Triple-helix collagen with thermal stability as well as preparation method and application of triple-helix collagen

Abstract

The invention belongs to the technical field of medical and cosmetic raw materials, and particularly relates to triple-helix collagen with thermal stability and a preparation method and application thereof. The structural formula of the triple helix collagen is as shown in a formula I, wherein the formula I is H2N-(G-P-P) n-COOH; wherein G-P-O is a peptide chain composed of three different amino acid residues; g is glycine residue, P is proline residue, and O is hydroxyproline residue. The weight-average molecular weight distribution is 5.0 * 10 < 3 >-1.5 * 10 < 7 >. The triple-helix collagen has a triple-helix structure, is good in thermal stability, has better effects of resisting wrinkles, tightening skin, repairing skin, removing freckles and whitening skin, can be better applied to the fields of cosmetics, medical instruments and the like, and is good in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medical and cosmetic raw materials, and particularly relates to a collagen with thermal stability, a preparation method thereof, and uses thereof. Background Art

[0002] Collagen is the most abundant functional protein in the human body, and 70% of the human skin components are composed of collagen. The application scenarios of collagen are very rich, and the terminal applications involve fields such as medical devices, beauty skin care, and functional foods.

[0003] In the beauty field, collagen is widely used in skin care products and cosmetics to improve the water retention capacity, elasticity, and glossiness of the skin, etc. Many functional skin care products, facial masks, essence liquids, facial cleansers, etc. are added with collagen. Collagen can supplement the nutrients required by the skin, improve the survival environment of skin cells, and promote the metabolism of skin tissues, thereby achieving the effects of delaying aging and nourishing the skin. Most of the current collagen skin care products on the market are prepared with high-content collagen raw materials and made into small molecules by scientific and technological means for easier skin absorption. In the medical plastic surgery industry and the field of medical materials, collagen also has a wide range of applications. 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 for clinical treatment of pathological changes such as skin burns and wounds to improve the skin condition. In tissue engineering, collagen, as a biodegradable material, can be used to manufacture various tissue engineering scaffolds for supporting cell growth and tissue repair. At the same time, due to its good biocompatibility and biodegradability, it can also be used for the preparation of medical devices such as medical sutures and skin grafts. In the field of bionics, it has great application prospects for treating diseases such as osteoporosis and bone defects with bone tissue engineering scaffolds, and as artificial blood vessels and flexible materials, etc.

[0004] Currently, the mainstream methods for extracting and preparing collagen include three categories: animal source extraction, genetic engineering method, and synthetic collagen. The extraction of animal source collagen is mainly carried out by acid method and enzyme method for industrial production, with relatively lower costs, but there are problems such as animal source disease infection, possible immune rejection or allergic reactions caused by allogeneic collagen, and production capacity limitations.

[0005] The production of recombinant collagen by genetic engineering technology is a hot topic in the industry. Recombinant collagen is produced by genetic engineering technology. The human collagen gene is subjected to specific sequence design, enzymatic digestion and splicing, and then ligated to a vector and transferred into engineering cells. Collagen is produced through fermentation expression. The products obtained by this method have the advantages of good safety, strong processability, and stable quality. However, the technical difficulties lie in how to achieve high-density expression of bacterial strains and yeast strains, ensure biological activity, and separate and purify recombinant proteins. The biggest difficulty is that a stable triple helix structure cannot be formed. There has been no major breakthrough in the chemically synthesized collagen-like peptides. First, although the synthesis of polypeptides mimicking the amino acid sequence of human collagen can be theoretically achieved, how to correctly fold these protein molecules to form an active triple helix structure is a key technical difficulty. In addition, how to prepare collagen with an excellent thermostable triple helix structure is also one of the major challenges currently faced.

[0006] In the prior art, a peptide synthesizer can be used to synthesize polypeptide molecules of collagen-like peptides. A peptide synthesizer is an automated laboratory device specifically designed for synthesizing polypeptide chains in vitro. It is based on solid-phase peptide synthesis (SPPS) technology. By precisely controlling reaction conditions and steps, it can efficiently construct polypeptide chains composed of several amino acids to hundreds of amino acids. However, experimental studies have shown that the polypeptide molecules prepared using a peptide 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 is a certain requirement for tolerating environmental temperature or performing high-temperature disinfection. The unstable triple helix structure is difficult to maintain at a higher temperature, which causes 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] Aiming at the problems of the prior art, the present invention provides a thermostable triple helix collagen, its preparation method and uses.

