 0.1-1% neutral protease. The temperature of the enzymatic reaction is 4-8°C. The enzymatic reaction is static, and the time of the enzymatic reaction is 24-48 h.

[0075] After the enzymatic reaction is completed, EDTA-containing PBS termination solution is added in a volume ratio of 1:1 to end the enzymatic reaction, and the enzymatic solution is centrifuged to remove the detached cell precipitate. The centrifugal speed is 2000-3000 rpm, and the centrifugal time is 20-30 min.

[0076] Through centrifugal separation, the separated tissue cells are removed to obtain a thick decellularized extracellular matrix supernatant.

[0077] (3) Depeptidation

[0078] To remove the telopeptide of collagen and reduce immunogenicity, the decellularized extracellular matrix supernatant obtained above is added into a citric acid buffer solution (pH 2.5) containing 0.05-0.1% pepsin. The enzymatic reaction is carried out at a temperature of 4-8°C. The enzymatic reaction is accelerated by continuously stirring. The enzymatic reaction is carried out for 12-24 hours.

[0079] After the enzymatic reaction is completed, the enzymatic solution is centrifuged to remove the thick mucus that is not completely digested. The centrifugation is carried out at a speed of 8000-10000 rpm for 20-30 minutes.

[0080] By centrifugation, the impurities and cell fragments that are not digested are removed to obtain a supernatant.

[0081] (4) Purification

[0082] The supernatant obtained above is filtered by using a polyacrylamide filter to further remove particulate impurities. The collagen is precipitated by using 15% sodium chloride, and the collagen precipitate is washed three times with 3-5% high-salt solution. The collagen precipitate is then dialyzed in a 30KD dialysis bag overnight to remove excess salt, small molecular impurities, and enzymes to obtain a collagen solution.

[0083] The polyacrylamide filter has a particle size of 0.45um.

[0084] The absorbance of the collagen solution at 230nm and 280nm is measured by using a UV spectrophotometer to calculate the purity of the collagen. The purity of the collagen solution is detected to be more than 90%.

[0085] (5) Self-assembly of collagen

[0086] The concentration of the collagen solution is adjusted to 1%, and the transdermal peptide is added at a weight percentage of 0.01-0.5% and mixed. EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) is added at a weight percentage of 1%, and the crosslinking is carried out at 4°C for 24 hours to obtain a transdermal peptide-collagen crosslinking product. The residual EDC is removed by using a 30K dialysis bag.

[0087] The transdermal peptide-collagen crosslinking product is quantified, mixed with a collagen solution at a weight percentage of 5% at a weight ratio of 1:(1-20), and the pH of the mixture is adjusted to 7 by using 5xPBS to obtain a collagen mixture. The collagen mixture is placed at 37°C for 30 minutes, and the collagen mixture is self-assembled to form a jelly-like gel at this time.

[0088] (6) Preparation of nanocollagen

[0089] Slowly stir the gel using a stirrer at a stirring speed of 200-500 rpm, and after the gel is evenly dispersed and no obvious particles are present, use a pressure nano-extruder to further nanoize, at an extrusion pressure of 50-100 bar and an extrusion cycle number of 1-3. The extruder filter membrane is a 50 nm and 100 nm carbonate membrane, and the transdermal absorption nano-ovine placenta collagen is obtained.

[0090] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application and cannot be understood as limiting the present application.

[0091] Unless otherwise specified in the embodiments, the technology or conditions are carried out according to the technology or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained commercially.

[0092] The present application will be further described in detail below in combination with the embodiments and test results.

[0093] Embodiment 1

[0094] The present embodiment provides a preparation method of transdermal absorption nano-ovine placenta collagen.

[0095] The preparation method specifically includes the following steps:

[0096] (1) Raw material pretreatment

[0097] Fresh postpartum ovine placenta tissue material is selected to ensure its reliable source and no disease contamination. The ovine placenta tissue material is subjected to strict quality testing, including appearance inspection, microbial testing, etc. The material is washed with pure water for 3 times, each time for 5 min. to remove impurities and possible contaminants.

