Polypeptide, skin care substance and application
By providing polypeptides with specific amino acid sequences for the preparation of skin care products, the problem of difficult prevention and treatment of UVB photoaging is solved, and the effects of anti-photoaging, anti-inflammatory and pro-collagen expression are achieved.
Patent Information
- Application Number
- CN202411680397.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to effectively prevent and control skin photoaging caused by ultraviolet rays (especially UVB), and there are fewer applications of polypeptides in related studies.
A specific polypeptide is provided, and its amino acid sequence is any one of SEQ ID NO: 1 to 4, which is used to prepare skin care products, and has functions such as anti-UVB photoaging, anti-inflammatory, and promoting collagen expression.
This polypeptide can effectively avoid or reduce the damage caused by UVB, has anti-inflammatory effects, and promotes skin collagen expression, so as to prevent and treat skin photoaging and repair scratches.
Smart Images
Figure CN119462848A_ABST
Abstract
Description
[0001] This application is a divisional application based on the Chinese patent application with application number 2024116352678, application date November 15, 2024, and invention name “Skin care products, polypeptides and their uses”. Technical Field
[0002] The present invention relates to the field of peptide technology, in particular to polypeptides, skin care products and uses. Background Art
[0003] Skin will age prematurely when exposed to ultraviolet radiation (such as UVB) for a long time. This is a phenomenon called photoaging, which is different from natural aging. For the prevention and treatment of this photoaging, current research mainly focuses on vitamins; while in other aspects, such as using peptides to prevent the damage of photoaging, there are fewer related studies.
[0004] Therefore, it can be said that there are currently many difficulties that are difficult to overcome in terms of how to obtain a new peptide that can be used for anti-photoaging. Summary of the invention
[0005] One purpose of the embodiments of the present application is to solve at least one problem in the above background and provide corresponding beneficial effects.
[0006] Another purpose of the embodiments of the present application is to provide a polypeptide, a skin care product and uses, wherein the polypeptide can be used to solve the technical problem of anti-UVB photoaging.
[0007] The embodiments of the present application mainly achieve the above objectives through the following technical solutions.
[0008] In a first aspect, the present invention provides a polypeptide having any one of the amino acid sequences shown in SEQ ID NOs: 1 to 4:
[0009] SEQ ID NO: 1: KRIIKRIKKLLR;
[0010] SEQ ID NO:2: KRIIRRIKRLLR;
[0011] SEQ ID NO:3:RRIIRRIRRLLR;
[0012] SEQ ID NO:4: RRIIRRIRRWLR.
[0013] In some technical solutions, the amino acid sequence of the polypeptide is selected from any one of SEQ ID NOs: 1 to 4.
[0014] In a second aspect, the present application provides a use of the polypeptide described in the first aspect in preparing a skin care product.
[0015] In a third aspect, the present invention provides a skin care product, comprising:
[0016] The first aspect is the polypeptide described in the embodiment.
[0017] In a fourth aspect, an embodiment of the present application provides a use of the skin care product as an anti-photoaging cosmetic and / or an active oxygen inhibiting cosmetic and / or an anti-inflammatory cosmetic and / or a collagen expression promoting cosmetic.
[0018] The beneficial effects of the embodiments of the present invention include:
[0019] The polypeptide provided in the examples of the present application has a specific amino acid sequence, which can avoid or reduce the photoaging damage caused by UVB, and can therefore be used to prevent and treat photoaging of the skin.
[0020] In some embodiments, the polypeptides provided in the embodiments of the present application can also prevent or reduce photoaging damage caused by UVA.
[0021] In some embodiments, the polypeptides provided in the embodiments of the present application can also be used for anti-inflammatory purposes.
[0022] In some embodiments, the polypeptides provided in the embodiments of the present application can also be used to repair skin scratches. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a schematic diagram of relative cell survival rate in Experimental Example 2 of this application;
[0024] Figure 2 This is a schematic diagram of relative cell survival rate in Experimental Example 3 of this application;
[0025] Figure 3 This is a schematic diagram of active oxygen content in Experimental Example 4 of this application;
[0026] Figure 4 This is a schematic diagram of active oxygen content in Experimental Example 5 of this application;
[0027] Figure 5 This is a schematic diagram of a gene expression level in Experimental Example 6 of this application;
[0028] Figure 6 This is a schematic diagram of a gene expression level in Experimental Example 7 of this application;
[0029] Figure 7 This is a schematic diagram of a cell scratch area in Experimental Example 8 of this application;
[0030] Figure 8This is a schematic diagram of cell migration rate in Experimental Example 8 of this application. DETAILED DESCRIPTION
[0031] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.
[0032] The terms "first", "second", "third", etc. in the embodiments of the present application are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", etc. may explicitly or implicitly include at least one of the features.
