Linear polypeptides and their use in cosmetics

Abstract

This invention provides a linear polypeptide and its application in cosmetics, belonging to the field of active molecule development technology. The polypeptide provided by this invention has a higher binding affinity to integrin receptors, significantly promoting fibroblast proliferation, increasing the content of elastin and type III collagen in the skin, and effectively scavenging DPPH free radicals and reducing reactive oxygen species levels. This polypeptide can be widely used in the preparation of cosmetics or topical skin formulations with anti-aging, antioxidant, and skin-repair promoting functions. It features high activity, good stability, and excellent safety, making it suitable as an active ingredient in functional skincare products.

Description

Technical Field

[0001] This invention relates to the field of active molecule development technology, and in particular to a linear polypeptide and its application in cosmetics. Background Technology

[0002] Peptides are increasingly used in the fields of biomedicine and cosmetics. Among them, peptides containing the arginine-glycine-aspartic acid (RGD) sequence have attracted attention due to their ability to be specifically recognized by cell surface integrin receptors. Integrins are important receptor molecules that mediate cell-extracellular matrix adhesion and participate in regulating various biological processes such as cell proliferation, migration, differentiation, and apoptosis. The RGD sequence was first discovered in fibronectin and subsequently confirmed to exist in various extracellular matrix proteins such as hyalin, collagen, and laminin. Based on its well-defined recognition characteristics, RGD peptides are widely used in areas such as biomaterial surface modification, tissue engineering scaffold construction, and targeted drug delivery.

[0003] In recent years, with the rapid development of the functional cosmetics industry, RGD peptides have gradually been introduced into skin care products. Studies have shown that RGD sequences can influence cell adhesion behavior by binding to integrins on the surface of dermal fibroblasts and keratinocytes, thereby participating in processes such as skin barrier repair, collagen synthesis, and anti-inflammation. However, existing RGD peptides still face some challenges in practical applications. For example, their conformational stability in complex environments is limited, and they are prone to conformational changes, thus affecting their binding efficiency and duration of action with integrin receptors. In addition, peptides are easily degraded by proteases in physiological or physiological-like environments, leading to a shortened effective duration and reduced bioavailability. Furthermore, the binding selectivity and affinity of existing RGD peptides with different integrin isoforms still have room for improvement, making it difficult to meet diverse application needs.

[0004] Therefore, developing RGD-related peptides with better structural stability and higher receptor binding activity still has significant research value and market prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a linear polypeptide that solves the technical problems of low binding efficiency and insufficient stability of existing short peptides containing RGD motifs to integrin receptors due to their high conformational flexibility.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a polypeptide composed of the amino acid sequence shown in SEQ ID NO.1, wherein the N-terminus of the polypeptide is a free amino group and the C-terminus is a free carboxyl group.

[0008] The present invention also provides a cosmetic composition comprising the above-mentioned polypeptide.

[0009] Preferably, the cosmetic composition further comprises at least one of a moisturizer, emulsifier, preservative, thickener, antioxidant, pH adjuster, or solvent.

[0010] This invention also provides the application of the above-mentioned polypeptide in the preparation of topical skin preparations with anti-aging effects.

[0011] This invention also provides the application of the above-mentioned polypeptide in the preparation of topical skin preparations with antioxidant effects.

[0012] The present invention also provides the application of the above-mentioned polypeptide in the preparation of topical skin formulations for increasing the content of elastin in the skin.

[0013] The present invention also provides the application of the above-mentioned polypeptide in the preparation of topical skin formulations for increasing the content of type III collagen in the skin.

[0014] The present invention also provides the use of the above-mentioned polypeptide in the preparation of topical skin formulations for reducing the level of reactive oxygen species in skin cells.

[0015] The present invention also provides the use of the above-mentioned polypeptide in the preparation of topical skin formulations for promoting fibroblast proliferation.

[0016] The present invention also provides an anti-aging topical product, using the above-mentioned polypeptide as an active ingredient; the dosage form of the topical product includes solution, gel, cream, lotion, serum, facial mask liquid or lyophilized powder.

