Triple-helix collagen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical and cosmetic raw materials, and particularly relates to triple helix collagen as well as a preparation method and application thereof. The structural formula of the triple helix collagen disclosed by the invention is as shown in formula I: H2N-(G-P) n-COOH; wherein the 01-G-P is a peptide chain composed of three different amino acid residues; g is glycine residue, P is proline residue, and O is hydroxyproline residue. And the weight-average molecular weight distribution of the triple helix collagen is 3.0 * 10 < 3 >-8.0 * 10 < 4 >. The triple-helix collagen has a triple-helix structure, is good in thermal stability, has better effects of moisturizing, resisting oxidation, repairing damaged hair, growing hair and the like, 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 triple-helix collagen and its preparation method and use. Background Art

[0002] Collagen, as the most abundant functional protein in the human body, weaves a strong network structure with its unique triple-helix structure and plays a crucial role in physiological functions. This protein is widely distributed in various tissues such as skin, bones, and ligaments, and is essential for ensuring the stability of the structures of various tissues and organs and the normal operation of their functions. With the rapid development of technology, artificially synthesized collagen has been widely incorporated into the research, development, and application of cosmetic formulations, medical plastic surgeries, and medical materials, greatly expanding its scope of use in various health and beauty fields.

[0003] In the beauty and skincare industry, collagen is widely adopted in various skincare and makeup products due to its key role in skin health, aiming to enhance core properties such as skin's water-locking and moisturizing ability, elasticity, and luster. Many high-quality skincare products such as creams, masks, serums, and facial cleansers have incorporated collagen into their formulations to supply the nutrients needed by the skin, optimize the cell survival conditions, and stimulate skin metabolism, thereby achieving the goals of anti-aging and deeply nourishing the skin. Currently, numerous collagen skincare brands on the market have launched collagen raw materials with high-concentration extraction and small-molecule treatment to ensure more efficient penetration into the skin. Meanwhile, in the medical plastic surgery and medical materials industries, collagen also shows broad application prospects. In the fields of facial contour shaping, anti-aging treatment, and skin repair and reconstruction, collagen, as the primary biological filling material, plays an irreplaceable role. Moreover, for skin burns, wounds, and other conditions, collagen can effectively participate in clinical treatment to improve the state of damaged skin. In tissue engineering technology, collagen is used to make various tissue engineering scaffolds due to its biodegradability, which provides an ideal supporting environment for cell proliferation and tissue regeneration. In addition, collagen, with its excellent biocompatibility and biodegradability, is also applied to the research and production of medical devices such as medical sutures and skin grafts. Furthermore, collagen has also achieved remarkable results in bionics and regenerative medicine. For example, in the field of bone tissue engineering, it can be used as a scaffold material to assist in the treatment of diseases such as osteoporosis and bone defects, and even extended to the research and development of artificial blood vessels and flexible implant materials, indicating that collagen has unlimited potential and broad application prospects in future biomedical materials and tissue regeneration science.

[0004] Currently, artificially synthesized collagen mainly covers two major source categories: one is derived from animal tissues, such as cows, pigs, chickens, and fish, which is animal-derived collagen obtained through specific extraction processes; the other is recombinant collagen prepared by using modern biotechnological means, such as bioconversion, purification, and genetic engineering technologies. However, both the preparation and application of these two types of artificial collagen face technical challenges. Firstly, the extraction of collagen from animal tissues inevitably comes with potential immune risks. For example, it may carry animal-derived infectious diseases such as mad cow disease, which poses a threat to human health. Secondly, there are certain differences in the amino acid sequences between animal-derived collagen and human collagen, which may lead to unsatisfactory bioactivity, stability, and safety, and it cannot perfectly mimic the functional performance of collagen in the human body. On the other hand, although recombinant collagen can theoretically ensure a closer amino acid arrangement order to human collagen, due to its complex production process, high cost, and the fact that it may not be able to ensure the formation of a complete triple helix structure in each batch during the actual production process, its bioactivity effectiveness is affected. Therefore, the bioactivity performance of recombinant collagen also needs to be further verified and improved.

[0005] However, there has been no major breakthrough in the chemically synthesized collagen-like peptides. Firstly, 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. Additionally, how to prepare collagen with an excellent thermally stable triple helix structure is also one of the major challenges currently faced.