[0098] The tissue material is soaked in 10% saline for 48 h to allow the tissue fluid to fully exude and effectively remove the fishy smell of the tissue material. The tissue material is then soaked in physiological saline for 10 min, repeated for 5 times to fully wash out the salt in the tissue material.

[0099] The treated tissue material is placed in a freeze dryer for freeze drying. The dried ovine placenta tissue material is placed in a pulverizer for pulverization to obtain ovine placenta tissue powder.

[0100] (2) Decellularization treatment

[0101] The sheep placenta tissue powder was added to a PBS buffer solution (pH 6.5-7.5) containing 0.5% neutral protease. The enzymatic reaction was carried out at 4°C. The enzymatic reaction was carried out for 36 hours.

[0102] After the enzymatic reaction was completed, a PBS solution containing EDTA was added to terminate the enzymatic reaction. The enzymatic solution was centrifuged to remove the precipitated cells. The centrifugation was carried out at 2500 rpm for 25 minutes.

[0103] The separated tissue cells were removed by centrifugation to obtain a thick acellular extracellular matrix supernatant.

[0104] (3) Removal of telopeptide

[0105] To remove the telopeptide of the collagen and thus reduce the immunogenicity, the acellular extracellular matrix supernatant obtained above was added to a citric acid buffer solution (pH 2.5) containing 0.05% pepsin. The enzymatic reaction was carried out at 4°C. The enzymatic reaction was carried out for 18 hours with continuous stirring.

[0106] After the enzymatic reaction was completed, the enzymatic solution was centrifuged to remove the thick mucus that was not completely digested. The centrifugation was carried out at 10000 rpm for 30 minutes.

[0107] The un-digested impurities and cell fragments were removed by centrifugation to obtain a supernatant.

[0108] (4) Purification

[0109] The supernatant obtained above was filtered using a 0.45 um polyacrylamide filter to further remove particulate impurities. The collagen was precipitated using 15% sodium chloride, and the precipitated collagen was washed three times with 5% high-salt solution. The collagen was then dialyzed overnight in a 30KD dialysis bag to remove excess salt, small molecular impurities, and enzymes, thereby obtaining a collagen solution.

[0110] The absorbance of the collagen solution at 230 nm and 280 nm was measured using a UV spectrophotometer to calculate the purity of the collagen. The purity of the collagen solution was detected to be more than 90%.

[0111] (5) Self-assembly of collagen

[0112] A 5% by weight collagen solution was adjusted to pH 7 using 5×PBS. The collagen solution was left to stand at 37°C for 30 minutes, during which the collagen solution was self-assembled to form a jelly-like gel.

[0113] (6) Preparation of nano-collagen

[0114] The gel was stirred slowly using a stirrer at a stirring speed of 250 rpm, and after being uniformly dispersed without obvious particles, further nanization was performed using a pressure nanometer extruder at an extrusion pressure of 70 bar and 2 times of extrusion cycles. A 100 nm carbonate membrane was used as the filter membrane of the extruder to obtain the transdermal absorption nanometer sheep placenta collagen.

[0115] Example 2

[0116] This example provides a preparation method of transdermal absorption nanometer sheep placenta collagen. The difference between this example and example 1 is that a transdermal peptide is added in the collagen self-assembly step of step (5).

[0117] The preparation method specifically includes the following steps:

[0118] (1) Raw material pretreatment: according to the method of the corresponding step in example 1.

[0119] (2) Decellularization treatment: according to the method of the corresponding step in example 1.

[0120] (3) Telopeptide removal: according to the method of the corresponding step in example 1.

[0121] (4) Purification: according to the method of the corresponding step in example 1.

[0122] (5) Collagen self-assembly

[0123] The concentration of the collagen protein solution was adjusted to 1%, a transdermal peptide (such as the sequence shown in SEQ ID NO. 1) was added at a weight percentage of 0.05% and mixed, and then EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) was added at a weight percentage of 1%, and crosslinked at 4°C for 24h to obtain a transdermal peptide-collagen protein crosslinking product. The residual EDC was dialyzed using a 30K dialysis bag.

