Transdermal absorption nano sheep placenta collagen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to transdermal absorption nano sheep placenta collagen as well as a preparation method and application thereof. The preparation method comprises the following steps: raw material pretreatment, decellularization treatment, telopeptide removal, purification, collagen self-assembly and nano collagen preparation. In the collagen self-assembly step, a collagen solution, transdermal peptide and EDC are cross-linked for 24 h at the temperature of 4 DEG C, and residual EDC is dialyzed away through a dialysis bag; mixing the obtained transdermal peptide-collagen cross-linked product with a collagen solution with the weight percentage of 5%, and adjusting the pH value to 7 by using PBS to obtain a collagen mixed solution; and placing at 37 DEG C for 30 minutes, so that the collagen mixed solution is self-assembled to form jelly-like gel. By means of the self-assembly performance of the collagen, the hydrophilic nano collagen can effectively permeate into the skin in a self-assembly collagen network by utilizing a high-pressure nanocrystallization technology after the collagen is embedded with the transdermal peptide.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to a transdermal absorbable nano sheep placenta collagen and a preparation method and application thereof. Background Art

[0002] As an important biological macromolecule, collagen is widely present in animal skin, bones, tendons and other tissues. Due to its good biocompatibility, biodegradability and biological activity, it has shown broad application prospects in many fields such as biomedicine, cosmetics, and food. As a natural biomaterial, sheep placenta is rich in collagen, especially type III and type IV collagen, and has extremely high nutritional value and medicinal value.

[0003] At present, traditional animal collagen extraction mainly comes from the skin or tendons. These parts have relatively few tissue cells, and cell impurities are easier to handle during extraction. However, sheep placenta contains a large amount of placental cell tissue and rich metabolic substances such as cell secretions, which makes the traditional sheep placental collagen preparation method have many disadvantages. The preparation process of traditional sheep placental collagen is relatively extensive and easily contaminated by a large amount of tissue cell fragments and cell lysis products, which leads to low collagen purity, high cost, and poor biological activity. In addition, traditional animal collagen has a fibrous macromolecular structure and cannot be effectively absorbed by the skin when applied to the skin surface.

[0004] In view of this, it is of vital practical significance to develop a method for preparing nano sheep placenta collagen with high efficiency, low cost, high purity and high biological activity. Summary of the invention

[0005] The present application provides a transdermal absorbable nano sheep placenta collagen and its preparation method and application. The present application makes use of the self-assembly property of collagen, and after being chimeric with transdermal peptides, in the self-assembled collagen network, high-pressure nano-crystallization technology is used to enable the hydrophilic nano collagen to effectively penetrate into the skin.

[0006] In the first aspect, the present application provides a method for preparing transdermal absorption nano sheep placenta collagen, using the following technical scheme:

[0007] A method for preparing transdermal absorption nano sheep placenta collagen, the method specifically comprising the following steps: raw material pretreatment, decellularization, atelo-peptide removal, purification, collagen self-assembly, and nano collagen preparation;

[0008] In the step of self-assembly of collagen, the collagen solution is cross-linked with the transdermal peptide and EDC at 4°C for 24 hours to obtain a transdermal peptide-collagen cross-linked product, and the residual EDC is dialyzed away using a dialysis bag;

[0009] The transdermal peptide-collagen crosslinked product was mixed with a 5% by weight collagen solution, and the pH was adjusted to 7 with PBS to obtain a collagen mixed solution; the mixed solution was placed at 37° C. for 30 minutes, and the collagen mixed solution was self-assembled to form a jelly-like gel.

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

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

[0012] In some specific embodiments, the amount of the transdermal peptide added is 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%, and 0.25-0.5%.

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

[0014] In a specific embodiment, the weight ratios of the transdermal peptide-collagen crosslinked product and the 5% by weight collagen solution are 1:1, 1:10, and 1:20, respectively.