[0009] A thermostable triple helix collagen, its structural formula is shown in Formula I:

[0010] Formula I: H2N-(G-P-О) n -COOH;

[0011] Wherein, G-P-О is a peptide chain composed of three different amino acid residues; G is a glycine residue, P is a proline residue, and О is a hydroxyproline residue; the weight-average molecular weight distribution of the triple helix collagen is 5.0×103 ~1.5×10 7 。

[0012] Preferably, the value of n satisfies 5.26×10 4 ≧n≧17.

[0013] Preferably, the triple-helix collagen is obtained by condensation through a liquid-phase reaction using a tripeptide with the structural formula H2N-G-P-О-COOH as a raw material.

[0014] The present invention also provides a method for preparing the above triple-helix collagen with thermal stability, comprising the following steps:

[0015] Step 1, preparing a tripeptide solution by using a tripeptide with the structural formula H2N-G-P-О-COOH and a condensation assistant with phosphate buffer solution as a solvent; preparing a dehydrating condensing agent solution by using a dehydrating condensing agent with phosphate buffer solution as a solvent;

[0016] Among them, the phosphate buffer solution is prepared with the following raw materials in parts by weight:

[0017] Potassium chloride 0.18 - 0.22 parts,

[0018] Sodium dihydrogen phosphate 1.0 - 1.2 parts,

[0019] Potassium dihydrogen phosphate 0.18 - 0.22 parts,

[0020] Water 995 - 1005 parts;

[0021] The tripeptide solution is prepared with the following raw materials in the following ratio:

[0022] Phosphate buffer solution 495 - 505 volume parts,

[0023] Tripeptide 49.5 - 50.5 parts by weight,

[0024] Condensation assistant 4.9 - 5.1 parts by weight;

[0025] The dehydrating condensing agent solution is prepared with the following raw materials in the following ratio:

[0026] Phosphate buffer solution 495 - 505 volume parts,

[0027] Dehydrating condensing agent 156 - 160 parts by weight;

[0028] Step 2: Add the dehydrating condensing agent solution to the tripeptide solution for polycondensation reaction. In the polycondensation reaction, the weight ratio of tripeptide, condensation aid, and dehydrating condensing agent 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.

[0029] Step 3: Separate and purify to obtain the product.

[0030] Preferably, in Step 1, the condensation aid is selected from at least one of 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole.

[0031] The dehydrating condensing agent is selected from at least one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or 1,3-dicyclohexylcarbodiimide.

[0032] The phosphate buffer solution is prepared with the following raw materials by weight:

[0033] Potassium chloride 0.2 parts,

[0034] Sodium dihydrogen phosphate 1.15 parts,

[0035] Potassium dihydrogen phosphate 0.2 parts,

[0036] Water 1000 parts;

[0037] The tripeptide solution is prepared with the following raw materials in the following ratio:

[0038] Phosphate buffer solution 500 volume parts,

[0039] Tripeptide 50 weight parts,

[0040] Condensation aid 5 weight parts;

[0041] The dehydrating condensing agent solution is prepared with the following raw materials in the following ratio:

[0042] Phosphate buffer solution 500 volume parts,

[0043] Dehydrating condensing agent 158 weight parts.

[0044] Preferably, in Step 2, in the dehydrating condensing agent solution and the tripeptide solution, the weight ratio of tripeptide, condensation aid, and dehydrating condensing agent is 50:5:158.

[0045] The time of the polycondensation reaction is 24 hours.

[0046] Preferably, in Step 3, the specific steps of the separation and purification are: Circulating and grinding with a homogenizer, and filtering through a 50,000 molecular weight cut-off membrane to obtain a solution containing the triple helix collagen.

[0047] The present invention also provides the use of the above-mentioned triple-helix collagen with thermal stability in the preparation of cosmetics, drugs, beauty and plastic surgery materials or medical devices.

[0048] Preferably, the cosmetic is a functional skin care product;

[0049] The medical device is a filling material or engineering scaffold for skin, bone, and ligament.