[0124] After quantifying the transdermal peptide-collagen protein crosslinking product, it was mixed with a collagen protein solution at a weight percentage of 5% at a weight ratio of 1:10, and the pH of the mixture was adjusted to 7 using 5xPBS to obtain a collagen protein mixture. The collagen protein mixture was placed at 37°C for 30 min, at which time the collagen protein mixture self-assembled to form a jelly-like gel.

[0125] (6) Preparation of nanometer collagen: according to the method of the corresponding step in example 1.

[0126] Examples 3-8

[0127] The above examples respectively provide a preparation method of transdermal absorption nanometer sheep placental collagen. The difference between the above examples and example 2 is that the control of the extrusion pressure, the cycle number or the filter membrane particle size of the extruder in the preparation step of the nanometer collagen in step (6) is different, and the specific is shown in Table 1. The rest is consistent with the steps of example 2.

[0128] Table 1 Partial parameters in the preparation step of nanometer collagen and particle size analysis results

[0129]

[0130] Test one-particle size analysis

[0131] The particle size of the transdermal absorption nanometer sheep placental collagen prepared in the above examples 2-8 and the nanometer sheep placental collagen prepared in example 1 is analyzed by using a Zetasizer Nano ZS-90 type laser particle size instrument. The analysis results are shown in Table 1.

[0132] During analysis, the sample to be tested is first diluted appropriately to ensure that it is within the measurement range, and then it is loaded into a special sample cell. After inputting the relevant parameters of the sample and the medium in the instrument software, starting the measurement program, the instrument will emit laser and analyze the fluctuation of scattered light, so as to calculate the particle size distribution of the particles. After the measurement is completed, the software provides particle size distribution statistical data, and the average value is taken through multiple measurements to ensure the accuracy of the results, and finally the data is arranged and the analysis report is output.

[0133] By comparing the results of examples 2-8, it can be known from the particle size analysis results in Table 1 that the particle size of the transdermal absorption nanometer sheep placental collagen prepared under the conditions of 2 cycles, 70-80 bar of extrusion pressure and 100 nm of extruder filter membrane is uniform, the particle size distribution is narrow, and the particle size is between 90-120 nm, and the average particle size is 105 nm.

[0134] At the same time, by comparing the particle size analysis results of example 1 and example 3, it can be known that the presence of the transdermal peptide does not affect the particle size range of the nanometer sheep placental collagen.

[0135] Examples 9-15

[0136] The above examples provide a preparation method of transdermal absorption nanometer sheep placental collagen. The difference between the above examples and example 2 is that the addition amount of the transdermal peptide in the collagen self-assembly step in step (5) or the weight ratio of the transdermal peptide-collagen protein crosslinker and the collagen protein solution with a weight percentage of 5% is different, and the specific is shown in Table 2. The rest is consistent with the steps of example 2.

[0137] Table 2 Partial parameters in the collagen self-assembly step and analysis results

[0138]

[0139] Test experiment two - transdermal absorption rate test

[0140] The transdermal absorption rate of the transdermal absorption nanometer sheep placenta collagen prepared in Example 2 and Examples 9-14 was tested by using the Franz diffusion test method. The test results are shown in Table 2.

[0141] During the test, first, the transdermal absorption nanometer sheep placenta collagen prepared in Example 2 and Examples 9-14 was diluted into a solution of 1 mg / ml with PBS, then the ex vivo depilated rat skin was placed in the donor chamber of the Franz diffusion cell, the solution to be tested was added to the donor chamber, and good contact between the sample and the membrane was ensured. The receptor chamber was filled with appropriate PBS solution as the receiving medium. The Franz diffusion cell device was placed in a 37°C constant temperature water bath, and a certain stirring speed was set to maintain the temperature and uniformity of the receiving medium. After 24 h, a certain amount of receiving medium was taken out from the receptor chamber for analysis, and the transdermal absorption of the transdermal absorption nanometer sheep placenta collagen was evaluated by determining the protein concentration.

[0142] By analyzing the transdermal absorption rate test results of Table 2, by comparing the results of Examples 1 and 2, 9-14, it can be seen that the transdermal peptide can effectively improve the transdermal absorption amount of the transdermal absorption nanometer sheep placenta collagen. In particular, when the amount of transdermal peptide added is 0.1-0.5% and the weight ratio of transdermal peptide-collagen crosslinking material to collagen protein solution with a weight percentage of 5% is 1:10, the transdermal absorption amount of the transdermal absorption nanometer sheep placenta collagen reaches more than 28 ug / ml, which is much larger than the transdermal absorption amount of the nanometer sheep placenta collagen without transdermal peptide in Example 1.

