A triple helical collagen with thermal stability and promoting self-collagen production, and preparation method and use thereof

Abstract

The present invention belongs to the technical field of medical and cosmetic raw materials, and specifically relates to a triple helical collagen protein with thermal stability and the ability to promote self-collagen production, and a preparation method and use thereof. The structural formula of the triple helical collagen protein of the present invention is shown in Formula I: Formula I: H2N-(X-Y-Z) n ‑COOH; wherein X‑Y‑Z is a peptide chain composed of three different amino acid residues; X, Y, and Z are independently selected from proline residues, hydroxyproline residues, or glycine residues. Its weight average molecular weight distribution is 5.0×10 4 ~3.0×10 8 The collagen of the present invention has a triple helix structure, good thermal stability, better anti-wrinkle and firming effects and injection filling effects, can be better applied to the fields of cosmetics, medical devices, etc., and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of medical and cosmetic raw materials, and specifically relates to a triple-helix collagen protein with thermal stability and the ability to promote self-collagen production, as well as a preparation method and use thereof. Background Art

[0002] Collagen is the most abundant functional protein in the human body. It has a stable triple helical network structure and very important physiological functions. It is mainly found in skin, bones, ligaments and other tissues, maintaining the stability and normal function of various tissues and organs. In recent years, with the advancement of science and technology, artificial collagen has been used in many fields such as cosmetics, medical plastic surgery, and medical materials.

[0003] In the field of beauty, collagen is widely used in skin care products and cosmetics to improve the skin's moisture retention, elasticity and glossiness. Many functional skin care products, facial masks, essences, facial cleansers, etc. have added collagen. Collagen can supplement the nutrients needed by the skin, improve the living environment of skin cells, and promote the metabolism of skin tissue, thereby achieving the effect of delaying aging and nourishing the skin. At present, most of the collagen skin care products on the market are prepared with high-content collagen raw materials, and use scientific and technological means to make them small molecules, which are easier for skin absorption. In the medical plastic surgery industry and the field of medical materials, collagen is also widely used. For example, in facial filling, anti-aging plastic surgery, skin repair, etc., collagen is used as an important biological filling material. In addition, it can also be used in the clinical treatment of pathological changes such as skin burns and trauma to improve skin conditions. In tissue engineering, collagen, as a biodegradable material, can be used to manufacture various tissue engineering scaffolds to support cell growth and tissue repair. At the same time, due to its good biocompatibility and biodegradability, it can also be used to prepare medical sutures, skin transplants and other medical devices. As a core component of regenerative medicine, bionic materials have greatly enriched the means of disease treatment. Collagen accounts for more than 30% of the total amount of human protein. Collagen can be used to develop artificial skin, artificial bone, artificial cornea, artificial tendon and ligament, artificial cartilage and other organoids and tissues, and related developments have achieved varying degrees of successful preparation and research results. Because collagen has advantages in cell adhesion, proliferation, biocompatibility, bioabsorbability, etc., and is a biomaterial with sufficient mechanical strength, it is the best choice in the treatment of medical materials that are in direct contact with cells and tissues. It can also be used as a cell material, or as a key technology for the large-scale preparation of cell therapy drugs, cell tissue sheets, etc., and the market size is expected to expand significantly in the future. At the same time, collagen also hopes to carry out more research and have more practical application results and cases in the regeneration and repair of tissues and organs of the elderly. Therefore, through the integration of industry and hospitals to study the clinical value and application scenarios of bionic biomaterials, the high-quality development of the bionic biomaterials industry is promoted, which has realistic clinical value and innovative industrial value.

[0004] At present, artificial collagen is mainly divided into animal collagen extracted from animal tissues such as cattle, pigs, chickens, and fish, and recombinant collagen produced by methods such as biological fermentation, purification, and gene editing. However, there are some technical problems in the production of artificial collagen. First, extracting collagen from animals may cause immune risks, such as the spread of viruses and diseases led by mad cow disease. Secondly, due to certain differences between the amino acid sequence of animal-derived collagen and that of human collagen, it cannot be guaranteed to have sufficient biological activity, stability, and safety. Although recombinant collagen can guarantee a more complete amino acid sequence, its process is cumbersome and costly, and it is impossible to determine whether it has a complete triple helix structure, and its biological activity is questionable.

[0005] There has been no major breakthrough in the preparation of collagen-like proteins by chemical synthesis. First, although the synthesis of peptides that mimic the amino acid sequence of human collagen can be achieved in theory, how to correctly fold these protein molecules to form an active triple helical structure is a key technical difficulty. In addition, how to prepare collagen with a triple helical structure with excellent thermal stability is also one of the major challenges currently faced.

