Application of polypeptide in increasing contents of collagen, fibroblast and fibrillar protein

Abstract

The invention belongs to the technical field of biological polypeptide research, and particularly relates to application of polypeptide in increasing the content of collagen, fibroblast and fibrillar protein. The polypeptide is a Taiwan mitochondrial energy sphere peptide, and the sequence of the polypeptide is nicotinic acid-C-C-C-A-r-B-r-B-r-B-r-A-R-G-D-A-NH2, and the sequence of the Taiwan mitochondrial energy Wherein G is glycine, R and r are arginine, D is aspartic acid, A is tyrosine, B is tryptophan, C is phenylalanine, in the polypeptide, r is D configuration, and other amino acids are L configuration. The Thai mitochondrial energy sphere peptide disclosed by the invention can remarkably improve the contents of various collagens and the contents of elastin and fibrillar protein, so that the Thai mitochondrial energy sphere peptide has a wide application prospect in the aspects of aging resistance and oxidation resistance.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide research technology, specifically involving the application of peptides in increasing the content of collagen, fibroblasts and fibrils. Background Technology

[0002] The essence of skin rejuvenation lies in maintaining and repairing its structural protein network, which together constitute the skin's support system. In the dermis, various structural proteins intertwine to form a complex three-dimensional network that determines the skin's firmness, elasticity, and smoothness. Type IV collagen, elastin, Type I collagen, and fibrillin 1 play indispensable roles in this network. Each has a unique structural function, yet they work synergistically to maintain youthful skin. With age and environmental factors, these key proteins in the skin gradually diminish or become dysfunctional, leading to wrinkles, skin laxity, and decreased elasticity. Therefore, increasing the levels of these proteins is crucial for maintaining youthful skin. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention aims to provide an application of peptides in increasing the content of collagen, fibroblasts, and fibrils.

[0004] The present invention specifically adopts the following technical solution: The application of Taishu mitochondrial energy globulins is selected from any of the following: Used to increase collagen content in fibroblasts for non-therapeutic purposes; Used to increase elastin content in fibroblasts for non-therapeutic purposes; Used to increase the fibrin content in fibroblasts for non-therapeutic purposes; It is used as an effective ingredient to increase the content of collagen, fibroblasts and fibrils in the preparation of topical products; The sequence of the Taishu mitochondrial energy globulin is nicotinic acid-CCArBrBrBrARGDA-NH2; wherein G is glycine, R and r are arginine, D is aspartic acid, A is tyrosine, B is tryptophan, C is phenylalanine, and in the polypeptide, r is in the D configuration, and the remaining amino acids are in the L configuration.

[0005] The inventors previously designed and synthesized a polypeptide that combines the functions of scavenging reactive oxygen species and upregulating the expression of SIRT family genes, the results of which have been published in Chinese patent application 202411760429.0. Building upon this previous research, this invention further reveals that the polypeptide also possesses biological activity that significantly increases the content of collagen, elastin, and fibrillary proteins in skin fibroblasts. This invention aims to provide a novel polypeptide active ingredient that achieves anti-skin aging effects through multi-target synergistic action, providing technical support for the development of anti-aging cosmetics and topical skin preparations.

[0006] Furthermore, the topical product is a drug or cosmetic.

[0007] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0008] Furthermore, the cosmetic contains moisturizers, surfactants, ultraviolet absorbers, fragrances, antioxidants, preservatives and mildew inhibitors, colorants, pH adjusters, solvents, or ingredients commonly used in cosmetics.

[0009] Furthermore, the cosmetic is an aqueous solution, emulsion, cream, serum, or mask.

[0010] Furthermore, the skin in question includes both normal and aged skin.

[0011] Furthermore, the aging process includes both natural aging and photoaging.

[0012] Furthermore, the skin aging described herein is caused by insufficient levels of collagen, elastin, and fibrils.

[0013] The present invention has the following beneficial effects: The polypeptides provided by this invention can significantly increase the content of various collagens, elastin, and fibrils, thus having broad application prospects in aging caused by insufficient collagen. They can be used to prepare cosmetics or drugs that combat human skin cell aging. Attached Figure Description

[0014] Figure 1 MS image of mitochondrial energy globulin from the Taishu (a type of mitochondrial peptide).

[0015] Figure 2 This is an HPLC chromatogram of the mitochondrial energy globulin from the Taishu.

[0016] Figure 3 The cell viability curve of mitochondrial energy globulin for Taishu.

