Hydrolyzed extracellular matrix as well as preparation method and application thereof

Abstract

The invention provides a hydrolyzed extracellular matrix and a preparation method and application thereof, and relates to the technical field of biological materials.The hydrolyzed extracellular matrix prepared through the method comprises hydrolyzed collagen, hydrolyzed elastin, hydrolyzed fibronectin and hydrolyzed laminin, in the hydrolyzed extracellular matrix, the number of polypeptide molecules with the molecular weight of 500-3000 Daltons accounts for not less than 96%, a good transdermal absorption effect can be achieved, and the hydrolyzed extracellular matrix has a good effect of promoting cell growth and proliferation and also has good functions of resisting wrinkles and enhancing skin elasticity.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a hydrolyzed extracellular matrix and a preparation method and application thereof. Background Art

[0002] The extracellular matrix is ​​a complex biological network structure composed of a variety of substances secreted by cells, mainly including collagen, elastin, proteoglycans, glycosaminoglycans and other bioactive substances. These substances regulate cell adhesion, migration, growth, proliferation, differentiation and functional expression through interaction with cells, thereby achieving tissue stability and balance. Therefore, the extracellular matrix plays an important role in promoting tissue repair. Studies have shown that the interaction between protein components in the extracellular matrix and cells is mainly achieved through the specific binding of specific amino acid sequence sites with integrins on the cell surface, such as the GFOGER sequence of collagen, the GRKRK sequence of elastin, the RGDS sequence of fibronectin, and the IKVAV sequence of laminin, which are typical sites for binding to integrins. Retaining amino acid sequences in the cell matrix that can interact with cells is of great significance for regulating cells.

[0003] As people's skin increases with age and ultraviolet radiation, it is a variety of problems and aging phenomena. This is essentially the degradation and loss of the extracellular matrix in the skin, which also loses the regulation of cells. Skin aging can be effectively slowed down by supplementing the main components of the extracellular matrix, and the healthy state of the skin can be reshaped. However, conventional extracellular matrix materials are insoluble substances obtained after tissue decellularization, and cannot enter the skin in a non-invasive manner. Based on this, there is currently a technology that processes the decellularized matrix into a soluble state, such as patent CN108812639A provides an extracellular matrix freeze-dried powder, which can be dissolved in water to form a solution, but the extracellular matrix components contained therein are still macromolecular states with a molecular weight of thousands to tens of thousands, and such a large molecular weight makes its transdermal absorption effect not good. On the other hand, the existing hydrolyzed collagen, although the molecular weight can reach the degree of transdermal absorption, does not substantially contain other hydrolyzed extracellular matrix proteins, and the method for preparing hydrolyzed collagen can not well retain the sites that can interact with cells, so that its biological activity is greatly reduced. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a hydrolyzed extracellular matrix and a preparation method and application thereof.

[0005] In one aspect, the present invention provides a method for preparing a hydrolyzed extracellular matrix.

[0006] Specifically, the preparation method comprises the following steps:

[0007] (1) taking peripheral tissue of a mammal and removing fat and adventitia tissue;

[0008] (2) cutting the tissue obtained in step (1) into segments, soaking them in ethyl acetate and shaking them;

[0009] (3) immersing the tissue fragments treated in step (2) in a peracetic acid solution for shaking;

[0010] (4) crushing the tissue fragments processed in step (3);

[0011] (5) treating the sample obtained in step (4) with a sodium periodate solution, wherein the concentration of the sodium periodate solution is 10-50 mmol / L;

[0012] (6) treating the sample treated in step (5) with a potassium iodide solution having a pH of 4.0-6.0, wherein the concentration of the potassium iodide solution is 0.5-2 mol / L;

[0013] (7) adding the sample treated in step (6) to a mixed solution containing dithiothreitol and urea; the concentration of dithiothreitol in the mixed solution is 2-20 mmol / L, and the concentration of urea is 4-10 mol / L;

[0014] (8) dialyzing the sample obtained in step (7) using purified water at a certain temperature to remove dithiothreitol and urea;