[0006] In the prior art, a peptide synthesizer can be used to synthesize collagen-like polypeptide molecules. A peptide synthesizer is an automated laboratory device specially designed for synthesizing polypeptide chains in vitro. It is based on solid phase peptide synthesis (SPPS) technology, and can efficiently construct polypeptide chains consisting of several amino acids to hundreds of amino acids by precisely controlling reaction conditions and steps. However, experimental studies have shown that it is difficult for polypeptide molecules prepared using a peptide synthesizer to form a triple helix structure, and even if a triple helix structure is formed, it has the problem of insufficient thermal stability. When collagen is used to make cosmetics or medical devices, there will be a certain need to tolerate ambient temperatures or perform high-temperature sterilization. The unstable triple helix structure is difficult to maintain at higher temperatures, which creates certain difficulties for the application of the above-mentioned polypeptides in cosmetics or medical devices.

[0007] Therefore, developing a collagen substitute with better thermal stability is an urgent problem to be solved in this field. Summary of the invention

[0008] In view of the problems of the prior art, the present invention provides a triple-helix collagen protein having thermal stability and promoting self-collagen production, and a preparation method and use thereof.

[0009] A triple-helical collagen protein with thermal stability and promoting self-collagen production, the structural formula of which is shown in Formula I:

[0010] Formula I: H2N-(XYZ)n -COOH;

[0011] Wherein, XYZ is a peptide chain composed of three different amino acid residues; the weight average molecular weight distribution of the triple helical collagen is 5.0×10 4 ~3.0×10 8

[0012] X, Y, Z are selected from one of the following combinations:

[0013] X is a glycine residue, Y is a hydroxyproline residue, and Z is a proline residue;

[0014] X is a hydroxyproline residue, Y is a proline residue, and Z is a glycine residue;

[0015] X is a proline residue, Y is a hydroxyproline residue, and Z is a glycine residue;

[0016] X is a proline residue, Y is a glycine residue, and Z is a hydroxyproline residue.

[0017] Preferably, the value of n satisfies 1.05×10 6 ≧n≧175.

[0018] Preferably, the triple helix collagen is obtained by condensing a tripeptide having a structural formula of H2N-XYZ-COOH as a raw material through a liquid phase reaction.

[0019] The present invention also provides a method for preparing the triple-helix collagen having thermal stability and promoting self-collagen production, comprising the following steps:

[0020] Step 1, a tripeptide having a structural formula of H2N-XYZ-COOH and a condensation aid are prepared into a tripeptide solution using a phosphate buffer as a solvent; a dehydration condensation agent is prepared into a dehydration condensation agent solution using a phosphate buffer as a solvent;

[0021] Wherein, the phosphate buffer is prepared using the following raw materials in parts by weight:

[0022] Potassium chloride 0.18-0.22 parts,

[0023] 1.0-1.2 parts of sodium dihydrogen phosphate,

[0024] 0.18-0.22 parts of potassium dihydrogen phosphate,

[0025] Water 995-1005 parts;

[0026] The tripeptide solution is prepared using raw materials in the following proportions:

[0027] Phosphate buffer 495-505 parts by volume,

[0028] 49.5 to 50.5 parts by weight of tripeptide,

[0029] Condensation aid 4.9-5.1 parts by weight;

[0030] The dehydration condensation agent solution is prepared using raw materials in the following proportions:

[0031] Phosphate buffer 495-505 parts by volume,

[0032] 156-160 parts by weight of a dehydration condensation agent;

[0033] Step 2, adding the dehydration condensation agent solution to the tripeptide solution to carry out a polycondensation reaction; the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent in the polycondensation reaction is 49.5-50.5:4.9-5.1:156-160; the temperature of the polycondensation reaction is less than or equal to 20° C., and the time of the polycondensation reaction is 20-26 hours;

[0034] Step 3, separation and purification.

[0035] Preferably, in step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole;

[0036] The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide;

[0037] The phosphate buffer is prepared using the following raw materials in parts by weight:

[0038] 0.2 parts of potassium chloride,

[0039] 1.15 parts of sodium dihydrogen phosphate,

[0040] 0.2 parts of potassium dihydrogen phosphate,

[0041] 1000 parts water;

[0042] The tripeptide solution is prepared using raw materials in the following proportions:

[0043] Phosphate buffer 500 parts by volume,

[0044] 50 parts by weight of tripeptide,

[0045] 5 parts by weight of condensation aid;

[0046] The dehydration condensation agent solution is prepared using raw materials in the following proportions:

[0047] Phosphate buffer 500 parts by volume,

[0048] 158 parts by weight of dehydration condensation agent.