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

[0016] Optionally, the preparation step of the nano collagen is: using a stirrer to slowly stir the gel at a stirring speed of 200-500rpm, and after being evenly dispersed without obvious particles, using a pressure nano extruder for further nano-sizing, the extrusion pressure is 50-100bar, and the number of extrusion cycles is 1-3 times. The extruder filter uses 50nm and 100nm carbonate membranes to obtain transdermal absorption nano sheep placenta collagen.

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

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

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

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

[0021] In a specific embodiment, the number of cycles is 1 time, 2 times, and 3 times respectively.

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

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

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

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

[0026] After the enzymatic hydrolysis reaction is completed, PBS stop solution containing EDTA is added at a volume ratio of 1:1 to terminate the enzymatic hydrolysis; the enzymatic hydrolyzate is centrifuged at a centrifugal speed of 2000-3000 rpm for 20-30 minutes to obtain a thick decellularized extracellular matrix supernatant.

[0027] Optionally, the step of removing the atelo-peptide is: adding the decellularized extracellular matrix supernatant to a citric acid buffer solution containing 0.05-0.1% pepsin and a pH value of 2.5; the temperature of the enzymatic reaction is 4-8°C; continuous and sufficient stirring accelerates the enzymatic reaction, and the enzymatic reaction time is 12-24h;

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

[0029] In a specific embodiment, a method for preparing transdermal absorption nano sheep placenta collagen comprises the following steps:

[0030] (1) Raw material pretreatment

[0031] Select fresh, post-delivery sheep placenta tissue materials to ensure that they are from reliable sources and free of disease contamination. Strict quality inspections are conducted on sheep placenta tissue materials, including appearance inspections, microbial inspections, etc. Rinse with pure water 2-3 times, each time for 5-10 minutes, to remove impurities and possible contaminants.

[0032] Soak the tissue material in 10% saline for 24-72 hours to allow the tissue fluid to fully seep out and effectively remove the fishy smell of the tissue material. Then soak it in physiological saline for 10-15 minutes, repeat 3-5 times, and fully wash out the salt in the tissue material.

[0033] The processed tissue material is placed in a freeze dryer for freeze drying. The dried sheep placenta tissue material is placed in a grinder for grinding to obtain sheep placenta tissue powder.

[0034] (2) Decellularization

[0035] The sheep placenta tissue powder is added into 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 enzymatic reaction time is 24-48 hours.

[0036] After the enzymatic hydrolysis reaction is completed, add PBS stop solution containing EDTA at a volume ratio of 1:1 to terminate the enzymatic hydrolysis, and centrifuge the enzymatic hydrolysis solution to remove the cell precipitate. The centrifugal speed is 2000-3000rpm, and the centrifugal time is 20-30min.

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

[0038] (3) De-telopeptide

[0039] In order to remove the telopeptide of collagen and thus reduce immunogenicity, the decellularized extracellular matrix supernatant obtained above was added to a citric acid buffer solution (pH 2.5) containing 0.05-0.1% pepsin. The temperature of the enzymatic reaction was 4-8° C. The enzymatic reaction was accelerated by continuous and sufficient stirring, and the enzymatic reaction time was 12-24 hours.

[0040] After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is centrifuged to remove the thick mucus at the bottom that has not been completely digested. The centrifugal speed is 8000-10000rpm and the centrifugal time is 20-30min.

[0041] Impurities and cell debris that have not been hydrolyzed by the enzyme are removed by centrifugation to obtain the supernatant.

[0042] (4) Purification

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

[0044] Among them, the particle size of the polyacrylamide filter membrane is 0.45um.

[0045] The absorbance of the collagen solution at 230nm and 280nm is measured by an ultraviolet spectrophotometer to calculate the purity of the collagen. The purity of the collagen solution can reach more than 90%.