[0143] Example 15

[0144] This example provides a preparation method of transdermal absorption nanometer sheep placenta collagen. The difference between this example and Example 2 is that the amount of transdermal peptide added in the collagen self-assembly step of step (5) is 1%. The rest is consistent with the steps of Example 2.

[0145] Test experiment three - triple helix structure analysis

[0146] Circular dichroism spectrum analysis was performed on the transdermal absorption nanometer sheep placenta collagen prepared in Example 15.

[0147] The specific analysis method is as follows: the sample was dissolved in a suitable buffer solution to prepare a sample solution to be tested, and then measured on a circular dichroism spectrometer, the baseline was corrected by comparing the spectral differences between the sample and the blank sample, and then the circular dichroism data was collected in the wavelength range of 190-250 nm, so as to judge the typical triple helix mechanism of the transdermal absorption nanometer sheep placenta collagen. The test results are shown in Table 3.Figure 1 as shown.

[0148] By Figure 1 It can be seen that the transdermal absorption nano sheep placental collagen prepared in the present application has a positive absorption peak of triple helix structure at a wavelength of 220 nm. It shows that the transdermal absorption nano sheep placental collagen prepared in the present application has a good triple helix structure of collagen protein, and the triple helix structure is still stable after forming nano collagen.

[0149] Test four - cell proliferation rate test

[0150] The transdermal absorption nano sheep placental collagen prepared in Example 10 was subjected to cell proliferation rate test.

[0151] The specific analysis method is as follows: first, the fibroblast cell suspension is divided into several groups and inoculated into 96-well plates, and multiple replicates are set for each group to ensure the accuracy of the results; then, the cells are cultured to adhere to the growth, and then the sample is diluted to a concentration of 10 ug / ml, and then added to the test wells; after 24h of treatment, the cell proliferation and toxicity detection kit (CCK-8) is added according to the instructions; finally, the absorbance of each well is measured by a microplate reader to reflect the cell survival rate.

[0152] The absorbance of the experimental group was compared with the control group, the cell survival rate was calculated, and the data was analyzed by statistical software to obtain the test results. The results are shown in Table 3.

[0153] Table 3 Test results of cell proliferation rate

[0154]

[0155]

[0156] As can be seen from Table 3, after the transdermal absorption nano sheep placental collagen of the present application is co-cultured with cells, it significantly promotes cell growth; compared with commercial collagen (type I, Zhejiang Chongshan Biological Products Co., Ltd.), the activity is equivalent; compared with DMEM medium, it is significantly better than DMEM medium. At the same time, the cells grow well in the environment containing transdermal absorption nano sheep placental collagen, which shows that the transdermal absorption nano sheep placental collagen of the present application has good biocompatibility.

[0157] Test five - application

[0158] The transdermal absorption nano sheep placental collagen prepared in Example 15 was used as a cosmetic raw material to prepare an essence milk with moisturizing and anti-wrinkle functions. The weight percentage content of the transdermal absorption nano sheep placental collagen was 1%. At the same time, an essence milk without adding transdermal absorption nano sheep placental collagen was used as a comparison.

[0159] Through the trial of 26 volunteers for 4 weeks, the skin data of the volunteers before and after use were collected and analyzed by using Visia skin detector and CK skin density softness detector probe. The test results are shown in Table 4.

[0160] Table 4 Skin data detection results of volunteers before and after use

[0161]

[0162] From the test results of Table 4, it can be seen that after using the essence cream containing transdermally absorbed nanometer sheep placenta collagen, the moisture content of the skin is significantly increased, the wrinkle depth and number are significantly reduced; at the same time, the elasticity of the skin is significantly increased, and the degree of skin relaxation is significantly reduced. It shows that the essence cream containing transdermally absorbed nanometer sheep placenta collagen has good moisturizing and anti-wrinkle properties, can increase the moisture content of the skin, reduce the generation of skin wrinkles, and at the same time can increase the elasticity of the skin and reduce the skin relaxation.