[0017] The beneficial effects of this invention are: The polypeptide provided by this invention has a higher binding affinity to integrin receptors, significantly promoting fibroblast proliferation, increasing the content of elastin and type III collagen in the skin, and effectively scavenging DPPH free radicals and reducing reactive oxygen species levels. This polypeptide can be widely used in the preparation of cosmetics or topical skin formulations with anti-aging, antioxidant, and skin-repair promoting functions. It features high activity, good stability, and excellent safety, making it suitable as an active ingredient in functional skincare products. Attached Figure Description

[0018] Figure 1 The images show the liquid chromatography (LC) and mass spectrometry (MS) spectra of the peptides. Figures 2-1 to 2-13 This is a fibroblast proliferation test report (report number: SHA01-25081184-JC-01), which includes: Figure 2-1 For the report cover; Figure 2-2 This is the report's basic information page; Figure 2-3 For testing purposes and test project pages; Figure 2-4 For testing materials; Figure 2-5 This is the cytotoxicity test page for the testing method; Figure 2-6 This is the cell proliferation test page for the testing method; Figure 2-7 Table of cytotoxicity test results; Figure 2-8 This is a graph showing the cytotoxicity results (KTRGD). Figure 2-9 This is a graph showing the cytotoxicity results (RGD). Figure 2-10 This is a graph showing the cytotoxicity results (KT). Figure 2-11 The results of the cell proliferation test are presented in a table and bar chart. Figure 2-12 This is the test conclusion page; Figure 2-13 This is the declaration page; Figures 3-1 to 3-14 This is a test report on the content of elastin and type III collagen (report number: SHA01-25081184-JC-13), which includes: Figure 3-1 For the report cover; Figure 3-2 This is the report's basic information page; Figure 3-3 For testing purposes and test project pages; Figure 3-4 For testing materials; Figure 3-5 This is the cytotoxicity test page for the testing method; Figure 3-6 This is the page for testing the content of Elastin and Collagen III using the test method; Figure 3-7 Table of cytotoxicity test results; Figure 3-8 This is a graph showing the cytotoxicity results (KTRGD). Figure 3-9 This is a graph showing the cytotoxicity results (RGD). Figure 3-10 This is a graph showing the cytotoxicity results (KT). Figure 3-11 Table and bar chart showing Elastin content results; Figure 3-12 Table and bar chart showing the Collagen III content results; Figure 3-13 This is the test conclusion page; Figure 3-14 This is the declaration page; Figures 4-1 to 4-8 This is a DPPH free radical scavenging rate test report (test report number: SHA01-25081190-JC-01), in which: Figure 4-1 For the report cover; Figure 4-2 This is the report's basic information page; Figure 4-3 For testing purposes and test material pages; Figure 4-4 For the test method page; Figure 4-5 Table and graph showing the DPPH scavenging rate of vitamin C, the reference standard for the system. Figure 4-6 Table showing the DPPH removal rate results for the samples; Figure 4-7 The bar chart shows the DPPH scavenging rate of the samples, and the conclusion page is also included. Figure 4-8 This is the declaration page; Figures 5-1 to 5-14This is a report on the detection of reactive oxygen species content in UVA-stimulated fibroblasts (report number: SHA01-25081190-JC-13), which includes: Figure 5-1 For the report cover; Figure 5-2 This is the report's basic information page; Figure 5-3 For testing purposes and test material pages; Figure 5-4 This is the cytotoxicity test page for the testing method; Figure 5-5 This is the ROS content test page for the testing method; Figure 5-6 For the cytotoxicity test results table (KTRGD); Figure 5-7 For the cytotoxicity test results (RGD); Figure 5-8 For the cytotoxicity test results table (KT); Figure 5-9 Cell viability graphs (KTRGD, RGD, KT); Figure 5-10 Fluorescence spectrum of ROS content (blank control, negative control, positive control, KTRGD-1ppm); Figure 5-11 Fluorescence diagrams for ROS content (RGD-1ppm, RGD-10ppm, KT-1ppm, KT-10ppm); Figure 5-12 This is a summary table of ROS data analysis. Figure 5-13 The bar chart shows the relative ROS content, and the conclusion page is also included. Figure 5-14 This is the declaration page; Figure 6 This is a superimposed diagram of representative conformations of KTRGD and RGD in the fibronectin receptor α5β1 integrin model (PDBID:4WK4); Figure 7 A comparative diagram of key interactions between KTRGD and RGD in the fibronectin receptor α5β1 integrin model (PDBID:4WK4); Figure 8 This represents the relative score results in a typical integrin receptor model; Figure 9 This is a diagram of the linear polypeptide structure of the present invention. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example This embodiment provides a linear polypeptide containing the RGD motif, with the amino acid sequence H-Lys-Thr-Arg-Gly-Asp-OH, as shown in SEQ ID NO.1.