[0033] Furthermore, the terms "comprises", "comprising", "including", "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or apparatuses.
[0034] It should also be noted that when it comes to amino acid sequences, unless otherwise clearly specified and limited, R represents arginine, Q represents glutamine, K represents lysine, D represents aspartic acid, I represents isoleucine, V represents valine, L represents leucine, F represents phenylalanine, and W represents tryptophan.
[0035] In addition to the above, it is still necessary to emphasize that the reference to "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0036] Ultraviolet light, in English, is called Ultraviolet or Ultraviolet Ray, abbreviated as UV or UVR. Ultraviolet light can be divided into different types such as UVA and UVB. When ultraviolet light (such as UVB) irradiates the skin, it will cause photoaging of the skin.
[0037] Peptide is an organic compound based on amino acids. KR-12 (amino acid sequence is KRIVQRIKDFLR) is a type of peptide. It is a short peptide that is easily absorbed by organisms, so it is a very valuable research object.
[0038] KR-12 is the 18-29 residues of the human LL-37 antimicrobial peptide, and is also a peptide fragment with antimicrobial activity in LL-37. It can be formed by cutting out a part of the sequence of LL-37. In the history of KR-12 research, the focus has been on its antimicrobial and immune functions, while its application in anti-photoaging has not yet been disclosed.
[0039] In the study of KR-12 polypeptide in the examples of the present application, it was unexpectedly found that certain specific improvements to the amino acid sequence of KR-12 can form a new polypeptide with anti-photoaging activity, as described below.
[0040] <Peptide>
[0041] The present application embodiment provides a polypeptide, wherein the polypeptide is selected from any one of KR12-M1, KR12-M2, KR12-M3, and KR12-M4; wherein the amino acid sequences of KR12-M1, KR12-M2, KR12-M3, and KR12-M4 are shown in SEQ ID NOs: 1 to 4, respectively:
[0042] SEQ ID NO: 1: KRIIKRIKKLLR;
[0043] SEQ ID NO:2: KRIIRRIKRLLR;
[0044] SEQ ID NO:3:RRIIRRIRRLLR;
[0045] SEQ ID NO:4: RRIIRRIRRWLR.
[0046] The polypeptides shown in the sequences of SEQ ID NOs: 1 to 4 provided in the examples of the present application have anti-photoaging effects and can be used to prevent and treat photoaging of the skin, and thus can be used to solve the problems raised in the background.
[0047] In addition to the above effects, the polypeptide has good solubility in water or aqueous solution (such as phosphate buffer), which is conducive to the performance of the polypeptide function.
[0048] In addition, compared with the long LL-37 peptide, the polypeptide of the embodiment of the present application, as a short peptide, has a lower production cost.
[0049] <Uses of polypeptides>
[0050] Based on the aforementioned polypeptide, the present application provides the following uses of the polypeptide.
[0051] In some embodiments, the polypeptide has an anti-photoaging effect and can therefore be used as a photoaging inhibitor.
[0052] In some embodiments, the polypeptide has an effect of inhibiting reactive oxygen species and can therefore be used as a reactive oxygen species inhibitor.
[0053] In some embodiments, the polypeptide has anti-inflammatory effects and thus can be used as an inflammation suppressant.
[0054] In some embodiments, the polypeptide has the effect of promoting collagen expression, and thus can be used as a collagen expression promoter.
[0055] In some embodiments, the collagen is selected from collagen-1 or collagen-3.
[0056] In some embodiments, the present application also provides a use of the polypeptide in preparing a skin care product.
[0057] In some embodiments, the skin care product has the purpose of anti-photoaging, that is, the skin care product is a skin care product with anti-photoaging function.
[0058] In some embodiments, the skin care product also has the purpose or function of inhibiting active oxygen.
[0059] In some embodiments, the skin care product also has anti-inflammatory use or function.
[0060] In some embodiments, the skin care product also has the purpose or function of promoting collagen expression.
[0061] In some embodiments, the skin care product also has the purpose or function of repairing scratches.
[0062] It is understandable that the polypeptides provided in the embodiments of the present application have a wide range of application scenarios in cosmetics because they have one or more of the above uses.
[0063] <Skin care products>
[0064] The present application embodiment provides a skin care product, comprising the polypeptide described in the above embodiment.
[0065] In some embodiments, the concentration of the polypeptide in the skin care product is 1-200 μM (μmol / L), optionally 1-100 μM.
[0066] In some embodiments, the skin treatment further comprises: a solvent.
[0067] In some embodiments, the solvent comprises water.
[0068] In some embodiments, the solvent is selected from water or an aqueous solution.
[0069] In some embodiments, the aqueous solution is phosphate buffered saline.
[0070] <Use of skin care products>
[0071] The embodiment of the present application provides a use of the skin care product as a cosmetic.