[0049] Preferably, in step 2, in the dehydration condensation agent solution and the tripeptide solution, the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent is 50:5:158;

[0050] The polycondensation reaction time is 24 hours.

[0051] Preferably, in step 3, the specific steps of separation and purification are: cyclic grinding with a homogenizer, filtering through a 50,000 molecular weight cutoff membrane to obtain a solution containing the triple helical collagen.

[0052] The present invention also provides the use of the triple helical collagen having thermal stability and promoting self-collagen production in the preparation of cosmetics, medicines, cosmetic and plastic surgery materials or medical devices.

[0053] Preferably, the cosmetics are functional skin care products;

[0054] The medical device is a filling material or an engineering scaffold for skin, bones, and ligaments.

[0055] Preferably, the functional skin care product is a facial mask, an essence or a facial cleanser.

[0056] The present invention also provides a cosmetic, which is prepared by using the triple helix collagen protein having thermal stability and promoting the self-collagen production as an active ingredient and adding auxiliary materials.

[0057] Preferably, the concentration of the triple helical collagen having thermal stability and promoting self-collagen production is 0.0005wt%-0.05wt%.

[0058] Preferably, the concentration of the triple helical collagen having thermal stability and promoting self-collagen production is 0.005 wt %.

[0059] The "triple helix collagen" provided by the present invention refers to a protein having a tripeptide repeating unit and a triple helix structure, which is a mixture of proteins with different polymerization degrees with a tripeptide repeating unit as a repeating structure. The "triple helix structure" is the basic structure of natural collagen, which is a triple helix structure formed by three polypeptide chains entangled with each other. The method for detecting whether a protein or polymer has a triple helix structure similar to that of natural collagen is to use circular dichroism to detect whether the polymer has a positive peak at 220-230nm. The ratio of the "volume parts" and "weight parts" is determined as follows:

[0060] 1 part by volume: 1 part by weight = 1 ml: 1 g.

[0061] The present invention provides a new collagen with thermal stability, which is a liquid-phase synthesized polypeptide chain obtained by condensation polymerization with three amino acids as a unit. Existing collagen products will gel at a temperature exceeding 40°C and cannot maintain a triple helix structure. In cosmetics (except for cosmetics used in refrigeration), it cannot be used as a raw material. In addition, if used as a medical device, it cannot be heated and sterilized (above 60°C) or disinfected (autoclave). The collagen polymer (polypeptide chain) provided by the present invention can maintain a triple helix structure under normal temperature and high temperature (40-60°C), and its thermal stability is better than that of existing similar synthetic collagen products, so that it can be better used in cosmetics or medical devices. In addition, the collagen polymer provided by the present invention has a better injection filling effect, can promote the expression of CollagenⅠ gene and CollagenⅢ gene in fibroblasts, has anti-wrinkle and firming effects, and has the best effect at a concentration of 0.005wt%.

[0062] Therefore, the collagen polymer of the present invention has a good application prospect.

[0063] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0064] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The circular dichroism spectrum of collagen after heat treatment at 20-60°C in Experimental Example 1;

[0066] Figure 2 This is the circular dichroism spectrum of the commercial polypeptide in Experimental Example 1 after heat treatment at 20-60°C;

[0067] Figure 3 This is a graph showing the molecular weight test results of collagen in Experimental Example 1;

[0068] Figure 4 This is the experimental result diagram of the relative expression of Collagen Ⅰ gene in Experimental Example 2;

[0069] Figure 5 This is the experimental result diagram of the relative expression of CollagenⅢ gene in Experimental Example 2;

[0070] Figure 6This is a schematic diagram of the drug administration area for mice in Experimental Example 3;

[0071] Figure 7 The imaging results after one week of administration in Experimental Example 3, wherein A is a control group injected with PBS, B is an experimental group injected with a low concentration of 0.6wt.% recombinant humanized type III collagen, C is an experimental group injected with a high concentration of 3wt.% recombinant humanized type III collagen, and D is an experimental group injected with 0.5wt.% collagen;

[0072] Figure 8 These are the imaging results after 3 weeks of administration in Experimental Example 3, wherein A is the control group injected with PBS, B is the experimental group injected with a low concentration of 0.6wt.% recombinant humanized type III collagen, C is the experimental group injected with a high concentration of 3wt.% recombinant humanized type III collagen, and D is the experimental group injected with 0.5wt.% collagen. DETAILED DESCRIPTION

[0073] The reagents and raw materials used in the following examples and experimental examples are all commercially available unless otherwise specified.

