Triple helix collagen having thermal stability and promoting self-collagen generation, and preparation method therefor and use thereof

Abstract

A triple helix collagen having thermal stability and promoting self-collagen generation, and a preparation method therefor and the use thereof. The structural formula of the triple helix collagen is as 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 respectively and independently selected from proline residue, hydroxyproline residue or glycine residue. The weight-average molecular weight distribution of the triple helix collagen is 5.0×104-3.0×108. The collagen has a triple helix structure, has good thermal stability, has a relatively good anti-wrinkle and tightening effect and injection filling effect, can be better applied to the fields of cosmetics, medical instruments etc., and has good application prospects.

Description

A triple-helix collagen with thermal stability and promoting self-collagen production, as well as preparation method and use thereof Technical Field

[0001] The present invention belongs to the technical field of medical and cosmetic raw materials, and particularly relates to a triple-helix collagen protein that has thermal stability and promotes self-collagen production, as well as a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant functional protein in the human body, possessing a stable triple-helical network structure and possessing crucial physiological functions. Predominantly found in tissues such as skin, bones, and ligaments, it maintains the stability and normal function of these tissues and organs. In recent years, advances in science and technology have led to the application of artificial collagen in numerous fields, including cosmetics, plastic surgery, and medical materials.

[0003] In the beauty industry, collagen is widely used in skincare and cosmetics to improve skin moisture retention, elasticity, and radiance. Many functional skincare products, facial masks, serums, and cleansers contain collagen. Collagen replenishes nutrients needed by the skin, improves the living environment of skin cells, and promotes skin tissue metabolism, thereby delaying aging and nourishing the skin. Most collagen skincare products currently on the market are manufactured from high-content collagen raw materials, which are then reduced to smaller molecules through technological means for easier skin absorption. Collagen also has a wide range of applications in the medical plastic surgery industry and medical materials. For example, collagen is used as a key biofiller in facial augmentation, anti-aging plastic surgery, and skin repair. It can also be used clinically to treat pathological changes such as burns and trauma, improving skin condition. In tissue engineering, collagen, as a biodegradable material, can be used to create various tissue engineering scaffolds to support cell growth and tissue repair. Furthermore, due to its excellent biocompatibility and biodegradability, it can also be used to manufacture medical devices such as medical sutures and skin grafts. As a core component of regenerative medicine, biomimetic materials have greatly enriched disease treatment options. Collagen, which accounts for over 30% of the body's total protein content, can be used to develop organoids and tissues, including artificial skin, artificial bone, artificial cornea, artificial tendons and ligaments, and artificial cartilage. These developments have achieved varying degrees of success. Collagen offers advantages in cell adhesion, proliferation, biocompatibility, and bioabsorbability, and is a biomaterial with sufficient mechanical strength. It is an ideal choice for medical treatments involving direct contact with cells and tissues. It can also be used as a cell material or as a key technology for the large-scale production of cell therapy drugs and cell tissue sheets. The market is expected to expand significantly in the future. Furthermore, further research and practical application results and case studies are expected for collagen in the regeneration and repair of tissues and organs in the elderly. Therefore, by integrating industry and hospitals, research is being conducted on the clinical value and application scenarios of biomimetic biomaterials, thereby promoting the high-quality development of the biomimetic biomaterials industry, which possesses both practical clinical value and innovative industrial value.

[0004] Currently, artificial collagen is mainly divided into animal-derived collagen extracted from animal tissues such as cattle, pigs, chickens, and fish, and recombinant collagen produced through methods such as biological fermentation, purification, and gene editing. However, the production of artificial collagen presents several technical challenges. First, extracting collagen from animals may pose immune risks, such as the spread of viruses and diseases, such as mad cow disease. Second, because the amino acid sequence of animal-derived collagen differs from that of human collagen, sufficient biological activity, stability, and safety cannot be guaranteed. While recombinant collagen can guarantee a more complete amino acid sequence, the process is cumbersome and costly, and it is not possible to determine whether it possesses a complete triple helical structure, raising questions about its biological activity.

[0005] However, there have been no significant breakthroughs in the chemical synthesis of collagen-like proteins. First, while the synthesis of peptides that mimic the amino acid sequence of human collagen is theoretically feasible, correctly folding these protein molecules into an active triple-helical structure remains a key technical challenge. Furthermore, the production of collagen with a triple-helical structure that exhibits excellent thermal stability is also a major challenge.