[0006] In the prior art, polypeptide molecules of collagen-like peptides can be synthesized using a peptide synthesizer. A peptide synthesizer is an automated laboratory device specifically designed for synthesizing polypeptide chains in vitro. Based on solid-phase peptide synthesis (SPPS) technology, by precisely controlling the 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 it is difficult for the polypeptide molecules prepared using a peptide synthesizer to form a triple helix structure, and even if a triple helix structure is formed, it has insufficient thermal stability. When collagen is used to make cosmetics or medical devices, there is a certain requirement for tolerating environmental temperatures or undergoing high-temperature disinfection. The unstable triple helix structure is difficult to maintain at a relatively high 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] In view of the problems of the prior art, the present invention provides a triple-helix collagen, a preparation method thereof and uses thereof.

[0009] A triple-helix collagen, the structural formula of which is shown in Formula I:

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

[0011] Wherein, О-G-P 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 3.0×10 3 ~8.0×10 4 .

[0012] Preferably, the triple-helix collagen is obtained by condensation through a liquid-phase reaction using a tripeptide with the structural formula H2N-О-G-P-COOH as a raw material.

[0013] The present invention also provides a preparation method of the above-mentioned triple-helix collagen, comprising the following steps:

[0014] Step 1, preparing a tripeptide solution by using a tripeptide with the structural formula H2N-О-G-P-COOH and a condensation aid with phosphate buffer as a solvent; preparing a dehydrating condensing agent solution by using a dehydrating condensing agent with phosphate buffer as a solvent;

[0015] Wherein, the phosphate buffer is prepared from the following raw materials in parts by weight:

[0016] Potassium chloride 0.18 - 0.22 parts,

[0017] Sodium dihydrogen phosphate 1.0 - 1.2 parts,

[0018] Potassium dihydrogen phosphate 0.18 - 0.22 parts,

[0019] Water 995 - 1005 parts;

[0020] The tripeptide solution is prepared from the following raw materials in the following ratio:

[0021] Phosphate buffer 495 - 505 volume parts,

[0022] Tripeptide 49.5 - 50.5 parts by weight,

[0023] Condensation aid 4.9 - 5.1 parts by weight;

[0024] The dehydrating condensing agent solution is prepared from the following raw materials in the following ratio:

[0025] Phosphate buffer 495 - 505 volume parts,

[0026] 156 - 160 parts by weight of a dehydrating condensing agent;

[0027] Step 2: Add the dehydrating condensing agent solution to the tripeptide solution for polycondensation reaction; in the polycondensation reaction, the feeding weight ratio of tripeptide, condensation assistant, 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;

[0028] Step 3: Separate and purify to obtain the product.

[0029] Preferably, in Step 1, the condensation assistant is selected from at least one of 1 - hydroxybenzotriazole or 1 - hydroxy - 7 - azabenzotriazole;

[0030] The dehydrating condensing agent is selected from at least one of 1 - ethyl - 3 - (3 - dimethylaminopropyl) - pentanediamide or 1,3 - dicyclohexylcarbodiimide;

[0031] The phosphate buffer solution is prepared with raw materials in the following parts by weight:

[0032] 0.2 part of potassium chloride,

[0033] 1.15 parts of sodium dihydrogen phosphate,

[0034] 0.2 part of potassium dihydrogen phosphate,

[0035] 1000 parts of water;

[0036] The tripeptide solution is prepared with raw materials in the following ratio:

[0037] 500 volume parts of phosphate buffer solution,

[0038] 50 parts by weight of tripeptide,

[0039] 5 parts by weight of condensation assistant;

[0040] The dehydrating condensing agent solution is prepared with raw materials in the following ratio:

[0041] 500 volume parts of phosphate buffer solution,

[0042] 158 parts by weight of dehydrating condensing agent.

[0043] Preferably, in Step 2, in the dehydrating condensing agent solution and the tripeptide solution, the feeding weight ratio of tripeptide, condensation assistant, and dehydrating condensing agent is 50:5:158;

[0044] The time of the polycondensation reaction is 24 hours.

[0045] Preferably, in step 3, the specific steps of the separation and purification are as follows: circularly grinding with a homogenizer and filtering through a 50,000 molecular weight cut-off membrane to obtain a solution containing the triple helix collagen.

[0046] The present invention also provides the use of the above-mentioned triple helix collagen in the preparation of cosmetics, drugs, beauty and plastic surgery materials or medical devices.