[0074] Example 1 Triple helical collagen with thermal stability and promoting self-collagen production

[0075] This embodiment provides a triple helical collagen protein that has thermal stability and promotes self-collagen production. It is a protein with a triple helical structure, and its structural formula is as follows:

[0076] H2N-(XYZ) n -COOH

[0077] X, Y and Z in the formula are composed of the following amino acid residues:

[0078] X: Proline (hereinafter referred to as P),

[0079] Y: Hydroxyproline (hereinafter referred to as O),

[0080] Z: glycine (hereinafter referred to as G);

[0081] n≧4. Since the triple-helical collagen in this embodiment is a mixture of molecules with different degrees of polymerization, there are many possible values ​​of n, and its range can be estimated based on the characterization of the molecular weight.

[0082] The preparation method of the triple helix collagen is as follows:

[0083] 1. Preparation of phosphate buffer

[0084] Add 0.2 g of potassium chloride (KCl), 1.15 g of sodium dihydrogen phosphate (Na2HPO4), and 0.2 g of potassium dihydrogen phosphate (KH2PO4) to 1000 ml of high-purity water, and stir to prepare phosphate buffer solution (Buffer-PB, hereinafter referred to as PB solution).

[0085] 2. Preparation of tripeptide solution

[0086] 50 g of tripeptide POG (manufactured by Uniqs Inc.) and 5 g of condensation aid 1-hydroxybenzotriazole (HOBt) were added to 500 ml of PB solution, and the mixture was stirred to prepare a tripeptide solution (hereinafter referred to as POG solution).

[0087] 3. Preparation of dehydration condensation agent solution

[0088] 158 g of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide (EDC) was added to 500 ml of the PB solution, and the mixture was stirred to prepare a dehydration condensation agent solution (hereinafter referred to as EDC solution).

[0089] 4. Polycondensation reaction

[0090] The prepared POG solution was poured into a 2L separatory funnel, cooled to 2°C in a thermostatic bath, and then gradually added with EDC solution cooled to 2°C (the volume ratio of POG solution to EDC solution was 1:1), and the reaction temperature was controlled to be kept below 20°C for 24 hours of polycondensation reaction. The reaction solution was transferred to a 10L container, circulated and ground with a homogenizer, and filtered with a rotary pump through a 50,000 molecular weight cutoff membrane (Asahi Kasei AHP-1013D) to obtain an aqueous solution containing the target product triple helical collagen.

[0091] Example 2 Triple helical collagen with thermal stability and promoting self-collagen production

[0092] The triple helical collagen and its preparation method in this embodiment are the same as those in embodiment 1, except that:

[0093] X is a glycine residue (G), Y is a hydroxyproline residue (O), and Z is a proline residue (P), and the tripeptide used in the preparation raw material is replaced with GOP accordingly.

[0094] Example 3: Triple helical collagen with thermal stability and promoting self-collagen production

[0095] The triple helical collagen and its preparation method in this embodiment are the same as those in embodiment 1, except that:

[0096] When X is a hydroxyproline residue (O), Y is a proline residue (P), and Z is a glycine residue (G), the tripeptide used in the preparation raw material is correspondingly replaced with OPG.

[0097] Example 4: Triple helical collagen with thermal stability and promoting self-collagen production

[0098] The triple helical collagen and its preparation method in this embodiment are the same as those in embodiment 1, except that:

[0099] When X is a proline residue (P), Y is a glycine residue (G), and Z is a hydroxyproline residue (O), the tripeptide used in the preparation raw material is correspondingly replaced with PGO.

[0100] Example 5 Moisturizing lotion for anti-wrinkle and firming

[0101] This embodiment provides a moisturizing lotion, the composition of which includes:

[0102] 0.005 wt% of triple helical collagen prepared in Example 1,

[0103] Glycerol 10wt%,

[0104] Polyethylene glycol 15wt%,

[0105] Hyaluronic acid 5wt%,

[0106] The rest is water.

[0107] The technical solution of the present invention is further illustrated by experiments below.

[0108] Experimental Example 1 Characterization of Products

[0109] 1. Experimental Methods

[0110] This experimental example characterizes the triple helical collagen prepared in Example 1, including:

[0111] 1. Circular dichroism spectroscopy characterization

[0112] A commercial peptide was selected as the reference substance, and its molecular formula is H2N-(POG) 10 -COOH (purchased from Anhui Guoping Pharmaceutical Co., Ltd.) The amino acid sequence of the reference substance is similar to that of the sample prepared in Example 1. The main difference between the reference substance and the sample prepared in Example 1 is that the reference substance is prepared using a peptide synthesizer.