[0072] In some embodiments, the skin care product can be used as an anti-photoaging cosmetic.
[0073] In some embodiments, the skin care product can be used as an active oxygen inhibiting cosmetic.
[0074] In some embodiments, the skin care products can be used as anti-inflammatory cosmetics.
[0075] In some embodiments, the skin care products can be used as cosmetics for promoting collagen expression.
[0076] <Preparation of polypeptide>
[0077] The polypeptide can be prepared by solid-phase peptide synthesis (SPPS).
[0078] Illustratively, the solid phase synthesis sequence can be synthesized from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus).
[0079] Accordingly, the present embodiment provides a method S10 for preparing a polypeptide, comprising the following steps:
[0080] Step 1: The first amino acid at the C-terminus of the peptide is linked to a specific resin through a chemical bond, the α-amino group of the amino acid is protected with Fmoc, and the side chain group is protected with a corresponding acid-sensitive protecting group;
[0081] Step 2: Use an alkaline solution (such as DMF solution of piperidine) to remove the protective group Fmoc of the α-amino group of the first amino acid. At this time, the first amino acid with the α-amino group exposed remains on the resin;
[0082] Step 3: add an excess of a second amino acid whose α-amino group is protected by Fmoc and whose side chain group is protected by a corresponding acid-sensitive protecting group, and allow it to undergo a condensation reaction with the first amino acid connected to the resin through a condensation agent;
[0083] Step 4: Detection. Detection is generally performed by detecting the exposed α-amino group of the previous amino acid, generally using ninhydrin. If an exposed α-amino group is detected, it means that the reaction is not complete, and the third step is repeated. If the reaction is complete, the second, third, and fourth steps are repeated to continue the condensation reaction with the next amino acid until all amino acids are condensed, and then the Fmoc on the α-amino group of the last amino acid is removed (the method is the same as the second step);
[0084] Step 5: Use acid (commonly trifluoroacetic acid) to cut the peptide chain from the resin. At the same time, because the amino acid side chain protecting group is an acid-sensitive group, this step will also remove the protecting group on the side chain to obtain the desired polypeptide.
[0085] Example 1
[0086] The polypeptide represented by the sequence of SEQ ID NO: 1, namely, KR12-M1 polypeptide, was prepared according to the polypeptide solid phase synthesis method.
[0087] Example 2
[0088] The polypeptide represented by the sequence of SEQ ID NO: 2, namely, KR12-M2 polypeptide, was prepared according to the polypeptide solid phase synthesis method.
[0089] Example 3
[0090] The polypeptide represented by the sequence of SEQ ID NO: 3, namely, KR12-M3 polypeptide, was prepared according to the polypeptide solid phase synthesis method.
[0091] Example 4
[0092] The polypeptide represented by the sequence of SEQ ID NO: 4, namely, KR12-M4 polypeptide, was prepared according to the polypeptide solid phase synthesis method.
[0093] Comparative Example 1
[0094] The KR12-M5 polypeptide was prepared according to the polypeptide solid phase synthesis method. The sequence of KR12-M5 is as follows: WRIVWRIKDFLR.
[0095] Comparative Example 2
[0096] The KR12-M6 polypeptide was prepared according to the polypeptide solid phase synthesis method. The sequence of KR12-M6 is as follows: WRICRRFKLFLR.
[0097] Comparative Example 3
[0098] The KR12-M7 polypeptide was prepared according to the polypeptide solid phase synthesis method. The sequence of KR12-M7 is as follows: KRIIKRIKKWLR.
[0099] <Experimental Example 1, Peptide Stability Experiment>
[0100] In many cases, polypeptides need to be dissolved before administration, so their efficacy is related to solubility. However, any improvement to KR12 may not necessarily result in a polypeptide with better solubility. Therefore, the properties of the polypeptides in the embodiments of the present application are discussed below through solubility experiments.
[0101] Experimental method: Take the polypeptides KR12-M1 to M7 corresponding to Examples 1 to 4 and Comparative Examples 1 to 3, respectively, mix them in phosphate buffer to form a peptide solution with a concentration of 50 μM (μmol / L) (the concentration of the polypeptide is 50 μM), and detect the solubility of the polypeptide in each peptide solution at 4 degrees Celsius.
[0102] Results and analysis of Experimental Example 1
[0103] KR12-M5-M6 of Comparative Examples 1-2 turned into turbid liquid within 12 hours, showing protein flocculent precipitation, while the remaining polypeptides could still maintain clear protein solutions within 7 days. This shows that KR12-M1-M4 corresponding to Examples 1-4 and KR12-M7 of Comparative Example 3 have good solubility in phosphate buffer, while KR12-M5-M 6 of Comparative Examples 1-2 have very poor solubility and are unstable.