[0050] Preferably, the functional skin care product is a facial mask, essence or facial cleanser.

[0051] The present invention also provides a cosmetic, which is prepared by using the above-mentioned triple-helix collagen with thermal stability as an active ingredient and adding excipients.

[0052] Preferably, the concentration of the triple-helix collagen with thermal stability is 0.0005 wt% - 0.05 wt%.

[0053] Preferably, the concentration of the triple-helix collagen with thermal stability is 0.0005 wt%.

[0054] The "triple-helix collagen" provided by the present invention refers to a protein having a tripeptide repeat unit and a triple-helix structure, which is a mixture of proteins with different degrees of polymerization having a tripeptide repeat unit as a repeating structure. The "triple-helix structure" is the basic structure of natural collagen, which is a triple-helix structure formed by the intertwining of three polypeptide chains. 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 spectroscopy to detect whether there is a positive peak at 220 - 230 nm for the polymer. The proportional relationship between "parts by volume" and "parts by weight" is determined as follows:

[0055] 1 part by volume: 1 part by weight = 1 ml: 1 g.

[0056] The present invention provides a new collagen polymer with thermal stability, which is a polypeptide chain synthesized in liquid phase and obtained by polycondensation with three amino acids as a unit. Existing collagen products will gel at temperatures above 40°C and cannot maintain the triple helix structure. In cosmetics (except for those used under refrigeration), they cannot be used as raw materials. In addition, if used as medical devices, they cannot be heat sterilized (above 60°C) or disinfected (autoclave). The collagen polymer (polypeptide chain) provided by the present invention can maintain the triple helix structure at normal and high temperatures (40 - 60°C), and its thermal stability is superior to existing similar synthetic collagen products, enabling it to be better applied in cosmetics or medical devices. In addition, the collagen polymer provided by the present invention can promote the increased expression of Elastin protein in fibroblasts, has anti-wrinkle and firming effects, and has the best effect when the concentration is 0.005wt%. In addition, the collagen polymer of the present invention also has the effects of repairing the skin and removing spots and whitening the skin.

[0057] Therefore, the collagen polymer of the present invention has good application prospects.

[0058] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0059] The following is a further detailed description of the above content of the present invention 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 belong to the scope of the present invention. Description of the Drawings

[0060] Figure 1 It is the circular dichroism spectrum diagram of triple helix collagen after heat treatment at 20 - 60°C in Experimental Example 1;

[0061] Figure 2 It is the circular dichroism spectrum diagram of commercialized polypeptide after heat treatment at 20 - 60°C in Experimental Example 1;

[0062] Figure 3 It is the molecular weight test result diagram of triple helix collagen in Experimental Example 1;

[0063] Figure 4 It is the experimental result diagram of the relative expression level of Elastin protein in Experimental Example 2;

[0064] Figure 5 It is the experimental result diagram of detecting the expression level of elastin in dermal fibroblasts HSF by cell immunofluorescence in Experimental Example 3;

[0065] Figure 6 It is the experimental result graph for detecting the relative content of melanin in B16 melanoma cells in Experimental Example 4;

[0066] Figure 7 It is the experimental result graph for detecting the activity of tyrosinase that promotes melanin production in B16 melanoma cells in Experimental Example 4;

[0067] Figure 8 It is the experimental result graph for detecting the expression of FLG gene in keratinocyte HaCaT in Experimental Example 5.

[0068] In the above experimental data graphs, **: corresponding to a significant level of 1%, that is, p < 0.01 for this experimental group compared to the control group; ***: corresponding to a significant level of 0.1%, that is, p < 0.001 for this experimental group compared to the control group. Detailed implementation manners

[0069] The reagents and raw materials used in the following examples and experimental examples are all commercially available products unless otherwise specified.

[0070] Example 1 Triple-helix collagen with thermal stability

[0071] This example provides triple-helix collagen with thermal stability, which is a protein with a triple-helix structure, and its structural formula is as follows:

[0072] H2N-(G-P-О) n -COOH

[0073] G, P, and O in the formula are composed of the following amino acid residues:

[0074] P: proline,

[0075] O: hydroxyproline,

[0076] G: glycine;

[0077] n ≧ 4. Since the triple-helix collagen with thermal stability in this example is a mixture of molecules with different degrees of polymerization, there are multiple possible values for n, and its range can be estimated according to the characterization of the molecular weight.