[0006] In the prior art, collagen-like polypeptide molecules can be synthesized using a polypeptide synthesizer. A polypeptide synthesizer is an automated laboratory device specifically designed for synthesizing polypeptide chains in vitro. It is based on solid phase peptide synthesis (SPPS) technology, and by precisely controlling the reaction conditions and steps, it can efficiently construct polypeptide chains consisting of several amino acids to hundreds of amino acids. However, experimental studies have shown that polypeptide molecules prepared using a polypeptide synthesizer are difficult 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 withstand ambient temperature or undergo 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.

[0008] Summary of the Invention

[0009] In view of the problems of the prior art, the present invention provides a triple-helical collagen protein with thermal stability and the ability to promote self-collagen production, as well as a preparation method and use thereof.

[0010] A triple-helical collagen protein with thermal stability and promoting self-collagen production, the structural formula of which is shown in Formula I:

[0011] Formula I: H2N-(XYZ) n -COOH;

[0012] 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

[0013] X, Y, Z are selected from one of the following combinations:

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

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

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

[0017] X is a proline residue, Y is a glycine residue, and Z is a hydroxyproline residue.

[0018] Preferably, the value of n satisfies 1.05×10 6 ≧n≧175.

[0019] 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.

[0020] The present invention also provides a method for preparing the triple-helical collagen having thermal stability and promoting self-collagen production, comprising the following steps:

[0021] 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;

[0022] The phosphate buffer solution is prepared using the following raw materials in parts by weight:

[0023] 0.18-0.22 parts of potassium chloride,

[0024] 1.0-1.2 parts of sodium dihydrogen phosphate,

[0025] 0.18-0.22 parts of potassium dihydrogen phosphate,

[0026] 995-1005 parts of water;

[0027] The tripeptide solution is prepared using the following raw materials:

[0028] 495-505 parts by volume of phosphate buffer,

[0029] 49.5-50.5 parts by weight of tripeptide,

[0030] 4.9-5.1 parts by weight of condensation aid;

[0031] The dehydration condensation agent solution is prepared using the following raw materials:

[0032] 495-505 parts by volume of phosphate buffer,

[0033] 156-160 parts by weight of a dehydration condensation agent;

[0034] 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;

[0035] Step 3, separation and purification to obtain.

[0036] Preferably, in step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole and 1-hydroxy-7-azabenzotriazole;

[0037] The dehydration condensation agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-glutarimide and 1,3-dicyclohexylcarbodiimide;

[0038] The phosphate buffer solution is prepared using the following raw materials in parts by weight:

[0039] 0.2 parts of potassium chloride,

[0040] 1.15 parts of sodium dihydrogen phosphate,

[0041] 0.2 parts of potassium dihydrogen phosphate,

[0042] 1000 parts water;

[0043] The tripeptide solution is prepared using the following raw materials:

[0044] 500 parts by volume of phosphate buffer,

[0045] 50 parts by weight of tripeptide,

[0046] 5 parts by weight of condensation aid;

[0047] The dehydration condensation agent solution is prepared using the following raw materials:

[0048] 500 parts by volume of phosphate buffer,

[0049] 158 parts by weight of dehydration condensation agent.

[0050] Preferably, in step 2, the weight ratio of the tripeptide, the condensation aid and the dehydration condensation agent in the dehydration condensation agent solution and the tripeptide solution is 50:5:158;

[0051] The polycondensation reaction time is 24 hours.

[0052] Preferably, in step 3, the specific steps of separation and purification are: cyclic grinding with a homogenizer, filtering through a 50,000 molecular weight cut-off membrane to obtain a solution containing the triple helical collagen.

[0053] The present invention also provides the use of the triple-helix collagen protein having thermal stability and promoting self-collagen production in the preparation of cosmetics, medicines, cosmetic and plastic surgery materials or medical devices.

[0054] Preferably, the cosmetics are functional skin care products;

[0055] The medical device is a filling material or an engineering scaffold for skin, bones, and ligaments.

[0056] Preferably, the functional skin care product is a facial mask, essence or facial cleanser.

[0057] The present invention also provides a cosmetic, which is prepared by using the triple helical collagen protein having thermal stability and promoting self-collagen production as an active ingredient and adding auxiliary materials.

[0058] Preferably, the concentration of the triple helical collagen protein having thermal stability and promoting self-collagen production is 0.0005 wt%-0.05 wt%.

[0059] Preferably, the concentration of the triple helical collagen protein having thermal stability and promoting self-collagen production is 0.005 wt%.