[0113] The triple helical collagen of Example 1 or a commercial polypeptide reference substance was prepared into an aqueous solution with a concentration of 0.25 mg / ml, and heat-treated at 20°C, 40°C, and 60°C for 1 hour. 5 ml of each solution was taken and analyzed on a circular dichroism spectrometer to confirm whether each sample had a positive peak unique to triple helical collagen.

[0114] 2. Molecular weight characterization

[0115] The measurement was performed by light scattering method, using Wyatt multi-angle light scattering detector DAWN HELEOS II and Wyatt differential refractive index detector Optilab T-rEX, and Shodex SB-806M column.

[0116] 2. Experimental Results

[0117] 1. Circular dichroism spectroscopy characterization

[0118] The characterization results of triple helical collagen are as follows Figure 1 As shown in the figure, it can be seen that after treatment at 20°C, 40°C, and 60°C, positive peaks in the range of 220-230nm can be detected in all three groups of samples, and the peak heights have almost no change, indicating that the triple helical collagen can remain stable in the temperature range of 20°C-60°C. This shows that the triple helical collagen provided by the present invention can form a stable triple helical structure in the temperature range of 20°C-60°C.

[0119] The characterization results of commercial peptide reference substances are as follows: Figure 2 As shown in the figure, it can be seen that after treatment at 20℃, 40℃, and 60℃, certain positive peaks can be detected in the range of 220-230nm, indicating that the commercial polypeptide can also form a triple helix structure. However, as the temperature increases, the height of the positive peak in the range of 220-230nm decreases, indicating that the triple helix structure formed by the commercial polypeptide molecule easily disappears under heat treatment conditions, and its triple helix structure has poor thermal stability.

[0120] The experimental results show that, for a polypeptide, whether it can form a stable triple helix structure is related to its preparation method, and the triple helix collagen prepared by the liquid phase synthesis method provided by the present invention can meet the requirements of forming a stable triple helix structure.

[0121] 2. Molecular weight characterization

[0122] like Figure 3 As shown in the results, the weight average molecular weight Mw of the triple helical collagen prepared in Example 1 is 5.0×10 4 ~3.0×10 8 (g / mol).

[0123] Experimental Example 2: Detection of the effect of promoting the expression of fibroblast-anti-wrinkle and firming related genes

[0124] 1. Test Purpose

[0125] Skin aging can be divided into endogenous aging caused by internal factors and exogenous aging secondary to external factors; oxidation is the biggest threat to skin aging, mainly caused by environmental stress factors such as ultraviolet radiation, environmental pollution, smog, life pressure, etc., among which ultraviolet radiation is the most important factor. When the skin is exposed to ultraviolet radiation, excessive reactive oxygen species will be produced in the cells, causing the expression of aging-related genes, inducing inflammatory cascade reactions and reducing the expression of elastin and collagen, resulting in sagging, wrinkles and other photoaging phenomena of the skin. Photoaging mainly occurs in the dermis.

[0126] This test uses fibroblasts as the test system, and evaluates whether the sample to be tested has anti-wrinkle and firming effects based on the changes in gene expression of Collagen I and Collagen III.

[0127] 2. Experimental Methods

[0128] 1. Main reagents

[0129] Low-glucose DMEM culture medium (Solarbio), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), cell lysis solution (Novozyme).

[0130] 2. Main equipment

[0131] CO2 incubator (Thermo, 160i), biological safety cabinet (Sujing Antai, BSC-1604ⅡA2), inverted microscope (Leica, DMi8), QPCR instrument (Roche), floor-standing half-body ultraviolet therapy device (Sigma High, SS-03AB).

[0132] 3. Test methods

[0133] The specific settings of the test group are as follows:

[0134] A blank control (BC), a negative control (NC), a positive control (PC) and three sample groups (0.0005%, 0.005%, 0.05%) were set up. The blank control was not treated with medication or radiation; the negative control was only treated with 9J / cm 2 UVA irradiation; positive control group was irradiated with 9J / cm 2 UVA irradiation, the drug was 100μg / mL vitamin C and 7μg / mL vitamin E; three sample groups (0.0005%, 0.005%, 0.05%) were irradiated with 9J / cm 2 UVA irradiation was performed, and the drugs were triple-helical collagen (prepared according to the method of Example 1) at concentrations of 0.0005wt%, 0.005wt%, and 0.05wt%.