[0046] (5) Collagen self-assembly

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

[0048] After the transdermal peptide-collagen crosslink is quantified, it is mixed with a 5% collagen solution at a weight ratio of 1: (1-20), and the pH of the mixture is adjusted to 7 using 5×PBS to obtain a collagen mixture. The mixture is placed at 37°C for 30 minutes, at which time the collagen mixture will self-assemble to form a jelly-like gel.

[0049] (6) Preparation of Nanocollagen

[0050] The gel was stirred slowly with a stirrer at a speed of 200-500 rpm. After being evenly dispersed without obvious particles, a pressure nano extruder was used for further nano-sizing at an extrusion pressure of 50-100 bar and 1-3 extrusion cycles. The extruder filter membrane used 50nm and 100nm carbonate membranes to obtain transdermal absorption of nano sheep placenta collagen.

[0051] Specifically, the preparation flow chart of nano sheep placenta collagen is as follows: Figure 2 As shown. After the transdermal peptide and placental collagen are cross-linked, a transdermal peptide-collagen cross-link is formed; after mixing with the collagen solution, a jelly-like gel (hydrogel) including the transdermal peptide-collagen cross-link and collagen is formed, and after being processed by the extruder filter membrane (nanofiltration membrane), a transdermal absorption nano sheep placental collagen of the type of "transdermal peptide-collagen cross-link wrapped collagen" is formed.

[0052] In a second aspect, the present application provides a transdermal absorbable nano sheep placenta collagen prepared by the above-mentioned preparation method.

[0053] In a third aspect, the present application provides a use of the above-mentioned transdermal absorption nano sheep 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. This application adopts nano-processing technology to prepare transdermal absorbable nano 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 decellularization and atelopeptification steps are prepared separately, which has a higher yield and can effectively obtain pure collagen and avoid contamination by cell debris.

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

[0060] 4. The transdermal absorbable nano sheep placenta collagen prepared in this application has good biocompatibility, biodegradability and biological activity, and has broad application prospects in the fields of biomedicine, cosmetics and food.

[0061] 5. This 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 THE DRAWINGS

[0062] Figure 1 Circular dichroism analysis of the transdermal absorption nano sheep placenta collagen prepared for this application.

[0063] Figure 2 This is a flow chart for the preparation of nano sheep placenta collagen in steps (5) and (6) of the preparation method of this application. DETAILED DESCRIPTION

[0064] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0065] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0066] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0067] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0068] The present application provides a method for preparing transdermal absorption nano sheep placenta collagen. The preparation method specifically comprises the following steps:

[0069] (1) Raw material pretreatment

[0070] Select fresh, post-delivery sheep placenta tissue materials to ensure that they are from reliable sources and free of disease contamination. Strict quality inspections are conducted on sheep placenta tissue materials, including appearance inspections, microbial inspections, etc. Rinse with pure water 2-3 times, each time for 5-10 minutes, to remove impurities and possible contaminants.

[0071] Soak the tissue material in 10% saline for 24-72 hours to allow the tissue fluid to fully seep out and effectively remove the fishy smell of the tissue material. Then soak it in physiological saline for 10-15 minutes, repeat 3-5 times, and fully wash out the salt in the tissue material.

[0072] The processed tissue material is placed in a freeze dryer for freeze drying. The dried sheep placenta tissue material is placed in a grinder for grinding to obtain sheep placenta tissue powder.

[0073] (2) Decellularization

[0074] The sheep placenta tissue powder is added into 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 enzymatic reaction time is 24-48 hours.

[0075] After the enzymatic hydrolysis reaction is completed, add PBS stop solution containing EDTA at a volume ratio of 1:1 to terminate the enzymatic hydrolysis, and centrifuge the enzymatic hydrolysis solution to remove the cell precipitate. The centrifugal speed is 2000-3000rpm, and the centrifugal time is 20-30min.

[0076] The separated tissue cells were removed by centrifugation to obtain a thick decellularized extracellular matrix supernatant.