[0017] Figure 4 This is a cell viability curve for a competitor's product.

[0018] Figure 5The results are for Collagen I immunofluorescence.

[0019] Figure 6 is a bar chart of Collagen I relative integrated optical density (IOD) / cell numerical value.

[0020] Figure 7 The results are for Collagen IV immunofluorescence.

[0021] Figure 8 This is a bar chart showing the relative integrated optical density (IOD) / cell numerical value of Collagen IV.

[0022] Figure 9 The results are for Elastin immunofluorescence.

[0023] Figure 10 is a bar chart of Elastin's relative integrated optical density (IOD) / cell numerical value.

[0024] Figure 11 The results are for the immunofluorescence of Fibrillin 1.

[0025] Figure 12 This is a bar chart showing the relative integrated optical density (IOD) / cell numerical value of Fibrillin 1. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0027] This invention provides a class of polypeptides that can significantly increase the content of various collagens, elastins, and fibrils. The general formula of the polypeptide sequence is nicotinic acid-CCArBrBrBrARGDA-NH2; wherein G is glycine, R is arginine, D is aspartic acid, and r is arginine; A is one of tryptophan, tyrosine, and phenylalanine; B is one of tryptophan, phenylalanine, tyrosine, and 3-cyclohexylalanine; and C is one of tryptophan, tyrosine, and phenylalanine. A, B, and C are different from each other. In the polypeptide, r is in the D configuration, and the remaining amino acids are in the L configuration.

[0028] The following examples use a polypeptide (hereinafter referred to as Taishu Mitochondrial Energy Globule Peptide) in which A is tyrosine, B is tryptophan, and C is phenylalanine as an example to verify its effect on significantly increasing the content of various collagens, elastin, and fibrils.

[0029] Example 1: Preparation and characterization of the polypeptide (Taisu Mitochondrial Energy Globule Peptide) of the present invention The peptide compound was synthesized using a solid-phase method with Fmoc-protected amino acids as the starting material, Rink Amide MBHA Resin (with a degree of substitution of 0.8 mmol / g) as the solid-phase support, and DIEA / HBTU as the condensing agent. The peptide was then cleaved from the resin using a mixed solution of trifluoroacetic acid and triisopropylsilane, followed by crystallization with methyl tert-butyl ether and drying to obtain the crude peptide. After dissolution, the crude peptide was purified multiple times by reversed-phase high-performance liquid chromatography (C18, 10µm, 100A packing material) using a gradient elution method with fractional sample collection. Through HPLC range detection and impurity analysis, a purification intermediate with a purity of over 90% was obtained. Finally, a 32 g / L ammonium acetate salt was used for conversion. After final verification, the organic solvent was replaced by nanofiltration to reduce residues and achieve enrichment. The resulting lyophilized peptide powder was 0.5 g.

[0030] The targeted peptide was analyzed by electrospray ionization mass spectrometry, and the molecular weight (e.g., ...) was displayed in the mass spectrum. Figure 1 , Figure 2 (As shown) is basically consistent with the theoretical molecular weight.

[0031] Example 2: Cytotoxicity and efficacy testing of the polypeptide of the present invention (Taisu Mitochondrial Energy Globule Peptide) 1. Testing Basis According to the "Method for Detecting the Content of Type I Collagen I, Type IV Collagen IV, Elastin, and Fibrillin 1 in Fibroblasts Based on UVA Irradiation".

[0032] 2. Purpose of Detection This test consists of two parts: the first part is based on fibroblasts, which conducts cytotoxicity testing to determine the drug concentration of the sample on fibroblasts; the second part uses UVA irradiation of fibroblasts to evaluate the firming and anti-wrinkle effects of the test sample by detecting changes in the content of type IV collagen (Collagen IV), elastin, type I collagen (Collagen I), and fibrillin 1.

[0033] 3. Test Materials 3.1 Test System The cells used in this test were fibroblasts, batch number: Fb19052002, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0034] 3.2 Sample Information Table 1 Sample Information 3.3 Main Reagents MTT (Sigma), DMEM (Gibco), PBS (Solepro), Trypsin (Gibco), Newborn calf serum (NBS, Lanzhou Rongye), Dimethyl sulfoxide / DMSO (Sigma), H2O2 (Sigma), Mitochondrial morphology kit (Thermo Fisher Scientific), JC-1 mitochondrial membrane potential detection kit (Beyotime).