[0104] Due to poor solubility, KR12-M5-6 are difficult to prepare into solution preparations for use, and it is even more difficult to exert the effect of the polypeptide in anti-photoaging. In contrast, the polypeptides of Examples 1-4 of the present application have excellent solubility and better stability.
[0105] <Experimental Example 2: Anti-photoaging experiment of HaCaT cells>
[0106] The experimental method includes the following steps:
[0107] HaCaT (human immortalized keratinocytes) were used as test cells and divided into a blank control group, a UVB irradiation control group, a drug administration control group 1, a drug administration control group 2, and experimental groups 1 to 4;
[0108] Except for the blank control group, the other groups were treated with 50 mJ / cm 2 UVB rays are used for irradiation treatment;
[0109] After the irradiation treatment, the drug treatment was performed; specifically, the corresponding peptide solution (mixed with water and polypeptide) was added to the HaCaT cells of the drug control group 1, the drug control group 2, and the experimental groups 1 to 4, and the HaCaT cells of the blank control group and the UVB irradiation control group were not added with the peptide solution. The specific conditions of the drug treatment are shown in Table 1, wherein the concentration of the polypeptide in each peptide solution was 50 μM (μmol / L);
[0110] Table 1. Dosing conditions
[0111] Group Peptide Liquid Blank control group / UVB irradiation control group / Control group 1 Aqueous solution containing KR12 Control group 2 Aqueous solution containing KR12-M7 Experimental Group 1 Aqueous solution containing KR12-M1 Experimental Group 2 Aqueous solution containing KR12-M2 Experimental Group 3 Aqueous solution containing KR12-M3 Experimental Group 4 Aqueous solution containing KR12-M4
[0112] After drug treatment, the relative cell viability of each group was measured.
[0113] Results and analysis of Experimental Example 2
[0114] The relative cell survival rates of each group were compared, and the results are shown in Table 2. In addition, a bar chart was formed based on the relative cell survival rates of the blank control group, UVB irradiation control group, drug administration control group 1, and experimental groups 1 to 4. For details, see Figure 1 , along the horizontal axis, from left to right correspond to the blank control group, UVB irradiation control group, drug treatment control group 1, and experimental groups 1 to 4. "+" represents the existence of corresponding experimental conditions, and "-" represents the lack of corresponding experimental conditions.
[0115] Table 2. Comparison of relative cell survival rates in each group
[0116]
[0117]
[0118] From the above results, we can know that:
[0119] 1. The relative cell survival rate of the blank control group was 100%, and the relative cell survival rates of the UVB irradiation control group, drug control group 1, drug control group 2, and experimental groups 1 to 4 were all lower than those of the blank control group, indicating that UVB irradiation caused photoaging damage to HaCaT cells.
[0120] 2. In the relative cell survival rate results, the drug control group 1 and experimental groups 1 to 4 were higher than the UVB irradiation control group; this shows that KR12, KR12-M1 to 4 and other polypeptides have anti-photoaging damage effects against UVB irradiation.
[0121] 3. The relative cell survival rate of the drug-treated control group 2 was lower than that of the UVB-irradiated control group, indicating that the KR12-M7 polypeptide is basically difficult to play a protective role against photoaging damage caused by UVB irradiation.
[0122] In summary, any improvement to KR12 may not necessarily result in a polypeptide with anti-photoaging effect. Only by making specific improvements to KR12 can a polypeptide with anti-photoaging effect be obtained, such as the polypeptides KR12-M1 to M4 provided in the examples of this application.
[0123] <Experimental Example 3: Anti-photoaging experiment of HDF cells>
[0124] The experimental method includes the following steps:
[0125] HDF (human dermal fibroblasts) were taken as the test cells and divided into blank control group, UVA irradiation control group, drug administration control group 1, drug administration control group 2, and experimental groups 1 to 4;
[0126] Except for the blank control group, the other groups were treated with 7mJ / cm 2 UVA rays for irradiation;
[0127] After the irradiation treatment, the drug treatment was performed; specifically, the corresponding peptide solution (mixed with water and polypeptide) was added to the HDF cells of the drug control group 1, the drug control group 2, and the experimental groups 1 to 4, and the HaCaT cells of the blank control group and the UVA irradiation control group were not added with the peptide solution. The specific conditions of the drug treatment are shown in Table 3, wherein the concentration of the polypeptide in each peptide solution was 50 μM;
[0128] Table 3. Dosing conditions
[0129] Group Peptide Liquid Blank control group / UVA irradiation control group / Control group 1 Aqueous solution containing KR12 Control group 2 Aqueous solution containing KR12-M7 Experimental Group 1 Aqueous solution containing KR12-M1 Experimental Group 2 Aqueous solution containing KR12-M2 Experimental Group 3 Aqueous solution containing KR12-M3 Experimental Group 4 Aqueous solution containing KR12-M4
[0130] After drug treatment, the relative cell survival rate of each group was determined.