[0077] (3) De-telopeptide

[0078] In order to remove the telopeptide of collagen and thus reduce immunogenicity, the decellularized extracellular matrix supernatant obtained above was added to a citric acid buffer solution (pH 2.5) containing 0.05-0.1% pepsin. The temperature of the enzymatic reaction was 4-8° C. The enzymatic reaction was accelerated by continuous and sufficient stirring, and the enzymatic reaction time was 12-24 hours.

[0079] After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is centrifuged to remove the thick mucus at the bottom that has not been completely digested. The centrifugal speed is 8000-10000rpm and the centrifugal time is 20-30min.

[0080] Impurities and cell debris that have not been hydrolyzed by the enzyme are removed by centrifugation to obtain the supernatant.

[0081] (4) Purification

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

[0083] Among them, the particle size of the polyacrylamide filter membrane is 0.45um.

[0084] The absorbance of the collagen solution at 230nm and 280nm is measured by an ultraviolet spectrophotometer to calculate the purity of the collagen. The purity of the collagen solution can reach more than 90%.

[0085] (5) Collagen self-assembly

[0086] The concentration of the collagen solution was adjusted to 1%, and transdermal peptide was added at a weight percentage of 0.01-0.5% and mixed, and EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) was added at a weight percentage of 1%, and cross-linked at 4°C for 24 hours to obtain transdermal peptide-collagen crosslinks. The residual EDC was dialyzed away using a 30K dialysis bag.

[0087] After the transdermal peptide-collagen crosslink is quantified, it is mixed with a 5% collagen solution at a weight ratio of 1: (1-20), and the pH of the mixture is adjusted to 7 using 5×PBS to obtain a collagen mixture. The mixture is placed at 37°C for 30 minutes, at which time the collagen mixture will self-assemble to form a jelly-like gel.

[0088] (6) Preparation of Nanocollagen

[0089] The gel was stirred slowly with a stirrer at a speed of 200-500 rpm. After being evenly dispersed without obvious particles, a pressure nano extruder was used for further nano-sizing at an extrusion pressure of 50-100 bar and 1-3 extrusion cycles. The extruder filter membrane used 50nm and 100nm carbonate membranes to obtain transdermal absorption of nano sheep placenta collagen.

[0090] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme 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 making creative work belong to 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 interpreted as limiting the present application.

[0091] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0092] The present application is further described in detail below in conjunction with the embodiments and test results.

[0093] Example 1

[0094] This embodiment provides a method for preparing transdermal absorbable nano sheep placenta collagen.

[0095] The above preparation method specifically comprises the following steps:

[0096] (1) Raw material pretreatment

[0097] Select fresh, post-delivery sheep placenta tissue materials to ensure that they are from reliable sources and free of disease contamination. Strict quality inspections are conducted on sheep placenta tissue materials, including appearance inspections, microbial inspections, etc. Rinse with pure water three times, each time for 5 minutes, to remove impurities and possible contaminants.

[0098] Soak the tissue material in 10% saline for 48 hours to allow the tissue fluid to fully seep out and effectively remove the fishy smell of the tissue material. Then soak it in physiological saline for 10 minutes, repeat 5 times, and fully wash out the salt in the tissue material.

[0099] The processed tissue material is placed in a freeze dryer for freeze drying. The dried sheep placenta tissue material is placed in a grinder for grinding to obtain sheep placenta tissue powder.

[0100] (2) Decellularization

[0101] The sheep placenta tissue powder was added into a PBS buffer solution (pH 6.5-7.5) containing 0.5% neutral protease. The temperature of the enzymatic reaction was 4° C. The enzymatic reaction was static and the enzymatic reaction time was 36 hours.

[0102] After the enzymatic hydrolysis reaction was completed, PBS stop solution containing EDTA was added at a volume ratio of 1:1 to terminate the enzymatic hydrolysis, and the enzymatic hydrolysis solution was centrifuged to remove the cell precipitate. The centrifugal speed was 2500 rpm and the centrifugal time was 25 min.