[0015] (9) adjusting the pH of the sample obtained in step (8) to 3.0-4.0, and adding pepsin for treatment;

[0016] (10) adjusting the pH of the sample obtained in step (9) to 7.0-9.0 to precipitate flocs, and separating the precipitate and the solution;

[0017] (11) heating the precipitate obtained in step (10) to obtain a solution;

[0018] (12) adding trypsin to the solution obtained in step (11);

[0019] (13) The solution separated in step (10) and the solution obtained in step (12) are mixed, the pH is adjusted to 6.0-8.0, and the mixture is filtered through a filter membrane.

[0020] More specifically, in step (3), the mass fraction of the peracetic acid solution is 0.1%-0.2%, and the shaking time is 1-2h.

[0021] More specifically, in step (5), the sodium periodate solution treatment time is 0.5-2h.

[0022] More specifically, in step (6), the potassium iodide solution treatment time is 2-4 hours.

[0023] More specifically, in step (7), the mixed solution is treated for 2-8 hours at a temperature of 4-20°C.

[0024] More specifically, in step (9), the pepsin concentration is 0.5-2 mg / mL, the treatment temperature is 25-35° C., and the treatment time is 6-24 h.

[0025] More specifically, in step (12), the trypsin concentration is 0.5-2 mg / mL, the treatment temperature is 30-37° C., and the treatment time is 12-24 h.

[0026] More specifically, the filtering conditions in step (13) are: filtering with 1000 mesh filter cloth, 1 μm filter membrane and 0.22 μm filter membrane in sequence.

[0027] More specifically, step (13) further includes a freeze-drying step, and the freeze-drying step has the following procedures: -55°C 4h, -50°C 2h, -40°C 2h, -20°C 2h, 0°C 2h, 10°C 4h, 20°C 4h, 30°C 24h.

[0028] In certain specific embodiments of the present invention, the preparation method comprises the following steps:

[0029] (1) Take the surrounding tissue of the mammal, remove the fat and adventitial tissue by physical methods, and clean it;

[0030] (2) cutting the tissue obtained in step (1) into segments, immersing them in ethyl acetate and shaking them for 2-6 hours, and then washing them with purified water;

[0031] (3) soaking the tissue fragments treated in step (2) in a peracetic acid solution for shaking, wherein the mass fraction of the peracetic acid solution is 0.1%-0.2%, the shaking time is 1-2 hours, and then washing with purified water;

[0032] (4) crushing the tissue fragments processed in step (3) using a crusher or a homogenizer;

[0033] (5) treating the sample obtained in step (4) with a sodium periodate solution having a concentration of 10-50 mmol / L for a treatment time of 0.5-2 h, and then washing with purified water;

[0034] (6) treating the sample treated in step (5) with a potassium iodide solution having a pH of 4.0-6.0, wherein the concentration of the potassium iodide solution is 0.5-2 mol / L, the treatment time is 2-4 h, and then washing with purified water;

[0035] (7) adding the sample treated in step (6) to a mixed solution containing dithiothreitol and urea, stirring evenly, and storing at 4-20° C. for 2-8 hours; the concentration of dithiothreitol in the mixed solution is 2-20 mmol / L, and the concentration of urea is 4-10 mol / L;

[0036] (8) dialyzing the sample obtained in step (7) with purified water at 4-20° C. to remove dithiothreitol and urea;

[0037] (9) adjusting the pH of the sample obtained in step (8) to 3.0-4.0, adding pepsin for treatment, the pepsin concentration is 0.5-2 mg / mL, the treatment temperature is 25-35° C., and the treatment time is 6-24 h;

[0038] (10) adjusting the pH of the sample obtained in step (9) to 7.0-9.0 to precipitate flocs, and separating the precipitate and the solution;

[0039] (11) heating the precipitate obtained in step (10) to 40-45° C. and maintaining for 1-2 h to obtain a solution;