[0131] Results and analysis of Experimental Example 3
[0132] The relative cell survival rates of each group were compared, and the results are shown in Table 3. In addition, a bar chart was formed based on the relative cell survival rates of the blank control group, UVA irradiation control group, drug administration control group 1, and experimental groups 1 to 4. For details, see Figure 2 , along the horizontal axis, from left to right correspond to the blank control group, UVA irradiation control group, drug treatment control group 1, and experimental groups 1 to 4. "+" represents the existence of corresponding experimental conditions, and "-" represents the lack of corresponding experimental conditions.
[0133] Table 4. Comparison of relative cell survival rates of each group
[0134] Group Relative cell viability Blank control group 100％ UVA irradiation control group Less than blank control group Control group 1 Smaller than the blank control group, larger than the UVA irradiation control group Control group 2 Less than the UVA irradiation control group Experimental Group 1 Less than blank control group Experimental Group 2 Less than blank control group Experimental Group 3 Smaller than the blank control group, larger than the UVA irradiation control group Experimental Group 4 Smaller than the blank control group, larger than the UVA irradiation control group
[0135] From the above results, we can know that:
[0136] 1. The relative cell survival rate of the blank control group was 100%, and the relative cell survival rates of the UVA irradiation control group, drug control group 1, drug control group 2, and experimental groups 1 to 4 were all lower than those of the blank control group, indicating that UVA irradiation caused photoaging damage to HDF cells.
[0137] 2. In the relative cell viability results, the drug-administered control group 1 and experimental groups 3-4 were all higher than the UVA-irradiated control group. This indicates that polypeptides such as KR12 and KR12-M3-4 have anti-photoaging damage effects against UVA irradiation.
[0138] 3. The relative cell viability of the drug-administered control group 2 was lower than that of the UVA-irradiated control group, indicating that the KR12-M7 polypeptide is difficult to play a protective role against photoaging damage caused by UVA irradiation.
[0139] <ROS Detection Experiment>
[0140] In a biological context, reactive oxygen species (英文名称为Reactive Oxygen Species, abbreviated as ROS) are natural by-products of the normal metabolism of oxygen and play important roles in cell signaling and homeostasis. During environmental stress (such as ultraviolet irradiation), the level of ROS will increase sharply, which may cause serious damage to cell structures.
[0141] Accordingly, the following ROS detection experiment is used to determine the properties of the polypeptides in the embodiments of the present application in terms of anti-ROS.
[0142] <Experimental Example 4. ROS Detection Experiment of HaCaT Cells>
[0143] The experimental method includes the following steps:
[0144] Take HaCaT cells as the cells to be tested and divide them into a blank control group, a UVB-irradiated control group, a drug-administered control group 1, and experimental groups 1-4;
[0145] Except for the blank control group, other groups were respectively irradiated with UVB light of 50 mJ / cm 2 for treatment;
[0146] After the irradiation treatment, drug administration treatment was carried out. Specifically, the corresponding peptide solution (mixed by water and polypeptide) was added to the HaCaT cells of the drug-administered control group 1 and experimental groups 1-4, and the HaCaT cells of the blank control group and the UVB-irradiated control group were not added with peptide solution. The conditions of the drug administration treatment are shown in Table 5 specifically, where the concentration of the polypeptide in each peptide solution is 50 μM;
[0147] Table 5. Drug Administration Conditions
[0148]
[0149]
[0150] After the drug administration treatment, the relative amount of reactive oxygen species generation (ROS generation) of each group was measured.
[0151] Results and analysis of Experimental Example 4
[0152] The results are shown in Figure 3 ; Figure 3 It is a bar chart comparison diagram of the relative ROS production of each group. In the figure, along the horizontal axis, from left to right are the bar charts of the blank control group, UVA irradiation control group, drug treatment control group 1, and experimental groups 1 to 4; "+" represents the existence of corresponding experimental conditions, and "-" represents the lack of corresponding experimental conditions.
[0153] according to Figure 3 It can be seen that:
[0154] 1. The relative ROS production of the blank control group was 100%, while the relative ROS production of the UVB irradiation control group, drug administration control group 1, and experimental groups 1 to 4 was much higher than that of the blank control group, indicating that UVB irradiation caused HaCaT cells to increase the production of reactive oxygen species (ROS).
[0155] 2. In the results of relative ROS production, the drug control group 1 and experimental groups 1 to 4 were lower than the UVB irradiation control group; this shows that KR12, KR12-M1 to 4 and other polypeptides have an inhibitory effect on the proliferation of reactive oxygen species (ROS) caused by UVB irradiation. In addition, KR12-M1 to 4 and other polypeptides are better than KR12 polypeptide in inhibiting the proliferation of reactive oxygen species.