[0135] The specific steps are as follows:

[0136] 1) Cell seeding: Human fibroblasts were plated at 8×10 4 Cells were seeded into 24-well plates / well and incubated in an incubator (37°C, 5% CO 2 ) overnight in DMEM medium.

[0137] 2) Administration: Administration was performed when the cell plating rate in the 24-well plate reached 40% to 60%. 1 mL of cell culture medium (DMEM medium) was added to each well of the blank control group and the negative control group; 1 mL of culture medium (DMEM medium) containing 100 μg / mL vitamin C and 7 μg / mL vitamin E was added to each well of the positive control group; 1 mL of culture medium (DMEM medium) containing the corresponding concentration of triple helical collagen was added to each well of the three sample groups.

[0138] 3) Radiation: 24 hours after the completion of the administration, the negative control group, the positive control group and the sample group received UVA radiation with a total dose of 9 J / cm2. At the same time, the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm2).

[0139] 4) Collecting cells: After incubation for 24 hours, add 0.5 mL of lysis buffer to each well and leave at room temperature for 5 minutes to allow for full lysis. Transfer the cells to a 1.5 mL RNase-free Eppendorf tube and extract RNA according to the operating procedures.

[0140] 5) Reverse transcription: Synthesize cDNA according to the instructions of the RNA reverse transcription kit.

[0141] 6) Perform qRT-PCR detection on CollagenⅠ and CollagenⅢ.

[0142] 3. Experimental Results

[0143] The results of the detection of Collagen I gene content are shown in Table 1 and Figure 4 As shown:

[0144] Table 1 Summary of relative expression data of CollagenⅠ genes

[0145]

[0146] Note: When using the t-test method for statistical analysis, the significance of the NC group compared with the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. The significance of the sample group and PC group compared with the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **

[0147] The results showed that compared with the BC group, the NC group fibroblasts received a total dose of 9J / cm 2 After UVA radiation, the expression of CollagenⅠ gene decreased significantly (p<0.01), indicating that the radiation stimulation was effective.

[0148] Compared with the NC group, vitamin C and vitamin E in the PC group could significantly increase the expression of Collagen Ⅰ gene (p<0.01), indicating that the positive control test was effective.

[0149] Compared with the NC group, the expression of Collagen I gene in fibroblasts was significantly increased when the sample was administered at a concentration of 0.0005wt%, 0.005wt% and 0.05wt% (p<0.05). In particular, when the administration concentration was 0.005wt%, the effect of increasing the expression of Collagen I gene in fibroblasts was the best, and the effect was significantly better than that of the PC group.

[0150] The results of the detection of CollagenⅢ gene content are shown in Table 2 and Figure 5 As shown:

[0151] Table 2 Summary of relative expression data of CollagenⅢ genes

[0152]

[0153] Note: When using the t-test method for statistical analysis, the significance of the NC group compared with the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##. The significance of the sample group and PC group compared with the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **

[0154] The results showed that compared with the BC group, the NC group fibroblasts received a total dose of 9J / cm 2 After UVA radiation, the expression of CollagenⅢ gene decreased significantly (p<0.01), indicating that the radiation stimulation was effective.

[0155] Compared with the NC group, vitamin C and vitamin E in the PC group could significantly increase the expression of elastin gene (p<0.01), indicating that the positive control test was effective.

[0156] Compared with the NC group, the expression of CollagenⅢ gene in fibroblasts was significantly increased when the sample was administered at a concentration of 0.0005wt%, 0.005wt% and 0.05wt% (p<0.05). In particular, when the concentration was 0.005wt%, the effect of increasing the expression of CollagenⅢ gene in fibroblasts was the best, and the effect was comparable to that of the PC group.

[0157] The above experimental results show that the triple-helical collagen of the present invention (especially when the dosage concentration is 0.005wt%) can promote the expression of CollagenⅠgene and CollagenⅢgene in fibroblasts, and has anti-wrinkle and firming effects.

[0158] Experimental Example 3: Verification of injection effect

[0159] 1. Purpose of the experiment

[0160] In the field of medical beauty, the injection of collagen products can fill and tighten the skin, shape the body, moisturize and repair, promote wound healing and improve skin quality. Two-photon imaging technology can use second harmonic generation (SHG) imaging to observe the collagen and muscle fibers of living animals (mice) without staining. Therefore, two-photon imaging can be used to observe the presence of collagen products in the animal body after they are injected subcutaneously, thereby examining the injection effect of collagen products.