[0060] The "triple helical collagen" provided by the present invention refers to a protein having a tripeptide repeating unit and a triple helical structure. It is a mixture of proteins with different degrees of polymerization with a tripeptide repeating unit as the repeating structure. The "triple helical structure" is the basic structure of natural collagen, which is a triple helical structure formed by three polypeptide chains entangled with each other. The method for detecting whether a protein or polymer has a triple helical structure similar to that of natural collagen is to use circular dichroism spectroscopy to detect whether the polymer has a positive peak at 220-230nm. The ratio of "parts by volume" to "parts by weight" is determined as follows:

[0061] 1 part by volume: 1 part by weight = 1 ml: 1 g.

[0062] 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 temperatures exceeding 40°C and cannot maintain the triple helix structure. They cannot be used as raw materials in cosmetics (except for cosmetics used in refrigerators). In addition, if used as medical devices, they cannot be heated and sterilized (above 60°C) or disinfected (autoclave). The collagen polymer (polypeptide chain) provided by the present invention can maintain the triple helix structure under normal temperature and high temperature (40-60°C) conditions, and its thermal stability is better than that of existing similar synthetic collagen products, making it better for use 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%.

[0063] Therefore, the collagen polymer of the present invention has good application prospects.

[0064] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0065] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 is a circular dichroism spectrum of collagen after heat treatment at 20-60° C. in Experimental Example 1;

[0067] FIG2 is a circular dichroism spectrum of the commercial polypeptide in Experimental Example 1 after heat treatment at 20-60° C.;

[0068] FIG3 is a graph showing the molecular weight test results of collagen in Experimental Example 1;

[0069] FIG4 is a graph showing the experimental results of the relative expression of the Collagen I gene in Experimental Example 2;

[0070] FIG5 is a graph showing the experimental results of the relative expression of Collagen III gene in Experimental Example 2;

[0071] Figure 6 is a schematic diagram of the drug administration area of ​​mice in Experimental Example 3;

[0072] Figure 7 shows the imaging results of Experimental Example 3 after one week of administration, wherein A is the experimental group injected with 0.5wt.% collagen, 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 control group injected with PBS;

[0073] Figure 8 shows the imaging results after 3 weeks of administration in Experimental Example 3, wherein A is the experimental group injected with 0.5wt.% collagen, B is the experimental group injected with 0.6wt.% low-concentration recombinant humanized type III collagen, C is the experimental group injected with 3wt.% high-concentration recombinant humanized type III collagen, and D is the control group injected with PBS. DETAILED DESCRIPTION

[0074] The reagents and raw materials used in the following examples and experimental examples are all commercially available unless otherwise specified.

[0075] Example 1: Triple Helical Collagen with Thermal Stability and Promoting Self-Collagen Production. This example provides a triple helical collagen with thermal stability and promoting 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] Wherein X, Y and Z 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 of 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 molecular weight.

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

[0083] 1. Preparation of phosphate buffer

[0084] To 1000 ml of high-purity water, 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), 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 1-hydroxybenzotriazole (HOBt) as a condensation aid were added to 500 ml of the 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 and cooled to 2°C in a thermostatic bath. EDC solution, which had also been cooled to 2°C, was then gradually added (the volume ratio of POG solution to EDC solution was 1:1). The reaction temperature was maintained below 20°C, and the polycondensation reaction was carried out for 24 hours. The reaction solution was transferred to a 10L container, circulated and ground using a homogenizer, and filtered through a 50,000 molecular weight cutoff membrane (Asahi Kasei AHP-1013D) using a rotary pump 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 protein and its preparation method in this embodiment are the same as those in Example 1, except that:

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

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

[0095] The triple helical collagen protein and its preparation method in this embodiment are the same as those in Example 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 promotion of self-collagen production

[0098] The triple helical collagen protein and its preparation method in this embodiment are the same as those in Example 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 a reference substance, and its molecular formula is H2N-(POG) 10 -COOH (purchased from Anhui Guoping Pharmaceutical Co., Ltd.) The amino acid sequence of this reference substance is similar to that of the sample prepared in Example 1. The main difference between this reference substance and the sample prepared in Example 1 is that this reference substance is prepared using a peptide synthesizer.

[0113] The triple-helical collagen from Example 1 or a commercially available peptide reference substance was prepared as a 0.25 mg / ml aqueous solution and heat-treated at 20°C, 40°C, and 60°C for 1 hour. 5 ml of each solution was sampled and analyzed on a circular dichroism spectrometer to confirm the presence of a positive peak unique to triple-helical collagen.