[0156] <Experimental Example 5, ROS detection experiment of HDF cells>
[0157] The experimental method includes the following steps:
[0158] HDF cells were taken as test cells and divided into blank control group, UVA irradiation control group, drug administration control group 1, and experimental groups 1 to 4;
[0159] Except for the blank control group, the other groups were treated with 7mJ / cm 2 UVA rays for irradiation;
[0160] After the irradiation treatment, the drug administration treatment was performed; specifically, the corresponding peptide solution (mixed with water and polypeptide) was added to the HaCaT cells of the drug administration control group 1 and the experimental groups 1 to 4, and the HaCaT cells of the blank control group and the UVB irradiation control group were not added with the peptide solution. The specific conditions of the drug administration treatment are shown in Table 6, wherein the concentration of the polypeptide in each peptide solution was 50 μM;
[0161] Table 6. Dosing conditions
[0162]
[0163]
[0164] After drug administration, the relative reactive oxygen species generation (ROS generation) of each group was measured.
[0165] Experimental results and analysis
[0166] The results are shown in Figure 4 ; Figure 4 It is a bar chart comparison diagram of the relative ROS production of each group. In the figure, along the horizontal axis, from left to right are the bar charts of the blank control group, UVA irradiation control group, drug treatment control group 1, and experimental groups 1 to 4; "+" represents the existence of corresponding experimental conditions, and "-" represents the lack of corresponding experimental conditions.
[0167] according to Figure 4 It can be seen that:
[0168] 1. The relative ROS production of the blank control group was 100%, and the relative ROS production of the UVA irradiation control group, the drug treatment control group 1, and the experimental groups 1 to 4 was much higher than that of the blank control group, indicating that UVA irradiation caused HaCaT cells to increase the production of reactive oxygen species (ROS).
[0169] 2. In the results of relative ROS production, the drug control group 1 and experimental groups 1 to 4 were lower than the UVA irradiation control group; this shows that KR12, KR12-M1 to 4 and other polypeptides have an inhibitory effect on the proliferation of reactive oxygen species (ROS) caused by UVA irradiation. In addition, KR12-M1 to 4 and other polypeptides are better than KR12 polypeptide in inhibiting the proliferation of reactive oxygen species.
[0170] <Gene expression detection experiment>
[0171] The polypeptides of the examples of the present application can also play an anti-inflammatory and collagen expression-promoting role through the regulation of cellular genes, which is verified by the following gene expression detection experiment.
[0172] <Experimental Example 6, Detection of Gene Expression in HaCaT Cells>
[0173] The experimental methods include:
[0174] HaCaT cell lines were used as test cells and divided into a blank control group (Control), a UVR irradiation control group, a drug administration control group 1, and experimental groups 1 to 4;
[0175] Except for the blank control group, the other groups were treated with 50 mJ / cm 2 UVB rays are used for irradiation treatment;
[0176] After the irradiation treatment, the drug administration treatment was performed; specifically, the corresponding peptide solution (mixed with water and polypeptide) was added to the HaCaT cells of the drug administration control group 1 and the experimental groups 1 to 4, and the HaCaT cells of the blank control group and the UVR irradiation control group were not added with the peptide solution. The specific conditions of the drug administration treatment are shown in Table 7, wherein the concentration of the polypeptide in each peptide solution was 50 μM;
[0177] Table 7. Dosing conditions
[0178] Group Peptide Liquid Blank control group / UVR irradiation control group / Control group 1 Aqueous solution containing KR12 Experimental Group 1 Aqueous solution containing KR12-M1 Experimental Group 2 Aqueous solution containing KR12-M2 Experimental Group 3 Aqueous solution containing KR12-M3 Experimental Group 4 Aqueous solution containing KR12-M4
[0179] The expression levels of genes such as TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and IL-6 (interleukin-6) in each group of cells were detected by qPCR (Real-time Quantitative PCR Detecting System).
[0180] Results and analysis of Experimental Example 6
[0181] 1. After UVR stimulation, the expression levels of TNF-α, IL-1β and IL-6 genes increased significantly, so the expression of these three inflammatory cytokines was upregulated. Among them, compared with the blank control group, the tumor necrosis factor-α (TNF-α) in the HaCaT cells of the UVR irradiation control group increased the most, increasing by about 10 times.
[0182] 2. Experimental groups 1 to 4 were similar to control group 1, and the gene expression levels of TNF-α, IL-1β, and IL-6 were significantly downregulated; this indicates that KR-12 analogs such as KR12-M1, KR12-M2, KR12-M3, and KR12-M4 also showed significant anti-inflammatory effects, which can reduce the TNF-α gene expression level in HaCaT cells by 2.7 to 5.3 times, the IL-1β gene expression level by 2.0 to 2.5 times, and the IL-6 gene expression level by 2.7 to 5.9 times.