[0040] (12) adding trypsin to the solution obtained in step (11), wherein the trypsin concentration is 0.5-2 mg / mL, the treatment temperature is 30-37° C., and the treatment time is 12-24 h;

[0041] (13) mixing the solution separated in step (10) and the solution obtained in step (12), adjusting the pH to 6.0-8.0, and filtering through a 1000 mesh filter cloth, a 1 μm filter membrane, and a 0.22 μm filter membrane in sequence;

[0042] (14) The solution obtained in step (13) is freeze-dried, and the freeze-drying procedure is: -55°C for 4 h, -50°C for 2 h, -40°C for 2 h, -20°C for 2 h, 0°C for 2 h, 10°C for 4 h, 20°C for 4 h, and 30°C for 24 h.

[0043] In another aspect, the present invention provides a hydrolyzed extracellular matrix prepared according to the above method.

[0044] Specifically, the hydrolyzed extracellular matrix includes: hydrolyzed collagen, hydrolyzed elastin, hydrolyzed fibronectin, and hydrolyzed laminin.

[0045] Specifically, the number of polypeptide molecules with a molecular weight of 500-3000 Daltons in the hydrolyzed extracellular matrix accounts for no less than 96%.

[0046] More specifically, the hydrolyzed collagen contains peptide segments whose amino acid sequences are GFOGER, GLOGER, GASGER, GLPGER, and GIPGEK, respectively.

[0047] More specifically, the hydrolyzed elastin contains peptide segments with amino acid sequences of AKPGVGGL and GRKRK.

[0048] The hydrolyzed fibronectin comprises peptide segments with amino acid sequences of RGDS and REDV.

[0049] The hydrolyzed laminin contains peptide segments with amino acid sequences of IKVAV, HQNMGSVNVSV, and SIKIRGTY.

[0050] In another aspect, the present invention provides the use of the hydrolyzed extracellular matrix in the preparation of products for promoting cell growth and proliferation, anti-wrinkle and enhancing skin elasticity.

[0051] Compared with the prior art, the present invention has the following advantages:

[0052] (1) The hydrolyzed extracellular matrix provided by the present invention has a suitable molecular weight and can achieve a good transdermal absorption effect.

[0053] (2) The hydrolyzed extracellular matrix provided by the present invention retains sites with specific amino acid sequences that can interact with cells in multiple main components of the extracellular matrix, including collagen, elastin, fibronectin and laminin, and retains the biological activity of the primary structure of the extracellular matrix protein.

[0054] (3) The hydrolyzed extracellular matrix provided by the present invention has a good effect of promoting cell growth and proliferation.

[0055] (4) The hydrolyzed extracellular matrix provided by the present invention has good anti-wrinkle and skin elasticity enhancing functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the appearance of the hydrolyzed extracellular matrix of the present invention in Example 1.

[0057] Figure 2 This is the result of transdermal absorption of the hydrolyzed extracellular matrix in Experimental Example 4.

[0058] Figure 3 This is the cell growth curve of the hydrolyzed extracellular matrix used for cell culture in Experimental Example 5. DETAILED DESCRIPTION

[0059] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0060] Example 1

[0061] Prepare hydrolyzed extracellular matrix as follows:

[0062] (1) Take the submucosal layer of the small intestine of the pig, scrape and cut off the fat and fascia attached to the surface, and wash it clean with tap water;

[0063] (2) Cut the tissue into segments, immerse in ethyl acetate and shake for 2 h, then rinse with purified water;

[0064] (3) Soak the tissue fragments in 0.1% peracetic acid solution and shake them for 1 hour;

[0065] (4) crushing the tissue fragments into particles using a blade crusher;

[0066] (5) Soak the tissue particles in 10 mmol / L sodium periodate solution, stir for 2 h, and wash with purified water;

[0067] (6) Soak the tissue particles in a potassium iodide solution with a pH of 4.0 and a concentration of 2 mol / L, stir for 2 h, and wash with purified water;

[0068] (7) Soak the tissue particles in a solution containing 2 mmol / L dithiothreitol and 10 mol / L urea, stir until the sample becomes a viscous liquid, and store at 4°C for 8 h;

[0069] (8) Place the viscous liquid into a dialysis bag at 4°C and dialyze it with 20 times the volume of purified water cooled to 4°C in advance. Replace the water every 6 hours. Dialyze for 24 hours until the sample is converted into a gel state.