[0047] Preferably, the cosmetics are functional skin care products;

[0048] The medical device is a filling material or an engineering scaffold for skin, bone, and ligament.

[0049] Preferably, the functional skin care products are facial masks, essence, or facial cleansers.

[0050] The present invention also provides a cosmetic, which is prepared by using the above-mentioned triple helix collagen as an active ingredient and adding excipients.

[0051] Preferably, the concentration of the triple helix collagen is 0.0005 wt% - 0.1 wt%.

[0052] 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 mutual winding of three polypeptide chains. The method for detecting whether a protein or a polymer has a triple helix structure similar to that of natural collagen is to use circular dichroism spectroscopy to detect whether the polymer has a positive peak at 220 - 230 nm. The proportional relationship between "volume parts" and "weight parts" is determined as follows:

[0053] 1 volume part: 1 weight part = 1 ml: 1 g.

[0054] The present invention provides a new collagen polymer with thermal stability, which is a polypeptide chain synthesized in liquid phase and obtained by polycondensation of a polypeptide chain 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), it cannot be used as a raw material. In addition, if used as a medical device, it 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 under normal temperature and high temperature (40 - 60 °C), and its thermal stability is better than that of 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 up-regulation of AQP3 expression in fibroblasts and has a moisturizing effect. In addition, the collagen polymer of the present invention also has functions such as antioxidant, repairing damaged hair quality, and promoting hair growth.

[0055] Therefore, the collagen polymer of the present invention has good application prospects.

[0056] 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.

[0057] 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 fall within the scope of the present invention. Description of the Drawings

[0058] Figure 1 It is the circular dichroism spectrum of triple helix collagen after heat treatment at 20 - 60 °C in Experimental Example 1;

[0059] Figure 2 It is the circular dichroism spectrum of commercialized polypeptide after heat treatment at 20 - 60 °C in Experimental Example 1;

[0060] Figure 3 It is the test result graph of the molecular weight of triple helix collagen in Experimental Example 1;

[0061] Figure 4 It is the experimental result graph of the relative expression level of AQP3 in Experimental Example 2;

[0062] Figure 5 It is the experimental result graph of the fluorescence image experiment of AQP3 expression in Experimental Example 3;

[0063] Figure 6Statistical result chart of fluorescence detection of AQP3 expression in Experimental Example 3;

[0064] Figure 7 Experimental result chart of fluorescence image of DCF in Experimental Example 4;

[0065] Figure 8 Statistical result chart of fluorescence detection of DCF in Experimental Example 4;

[0066] Figure 9 Electron microscopy detection result of collagen in repairing heat-damaged hair quality in Experimental Example 5.

[0067] In the above experimental data charts, **: corresponding to a significant level of 1%, that is, p < 0.01 for this experimental group compared with the control group; ***: corresponding to a significant level of 0.1%, that is, p < 0.001 for this experimental group compared with the control group. Detailed implementation mode

[0068] In the following examples and experimental examples, the reagents and raw materials used, unless otherwise specified, are all commercially available products.

[0069] Example 1 Triple-helix collagen

[0070] This example provides triple-helix collagen, which is a polypeptide with a triple-helix structure, and its structural formula is as follows:

[0071] H2N-(О-G-P) n -COOH

[0072] G, P, and O in the formula are composed of the following amino acid residues:

[0073] P: Proline,

[0074] O: Hydroxyproline,

[0075] G: Glycine;

[0076] n ≧ 4. Since the triple-helix collagen 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.

[0077] The preparation method of the above triple-helix collagen is as follows:

[0078] 1. Preparation of phosphate buffer

[0079] Add 0.2 g of potassium chloride (KCl), 1.15 g of sodium phosphate dibasic (Na2HPO4), and 0.2 g of potassium phosphate dibasic (KH2PO4) to 1000 ml of high-purity water, and stir to prepare phosphate buffer (Buffer-PB, hereinafter referred to as PB solution).

[0080] 2. Preparation of tripeptide solution

[0081] Add 50 g of the tripeptide O-G-P (manufactured by Uniqs Inc.) and 5 g of the condensation aid 1-hydroxybenzotriazole (HOBt) to 500 ml of PB solution, and prepare a tripeptide solution (hereinafter referred to as OGP solution) after stirring.

[0082] 3. Preparation of the dehydrating condensing agent solution

[0083] 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.