[0183] 3. In addition, for each gene's qPCR measurement value X, the corresponding common logarithm [Regulation (LgX)] is taken, and then a comparative diagram of gene expression levels is drawn based on the common logarithm. Figure 5 . Figure 5 This is a schematic diagram of the gene expression levels of TNF-α, IL-1β, and IL-6 in each group. In the figure, the redder the color corresponding to the gene, the greater the expression level, and the greener the color corresponding to the gene, the lower the expression level.
[0184] The common logarithm refers to the logarithm with base 10.
[0185] <Experimental Example 7: Detection of Gene Expression in HDF Cells>
[0186] The experimental methods include:
[0187] HDF cell lines were used as test cells and divided into a blank control group (Control), a UVR irradiation control group, a drug administration control group 1, and experimental groups 1 to 4;
[0188] Except for the blank control group, the other groups were treated with 7mJ / cm 2 UVA rays for irradiation;
[0189] After the irradiation treatment, the drug treatment was performed; specifically, the corresponding peptide solution (mixed with water and polypeptide) was added to the HDF cells of the drug control group 1 and the experimental groups 1 to 4, and the HDF cells of the blank control group and the UVB irradiation control group were not added with the peptide solution. The specific conditions of the drug treatment are shown in Table 8, wherein the concentration of the polypeptide in each peptide solution was 50 μM;
[0190] Table 8. Dosing conditions
[0191] Group Peptide Liquid Blank control group / UVR irradiation control group / Control group 1 Aqueous solution containing KR12 Experimental Group 1 Aqueous solution containing KR12-M1 Experimental Group 2 Aqueous solution containing KR12-M2 Experimental Group 3 Aqueous solution containing KR12-M3 Experimental Group 4 Aqueous solution containing KR12-M4
[0192] The expression levels of TNF-α gene, IL-1β gene, IL-6 gene, colgen-1 (collagen-1) gene and colgen-3 (collagen-3) gene in each group of cells were detected by qPCR (Real-time Quantitative PCR Detecting System).
[0193] Results and analysis of Experimental Example 7
[0194] 1. After UVR stimulation, the gene expression levels of TNF-α, IL-1β, and IL-6 increased significantly, so the expression of these three inflammatory cytokines was upregulated. Among them, compared with the blank control group, the increase of interleukin-6 (IL-6) in HDF cells of the UVR irradiation control group was the lowest, increasing by about 1.5 times.
[0195] 2. For anti-inflammatory effects, experimental groups 1 to 4 and control group 1 had similar results, and the gene expression levels of TNF-α, IL-1β, and IL-6 were significantly downregulated; this indicates that KR-12 analogs such as KR12-M1, KR12-M2, KR12-M3, and KR12-M4 also showed significant anti-inflammatory effects, which can approximately reduce the TNF-α gene expression level in HDF cells by about 3 times, the IL-1β gene expression level by 1.5 times, and the IL-6 gene expression level to a level close to that of the blank control group.
[0196] 3. According to the comparison between the UVR irradiation control group and the blank control group, UVA stimulation can reduce the expression level of collagen-1 gene in HDF cells by about 47.9% and the expression level of collagen-3 gene by 67.4%.
[0197] According to the results of the control group 1 and experimental groups 1 to 4, collagen was supplemented after the peptide treatment. Among them, KR-12-M1 and KR-12-M3 increased the expression of collagen-1 gene by approximately 12.6% and 28.0%, respectively; for collagen-3 gene, KR-12-M4 was the most effective, increasing by approximately 45.4%.
[0198] 4. Skin is a complex multi-layer connective tissue, mainly composed of the epidermis and dermis, separated by a basement membrane. The epidermis is mainly composed of keratinocytes, which are responsible for the protective function of the skin; while the dermis is rich in immune cells, fibroblasts and extracellular matrix (ECM), and the extracellular matrix of the dermis is mainly composed of various extracellular glycoproteins, including collagen, fibronectin, laminin, elastin and polysaccharides. Collagen I (collagen-1) is the most abundant structural protein in the dermal extracellular matrix, cross-linked and interwoven with collagen III (collagen-3) to form a fiber network, providing cell support and maintaining skin elasticity. Therefore, it can be understood that the polypeptide of the present application embodiment can promote the synthesis of collagen in the skin, which is conducive to increasing the collagen content and protecting the skin.