[0078] The preparation method of the above triple-helix collagen with thermal stability is as follows:

[0079] 1. Preparation of phosphate buffer

[0080] Add 0.2 g of potassium chloride (KCl), 1.15 g of sodium phosphate dibasic (Na2HPO4), and 0.2 g of potassium phosphate monobasic (KH2PO4) to 1000 ml of high-purity water, and prepare phosphate buffer (Buffer-PB, hereinafter referred to as PB solution) after stirring.

[0081] 2. Preparation of Tripeptide Solution

[0082] Add 50 g of tripeptide G-P-О (manufactured by Uniqs Inc.) and 5 g of condensation aid 1-hydroxybenzotriazole (HOBt) to 500 ml of PB solution, and prepare a tripeptide solution (hereinafter referred to as GPO solution) after stirring.

[0083] 3. Preparation of Dehydrating Condensing Agent Solution

[0084] Add 158 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to 500 ml of PB solution, and prepare a dehydrating condensing agent solution (hereinafter referred to as EDC solution) after stirring.

[0085] 4. Polycondensation Reaction

[0086] Pour the prepared GPO solution into a 2 L separatory funnel, cool it to 2 °C in a constant temperature bath, and then gradually add the EDC solution cooled to 2 °C (the volume ratio of GPO solution to EDC solution is 1:1). Control the reaction temperature to be kept below 20 °C and carry out a polycondensation reaction for 24 hours. Transfer the reaction solution to a 10 L container, circulate and grind it with a homogenizer, and filter it with a rotary pump through a 50,000 molecular weight cut-off membrane (Asahi Kasei AHP-1013D) to obtain an aqueous solution containing the target product, thermally stable triple-helix collagen.

[0087] Example 2 Moisturizing Lotion and Emulsion for Anti-Wrinkle and Firming

[0088] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0089] 0.0005 wt% of triple-helix collagen prepared in Example 1,

[0090] 10 wt% of glycerol,

[0091] 15 wt% of polyethylene glycol,

[0092] 5 wt% of hyaluronic acid,

[0093] The rest is water.

[0094] The technical solution of the present invention will be further described through experiments below.

[0095] Experimental Example 1 Characterization of the Product

[0096] I. Experimental Method

[0097] This experimental example characterizes the triple-helix collagen prepared in Example 1, including:

[0098] 1. Circular Dichroism Spectroscopy Characterization

[0099] A commercially available polypeptide was selected as a reference substance, with the molecular formula H2N-(P-O-G) 10 -COOH (purchased from Guoping Pharmaceutical Co., Ltd., Anhui Province). The amino acid sequence of this reference substance is similar to that of the sample prepared in Example 1. The main difference between it and the sample prepared in Example 1 is that it is prepared using a polypeptide synthesizer.

[0100] The triple-helix collagen of Example 1 or the commercially available polypeptide reference substance was prepared into an aqueous solution with a concentration of 0.25 mg / ml, and heat treatment was carried out at 20 °C, 40 °C, and 60 °C for 1 hour. Take 5 ml of each solution and analyze it on a circular dichroism spectrometer. Confirm whether there is a positive peak unique to triple-helix structure collagen in each sample.

[0101] 2. Molecular weight characterization

[0102] It was measured using light scattering method. A Wyatt multi-angle light scattering detector DAWN HELEOS II and a Wyatt differential refractive index detector Optilab T-rEX were used, and a Shodex SB-806M column was adopted.

[0103] II. Experimental results

[0104] 1. Circular dichroism spectroscopy characterization

[0105] The characterization results of triple-helix collagen are as Figure 1 shown. It can be seen from the figure that after treatment at 20 °C, 40 °C, and 60 °C, positive peaks in the range of 220 - 230 nm can be detected in all three groups of samples, and the peak heights hardly change, indicating that triple-helix collagen can remain stable in the temperature range of 20 °C - 60 °C. This shows that the triple-helix collagen provided by the present invention can form a stable triple-helix structure in the temperature range of 20 °C - 60 °C.