[0070] (9) The gel pH was adjusted to 3.0 with 1 mol / L hydrochloric acid, the sample was converted into a solution, pepsin was added to a concentration of 0.5 mg / mL, and the solution was stirred at 35 °C for 24 h.

[0071] (10) adjusting the pH of the solution to 7.0 with a 1 mol / L sodium hydroxide solution until the floccules are completely precipitated, and filtering the precipitate and the solution with a 300-mesh nylon cloth;

[0072] (11) The precipitate is heated to 45°C and maintained for 2 h, and the precipitate is converted into a solution;

[0073] (12) Cool the solution to 37°C, add trypsin to a concentration of 0.5 mg / mL, and maintain at 37°C with stirring for 24 h;

[0074] (13) Mix the solution obtained in the previous step and the solution obtained in step (10), adjust the pH to 6.0 with 1 mol / L sodium hydroxide, filter with 1000 mesh filter cloth, 1 μm filter membrane and 0.22 μm filter membrane in sequence, and collect the filtrate;

[0075] (14) The filtrate was freeze-dried. The freeze-drying procedure was: -55°C for 4 h, -50°C for 2 h, -40°C for 2 h, -20°C for 2 h, 0°C for 2 h, 10°C for 4 h, 20°C for 4 h, and 30°C for 24 h.

[0076] Example 2

[0077] Prepare hydrolyzed extracellular matrix as follows:

[0078] (1) Take the submucosal layer of the small intestine of the pig, scrape and cut off the fat and fascia attached to the surface, and wash it clean with tap water;

[0079] (2) Cut the tissue into segments, immerse in ethyl acetate and shake for 6 h, then rinse with purified water;

[0080] (3) Soak the tissue fragments in 0.2% peracetic acid solution and shake them for 2 h;

[0081] (4) crushing the tissue fragments into particles using a blade crusher;

[0082] (5) Soak the tissue particles in 50 mmol / L sodium periodate solution, stir for 0.5 h, and wash with purified water;

[0083] (6) Soak the tissue particles in a potassium iodide solution with a pH of 6.0 and a concentration of 2 mol / L, stir for 4 h, and wash with purified water;

[0084] (7) Soak the tissue particles in a solution containing 20 mmol / L dithiothreitol and 4 mol / L urea, stir until the sample becomes a viscous liquid, and store at 20°C for 8 h;

[0085] (8) Place the viscous liquid into a dialysis bag at 20°C and dialyze it with 20 times the volume of purified water cooled to 20°C in advance. Replace the water every 6 hours. Dialyze for 24 hours until the sample is converted into a gel state.

[0086] (9) The gel pH was adjusted to 4.0 with 1 mol / L hydrochloric acid, the sample was converted into a solution, pepsin was added to a concentration of 2 mg / mL, and the solution was stirred at 35 °C for 12 h.

[0087] (10) adjusting the pH of the solution to 9.0 with a 1 mol / L sodium hydroxide solution until the floccules are completely precipitated, and centrifuging at 8000 rpm for 30 min to separate the precipitate and the solution;

[0088] (11) The precipitate is heated to 45°C and maintained for 1 h, and the precipitate is converted into a solution;

[0089] (12) Cool the solution to 37°C, add trypsin to a concentration of 2 mg / mL, and maintain stirring at 37°C for 12 h;

[0090] (13) Mix the solution obtained in the previous step and the solution obtained in step (10), adjust the pH to 8.0 with 1 mol / L sodium hydroxide, filter with 1000 mesh filter cloth, 1 μm filter membrane and 0.22 μm filter membrane in sequence, and collect the filtrate;

[0091] (14) The filtrate was freeze-dried. The freeze-drying procedure was: -55°C for 4 h, -50°C for 2 h, -40°C for 2 h, -20°C for 2 h, 0°C for 2 h, 10°C for 4 h, 20°C for 4 h, and 30°C for 24 h.