[0021] The polypeptide was synthesized based on the above sequence: (1) Solid-phase synthesis of peptide chains: Weigh an appropriate amount of 2-chlorotriphenylmethylchloro resin (2-CTC resin, loading 0.98 mmol / g), place it in a reaction tube, add dichloromethane (DCM), and allow it to swell at room temperature for 1 hour. After swelling, remove the solvent, then dissolve the first amino acid monomer at the C-terminus (1.5 equiv) and N,N-diisopropylethylamine (DIEA, 5 equiv) in dichloromethane, add it to the reaction tube, and allow it to react with shaking at room temperature for 1 hour. After the reaction, wash the resin sequentially with N,N-dimethylformamide (DMF) and dichloromethane.

[0022] The Fmoc protecting group was then removed: a 20% piperidine / DMF solution was added to the resin, and deprotection was performed twice, 10 minutes each time, followed by washing with DCM and DMF. Next, the second amino acid was coupled. The second amino acid monomer (5 equiv), condensing agent HBTU (4.75 equiv), and DIEA (5 equiv) were dissolved in DMF, activated, and added to the reaction tube. The reaction was carried out at room temperature with shaking for 1 hour, followed by washing the resin with DMF and DCM. This process of "deprotection—washing—coupling—washing" was repeated until all amino acid monomers were coupled sequentially.

[0023] After the last amino acid coupling is completed, the N-terminal Fmoc protecting group is removed again, following the same procedure as before: add 20% piperidine / DMF solution and treat twice, 10 minutes each time, then wash the resin with DCM, DMF and DCM in sequence, and finally dry the resin.

[0024] (2) Peptide cleavage: A cleavage solution was prepared by mixing hexafluoroisopropanol and dichloromethane at a volume ratio of 1:4. The solution was added to the dried resin and the mixture was shaken at room temperature for 30 minutes. This process was repeated once. The filtrates collected from the two reactions were combined in a centrifuge tube and concentrated to dryness by rotary evaporation to obtain the crude peptide product.

[0025] (3) Purification: The obtained crude peptide was dissolved in an appropriate amount of acetonitrile-water (1:1, containing 0.1% trifluoroacetic acid), filtered through a 0.22 μm filter membrane, and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The target product fraction was collected, combined, and freeze-dried to obtain a white flocculent solid, which was the pure peptide.

[0026] (5) Detection of peptide samples: The prepared products were analyzed by LCMS, and the results are shown in the figure. Figure 1 .

[0027] The bioactivity of the polypeptides of this invention will be described below in conjunction with the test report.

[0028] I. Fibroblast proliferation test (Test report SHA01-25081184-JC-01, 13 pages in total, corresponding to...) Figures 2-1 to 2-13 ) like Figures 2-1 to 2-13 As shown, this test, based on a human fibroblast model, examined the effects of the pentapeptide KTRGD, control RGD, and control KT at concentrations of 1 ppm and 10 ppm on cell viability after 48 hours of treatment. The results showed that the pentapeptide KTRGD significantly promoted fibroblast proliferation at both 1 ppm and 10 ppm concentrations, with a significant increase in cell viability at 1 ppm (p<0.05) and a highly significant increase at 10 ppm (p<0.01). Control RGD showed a highly significant proliferative effect at both concentrations (p<0.01). Control KT only showed a highly significant effect at a concentration of 10 ppm (p<0.01), with no significant difference at 1 ppm. This indicates that the pentapeptide of this invention has a firming effect.

[0029] II. Determination of Elastin and Type III Collagen Content (Test Report SHA01-25081184-JC-13, 14 pages in total, corresponding to...) Figures 3-1 to 3-14 ) like Figures 3-1 to 3-14 As shown, this test, based on a UVA-stimulated human fibroblast model, examined the effects of the pentapeptide KTRGD of this invention, control RGD, and control KT at concentrations of 1 ppm and 10 ppm on the content of elastin and type III collagen. After UVA stimulation, the content of elastin and type III collagen in the negative control group decreased significantly (p<0.01), indicating successful model establishment. The positive control group (VC+VE) significantly increased the content of both proteins (p<0.01). The pentapeptide KTRGD of this invention significantly increased the content of elastin at both 1 ppm and 10 ppm concentrations (p<0.01), and also significantly increased the content of type III collagen (p<0.01). Control RGD also showed a significantly increased effect at both concentrations (p<0.01). The control group KT showed a significant increase in elastin production at 1 ppm (p<0.05) and a highly significant increase at 10 ppm (p<0.01); it also showed a significant increase in type III collagen production at 1 ppm (p<0.05) and a highly significant increase at 10 ppm (p<0.01). This indicates that the pentapeptide of the present invention promotes the synthesis of elastin and type III collagen.