[0106] The characterization results of the commercially available polypeptide reference substance are as Figure 2 shown. It can be seen from the figure that after treatment at 20 °C, 40 °C, and 60 °C, certain positive peaks can also be detected in the range of 220 - 230 nm, indicating that the commercially available polypeptide can also form a triple-helix structure. However, as the temperature increases, the height of the positive peak in the range of 220 - 230 nm decreases, indicating that the triple-helix structure formed by this commercially available polypeptide molecule is prone to disappear under heat treatment conditions, and its thermal stability of the triple-helix structure is poor.

[0107] The experimental results of this experiment show that for polypeptides, whether they can form a stable triple-helix structure is related to their preparation method and amino acid sequence, and the triple-helix collagen prepared by the liquid-phase synthesis method provided by the present invention can meet the requirements for forming a stable triple-helix structure.

[0108] 2. Molecular weight characterization

[0109] As Figure 3 shown, the results show that the weight-average molecular weight Mw of the triple-helix collagen prepared in Example 1 is 5.0×10 3 ~1.5×10 7 (g / mol).

[0110] Experimental Example 2 Detection of protein expression of anti-wrinkle and firming-related protein (elastin) promoting fibroblasts

[0111] I. Test purpose

[0112] Elastin is the main component of elastic fibers. Elastic fibers mainly exist in ligaments and blood vessel walls. Elastic fibers coexist with collagen fibers, endowing tissues with elasticity and tensile strength. Although elastin only accounts for 2% of the total dermal protein, it plays an important role in skin elasticity. Elastin is the main cause of skin aging, resulting in relaxation, sagging and fine wrinkles. As the skin naturally ages, the decomposition of elastic fibers becomes more and more significant. Elastin protein is a protein encoded by the ELN gene and is one of the main components of elastic fibers. Characterizing the expression of elastin and other elastic proteins can detect whether the product has anti-wrinkle and firming effects.

[0113] II. Experimental method

[0114] 1. Cell sample treatment

[0115] Adherent cells: After rinsing the cells twice with PBS, carefully scrape the cells with a cell scraper, centrifuge the culture medium at 3000 rpm for 10 min. Discard the supernatant and retain the cell pellet, and transport it on dry ice.

[0116] Suspension cells: Centrifuge the culture medium at 3000 rpm for 10 min. Discard the supernatant and retain the cell pellet, and transport it on dry ice.

[0117] Cell supernatant: Centrifuge the specimen at 3000 rpm for 15 min at 2-8°C to obtain the supernatant. The supernatant is immediately used for the experiment, or aliquoted and stored at -20°C or -80°C. Avoid repeated freezing and thawing.

[0118] 2. The specific test groups are set as follows:

[0119] Set up a blank control (BC), a negative control (NC), a positive control (PC) and three sample groups (0.0005%, 0.005%, 0.05%). Among them, the blank control is not treated with drug administration and radiation; the negative control is only irradiated with 9 J / cm 2 UVA; the positive control group is irradiated with 9 J / cm after drug administration2 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%.

[0120] 6. ELISA experimental steps

[0121] Follow the instructions of the kit. The operation of different indicators is different. Please refer to the instructions of the ordered kit for details. The basic steps are as follows:

[0122] 1) Coating: Dilute the antibody with carbonate coating buffer to a protein content of 1-10μg / ml. Add 100μl to each well of the polystyrene ELISA plate and incubate at 4℃ overnight. The next day, discard the solution in the wells and wash 3 times with washing buffer, 3 minutes each time. (Generally, the commercial kit has already coated the antibody, so this step can be omitted)

[0123] 2) Blocking: Add 200ul of blocking solution to each well and incubate at 37℃ for 1-2h.

[0124] 3) Washing: Carefully peel off the sealing film, put it into the plate washer, and wash it 3-5 times. You can also wash the plate manually: discard the liquid, add 300ul of washing solution to each well, soak for 1-2 minutes, pat dry on absorbent paper, and repeat 3-5 times. (Generally, the commercial kit has been coated with antibodies, and the first three steps can be omitted)

[0125] 4) Sample addition: Add 100 μl of appropriately diluted sample to the coated wells. (At the same time, make blank wells, standard wells with multiple dilutions, and negative control wells and positive control wells as quality control points if conditions permit).