[0035] 3.4 Main Equipment Microplate reader (Bio-tek), CO2 incubator (Thermo, 150I), inverted microscope (Olympus, CKX53), clean bench (Sujing Antai), fluorescence microscope (Olympus, BX43), super-resolution microscope (NanoInsights-Tech Co., Multi-SIM).

[0036] 4. Cytotoxicity test 4.1 Test Method 4.1.1 Cell viability testing method 1) Cell seeding: After cell resuscitation, when the cell seeding rate reaches about 60%, seed the cells into 96-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0037] 2) Experimental Groups: The experiment included a zeroing group, a solvent control group, a positive control group, and a sample group. In the sample group, each sample had 8 concentration gradients, and each concentration gradient had 3 replicate wells.

[0038] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 2).

[0039] Table 2 Test Concentration Setting Table 4) Drug administration: Drug administration was performed when the cell seeding rate in the 96-well plate reached 50%–60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of the test sample was added to each well; for the zeroing group, no cells were seeded, only 200 μL of cell culture medium was added. After drug administration, the 96-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0040] 5) Detection: After culturing cells for 24 hours, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 150 μL of DMSO to each well, and read the OD value at 490 nm.

[0041] 6) Calculation of relative cell viability: 4.1.2 Cell morphology testing methods 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 24-well plates and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0042] 2) Test grouping: The experiment was set up with a solvent control group and a sample group.

[0043] 3) Drug administration: Drug administration was performed when the cell deposition rate in the 24-well plate reached 50%–60%. For the solvent control group, 1 mL of culture medium was added to each well; for the sample group, 1 mL of culture medium containing the corresponding concentration of the test sample was added to each well. After drug administration, the 24-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 hours.

[0044] 4) Take photos: After incubation, discard the supernatant and take photos under an inverted microscope.

[0045] 4.2 Test Results Table 3. Results of MTT assay for mitochondrial energy globulin from Taishu. As shown in Table 3, the MTT results and Figure 3 Morphological results indicate that the mitochondrial energy globulin based on fibroblasts did not exhibit significant cytotoxicity at concentrations up to 10% (v / v).

[0046] Table 4 Competitor MTT Test Results As shown in Table 4, the MTT results and Figure 4 Morphological results indicate that the competing product, based on fibroblasts, did not exhibit significant cytotoxicity at a concentration range of 1.25% (v / v).

[0047] 5. Assays based on UVA irradiation of fibroblasts 5.1 Test Method 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 24-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0048] 2) Solution preparation: Prepare the working solution of the test substance according to the test group (Table 5).

[0049] Table 5 Test Groups 3) Drug administration: According to the test groups, when the cell deposition rate in the 24-well plate reached 30%~50%, drug administration was performed in groups, with 3 replicates per group. 1 mL of culture medium was added to each well of the blank control group and negative control group, 1 mL of culture medium containing quercetin was added to each well of the positive control group, and 1 mL of culture medium containing the corresponding test sample was added to each well of the sample group. After drug administration, the 24-well plate was incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours.

[0050] 4) UVA irradiation: Except for the blank control group, all other groups were subjected to UVA irradiation at a dose of 30 J / cm². After irradiation, they were placed in a CO2 incubator (37℃, 5% CO2) for 24 hours.

[0051] 5) Immunofluorescence detection: Cells were fixed with 4% paraformaldehyde for 30 min, and then immunofluorescence detection was performed. The staining results were observed and photographed under a fluorescence microscope, and the images were collected and analyzed using Image-Pro®Plus software.

[0052] 6) Calculation of lift rate: 7) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, P < 0.01 was considered significantly significant, and P < 0.001 was considered highly significant.

[0053] 5.2 Test Results 5.2.1 Type I Collagen Test Results The results are as follows Figure 5 As shown in Table 6, Figure 5 Images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents Collagen I. The stronger the green fluorescence, the higher the Collagen I content.

[0054] Table 6 Summary of Collagen I Immunofluorescence Analysis Results Note 1: Integrated optical density (IOD) / cell number reflects the content of Collagen I. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 = #, P-value < 0.01 = ##, P-value < 0.001 = ###); significance compared to the NC group is indicated by * (P-value < 0.05 = *, P-value < 0.01 = **, P-value < 0.001 = ***); significance compared to the competitor's -1.25% group is indicated by ▲ (P-value < 0.05 = ▲, P-value < 0.01 = ▲▲, P-value < 0.001 = ▲▲▲).