[0084] 4. Polycondensation reaction

[0085] Pour the prepared OGP solution into a 2 L separatory funnel, cool it to 2 °C in a thermostatic bath, and then gradually add the EDC solution cooled to 2 °C (the volume ratio of the OGP solution to the 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 triple helix collagen.

[0086] Example 2 Moisturizing lotion and emulsion for anti-wrinkle and firming

[0087] This example provides a moisturizing lotion and emulsion, and its composition includes:

[0088] 0.1 wt% of the triple helix collagen prepared in Example 1,

[0089] 10 wt% of glycerol,

[0090] 15 wt% of polyethylene glycol,

[0091] 5 wt% of hyaluronic acid,

[0092] The balance is water.

[0093] The technical solution of the present invention will be further described through experiments below.

[0094] Experimental Example 1 Characterization of the product

[0095] I. Experimental method

[0096] This experimental example characterizes the triple helix collagen prepared in Example 1, including:

[0097] 1. Circular dichroism spectroscopy characterization

[0098] Select a commercial polypeptide as a reference substance, and its molecular formula is H2N-(P-O-G)10 -COOH (purchased from Anhui Guoping Pharmaceutical Co., Ltd.). The amino acid sequence of this reference substance is similar to 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 peptide synthesizer.

[0099] Prepare the triple-helix collagen of Example 1 or the commercial peptide reference substance into an aqueous solution with a concentration of 0.25 mg / ml, and perform heat treatment 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.

[0100] 2. Molecular weight characterization

[0101] Determination is carried out using light scattering method. Use Wyatt multi-angle light scattering detector DAWN HELEOS II and Wyatt differential refractive index detector Optilab T-rEX, and the column is Shodex SB-806M.

[0102] II. Experimental results

[0103] 1. Circular dichroism spectroscopy characterization

[0104] 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 height hardly changes, 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.

[0105] The characterization results of the commercial peptide 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 commercial peptide 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 commercial peptide molecule is prone to disappear under heat treatment conditions, and its thermal stability of the triple-helix structure is poor.

[0106] The experimental results of this experiment show that for peptides, 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.

[0107] 2. Molecular weight characterization

[0108] As Figure 3 shown, the results show that the weight-average molecular weight Mw of the triple-helix collagen prepared in Example 1 is 3.0×10 3 ~8.0×10 4 (g / mol).

[0109] Detection of the effect of promoting the expression of skin moisturizing-related proteins in Experimental Example 2

[0110] I. Test purpose

[0111] Aquaporin 3 (AQP3) is a substance on the cell membrane, located in the basal layer of the epidermis, regulating the biological functions of the skin, such as water retention and trans-epidermal water loss. It is responsible for the transport of substances such as water, glycerol, and urea, and belongs to a kind of transport protein factor, mainly expressed in keratinocytes and skin fibroblasts. AQP3 not only participates in skin hydration and barrier function, but also plays an important role in skin injury, repair, and healing, and is an important guarantee for skin moisturization, normal morphology, and function maintenance.

[0112] II. Experimental method

[0113] 1. Main reagents

[0114] Low-glucose DMEM culture medium (Solarbio), fetal bovine serum (Gibco), PBS (VivaCell), trypsin (Gibco), cell lysate (Novoprotein).

[0115] 2. Main equipment

[0116] CO2 incubator (Thermo, 160i), biological safety cabinet (Suzhou Jing'an Antai, BSC-1604ⅡA2), inverted microscope (Leica, DMi8), QPCR instrument (Roche), floor-standing half-body ultraviolet therapy instrument (Sigma High, SS-03AB).

[0117] 3. Test method

[0118] The specific settings of the test groups are as follows:

[0119] Use serum-containing DMEM medium, seed 1*10^5 cells per well in a six-well plate. After culturing Hacat cells for 24 h until they adhere to the wall, treat the cells with 200 μmol / L hydrogen peroxide for 2 h to establish a model. Then, change to clean DMEM containing calf serum and continue to culture the cells (add 0.1 wt% collagen prepared in Example 1 to the sample group). After 2 days, collect the cells and extract proteins for ELISA experiments. Among them, the blank control does not add hydrogen peroxide and collagen, and the other treatments are the same; the negative control does not add collagen, and the other treatments are the same.

[0120] 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 min, on an ELISA reader, at 450 nm, after zeroing with the blank control well, measure the OD value of each well.