[0126] 5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1-2 hours.

[0127] 6) Washing: Same as step 3.

[0128] 7) Add antibody: Add 100 μl of diluted biotinylated antibody working solution to each well.

[0129] 8) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour.

[0130] 9) Washing: Same as step 3.

[0131] 10) Add enzyme conjugate: Add 100 μl of diluted enzyme conjugate working solution to each well.

[0132] 11) Incubation: Seal the plate with a sealing film and incubate at 37°C in the dark for 30 min.

[0133] 12) Washing: Same as step 3.

[0134] 13) Add color substrate: Add 100 μl of TMB substrate solution to each well and react at 37°C in the dark for 10 to 30 minutes until a clear color gradient appears in the wells of the diluted standard.

[0135] 14) Termination of reaction: Add 100 μl of 2 M sulfuric acid to each reaction well, and the color changes from blue to yellow.

[0136] 15) Result determination: Within 10 minutes, measure the OD value of each well on a microplate reader at 450 nm after adjusting the blank control well to zero.

[0137] 3. Experimental Results

[0138] Elastin protein expression test results Figure 4 As shown:

[0139] 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 Elastin protein decreased significantly (p < 0.01), indicating that the radiation stimulation was effective.

[0140] Compared with the NC group, vitamin C and vitamin E in the PC group could significantly increase the expression of Elastin protein (p<0.01), indicating that the positive control test was effective.

[0141] Compared with the NC group, the Elastin protein expression of 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 0.0005wt%, the Elastin protein expression of fibroblasts was the best, and the increase effect was significantly better than that of the PC group.

[0142] When the t-test method was used for statistical analysis, the significance of the NC group compared with the BC group was indicated by #, p-value < 0.05 was indicated by #, and p-value < 0.01 was indicated by ##. The significance of the sample group and PC group compared with the NC group was indicated by *, p-value < 0.05 was indicated by *, and p-value < 0.01 was indicated by **.

[0143] The above experimental results show that the triple-helical collagen of the present invention (especially when the dosage concentration is 0.0005wt%) can promote the expression of Elastin protein in fibroblasts and has anti-wrinkle and firming effects.

[0144] Experimental Example 3 Detection of the Expression Level of Elastin in Dermal Fibroblasts HSF

[0145] I. Experimental Method

[0146] Add the sample to human skin fibroblasts for culture, and detect the effect of the sample on the expression of elastin in ESF cells by immunofluorescence cytochemistry technique.

[0147] 1. Sample Preparation

[0148] Three types of collagen ((GPO)n-1, (GPO)n-2, and (GPO)n-3) samples were used in this experimental example, and their preparation methods were as follows:

[0149] (GPO)n-1: Prepared according to the method of Example 1, except that the polycondensation reaction time was adjusted to 5 h;

[0150] (GPO)n-2: Prepared according to the method of Example 1;

[0151] (GPO)n-3: Prepared according to the method of Example 1, except that the polycondensation reaction time was adjusted to 36 h.

[0152] Dilute the sample with DMEM complete medium to the required detection concentration (V / V), and a total of 1 mL of sample working solution was prepared.

[0153] 2. Experimental Steps

[0154] (1) Take out the sterile cell slide and place it in the culture plate;

[0155] (2) Collect the ESF cell suspension with a confluence of 80% - 90% in a T25 flask, count, adjust the cell density and inoculate it in the culture plate, and culture it in a carbon dioxide incubator;

[0156] (3) Take out the culture plate, discard the culture solution in the wells, add the working solutions with different concentrations according to the test grouping list respectively, and continue to culture in a carbon dioxide incubator.

[0157] (4) Take out the culture plate, discard the culture solution in the wells, and add PBS for washing;

[0158] (5) Add 4% paraformaldehyde fixative to fix the cells, and add PBS for washing;

[0159] (6) Add 0.2% Triton X-100 solution for permeabilization treatment, and add PBS for washing;

[0160] (7) Add bovine serum albumin solution for blocking treatment;

[0161] (8) Add Elastin Rabbit PAb and Goat Anti-Rabbit IgG H&L (Alexa 488) for staining incubation, and add PBS for washing;

[0162] (9) Counterstain with DAPI staining solution, and add PBS for washing;

[0163] (10) Add a drop of anti-fluorescence quenching agent on the glass slide for mounting, and observe and take pictures under a fluorescence microscope.