[0092] Example 3

[0093] Prepare hydrolyzed extracellular matrix as follows:

[0094] (1) Take the submucosal layer of the small intestine of the pig, scrape and cut off the fat and fascia attached to the surface, and wash it clean with tap water;

[0095] (2) Cut the tissue into segments, immerse in ethyl acetate and shake for 2 h, then rinse with purified water;

[0096] (3) Soak the tissue fragments in 0.1% peracetic acid solution and shake them for 2 h;

[0097] (4) crushing the tissue fragments into particles using a blade crusher;

[0098] (5) Soak the tissue particles in 20 mmol / L sodium periodate solution, stir for 2 h, and wash with purified water;

[0099] (6) Soak the tissue particles in a potassium iodide solution with a pH of 5.0 and a concentration of 2 mol / L, stir for 2 h, and wash with purified water;

[0100] (7) Soak the tissue particles in a solution containing 10 mmol / L dithiothreitol and 10 mol / L urea, stir until the sample becomes a viscous liquid, and store at 4°C for 2 h;

[0101] (8) Place the viscous liquid into a dialysis bag at 4°C and dialyze it with 20 times the volume of purified water cooled to 4°C in advance. Replace the water every 6 hours. Dialyze for 24 hours until the sample is converted into a gel state.

[0102] (9) The gel pH was adjusted to 3.0 with 1 mol / L hydrochloric acid, the sample was converted into a solution, pepsin was added to a concentration of 2 mg / mL, and the solution was stirred at 35 °C for 6 h.

[0103] (10) adjusting the pH of the solution to 7.0 with a 1 mol / L sodium hydroxide solution until the floccules are completely precipitated, and filtering the precipitate and the solution with a 300-mesh nylon cloth;

[0104] (11) The precipitate is heated to 40°C and maintained for 2 h, and the precipitate is converted into a solution;

[0105] (12) Cool the solution to 30°C, add trypsin to a concentration of 2 mg / mL, and maintain stirring at 30°C for 24 h;

[0106] (13) Mix the solution obtained in the previous step and the solution obtained in step (10), adjust the pH to 7.0 with 1 mol / L sodium hydroxide, filter with 1000 mesh filter cloth, 1 μm filter membrane and 0.22 μm filter membrane in sequence, and collect the filtrate;

[0107] (14) The filtrate was freeze-dried. The freeze-drying procedure was: -55°C for 4 h, -50°C for 2 h, -40°C for 2 h, -20°C for 2 h, 0°C for 2 h, 10°C for 4 h, 20°C for 4 h, and 30°C for 24 h.

[0108] Example 4

[0109] Prepare hydrolyzed extracellular matrix as follows:

[0110] (1) Take the submucosal layer of the small intestine of the pig, scrape and cut off the fat and fascia attached to the surface, and wash it clean with tap water;

[0111] (2) Cut the tissue into segments, immerse in ethyl acetate and shake for 4 h, then rinse with purified water;

[0112] (3) Soak the tissue fragments in 0.1% peracetic acid solution and shake them for 1 hour;

[0113] (4) crushing the tissue fragments into particles using a blade crusher;

[0114] (5) Soak the tissue particles in 10 mmol / L sodium periodate solution, stir for 0.5 h, and wash with purified water;

[0115] (6) Soak the tissue particles in a potassium iodide solution with a pH of 4.0 and a concentration of 0.5 mol / L, stir for 4 h, and wash with purified water;

[0116] (7) Soak the tissue particles in a solution containing 20 mmol / L dithiothreitol and 6 mol / L urea, stir until the sample becomes a viscous liquid, and store at 4°C for 8 h;

[0117] (8) Place the viscous liquid into a dialysis bag at 20°C and dialyze it with 20 times the volume of purified water cooled to 20°C in advance. Replace the water every 6 hours. Dialyze for 24 hours until the sample is converted into a gel state.