[0199] 5. In addition, for each gene's qPCR measurement value X, the corresponding common logarithm [Regulation (LgX)] is taken, and then a comparative diagram of gene expression levels is drawn based on the common logarithm. For details, please refer to Figure 6 . Figure 6 This is a schematic diagram of the gene expression levels of TNF-α, IL-1β, IL-6, colgen-1, and colgen-3 in each group. In the figure, the redder the color corresponding to the gene, the greater the expression level, and the greener the color corresponding to the gene, the lower the expression level. Figure 6 COL-1 and COL-3 stand for colgen-1 and colgen-3, respectively.
[0200] The common logarithm refers to the logarithm with base 10.
[0201] <Experimental Example 8, Cell Repair Experiment of HaCaT Cells>
[0202] The experimental methods include:
[0203] HaCaT cells were taken as test cells and divided into a blank control group (Control), a drug-treated control group 1, and experimental groups 1 to 4;
[0204] The cells to be tested in each group were scratched;
[0205] After the scratch treatment, the drug treatment was performed; specifically, the corresponding peptide solution (mixed with water and polypeptide) was added to the HaCaT cells of the drug control group 1 and the experimental groups 1 to 4, and the HaCaT cells of the blank control group and the UVR irradiation control group were not added with the peptide solution. The specific conditions of the drug treatment are shown in Table 9, wherein the concentration of the polypeptide in each peptide solution was 50 μM;
[0206] Table 9. Dosing conditions
[0207] Group Peptide Liquid Blank control group / Control group 1 Aqueous solution containing KR12 Experimental Group 1 Aqueous solution containing KR12-M1 Experimental Group 2 Aqueous solution containing KR12-M2 Experimental Group 3 Aqueous solution containing KR12-M3 Experimental Group 4 Aqueous solution containing KR12-M4
[0208] The scratch area of each group of cells was detected at 0h and 12h after scratching, and the cell migration rate of each group was calculated after 12h.
[0209] Results and analysis of Experimental Example 8
[0210] The scratch results can be found in Figure 7 and 8 .
[0211] Figure 7 A schematic diagram of the scratch area of each group. Figure 7 In the table, Control represents the blank control group, KR12 represents the drug-treated control group 1, KR12-M1 represents the experimental group 1, KR12-M2 represents the experimental group 2, KR12-M3 represents the experimental group 3, and KR12-M4 represents the experimental group 4. Figure 7 The scale bar of the corresponding pictures in each group is 50 μm (micrometer).
[0212] Figure 8 A schematic diagram of cell migration rate in each group. Figure 8 In the figure, A represents the blank control group (Control), B represents the drug-treated control group 1, C represents the experimental group 1, D represents the experimental group 2, E represents the experimental group 3, and F represents the experimental group 4.
[0213] according to Figure 7 It can be seen that in the blank control group and the drug-treated control group 1, the scratch area changed slightly after the 12th hour, almost the same as at the 0th hour, and there was basically no repair effect.
[0214] However, in experimental groups 1 to 4, the scratch area at the 12th hour was significantly reduced, which indicates that the treatment of scratched cells with polypeptides such as KR12-M1 to M4 can increase cell migration rate and enhance the expression of cell growth factors, thereby promoting skin repair.
[0215] according to Figure 8It can be seen that after the cells are treated with the KR12-M1-M4 polypeptides of the examples of the present application, the cell migration rate can be increased by approximately 11% to 21%, which is much higher than that of KR12.
[0216] The above specific embodiments have been described in detail for the present invention, but these do not constitute limitations on the present invention. The protection scope of the present invention is not limited to the above embodiments, and any equivalent modifications or changes made by ordinary technicians in the field based on the content disclosed by the present invention should be included in the protection scope recorded in the claims.
Claims
1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 1 to 3, SEQ ID NO: 1: KRIIKRIKKLLR; SEQ ID NO:2: KRIIRRIKRLLR; SEQ ID NO:3:RRIIRRIRRLLR.
2. Use of the polypeptide according to claim 1 as a photoaging inhibitor and / or a reactive oxygen species inhibitor and / or an inflammation inhibitor and / or a collagen expression promoter.
3. Use of the polypeptide according to claim 1 in preparing a skin care product.
4. The use according to claim 3, characterized in that The skin care product has the functions of anti-photoaging and / or inhibiting active oxygen and / or anti-inflammatory.
5. The use according to claim 3, characterized in that: The skin care product has the function of promoting collagen expression; wherein the collagen is selected from collagen-1 or collagen-3.
6. The use according to claim 3, characterized in that: The skin care product has the function of repairing skin scratches.
7. A skin care product, characterized in that include: The polypeptide according to claim 1.
8. The skin care product according to claim 7, characterized in that Also includes: Solvent.
9. Use of the skin care product according to any one of claims 7 to 8 as an anti-photoaging cosmetic and / or an active oxygen inhibiting cosmetic and / or an anti-inflammatory cosmetic and / or a collagen expression promoting cosmetic.