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

[0104] (3) De-telopeptide

[0105] In order to remove the telopeptide of collagen and thus reduce immunogenicity, the decellularized extracellular matrix supernatant obtained above was added to a citric acid buffer solution (pH 2.5) containing 0.05% pepsin. The temperature of the enzymatic reaction was 4° C. The enzymatic reaction was accelerated by continuous and sufficient stirring, and the enzymatic reaction time was 18 h.

[0106] After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is centrifuged to remove the thick mucus at the bottom that has not been completely digested by the enzyme. The centrifugal speed is 10000 rpm and the centrifugal time is 30 minutes.

[0107] Impurities and cell debris that have not been hydrolyzed by the enzyme are removed by centrifugation to obtain the supernatant.

[0108] (4) Purification

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

[0110] The absorbance of the collagen solution at 230nm and 280nm is measured by an ultraviolet spectrophotometer to calculate the purity of the collagen. The purity of the collagen solution can reach more than 90%.

[0111] (5) Collagen self-assembly

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

[0113] (6) Preparation of Nanocollagen

[0114] The gel was slowly stirred with a stirrer at a speed of 250 rpm. After being evenly dispersed without obvious particles, a pressure nano extruder was used for further nano-sizing. The extrusion pressure was 70 bar and the number of extrusion cycles was 2. The extruder filter membrane used a 100 nm carbonate membrane to obtain transdermal absorption nano sheep placenta collagen.

[0115] Example 2

[0116] This embodiment provides a method for preparing transdermal absorbable nano sheep placenta collagen. The difference between this embodiment and embodiment 1 is that a transdermal peptide is added in step (5) of the collagen self-assembly step.

[0117] The above preparation method specifically comprises the following steps:

[0118] (1) Raw material pretreatment: proceed according to the corresponding steps in Example 1.

[0119] (2) Decellularization: Follow the corresponding steps in Example 1.

[0120] (3) De-telopeptide: proceed according to the corresponding steps in Example 1.

[0121] (4) Purification: Purify according to the corresponding steps in Example 1.

[0122] (5) Collagen self-assembly

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

[0124] After the transdermal peptide-collagen crosslink is quantified, it is mixed with a 5% collagen solution at a weight ratio of 1:10, and the pH of the mixture is adjusted to 7 using 5×PBS to obtain a collagen mixture. The mixture is placed at 37°C for 30 minutes, at which time the collagen mixture will self-assemble to form a jelly-like gel.

[0125] (6) Preparation of nano-collagen: proceed according to the corresponding steps in Example 1.

[0126] Embodiment 3-8

[0127] The above embodiments respectively provide a method for preparing transdermal absorption of nano sheep placenta collagen. The difference between the above embodiments and embodiment 2 is that: in step (6) of preparing nano collagen, the extrusion pressure, the number of cycles or the particle size of the extruder filter membrane are controlled, as shown in Table 1. The rest are consistent with the steps of embodiment 2.

[0128] Table 1 Some parameters in the preparation steps of nano-collagen and particle size analysis results

[0129]

[0130] Test 1 - Particle Size Analysis

[0131] The particle sizes of the transdermal absorbable nano sheep placenta collagen prepared in Examples 2-8 and the nano sheep placenta collagen prepared in Example 1 were analyzed using a Zetasizer Nano ZS-90 laser particle size analyzer. 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 loaded into a dedicated sample pool. After entering the relevant parameters of the sample and medium in the instrument software, the measurement program is started. The instrument will emit a laser and analyze the fluctuations of the scattered light to calculate the particle size distribution. After the measurement is completed, the software provides particle size distribution statistics, takes the average value through multiple measurements to ensure the accuracy of the results, and finally organizes the data and outputs an analysis report.

[0133] The particle size analysis results in Table 1 are analyzed. By comparing the results of Examples 2-8, it can be seen that the particle size uniformity of the transdermal absorption nano sheep placenta collagen prepared under the conditions of 2 cycles, 70-80 bar extrusion pressure, and 100 nm extruder filter membrane is high, the particle size distribution is narrow, 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 seen that the presence of transdermal peptide does not affect the particle size range of nano sheep placenta collagen.