[0118] (9) The gel pH was adjusted to 3.5 with 1 mol / L hydrochloric acid, the sample was converted into a solution, pepsin was added to a concentration of 1 mg / mL, and the solution was stirred at 30 °C for 24 h.

[0119] (10) adjusting the pH of the solution to 7.2 with a 1 mol / L sodium hydroxide solution until the floccules are completely precipitated, and filtering the precipitate and the solution with a 300-mesh nylon cloth;

[0120] (11) The precipitate is heated to 45°C and maintained for 2 h, and the precipitate is converted into a solution;

[0121] (12) Cool the solution to 37°C, add trypsin to a concentration of 1 mg / mL, and maintain stirring at 37°C for 12 h;

[0122] (13) Mix the solution obtained in the previous step and the solution obtained in step (10), adjust the pH to 7.0 with 1 mol / L sodium hydroxide, filter with 1000 mesh filter cloth, 1 μm filter membrane and 0.22 μm filter membrane in sequence, and collect the filtrate;

[0123] (14) The filtrate was freeze-dried. The freeze-drying procedure was: -55°C for 4 h, -50°C for 2 h, -40°C for 2 h, -20°C for 2 h, 0°C for 2 h, 10°C for 4 h, 20°C for 4 h, and 30°C for 24 h.

[0124] Comparative Example 1

[0125] As a method for preparing a hydrolyzed extracellular matrix as a comparative example of the present invention, the only difference between this comparative example and Example 1 is that the concentration of the sodium periodate solution used in step (5) is 5 mmol / L.

[0126] Comparative Example 2

[0127] As a method for preparing a hydrolyzed extracellular matrix as a comparative example of the present invention, the only difference between this comparative example and Example 1 is that the concentration of the potassium iodide solution used in step (6) is 0.2 mol / L.

[0128] Comparative Example 3

[0129] As a method for preparing a hydrolyzed extracellular matrix as a comparative example of the present invention, the only difference between this comparative example and Example 1 is that the concentration of urea in the solution used in step (7) is 2 mol / L.

[0130] Experimental Example 1

[0131] The weight average molecular weight, data molecular weight and molecular weight distribution coefficient of the hydrolyzed extracellular matrix and hydrolyzed collagen (purchased from Chengdu Kele Biology, sourced from pigskin) obtained by each embodiment and comparative example were measured using gel permeation chromatography-light scattering coupling instrument (Wyatt DAWN HELEOS II), and the measurement results are shown in Table 1. It can be seen that the molecular weight of the samples obtained by each embodiment is relatively small and close. The molecular weight of the three samples of the comparative example is relatively large, which may be because each comparative example retains a certain degree of glycosylation, cross-linking or folding when the corresponding treatment reagent concentration is low, shielding the site of partial enzyme cutting, so that the molecular weight of the final product is large. In addition, the molecular weight of the hydrolyzed collagen purchased is close to the molecular weight of each embodiment.

[0132] Table 1 Molecular weight information determination results

[0133]

[0134]

[0135] Experimental Example 2

[0136] The hydrolyzed extracellular matrix and hydrolyzed collagen obtained in each embodiment and comparative example were detected using a high performance liquid chromatography-mass spectrometry system (high performance liquid chromatograph (Agilent 1100), electrospray ion trap mass spectrometer (Thermal Fisher LCQ DecaXP)), and processed by Xcalibur 3.0 data processing software. The obtained mass-to-nuclear ratio information was compared with the database established by the theoretical sequence of the target peptide segment to determine the presence of the target peptide segment. The results are shown in Table 2. It can be seen that all the target peptide segments are retained in each embodiment and comparative example, which is due to the very mild conditions maintained throughout the preparation process and the use of a multi-step enzymatic hydrolysis method with a reasonable order. However, only part of the target peptide segments of collagen and elastin can be detected in the hydrolyzed collagen, which is related to the random breakage of the peptide bonds of the amino acid chain and the inappropriate enzymatic cleavage method in the preparation process of the hydrolyzed collagen.