[0114] 2. Molecular weight characterization

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

[0116] 2. Experimental Results

[0117] 1. Circular dichroism spectroscopy characterization

[0118] The characterization results of triple-helical collagen are shown in Figure 1. As can be seen from the figure, after treatment at 20°C, 40°C, and 60°C, positive peaks in the 220-230nm range were detected in all three groups of samples, and the peak heights remained almost unchanged, indicating that triple-helical collagen can remain stable within 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 within the temperature range of 20°C-60°C.

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

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

[0121] 2. Molecular weight characterization

[0122] As shown in FIG3 , the results show that 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 greatest threat to skin aging, primarily caused by environmental stressors such as UV radiation, pollution, smog, and lifestyle stress, with UV radiation being the most prominent. When skin is exposed to UV radiation, excessive reactive oxygen species are produced within cells, triggering the expression of aging-related genes, inducing an inflammatory cascade, and reducing the expression of elastin and collagen, leading to photoaging phenomena such as sagging and wrinkles. Photoaging primarily occurs in the dermis.

[0126] This test uses fibroblasts as the test system and evaluates whether the test sample 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 (Solebro), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), and cell lysis buffer (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, 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. Among them, the blank control was not treated with drug and 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 0.0005 wt%, 0.005 wt%, and 0.05 wt%, respectively.

[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 a 37°C, 5% CO 2 ) and incubated overnight in DMEM culture medium.

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

[0138] 3) Radiation: 24 hours after the completion of the drug 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) Cell collection: After 24 h of incubation, add 0.5 mL of lysis buffer to each well and incubate at room temperature for 5 min to allow for complete lysis. Transfer the cells to a 1.5 mL RNase-free Eppendorf tube and extract RNA according to the protocol.

[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 Collagen I gene content detection are shown in Table 1 and Figure 4:

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

[0145] Note: When using the t-test method for statistical analysis, the significance of the comparison between the NC group and 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 comparison between the sample group and the PC group and the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0146] 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.

[0147] 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.

[0148] Compared with the NC group, the expression of Collagen I gene in fibroblasts was significantly increased at the dose concentrations of 0.0005wt%, 0.005wt%, and 0.05wt% (p<0.05). In particular, the 0.005wt% dose had the best effect in increasing Collagen I gene expression in fibroblasts, and the effect was significantly better than that in the PC group.

[0149] The results of Collagen III gene content detection are shown in Table 2 and Figure 5:

[0150] Table 2 Summary of relative expression data of Collagen III genes

[0151] Note: When using the t-test method for statistical analysis, the significance of the comparison between the NC group and 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 comparison between the sample group and the PC group and the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0152] 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.

[0153] 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.

[0154] Compared with the NC group, the expression of Collagen III genes in fibroblasts was significantly increased at concentrations of 0.0005wt%, 0.005wt%, and 0.05wt% (p<0.05). In particular, the 0.005wt% concentration showed the best effect in increasing Collagen III gene expression in fibroblasts, and the effect was comparable to that in the PC group.

[0155] 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 Collagen I gene and Collagen III gene in fibroblasts, and has anti-wrinkle and firming effects.

[0156] Experimental Example 3: Verification of injection effect

[0157] 1. Experimental Purpose

[0158] In the field of medical aesthetics, injectable collagen products can provide benefits such as skin filling and firming, contouring, moisturizing and repairing, promoting wound healing, and improving skin texture. Two-photon imaging technology uses second harmonic generation (SHG) imaging to observe collagen and muscle fibers in living animals (mice) without staining. Therefore, two-photon imaging can be used to observe the presence of collagen products in animals after subcutaneous injection, thereby examining the effectiveness of collagen injections.

[0159] 2. Experimental Methods

[0160] As shown in Figure 6, the dosing area of ​​the mouse back skin was set up, with 4 injection areas on the back of each mouse. According to whether collagen was injected, 6-week-old male BALB / c mice were 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 was hyaluronic acid injection, with 0.2ml administered to each injection area (1*1cm). The injection layers were the superficial dermis and the deep dermis. There were 12 mice in each group, and two-photon imaging was performed 1 week and 3 weeks after injection.

[0161] 3. Experimental Results

[0162] The imaging results 1 week and 3 weeks after injection are shown in Figures 7 and 8, respectively. As can be seen from the figures, one week after injection, collagen accumulation can be observed in the experimental groups, and the amount of collagen in the 0.5wt.% triple helical 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, further accumulation of collagen can be observed in each experimental group, indicating that collagen injection can promote the regeneration of the mouse's own collagen. At the same time, similar to the experimental data 1 week after injection, the amount of collagen in the 0.5wt.% triple helical 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.