[0135] Examples 9-15

[0136] The above embodiment provides a method for preparing transdermal absorption nano sheep placenta collagen. The difference between the above embodiment and embodiment 2 is that the amount of transdermal peptide added in the collagen self-assembly step (5) or the weight ratio of the transdermal peptide-collagen crosslinked product mixed with the collagen solution with a weight percentage of 5% is specifically shown in Table 2. The rest are consistent with the steps of embodiment 2.

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

[0138]

[0139] Test 2-Transdermal absorption rate test

[0140] The Franz diffusion test method was used to test the transdermal absorption rate of the transdermal absorbable nano sheep placenta collagen prepared in Example 2 and Examples 9-14. The test results are shown in Table 2.

[0141] During the test, the transdermal absorption nano sheep placenta collagen prepared in Example 2 and Examples 9-14 was first diluted with PBS to a solution of 1 mg / ml, and then the depilated rat skin was placed in the donor chamber of the Franz diffusion cell, and the solution to be tested was added to the donor chamber to ensure good contact between the sample and the membrane. The receptor chamber was filled with a suitable PBS solution as a 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 hours, a certain amount of receiving medium was taken out from the receptor chamber for analysis, and the transdermal absorption of the transdermal absorption nano sheep placenta collagen was evaluated by measuring the protein concentration.

[0142] Analyzing the transdermal absorption rate test results in Table 2, by comparing the results of Examples 1 and 2, 9-14, it can be seen that the transdermal peptide can effectively increase the transdermal absorption of the transdermal absorption nano sheep placenta collagen. In particular, when the amount of transdermal peptide added is 0.1-0.5% and the weight ratio of the transdermal peptide-collagen cross-linked product to the collagen solution with a weight percentage of 5% is 1:10, the transdermal absorption of the transdermal absorption nano sheep placenta collagen reaches more than 28ug / ml, which is much greater than the transdermal absorption of the nano sheep placenta collagen that does not contain the transdermal peptide in Example 1.

[0143] Embodiment 15

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

[0145] Detection test Triple-triple helix structure analysis

[0146] The transdermal absorbable nano sheep placenta collagen prepared in Example 15 was subjected to circular dichroism analysis.

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

[0148] Depend on Figure 1 It can be seen that the transdermal absorption nano sheep placenta collagen prepared in the present application has a positive absorption peak of a triple helix structure at a wavelength of 220nm, indicating that the transdermal absorption nano sheep placenta collagen prepared in the present application has a good collagen triple helix structure, and the triple helix structure remains stable after forming nano collagen.

[0149] Test 4 - Cell proliferation rate test

[0150] The transdermal absorbable nano sheep placenta collagen prepared in Example 10 was tested for cell proliferation rate.

[0151] The specific analysis method is as follows: First, the fibroblast suspension is divided into several groups and inoculated into a 96-well plate, with multiple replicate wells in each group to ensure the accuracy of the results; then, the cells are cultured until they grow adherently, and then the samples are diluted to a concentration of 10ug / ml and added to the wells to be tested; after 24 hours of treatment, the cell proliferation and toxicity detection kit (CCK-8) is used to add CCK-8 solution according to the instructions; finally, the absorbance of each well is measured by an ELISA instrument to reflect the cell survival rate.

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

[0153] Table 3 Cell proliferation rate test results

[0154]

[0155]

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

[0157] Test 5 - Application

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

[0159] Through a trial of 26 volunteers for 4 consecutive weeks, the Visia skin detector and CK skin density and softness detection probe were used to collect and analyze the skin data of the volunteers before and after use. The test results are shown in Table 4.