[0137] Table 2 Presence of target peptides

[0138]

[0139]

[0140] Experimental Example 3

[0141] The Franz-Cell diffusion cell method was used to determine the transdermal absorption rate of the sample in Example 3, the sample in Comparative Example 1, and the hydrolyzed collagen. The sample in Example 3, the sample in Comparative Example 1, and the hydrolyzed collagen were respectively formulated into solutions with a content of 1.0%. In addition, 5% glycerol (purchased from Shandong Ruisheng Pharmaceutical Excipients Co., Ltd.) and 1% 1,3-propylene glycol (purchased from Shanghai Ronghai Biological Co., Ltd.) were added to each solution as a test solution. The ex vivo pig ear skin was used as a skin model and placed between the diffusion cell and the receiving cell. The test solution was dripped onto the pig ear skin. The receiving solution was physiological saline and magnetic stirring was continued. Samples were taken at 0.5h, 1h, 2h, and 4h after the start. The sampled solution was hydrolyzed into amino acids with hydrochloric acid and the hydroxyproline content therein was determined using a high performance liquid chromatograph (Agilent 1100). The hydroxyproline content in the test solution was measured using the same method. The transdermal absorption rate was calculated based on the hydroxyproline content. The results are as follows: Figure 2 As shown, it can be seen that the transdermal absorption rate of the sample of Example 3 and the hydrolyzed collagen is much higher than that of the sample of Comparative Example 1, which indicates that the molecular weight has an important influence on the transdermal absorption rate, and the transdermal absorption rate of the sample with a high molecular weight is poor.

[0142] Experimental Example 4

[0143] Test the promotion of cell growth by the sample of Example 1 and hydrolyzed collagen. NIH 3T3 cells were seeded in a cell culture plate, and the sample of Example 1 and hydrolyzed collagen were added to DMEM culture medium (purchased from Solebao Biotechnology) to a concentration of 5 mg / mL for cell culture. DMEM culture medium without additional substances was used as a blank control. Fresh culture medium was replaced every two days during the culture period, and samples were taken on the 1st, 4th, 7th, and 10th days for detection using the CCK-8 method. The obtained OD value represents the relative number of living cells. The results are as follows: Figure 3 As shown, it can be seen that although hydrolyzed collagen can also promote cell proliferation, the promoting effect of the sample in Example 1 is more significant.

[0144] Experimental Example 5

[0145] Test of anti-wrinkle and skin elasticity enhancement efficacy. The sample of Example 1, the sample of Comparative Example 1 and the hydrolyzed collagen were respectively formulated into a solution with a content of 0.5%. In addition, 3% glycerol, 1% 1,3-propylene glycol, 1% Spectrastat PHL (purchased from Shanghai Haoyun Trading) and 0.3% sodium hyaluronate (purchased from Shandong Focus Biology) were added to each solution as test solution. 18 women aged 23-48 were selected as subjects and randomly divided into 3 groups, with 6 people in each group. Each group used a test solution to apply after washing their face every morning and evening for 1 month. A skin detector (Canfield Visia) was used to detect the wrinkles in the 2×2cm area of ​​the corners of the eyes of the subjects before and after the use of the test solution. The wrinkle index before use was calculated according to the analysis software provided by the instrument as W0, and the wrinkle index after using the test solution for 1 month was W1. The wrinkle improvement rate was calculated as (W0-W1) / W0×100%, and the wrinkle improvement rate of 6 people in each group was averaged. The skin elasticity of the cheek area of ​​the subjects before and after using the test solution was tested using a skin tester (CK MPA580). The elasticity index before use was measured as R0, and the elasticity index after one month of using the test solution was measured as R1. The skin elasticity increase rate was calculated as (R1-R0) / R0×100%, and the average value of the skin elasticity increase rate of 6 people in each group was taken. The results of the wrinkle improvement rate and the skin elasticity increase rate are shown in Table 3. It can be seen that the wrinkle improvement rate and the skin elasticity increase rate of the sample in Example 1 are significantly improved compared with the sample in Comparative Example 1 and the hydrolyzed collagen, indicating that the sample in Example 1 has better anti-wrinkle and skin elasticity increase effects.