[0055] Note 2: P-value < 0.05 is represented by #, indicating a significant difference; P-value < 0.01 is represented by ##, indicating a significant difference; P-value < 0.001 is represented by ##, indicating a highly significant difference; P-value < 0.05 is represented by *, indicating a significant difference; P-value < 0.01 is represented by **, indicating a significant difference; P-value < 0.001 is represented by ***, indicating a highly significant difference; P-value < 0.05 is represented by ▲, indicating a significant difference; P-value < 0.01 is represented by ▲▲, indicating a significant difference; P-value < 0.001 is represented by ▲▲▲, indicating a highly significant difference.

[0056] like Figure 6 As shown, compared with the BC group, the Collagen I content in the NC group was significantly lower, indicating that the stimulation conditions in this test were effective.

[0057] Compared with the NC group, the Collagen I content in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0058] Compared with the NC group, the CollagenI content of Taishu Mitochondrial Energy Globule Peptide-1.25% (denoted as sample / 1.25%) and competitor-1.25% (denoted as competitor / 1.25%) both increased significantly, with increases of 192.11% and 176.32%, respectively.

[0059] 5.2.2 Results of Type IV Collagen Test Figure 7 Images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents the cell nucleus, and green fluorescence represents Collagen IV. The stronger the green fluorescence, the higher the Collagen IV content.

[0060] Table 7 Summary of Collagen IV Immunofluorescence Analysis Results Note 1: Integrated optical density (IOD) / cell number reflects the content of CollagenIV. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 = #, P-value < 0.01 = #, P-value < 0.001 = ###); significance compared to the NC group is indicated by * (P-value < 0.05 = *, P-value < 0.01 = **, P-value < 0.001 = ***); significance compared to the competitor's -1.25% group is indicated by ▲ (P-value < 0.05 = ▲, P-value < 0.01 = ▲▲, P-value < 0.001 = ▲▲▲).

[0061] Note 2: P-value < 0.05 is represented by #, indicating a significant difference; P-value < 0.01 is represented by #, indicating a significant difference; P-value < 0.001 is represented by ###, indicating a highly significant difference; P-value < 0.05 is represented by *, indicating a significant difference; P-value < 0.01 is represented by **, indicating a significant difference; P-value < 0.001 is represented by ***, indicating a highly significant difference; P-value < 0.05 is represented by ▲, indicating a significant difference; P-value < 0.01 is represented by ▲▲, indicating a significant difference; P-value < 0.001 is represented by ▲▲▲, indicating a highly significant difference.

[0062] like Figure 8 As shown, compared with the BC group, the Collagen IV content in the NC group was significantly lower, indicating that the stimulation conditions in this test were effective.

[0063] Compared with the NC group, the Collagen IV content in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0064] Compared with the NC group, the Collagen IV content of Taishu Mitochondrial Energy Globule Peptide-1.25% and the competing product-1.25% were significantly increased, with increases of 261.54% and 115.38%, respectively.

[0065] Compared with the competing product group (-1.25%), the Collagen IV content of Taishu Mitochondrial Energy Globule Peptide (-1.25%) was significantly increased.

[0066] 5.2.3 Elastin Test Results The results are as follows Figure 9 As shown in Table 8, Figure 9 Images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. Blue fluorescence represents cell nuclei, and green fluorescence represents elastin. The stronger the green fluorescence, the higher the elastin content.

[0067] Table 8 Summary of Elastin Immunofluorescence Analysis Results Note 1: Integrated optical density (IOD) / cell number reflects the elastin content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 = #, P-value < 0.01 = #, P-value < 0.001 = ###); significance compared to the NC group is indicated by * (P-value < 0.05 = *, P-value < 0.01 = **, P-value < 0.001 = ***); significance compared to the competitor's -1.25% group is indicated by ▲ (P-value < 0.05 = ▲, P-value < 0.01 = ▲▲, P-value < 0.001 = ▲▲▲).

[0068] Note 2: P-value < 0.05 is represented by #, indicating a significant difference; P-value < 0.01 is represented by #, indicating a significant difference; P-value < 0.001 is represented by ###, indicating a highly significant difference; P-value < 0.05 is represented by *, indicating a significant difference; P-value < 0.01 is represented by **, indicating a significant difference; P-value < 0.001 is represented by ***, indicating a highly significant difference; P-value < 0.05 is represented by ▲, indicating a significant difference; P-value < 0.01 is represented by ▲▲, indicating a significant difference; P-value < 0.001 is represented by ▲▲▲, indicating a highly significant difference.