[0163] The above experimental results show that the triple helix collagen of the present invention can more effectively promote the regeneration of one's own collagen than the recombinant collagen in the prior art, and thus has a better injection effect.

[0164] Through the above embodiments and experimental examples, it can be seen that the present invention provides a triple-helix collagen protein with thermal stability and promoting self-collagen production. The triple-helix collagen protein has the same triple-helix structure as natural collagen protein, 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. It has good thermal stability and can be better applied in cosmetics, medical devices and other fields, and has good application prospects.

Claims

1. A triple helical collagen protein having thermal stability and promoting self-collagen production, characterized in that: Its structural formula is shown in Formula I: Formula I: H2N-(X-Y-Z) n -COOH; 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 one of the following combinations: X is a glycine residue, Y is a hydroxyproline residue, and Z is a proline residue; X is a hydroxyproline residue, Y is a proline residue, and Z is a glycine residue; X is a proline residue, Y is a hydroxyproline residue, and Z is a glycine residue; X is a proline residue, Y is a glycine residue, and Z is a hydroxyproline residue.

2. The triple-helical collagen protein having thermal stability and promoting self-collagenization according to claim 1, characterized in that: The value of n satisfies 1.05×10 6 ≧n≧175.

3. The triple-helical collagen protein having thermal stability and promoting autocollagenization according to claim 1, characterized in that: The triple helix collagen is obtained by condensing a tripeptide with a structural formula of H2N-XYZ-COOH as a raw material through a liquid phase reaction.

4. The method for preparing triple-helical collagen having thermal stability and promoting self-collagen production according to any one of claims 1 to 3, 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; The phosphate buffer solution is prepared using the following raw materials in parts by weight: 0.18-0.22 parts of potassium chloride, 1.0-1.2 parts of sodium dihydrogen phosphate, 0.18-0.22 parts of potassium dihydrogen phosphate, 995-1005 parts of water; The tripeptide solution is prepared using the following raw materials: 495-505 parts by volume of phosphate buffer, 49.5-50.5 parts by weight of tripeptide, 4.9-5.1 parts by weight of condensation aid; The dehydration condensation agent solution is prepared using the following raw materials: 495-505 parts by volume of phosphate buffer, 156-160 parts by weight of a 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 20-26 hours; Step 3: separation and purification to obtain.

5. The method for preparing triple-helical collagen having thermal stability and promoting self-collagenization according to claim 4, characterized in that: In step 1, the condensation auxiliary agent is selected from at least one of 1-hydroxybenzotriazole and 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 solution 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 the following raw materials: 500 parts by volume of phosphate buffer, 50 parts by weight of tripeptide, 5 parts by weight of condensation aid; The dehydration condensation agent solution is prepared using the following raw materials: 500 parts by volume of phosphate buffer, 158 parts by weight of dehydration condensation agent.

6. The method for preparing triple-helical collagen having thermal stability and promoting autocollagen formation according to claim 4, characterized in that: In step 2, the weight ratio of the tripeptide, condensation aid and dehydration condensation agent in the dehydration condensation agent solution and the tripeptide solution is 50:5:158; The polycondensation reaction time is 24 hours.

7. The method for preparing triple helical collagen with thermal stability and promoting self-collagen production according to claim 4, 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 cut-off membrane to obtain a solution containing the triple helical collagen.

8. Use of the triple-helical collagen protein having thermal stability and promoting endogenous collagen production according to any one of claims 1 to 3 in the preparation of cosmetics, medicines, cosmetic and plastic surgery materials or medical devices.

9. The use according to claim 8, 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.

10. The use according to claim 9, characterized in that: The functional skin care product is a facial mask, essence or facial cleanser.

11. A cosmetic, characterized in that: The invention is prepared by using the triple helix collagen with thermal stability and the ability to promote self-collagen production as claimed in any one of claims 1 to 3 as an active ingredient and adding auxiliary materials.

12. The cosmetic according to claim 11, characterized in that: The concentration of the triple helical collagen protein having thermal stability and promoting self-collagen production is 0.0005 wt%-0.05 wt%.

13. The cosmetic according to claim 12, characterized in that: The concentration of the triple helical collagen protein having thermal stability and promoting self-collagen production is 0.005 wt %.

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