[0163] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0164] Finally, it should be pointed out that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing transdermally absorbable nanoscale sheep placental collagen, characterized by, The steps of the preparation method are as follows: (1) raw material pretreatment Fresh, postpartum sheep placenta tissue materials are selected, and the materials are washed with pure water for 2-3 times, each time for 5-10 min; the tissue materials are soaked in 10% saline for 24-72 h; the tissue materials are soaked in normal saline for 10-15 min, repeated for 3-5 times; the treated tissue materials are freeze-dried; the dried sheep placenta tissue materials are crushed to obtain sheep placenta tissue powder; (2) decellularization treatment The sheep placenta tissue powder is added to a PBS buffer solution containing 0.1-1% neutral protease and having a pH value of 6.5-7.5; the enzymolysis reaction is carried out at a temperature of 4-8°C, and the enzymolysis is static; the enzymolysis reaction is carried out for 24-48 h; after the enzymolysis reaction is completed, a PBS solution containing EDTA is added in a volume ratio of 1:1 to terminate the enzymolysis, and the enzymolysis solution is centrifuged to remove the precipitated cells, at a centrifugal speed of 2000-3000 rpm for 20-30 min, to obtain a concentrated decellularized extracellular matrix supernatant; (3) removing telopeptide The decellularized extracellular matrix supernatant is added to a citric acid buffer solution containing 0.05-0.1% pepsin and having a pH value of 2.5; the enzymolysis reaction is carried out at a temperature of 4-8°C, and the enzymolysis is continuously accelerated by stirring; the enzymolysis reaction is carried out for 12-24 h; after the enzymolysis reaction is completed, the enzymolysis solution is centrifuged at a centrifugal speed of 8000-10000 rpm for 20-30 min to obtain a supernatant; (4) purification The supernatant is filtered by using a polyacrylamide filter membrane, and the filtrate is obtained; the collagen in the filtrate is precipitated by using 15% sodium chloride, and the collagen precipitate is washed with a 3-5% high-salt solution for 3 times; then the collagen precipitate is dialyzed in a 30KD dialysis bag overnight to obtain a collagen solution; (5) collagen self-assembly The collagen solution, the transdermal peptide, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are crosslinked at 4°C for 24 h to obtain a transdermal peptide-collagen crosslinking product, and the residual 1-ethyl-(3-dimethylaminopropyl) carbodiimide is removed by using a dialysis bag; the transdermal peptide-collagen crosslinking product is mixed with a collagen solution having a weight percentage of 5%, and the pH value is adjusted to 7 by using PBS to obtain a collagen mixture; the collagen mixture is placed at 37°C for 30 min, and the collagen mixture is self-assembled to form a jelly-like gel; the transdermal peptide is an amino acid sequence as shown in SEQ ID NO. 1, and the addition amount is 1%; (6) preparation of nano collagen The gel is slowly stirred by using a stirrer at a stirring speed of 200-500 rpm, and after the gel is uniformly dispersed without obvious particles, the gel is further nanoized by using a pressure nano-extruder at an extrusion pressure of 50-100 bar and for 1-3 cycles; a carbonate membrane having a particle size of 100 nm is used as an extruder filter membrane to obtain a transdermally absorbable nano sheep placenta collagen. ​ 2. The production method according to claim 1, characterized by, The weight ratio of the transdermal peptide-collagen cross-linking product and the collagen solution with a weight percentage of 5% is 1: (1-20).

3. The preparation method according to claim 1, characterized in that, The weight ratio of the transdermal peptide-collagen cross-linking product and the collagen solution with a weight percentage of 5% is 1: (10-20).

4. The preparation method according to claim 1, characterized in that, The extrusion pressure is 70-80 bar.

5. The preparation method according to claim 1, characterized in that, The number of cycles is 2-3, respectively.

6. A transdermal absorption nano sheep placental collagen prepared by the preparation method of any one of claims 1-5.

7. Use of the transdermal absorption nano sheep placental collagen of claim 6 in the preparation of a cosmetic composition for moisturizing and / or anti-wrinkle.

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