[0069] like Figure 10 As shown, compared with the BC group, the NC group had a significantly lower Elastin content, indicating that the stimulation conditions in this test were effective.

[0070] Compared with the NC group, the PC group showed a significant increase in Elastin levels, indicating that the positive control in this test was effective.

[0071] Compared with the NC group, the elastin content of Taishu Mitochondrial Energy Globule Peptide-1.25% and the competing product-1.25% were significantly increased, with increases of 260.61% and 239.39%, respectively.

[0072] 5.5.4 Fibrillin 1 Test Results The results are as follows Figure 11 As shown in Table 9, the above images were taken using a fluorescence microscope (Olympus, BX43) at a magnification of 20×. The blue fluorescence represents the cell nucleus, and the green fluorescence represents Fibrillin1. The stronger the green fluorescence intensity, the higher the Fibrillin1 content.

[0073] Table 9 Summary of Fibrillin 1 Immunofluorescence Analysis Results Note 1: Integrated optical density (IOD) / cell number reflects the content of Fibrillin1. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 = #, P-value < 0.01 = ##, P-value < 0.001 = ##); significance compared to the NC group is indicated by * (P-value < 0.05 = *, P-value < 0.01 = **, P-value < 0.001 = ***); significance compared to the competitor's -1.25% group is indicated by ▲ (P-value < 0.05 = ▲, P-value < 0.01 = ▲▲, P-value < 0.001 = ▲▲▲).

[0074] Note 2: P-value < 0.05 is represented by #, indicating a significant difference; P-value < 0.01 is represented by #, indicating a significant difference; P-value < 0.001 is represented by ###, indicating a highly significant difference; P-value < 0.05 is represented by *, indicating a significant difference; P-value < 0.01 is represented by **, indicating a significant difference; P-value < 0.001 is represented by ***, indicating a highly significant difference; P-value < 0.05 is represented by ▲, indicating a significant difference; P-value < 0.01 is represented by ▲▲, indicating a significant difference; P-value < 0.001 is represented by ▲▲▲, indicating a highly significant difference.

[0075] like Figure 12 As shown, compared with the BC group, the Fibrillin 1 content in the NC group was significantly reduced, indicating that the stimulation conditions in this test were effective.

[0076] Compared with the NC group, the Fibrillin 1 content in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0077] Compared with the NC group, the content of Fibrillin 1 in Taishu ⑧ Mitochondrial Energy Globule Peptide-1.25% and the competing product-1.25% was significantly increased, with an increase rate of 78.85% and 61.54%, respectively.

[0078] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0079] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. The application of Taishu mitochondrial energy globulin, characterized in that, Choose from any of the following: Used to increase collagen content in fibroblasts for non-therapeutic purposes; Used to increase elastin content in fibroblasts for non-therapeutic purposes; Used to increase the fibrin content in fibroblasts for non-therapeutic purposes; It is used as an active ingredient in the preparation of topical products for the prevention and / or improvement of skin aging; The sequence of the Taishu mitochondrial energy globulin is nicotinic acid-CCArBrBrBrARGDA-NH2; wherein G is glycine, R and r are arginine, D is aspartic acid, A is tyrosine, B is tryptophan, C is phenylalanine, and in the polypeptide, r is in the D configuration, and the remaining amino acids are in the L configuration.

2. The application of the mitochondrial energy globulin according to claim 1, characterized in that, The external product is a medicine or cosmetic.

3. The application of the mitochondrial energy globulin according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application of the mitochondrial energy globulin according to claim 2, characterized in that, The cosmetic contains moisturizers, surfactants, ultraviolet absorbers, fragrances, antioxidants, preservatives and mildew inhibitors, colorants, pH adjusters, solvents, or ingredients commonly used in cosmetics.

5. The application of the mitochondrial energy globulin according to claim 4, characterized in that, The cosmetics mentioned are aqueous solutions, emulsions, creams, serums, or masks.

6. The application of the mitochondrial energy globulin according to claim 1, characterized in that, The skin in question includes both normal and aging skin.

7. The application of the mitochondrial energy globulin according to claim 6, characterized in that, The aging process includes natural aging and photoaging.

8. The application of the mitochondrial energy globulin according to claim 1, characterized in that, Skin aging is caused by insufficient levels of collagen, elastin, and fibrils.

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