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

[0161]

[0162] From the test results in Table 4, it can be seen that after using the essence milk containing transdermal absorption of nano sheep placenta collagen, the moisture content of the skin is significantly increased, and the depth and number of wrinkles are significantly reduced; at the same time, the elasticity of the skin is significantly increased, and the degree of skin sagging is significantly reduced. This shows that the essence milk containing transdermal absorption of nano 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 skin sagging.

[0163] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing transdermal absorption nano sheep placenta collagen, characterized in that: The preparation method specifically comprises the following steps: raw material pretreatment, decellularization, atelo-peptide removal, purification, collagen self-assembly, and preparation of nano-collagen; In the step of self-assembly of collagen, the collagen solution is cross-linked with the transdermal peptide and EDC at 4°C for 24 hours to obtain a transdermal peptide-collagen cross-linked product, and the residual EDC is dialyzed away using a dialysis bag; The transdermal peptide-collagen crosslinked product was mixed with a 5% by weight collagen solution, and the pH was adjusted to 7 with PBS to obtain a collagen mixed solution; the mixed solution was placed at 37° C. for 30 minutes, and the collagen mixed solution was self-assembled to form a jelly-like gel.

2. The preparation method according to claim 1, characterized in that: In the collagen self-assembly step, the amount of the transdermal peptide added is 0.01-0.5%; Optionally, the amount of the transdermal peptide added is 0.1-0.5%.

3. The preparation method according to claim 1, characterized in that: The weight ratio of the transdermal peptide-collagen crosslinked product to the 5% by weight collagen solution is 1:(1-20); Optionally, the weight ratio of the transdermal peptide-collagen crosslinked product to the 5% by weight collagen solution is 1:(10-20).

4. The preparation method according to claim 1, characterized in that: The preparation steps of the nano collagen are as follows: using a stirrer to slowly stir the gel at a stirring speed of 200-500rpm, and after being evenly dispersed without obvious particles, using a pressure nano extruder for further nano-sizing, the extrusion pressure is 50-100bar, and the number of extrusion cycles is 1-3 times; the extruder filter membrane uses 50nm and 100nm carbonate membranes to obtain transdermal absorption nano sheep placenta collagen.

5. The preparation method according to claim 1, characterized in that: The extrusion pressure is 70-80 bar; Optionally, the number of cycles is 2-3 times respectively; Optionally, the particle size of the extruder filter membrane is 100 nm.

6. The preparation method according to claim 1, characterized in that: The decellularization treatment steps are: adding sheep placenta tissue powder into a PBS buffer solution containing 0.1-1% neutral protease and a pH value of 6.5-7.5; the temperature of the enzymatic reaction is 4-8°C; static enzymatic reaction; the enzymatic reaction time is 24-48h; After the enzymatic hydrolysis reaction is completed, PBS stop solution containing EDTA is added at a volume ratio of 1:1 to terminate the enzymatic hydrolysis; the enzymatic hydrolyzate is centrifuged at a centrifugal speed of 2000-3000 rpm for 20-30 minutes to obtain a thick decellularized extracellular matrix supernatant.

7. The preparation method according to claim 6, characterized in that: The step of removing the atelo-peptide is as follows: adding the decellularized extracellular matrix supernatant to a citric acid buffer solution containing 0.05-0.1% pepsin and a pH value of 2.5; the temperature of the enzymatic hydrolysis reaction is 4-8°C; continuous and sufficient stirring accelerates the enzymatic hydrolysis, and the enzymatic hydrolysis reaction time is 12-24 hours; After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolyzate is centrifuged at a speed of 8000-10000 rpm for 20-30 min to obtain a supernatant.

8. A transdermal absorbable nano sheep placenta collagen prepared by the preparation method described in any one of claims 1 to 7.

9. Use of the transdermal absorbable nano sheep placenta collagen according to claim 8 in preparing a composition for moisturizing and / or anti-wrinkle.

10. The use according to claim 9, characterized in that: The composition is a cosmetic or a food; Optionally, the composition is a health product.

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