[0161] 2. Experimental Methods

[0162] like Figure 6 As shown, the dosing area of ​​the mouse back skin is set, and 4 injection areas are set on the back of each mouse. According to whether collagen is injected, 6-week-old male BALB / c mice are divided into an experimental group injected with 0.5wt.% triple helix collagen (prepared according to the method of Example 1), an experimental group injected with 0.6wt.% low-concentration recombinant humanized type III collagen (Ou Lifang, c36), an experimental group injected with 3wt.% high-concentration recombinant humanized type III collagen (Ou Lifang, c200), and a control group injected with PBS. The administration method is water light needle injection, and 0.2ml is administered to each injection area (1*1cm). The injection levels are the superficial dermis and the deep dermis. There are 12 mice in each group, and two-photon imaging is performed at 1 week and 3 weeks after injection.

[0163] 3. Experimental Results

[0164] The imaging results at 1 week and 3 weeks after injection were as follows: Figure 7 , Figure 8As shown, it can be seen from the figure that one week after injection, the accumulation of collagen can be observed in the experimental group, and the amount of collagen in the 0.5wt.% triple helix collagen experimental group is not only significantly higher than the 0.6wt.% low-concentration recombinant humanized type III collagen experimental group, but also significantly higher than the 3wt.% high-concentration recombinant humanized type III collagen experimental group. Three weeks after injection, it can be observed that the collagen in each experimental group further accumulates, indicating that the injection of collagen can promote the regeneration of the mouse's own collagen. At the same time, similar to the experimental data one week after injection, the amount of collagen in the 0.5wt.% triple helix collagen experimental group is not only significantly higher than the 0.6wt.% low-concentration recombinant humanized type III collagen experimental group, but also significantly higher than the 3% high-concentration recombinant humanized type III collagen experimental group.

[0165] The above experimental results show that the triple helix collagen of the present invention can more effectively promote the regeneration of own collagen than the recombinant collagen in the prior art, and thus has a better injection effect.

[0166] It can be seen from the above embodiments and experimental examples that the present invention provides a triple helix collagen with thermal stability and promoting self-collagen production, which has the same triple helix structure as natural collagen, can promote the expression of Collagen I gene and Collagen III gene in fibroblasts, has anti-wrinkle and firming effects, has better injection filling effects, and can maintain the triple helix structure in the temperature range of 20°C-60°C, has good thermal stability, can be better applied to cosmetics, medical devices and other fields, and has good application prospects.

Claims

1. A triple helical collagen composition having thermal stability and promoting self-collagen production, characterized in that: Its structural formula is shown in Formula I: Formula I: H2N-(XYZ) n -COOH; the value of n satisfies 1.05×10 6 ≧n≧175; Wherein, XYZ is a peptide chain composed of three different amino acid residues; the weight average molecular weight distribution of the triple helical collagen is 5.0×10 4 ~3.0×10 8 ; X, Y, Z are selected from: X is a proline residue, Y is a hydroxyproline residue, and Z is a glycine residue; The triple helical collagen composition is prepared according to the following method: Step 1, a tripeptide having a structural formula of H2N-XYZ-COOH and a condensation aid are prepared into a tripeptide solution using a phosphate buffer as a solvent; a dehydration condensation agent is prepared into a dehydration condensation agent solution using a phosphate buffer as a solvent; Wherein, the phosphate buffer is prepared using the following raw materials in parts by weight: Potassium chloride 0.18~0.22 parts, 1.0~1.2 parts of sodium dihydrogen phosphate, 0.18~0.22 parts of potassium dihydrogen phosphate, Water 995~1005 parts; The tripeptide solution is prepared using raw materials in the following proportions: Phosphate buffer 495~505 parts by volume, 49.5-50.5 parts by weight of tripeptide, Condensation aid 4.9-5.1 parts by weight; The dehydration condensation agent solution is prepared using raw materials in the following proportions: Phosphate buffer 495~505 parts by volume, 156-160 parts by weight of dehydration condensation agent; Step 2, adding the dehydration condensation agent solution to the tripeptide solution to carry out a polycondensation reaction; the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent in the polycondensation reaction is 49.5-50.5:4.9-5.1:156-160; the temperature of the polycondensation reaction is less than or equal to 20° C., and the time of the polycondensation reaction is 24 hours; Step 3, separation and purification.

2. The triple-helix collagen composition having thermal stability and promoting autocollagen production according to claim 1, characterized in that: In step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole; The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide; The phosphate buffer is prepared using the following raw materials in parts by weight: 0.2 parts of potassium chloride, 1.15 parts of sodium dihydrogen phosphate, 0.2 parts of potassium dihydrogen phosphate, 1000 parts water; The tripeptide solution is prepared using raw materials in the following proportions: Phosphate buffer 500 parts by volume, 50 parts by weight of tripeptide, 5 parts by weight of condensation aid; The dehydration condensation agent solution is prepared using raw materials in the following proportions: Phosphate buffer 500 parts by volume, 158 parts by weight of dehydration condensation agent.