[0146] Table 3 Wrinkle improvement rate and skin elasticity increase rate.

[0147] Test product Wrinkle improvement rate Skin elasticity increase rate Example 1 Sample 22.7％ 30.3％ Comparative Example 1 Sample 15.3％ 20.5％ Hydrolyzed collagen 16.8％ 23.2％

[0148] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a hydrolyzed extracellular matrix, characterized in that: The following steps are included: (1) taking peripheral tissue of a mammal and removing fat and adventitial tissue; (2) immersing the tissue obtained in step (1) in ethyl acetate and shaking; (3) immersing the tissue fragments treated in step (2) in a peracetic acid solution for shaking; (4) crushing the tissue fragments processed in step (3); (5) treating the sample obtained in step (4) with a sodium periodate solution, wherein the concentration of the sodium periodate solution is 10-50 mmol / L; (6) treating the sample treated in step (5) with a potassium iodide solution having a pH of 4.0-6.0, wherein the concentration of the potassium iodide solution is 0.5-2 mol / L; (7) adding the sample treated in step (6) to a mixed solution containing dithiothreitol and urea; the concentration of dithiothreitol in the mixed solution is 2-20 mmol / L, and the concentration of urea is 4-10 mol / L; (8) dialyzing the sample obtained in step (7) using purified water at a certain temperature to remove dithiothreitol and urea; (9) adjusting the pH of the sample obtained in step (8) to 3.0-4.0, and adding pepsin for treatment; (10) adjusting the pH of the sample obtained in step (9) to 7.0-9.0 to precipitate flocs, and separating the precipitate and the solution; (11) heating the precipitate obtained in step (10) to obtain a solution; (12) adding trypsin to the solution obtained in step (11); (13) The solution separated in step (10) and the solution obtained in step (12) are mixed, the pH is adjusted to 6.0-8.0, and the mixture is filtered through a filter membrane.

2. The preparation method according to claim 1, characterized in that: In step (3), the mass fraction of the peracetic acid solution is 0.1%-0.2%.

3. The preparation method according to claim 1, characterized in that: In step (9), the pepsin concentration is 0.5-2 mg / mL.

4. The preparation method according to claim 1, characterized in that: In step (12), the trypsin concentration is 0.5-2 mg / mL.

5. The hydrolyzed extracellular matrix prepared by the method according to any one of claims 1 to 4, characterized in that: The hydrolyzed extracellular matrix comprises hydrolyzed collagen, hydrolyzed elastin, hydrolyzed fibronectin and hydrolyzed laminin, and the number of polypeptide molecules with a molecular weight of 500-3000 Daltons in the hydrolyzed extracellular matrix accounts for no less than 96%.

6. The hydrolyzed extracellular matrix according to claim 5, characterized in that The hydrolyzed collagen contains peptide segments with amino acid sequences of GFOGER, GLOGER, GASGER, GLPGER and GIPGEK respectively.

7. The hydrolyzed extracellular matrix according to claim 5, characterized in that The hydrolyzed elastin contains peptide segments with amino acid sequences of AKPGVGGL and GRKRK respectively.

8. The hydrolyzed extracellular matrix according to claim 5, characterized in that The hydrolyzed fibronectin contains peptide segments with amino acid sequences of RGDS and REDV respectively.

9. The hydrolyzed extracellular matrix according to claim 5, characterized in that The hydrolyzed laminin contains peptide segments with amino acid sequences of IKVAV, HQNMGSVNVSV, and SIKIRGTY respectively.

10. Use of the hydrolyzed extracellular matrix prepared by the preparation method according to any one of claims 1 to 5 or the hydrolyzed extracellular matrix according to any one of claims 6 to 9 in preparing products that promote cell growth and proliferation, resist wrinkles and enhance skin elasticity.

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