[0164] II. Experimental Results

[0165] The experimental results of detecting the elastin expression level of dermal fibroblasts HSF using cell immunofluorescence are as Figure 5 shown. It can be seen that all three types of collagen ((GPO)n-1, (GPO)n-2, (GPO)n-3) can promote elastin expression. Among them, judged by the fluorescence intensity, (GPO)n-2 has a significantly better promotion effect than (GPO)n-1 and (GPO)n-3. This result indicates that different polycondensation reaction times have a significant impact on the activity of the collagen of the present invention. Controlling the polycondensation reaction time at 24 hours can obtain a collagen product with the best biological activity.

[0166] Detection Results of Reducing the Relative Content of Melanin and Inhibiting Tyrosinase Activity in Experimental Example 4 B16 Melanocytes

[0167] I. Experimental Purpose

[0168] The color of the skin is mainly determined by the content of melanin and the content of heme, but the content and distribution of melanin play a key determining role. Melanin in the body is mainly produced by melanocytes located in the basal cell layer. The melanin formed in the melanosomes within the cells is transported to the stratum corneum through dendritic protrusions to form the skin color and pigment deposition. The skin color and pigment deposition may depend on an increase in the number of melanocytes, an increase in tyrosinase activity, or abnormal release of α-melanocyte-stimulating hormone (α-MSH), as well as factors such as ultraviolet radiation, chronic inflammation, and skin friction.

[0169] Mouse B16 melanoma cells are commonly used cell models for studying melanin and can be used to study the effects of whitening cosmetics on intracellular tyrosinase and the amount of cellular melanin synthesis. In this experimental example, α-melanocyte-stimulating hormone (α-MSH) is used to stimulate mouse B16 cells for cell modeling, and at the same time, the test sample is added for detection to evaluate whether the sample has an inhibitory effect on cellular melanin synthesis and tyrosinase activity.

[0170] II. Experimental Method

[0171] 1. Detection of relative content of cellular melanin

[0172] (1) Prepare cell suspension, adjust cell density, inoculate into a 6-well culture plate, and culture in a carbon dioxide incubator for 24 h;

[0173] (2) Take out the culture plate, discard the culture medium in the wells, add media with different concentrations of the test substance and α-MSH to the corresponding wells, and continue to culture in a carbon dioxide incubator for 72 h;

[0174] (3) Take out the culture plate, discard the culture medium, add PBS to each well and wash 2 times, collect the cells in each well with a cell scraper, and centrifuge to collect the cells;

[0175] (4) Add NaOH solution, place the cell solution in a water bath at 80 °C and heat for 1.5 h;

[0176] (5) Take the cell solution and measure the absorbance value at a wavelength of 405 nm;

[0177] 2. Detection of relative activity of cellular tyrosinase

[0178] (1) Prepare cell suspension, adjust cell density, inoculate into a 6-well culture plate, and culture in a carbon dioxide incubator for 24 h;

[0179] (2) Take out the culture plate, discard the culture medium in the wells, add media with different concentrations of the test substance and α-MSH to the corresponding wells, and continue to culture in a carbon dioxide incubator for 72 h;

[0180] (3) Take out the culture plate, discard the culture medium, add PBS to each well and wash 2 times;

[0181] (4) Add 1% TritonX-100 to the wells, collect the cells in each well with a cell scraper, freeze-thaw at -80 °C to lyse the cells, and centrifuge at 4 °C to obtain the cell supernatant;

[0182] (5) After adding the cell supernatant of each group to a 96-well plate, immediately add L-DOPA solution, gently mix, react at 37 °C in the dark for 2 h, and then measure the absorbance value at 490 nm.

[0183] III. Experimental results

[0184] The effect of the collagen prepared according to the method of Example 1 on the relative content of melanin is as Figure 6 shown. It can be seen that when the concentration of collagen reaches 0.00313 wt.% or more, it has a significant inhibitory effect on melanin production. At the same time, as Figure 7 shown, the collagen of the present invention can inhibit tyrosinase activity.