[0030] III. DPPH Free Radical Scavenging Rate Test (Test Report SHA01-25081190-JC-01, 8 pages in total, corresponding to...) Figures 4-1 to 4-8 ) like Figures 4-1 to 4-8As shown, the antioxidant activity of the pentapeptide KTRGD, control RGD, and control KT at concentrations of 10 ppm and 50 ppm was detected using the DPPH radical scavenging method. The results showed that the pentapeptide KTRGD exhibited significant DPPH radical scavenging ability at 10 ppm (p<0.05) and highly significant scavenging ability at 50 ppm (p<0.01). Control RGD showed highly significant scavenging ability only at 50 ppm (p<0.01), with no significant difference at 10 ppm. Control KT showed significant scavenging ability at both 10 ppm and 50 ppm concentrations (p<0.05 and p<0.01, respectively). This indicates that the pentapeptide of the present invention possesses antioxidant activity.

[0031] IV. Detection of Reactive Oxygen Species Content in UVA-Stimulated Fibroblasts (Detection Report SHA01-25081190-JC-13, 14 pages in total, corresponding to...) Figures 5-1 to 5-14 ) like Figures 5-1 to 5-14 As shown, this test was conducted using a UVA-stimulated human fibroblast model. A fluorescent probe method was employed to detect the effects of the pentapeptide KTRGD, control RGD, and control KT at concentrations of 1 ppm and 10 ppm on intracellular reactive oxygen species (ROS) levels. After UVA stimulation, the ROS level in the negative control group increased significantly (p<0.01), indicating successful model establishment. The positive control group (VC+VE) significantly reduced ROS levels (p<0.01), with a relative scavenging rate of 32.89%. The pentapeptide KTRGD significantly reduced ROS levels at both 1 ppm and 10 ppm concentrations (p<0.01), with relative scavenging rates of 16.45% and 28.29%, respectively. Control RGD and control KT also significantly reduced ROS levels at both concentrations (p<0.01). This indicates that the pentapeptide of this invention possesses antioxidant capabilities.

[0032] To illustrate the receptor-binding characteristics of the pentapeptide, the fibronectin receptor α5β1 integrin model (PDBID: 4WK4) was selected as the master structure validation system, the αvβ3 integrin model (PDBID: 1L5G) was selected as the trend validation system, and another structural model of α5β1 integrin (PDBID: 3VI4) was selected as the cross-validation system. Comparison objects included the target sequence KTRGD (as shown in SEQ ID NO.1), the basic control RGD, the sequence alignment control TKRGD (as shown in SEQ ID NO.2), and the extended controls KTRAD (as shown in SEQ ID NO.3), KGRGD (as shown in SEQ ID NO.4), and GTRGD (as shown in SEQ ID NO.5).

[0033] The verification method adopts a combination of relative docking scoring, model re-scoring, representative receptor binding conformation review, key interaction analysis, multiple independent repetitions and local conformation optimization verification, forming mutual verification from the aspects of receptor type, sequence composition, conformation rationality and result consistency.

[0034] Among them, 4WK4 was used to observe the specific recognition method in the main binding pocket, 1L5G was used to confirm the relative scoring trend of the sequence in another typical integrin background, 3VI4 was used to test the conformational consistency of the sequence in different α5β1 structural backgrounds, and multiple control sequences were used to examine the effects of N-terminal residue composition, sequence arrangement and overall conformation on receptor binding characteristics.

[0035] In the fibronectin receptor α5β1 integrin model (PDBID:4WK4), the representative conformation of KTRGD maintains the key coordination relationship between the Asp site and the metal ion, and forms a recognition anchor for the Arg site; simultaneously, the N-terminal residues introduced by Lys / Thr form additional contacts with the receptor pocket. The basic RGD sequence did not exhibit the same degree of binding mode stability in the same receptor model.

[0036] Figure 6 This is a superimposed diagram of representative conformations of KTRGD and RGD in the fibronectin receptor α5β1 integrin model (PDBID:4WK4). In the diagram, orange represents KTRGD, blue represents RGD, and magenta represents the metal site.

[0037] Two-dimensional interaction analysis of the master validation system revealed that KTRGD forms 9 hydrogen bonds and 2 salt bridges with the receptor, involving key residues such as ASPA227, ASPA228, ASNB224, SERB134, SERB227, and GLYB261. Simultaneously, its Asp site maintains metal coordination, its Arg site maintains recognition anchoring, and 7 new N-terminal contacts are formed. In contrast, RGD forms 6 hydrogen bonds under the same model, does not form salt bridges, and does not exhibit Asp site metal coordination, Arg site anchoring, or new N-terminal contacts.