[0137] III. Experimental results

[0138] The detection results of AQP3 content are as Figure 4 shown:

[0139] The results showed that compared with the blank control group, after Hacat cells in the negative control group were treated with hydrogen peroxide, the expression level of AQP3 was significantly increased (p < 0.01), indicating that under the condition of initial damage stimulation by hydrogen peroxide, AQP3 would be highly expressed to repair the damage, and the model establishment was effective.

[0140] Compared with the negative control group, when the dosing concentration of the sample group was 0.1 wt%, the AQP3 expression level of Hacat cells was further significantly increased (p < 0.05). This indicates that triple-helix collagen can promote the expression of AQP3, thereby enhancing the effect of repairing damage.

[0141] Among them, when performing statistical analysis using the t-test method, compared with the blank control group, the significance in the negative control group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **. Compared with the negative control group, the significance in the sample group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **.

[0142] The above experimental results show that after Hacat cells are stimulated by hydrogen peroxide, the expression of aquaporin AQP3 will increase to repair the damage, and by using the triple-helix collagen of the present invention, the AQP3 expression of Hacat cells can be promoted, enhancing the effect of repairing damage. At the same time, this also indicates that the triple-helix collagen provided by the present invention has a moisturizing effect.

[0143] Experimental Example 3 Detection of the expression of AQP3 protein in keratinocytes HaCaT by immunofluorescence assay I. Experimental purpose

[0144] Aquaporins (AQPs) are integral membrane proteins that penetrate biological membranes to form water channels. The main function of AQPs is to transport water, glycerol, and other small molecule solutes across biological membranes. Among them, AQP3 is highly expressed in the plasma membrane of keratinocytes and functions as a water and glycerol transporter in the basal layer of the skin epidermis. The glycerol content and water content of the skin epidermis lacking AQP3 will decrease. Therefore, theoretically, an effective way to hydrate is to activate aquaporins or increase the number of aquaporins to transport water from the dermis to the epidermis.

[0145] II. Experimental Methods

[0146] In this experimental example, the sample was added to human immortalized keratinocytes (HaCaT) for culture, and immunofluorescence cytochemistry was used to detect the effect of the sample on the expression of AQP3 in HaCaT cells, so as to illustrate whether the sample has a moisturizing effect.

[0147] 1. Sample Preparation

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

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

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

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

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

[0153] 2. Immunofluorescence Detection

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

[0155] (2) Collect the HaCaT 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, and add the working solutions of (OGP)n-1, (OGP)n-2, and (OGP)n-3 with a mass percentage of 0.025% 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) Fix the cells with 4% paraformaldehyde fixative and wash with PBS.

[0159] (6) Permeabilize the cells with 0.2% Triton X-100 solution and wash with PBS.

[0160] (7) Block the cells with bovine serum albumin solution.

[0161] (8) Incubate the cells with Anti-AQP3 Antibody and Goat Anti-Rabbit IgG H&L (Alexa 488) for staining, and then wash with PBS.

[0162] (9) Counterstain the cells with DAPI staining solution and wash with PBS.

[0163] (10) Mount the cells on a glass slide with a drop of anti-fluorescence quenching agent and observe and photograph them under a fluorescence microscope.

[0164] III. Experimental Results

[0165] The results are as Figure 5 and Figure 6 shown. It can be seen that all three types of collagen, (OGP)n-1, (OGP)n-2, and (OGP)n-3, can promote the expression of AQPs protein. Among them, (OGP)n-2 has a significantly better promoting effect than the other two types of collagen. Thus, it can be seen that the condensation reaction time has a significant impact on the biological activity of the collagen of the present invention. Controlling the condensation reaction time at 24 hours can obtain a collagen product with the best biological activity.

[0166] Experimental Example 4 uses immunofluorescence to detect intracellular ROS in dermal fibroblasts HSF

[0167] I. Experimental Purpose

[0168] The Reactive Oxygen Species Assay Kit (also known as the ROS Assay Kit) is a kit for detecting reactive oxygen species using the fluorescent probe DCFH-DA. DCFH-DA itself has no fluorescence and can freely cross the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH. DCFH cannot permeate the cell membrane, so the probe can be easily loaded into the cell. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to generate fluorescent DCF. Detecting the fluorescence of DCF can know the level of intracellular reactive oxygen species.

[0169] In this experimental example, cells were pretreated with a collagen sample, and a hydrogen peroxide-induced oxidative stress model of dermal fibroblasts was established. The intracellular ROS was detected using a fluorescent probe, which can be used to evaluate whether the collagen of the present invention has antioxidant effects.