3. The triple-helix collagen composition having thermal stability and promoting autocollagen production according to claim 1, characterized in that: In step 2, in the dehydration condensation agent solution and the tripeptide solution, the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent is 50:5:

158.

4. The triple-helix collagen composition having thermal stability and promoting self-collagen production according to claim 1, characterized in that: In step 3, the specific steps of separation and purification are: cyclic grinding with a homogenizer, filtering through a 50,000 molecular weight cutoff membrane to obtain a solution containing the triple helical collagen.

5. The method for preparing the triple helical collagen composition having thermal stability and promoting self-collagen production according to any one of claims 1 to 4, characterized in that: The steps include: Step 1, a tripeptide having a structural formula of H2N-XYZ-COOH and a condensation aid are prepared into a tripeptide solution using a phosphate buffer as a solvent; a dehydration condensation agent is prepared into a dehydration condensation agent solution using a phosphate buffer as a solvent; Wherein, the phosphate buffer is prepared using the following raw materials in parts by weight: Potassium chloride 0.18~0.22 parts, 1.0~1.2 parts of sodium dihydrogen phosphate, 0.18~0.22 parts of potassium dihydrogen phosphate, Water 995~1005 parts; The tripeptide solution is prepared using raw materials in the following proportions: Phosphate buffer 495~505 parts by volume, 49.5-50.5 parts by weight of tripeptide, Condensation aid 4.9-5.1 parts by weight; The dehydration condensation agent solution is prepared using raw materials in the following proportions: Phosphate buffer 495~505 parts by volume, 156-160 parts by weight of dehydration condensation agent; Step 2, adding the dehydration condensation agent solution to the tripeptide solution to carry out a polycondensation reaction; the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent in the polycondensation reaction is 49.5-50.5:4.9-5.1:156-160; the temperature of the polycondensation reaction is less than or equal to 20° C., and the time of the polycondensation reaction is 24 hours; Step 3, separation and purification.

6. The method for preparing the triple helical collagen composition having thermal stability and promoting self-collagen production according to claim 5, characterized in that: In step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole; The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide; The phosphate buffer is prepared using the following raw materials in parts by weight: 0.2 parts of potassium chloride, 1.15 parts of sodium dihydrogen phosphate, 0.2 parts of potassium dihydrogen phosphate, 1000 parts water; The tripeptide solution is prepared using raw materials in the following proportions: Phosphate buffer 500 parts by volume, 50 parts by weight of tripeptide, 5 parts by weight of condensation aid; The dehydration condensation agent solution is prepared using raw materials in the following proportions: Phosphate buffer 500 parts by volume, 158 parts by weight of dehydration condensation agent.

7. The method for preparing the triple helical collagen composition having thermal stability and promoting self-collagen production according to claim 5, characterized in that: In step 2, in the dehydration condensation agent solution and the tripeptide solution, the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent is 50:5:158; The polycondensation reaction time is 24 hours.

8. The method for preparing the triple helical collagen composition having thermal stability and promoting self-collagen production according to claim 5, characterized in that: In step 3, the specific steps of separation and purification are: cyclic grinding with a homogenizer, filtering through a 50,000 molecular weight cutoff membrane to obtain a solution containing the triple helical collagen.

9. Use of the triple-helix collagen composition having thermal stability and promoting autologous collagen production according to any one of claims 1 to 4 in the preparation of cosmetics, cosmetic materials or medical devices.

10. The use according to claim 9, characterized in that: The cosmetics are functional skin care products; The medical device is a filling material or an engineering scaffold for skin, bones, and ligaments.

11. The use according to claim 10, characterized in that: The functional skin care product is a facial mask, an essence or a facial cleanser.

12. A cosmetic, characterized in that: The invention is prepared by using the triple helix collagen composition with thermal stability and promoting self-collagen production as claimed in any one of claims 1 to 4 as an active ingredient and adding auxiliary materials.

13. The cosmetic according to claim 12, characterized in that: The concentration of the triple-helix collagen composition having thermal stability and promoting auto-collagen production is 0.0005 wt %-0.05 wt %.

14. The cosmetic according to claim 13, characterized in that: The concentration of the triple-helix collagen composition having thermal stability and promoting auto-collagen production is 0.005 wt %.

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