[0185] From the above experimental results, it can be seen that the collagen provided by the present invention has the effect of whitening.

[0186] Test results of gene expression of skin barrier gene FLG in keratinocyte HaCaT in Experimental Example 5

[0187] I. Experimental purpose

[0188] In this experiment, the effect of the sample on repairing the skin barrier was evaluated by detecting the expression of genes related to the skin barrier in human immortalized keratinocytes by collagen. Filaggrin (FLG) is an intermediate filament-related protein. When conditions are suitable, filaggrin is hydrolyzed into free amino acids under the action of caspases and other enzymes, constituting the amino acid components in the natural moisturizing factor NMF. It combines with keratin in keratinocytes and is very important for maintaining the water content and barrier function of the epidermis.

[0189] II. Experimental method

[0190] In this experimental example, the effect of the test substance on the expression of filaggrin gene (FLG) in human immortalized keratinocytes (HaCaT) was detected by real-time fluorescence PCR technology, so as to determine whether the sample has the effect of synergistically repairing the skin barrier.

[0191] The specific experimental steps are as follows:

[0192] (1) Collect the HaCaT cell suspension with a confluence of 80% - 90% in a T75 flask, count, adjust the cell density and inoculate it into a culture plate, and culture it in a carbon dioxide incubator.

[0193] (2) Take out the culture plate, discard the culture medium in the wells, add the collagen prepared in Example 1 to make the final concentration of collagen 0.0125%, 0.025% and 0.05% by mass percentage, and continue to culture in a carbon dioxide incubator.

[0194] (3) Take out the culture plate, discard the culture medium in the wells, add 1 mL of PBS to each well for rinsing and then discard, repeat once, suck out the liquid in the wells, place it on ice for standby, or use trypsin digestion to collect the cells and store them at -80 °C temporarily.

[0195] (4) Extract RNA with reference to the instruction manual of the Total RNA Extraction Kit for Cultured Cells / Bacteria.

[0196] (5) Reverse transcription was carried out with reference to the instruction manual of the 1st Strand cDNA Synthesis Kit (+gDNA wiper) to obtain cDNA.

[0197] (6) Detect the expression of related genes in cells by fluorescence quantitative PCR method.

[0198] III. Experimental Results

[0199] The collagen prepared according to the method of Example 1 has a significant promoting effect on the expression of FLG at a specific concentration (0.025% by mass). This indicates that the collagen provided by the present invention can effectively promote skin repair.

[0200] From the above examples and experimental examples, it can be seen that the present invention provides a triple-helix collagen with thermal stability, which has the same triple-helix structure as natural collagen, has the effects of anti-wrinkle and firming, whitening, and skin repair, and can maintain the triple-helix structure in the temperature range of 20°C - 60°C, with good thermal stability, and can be better applied to fields such as cosmetics and medical devices, and has good application prospects.

Claims

1. A triple-helical collagen with thermal stability, characterized in that Its structural formula is shown in Formula I: Formula I: H2N-(G-P-О) n -COOH; Among them, GP-О is a peptide chain composed of three different amino acid residues; G is a glycine residue, P is a proline residue, and О is a hydroxyproline residue; the weight average molecular weight distribution of the triple helical collagen is 5.0×10 3 ~1.5×10 7 ; The triple helical collagen is prepared according to the following steps: Step 1, a tripeptide having a structural formula of H2N-GP-О-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 to 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 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.

2. The triple-helical collagen with thermal stability 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-helical collagen with thermal stability 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; The polycondensation reaction time is 24 hours.

4. The triple-helical collagen with thermal stability 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 triple-helical collagen with thermal stability 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-GP-О-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 to 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 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.

6. Use of the triple-helical collagen with thermal stability according to any one of claims 1 to 4 in the preparation of cosmetics, medicines, cosmetic materials or medical devices.

7. The use according to claim 6, 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.

8. The use according to claim 7, characterized in that: The functional skin care product is a facial mask, an essence or a facial cleanser.

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

10. The cosmetic according to claim 9, characterized in that: The concentration of the triple-helical collagen with thermal stability is 0.0005wt%-0.05wt%.

11. The cosmetic according to claim 10, characterized in that: The concentration of the triple-helical collagen with thermal stability is 0.0005 wt %.

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