[0170] II. Experimental Methods

[0171] (1) ESF-M cells in good growth condition and in the mid-to-late logarithmic growth phase were taken, digested, and counted. The cell density was adjusted, and the cells were seeded in a 24-well cell culture plate and then cultured in a cell culture incubator for 18 - 24 h;

[0172] (2) The original culture medium in the wells was discarded. According to the grouping, collagen (prepared according to the method of Example 1) with mass percentages of 0.00039%, 0.00078%, and 0.00156% was added to each group, and the cells were continued to be cultured in a cell culture incubator for 24 h;

[0173] (3) The cell plate was taken, the supernatant was aspirated, and the cells were washed with PBS. The NC group was replaced with maintenance medium, and the other groups were replaced with maintenance medium containing H2O2 + sample according to the experimental grouping and continued to be cultured for 0.5 h.

[0174] (4) The cell culture medium was aspirated, and after washing with PBS, the diluted DCFH-DA probe was added, and the cells were incubated in a 37°C cell culture incubator for 20 minutes;

[0175] (5) The probe was aspirated, and the cells were washed three times with PBS, and then observed and photographed under a fluorescence microscope.

[0176] III. Experimental Results

[0177] The results are as Figure 7 、 Figure 8 shown. After adding the collagen prepared in Example 1, the fluorescence intensity representing the intracellular reactive oxygen species level decreased, indicating that the collagen of the present invention has the effect of reducing the intracellular reactive oxygen species level and has antioxidant efficacy. In addition, when the collagen concentration is 0.156% by mass fraction, it has the best antioxidant effect.

[0178] Experimental Example 5 Electron Microscopy Detection of the Efficacy of Collagen in Repairing Damaged Hair Quality in Vitro

[0179] I. Experimental Purpose

[0180] In daily life, the types of hair damage can be classified into light damage, heat damage, bleaching and dyeing damage, etc. according to different external factors. These factors cause hair damage, resulting in hair quality problems such as dullness, easy breakage, frizz, and split ends. The hair structure from the outside to the inside is the cuticle, the cortex, and the medulla in turn. The cuticle is the outermost layer of the hair, protecting the medulla and cortex from various external damages. At the same time, the texture and luster of the hair are also closely related to the hair cuticle. Therefore, whether the cuticles of the outermost layer of the hair are neatly arranged and the structure is complete has become an important criterion for evaluating the quality of hair care effects.

[0181] In this experiment, the hair was damaged by heat damage, and then the state of the hair cuticles could be determined by scanning electron microscopy of the hair.

[0182] II. Experimental methods

[0183] (1) Collect hair with a length close to that of human hair, wash it thoroughly with clean water to remove impurities such as dandruff, place it under a microscope and observe until there are no large particle impurities, and then dry it completely.

[0184] (2) Turn on the hair straightener switch and preheat it for more than 3 minutes to ensure its temperature is stable.

[0185] (3) Use the hair straightener to create a heat damage model for the hair.

[0186] (4) Collagen (prepared according to the method of Example 1) was successively prepared into working solutions with mass percentages of 0.0125%, 0.025%, and 0.05%. The working solutions were evenly applied to the hair samples and massaged for 1 minute, left for 5 minutes, and then washed with water for 1 minute. This was continuously processed for three days, once a day.

[0187] (5) Use an electron microscope to scan the hair.

[0188] III. Experimental results

[0189] The results are as Figure 9 shown. After treating the heat-damaged hair with the collagen prepared in Example 1, the integrity and neatness of the cuticles in the sample group treated with collagen were significantly better than those in the model control group. It can be seen that the collagen product of the present invention has the effect of repairing damaged hair quality.

[0190] From the above examples and experimental examples, it can be seen that the present invention provides a triple-helix collagen, which has the same triple-helix structure as natural collagen, has the effects of moisturizing, antioxidant, and repairing damaged hair quality, and can maintain the triple-helix structure within 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, characterized in that Its structural formula is shown in Formula I: Formula I: H2N-(O-G-P) n -COOH; Among them, О-GP 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 3.0×10 3 ~8.0×10 4 ; The triple helical collagen is prepared according to the following steps: Step 1, a tripeptide having a structural formula of H2N-О-GP-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 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 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 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 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-О-GP-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 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 taking the triple helix collagen described 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 is 0.0005wt%-0.1wt%.

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