[0038] Among them, the coordination of the Asp site with the metal ion is conducive to stabilizing the localization of the acidic terminus in the integrin pocket, the recognition and anchoring of the Arg site is conducive to maintaining the directional binding of the RGD motif, and the addition of new contacts at the N-terminus and the increase of hydrogen bonds and salt bridges are conducive to increasing the local interaction area and enhancing the overall conformational stability.

[0039] Figure 7 This is a comparison diagram of the key interactions between KTRGD and RGD in the fibronectin receptor α5β1 integrin model (PDBID:4WK4).

[0040] Based on the conformational and interaction characteristics in the aforementioned main structure verification system, it can be inferred that KTRGD is more likely to form a stable and interpretable receptor recognition conformation in α5β1 integrin (PDBID:4WK4) compared to the basic RGD. Firstly, KTRGD retains the core recognition elements of the RGD motif; secondly, the N-terminal Lys / Thr residues provide additional contacts and a richer non-covalent interaction network; and thirdly, the increased number of hydrogen bonds and salt bridges contributes to improved local stability around the binding site.

[0041] In the αvβ3 integrin model (PDBID: 1L5G), KTRGD exhibited a lower docking score and a higher model re-score compared to the basic RGD, indicating that this sequence maintains a relative binding trend consistent with the main validation system even in the context of another typical RGD recognizing integrins. In another structural model of α5β1 integrin (PDBID: 3VI4), KTRGD was also able to form the corresponding receptor-binding conformation, providing supplementary support to the main validation system as cross-validation.

[0042] To facilitate the overall observation of the relative scoring results of the pentapeptide and basic RGD sequence of this invention from the main validation system and the trend validation system, the relative scoring results of the three typical integrin receptor models, 4WK4, 1L5G and 3VI4, are summarized as follows.

[0043] Table 1. Relative scores of the pentapeptide and basic RGD sequence of this invention in a typical integrin receptor model.

[0044] Note: Within the same receptor model, a lower Vina value indicates a more favorable predictive binding, while a higher CNNaffinity value indicates a more favorable model score. 4WK4 is the main structure validation system, 1L5G is the trend validation system, and 3VI4 is the cross-validation system.

[0045] As shown in Table 1, in the fibronectin receptor α5β1 integrin model (PDBID: 4WK4) and the αvβ3 integrin model (PDBID: 1L5G), KTRGD exhibited both a lower docking score and a higher model re-scoring score compared to the basic RGD. In another structural model of α5β1 integrin (PDBID: 3VI4), KTRGD still showed a more favorable docking score, while the model re-scoring scores differed, indicating that 3VI4 mainly served as a cross-validation and receptor-dependent supplementary explanation. Therefore, the main conclusion of this invention regarding the superior predictive value of KTRGD compared to the basic RGD is primarily based on the co-directional results of 4WK4 and 1L5G, and further supported by the supplementary results of 3VI4.

[0046] Meanwhile, the existing comparative results support the following further explanation: the receptor recognition characteristics are not determined solely by the RGD core motif, but are also related to the composition of N-terminal residues, the sequence arrangement, and whether the overall conformation can maintain Asp site coordination, Arg site anchoring, and additional contact. The overall results provide continuous support for the receptor binding characteristics from the perspectives of relative score distribution, the quality of the reviewed conformations, multiple independent replicates, and local optimization verification.

[0047] The results directly related to the above four sets of control sequences are summarized as follows: Table 2 Comparison results of control sequences

[0048] Figure 8 The figures show relative scores in typical integrin receptor models. 4WK4 and 3VI4 represent the α5β1 integrin model, and 1L5G represents the αvβ3 integrin model. Lower Vina values ​​indicate more favorable predicted binding, while higher CNNaffinity values ​​indicate a more favorable model score. Based on the combined results of receptor model setup, control sequence comparison, multiple independent replicates, representative conformation verification, key interaction analysis, and local conformation optimization, the pentapeptide H-Lys-Thr-Arg-Gly-Asp-OH of this invention exhibits a more stable receptor recognition conformation and a more favorable predicted binding trend compared to the basic RGD sequence.

[0049] As can be seen from the above embodiments, the present invention provides a polypeptide composed of Lys-Thr-Arg-Gly-Asp (structural diagram shown). Figure 9 Fibroblast proliferation experiments showed that the peptide significantly improved the cell viability of human fibroblasts within a safe concentration range. Elastin and type III collagen content assays showed that the peptide effectively increased the levels of elastin and type III collagen in fibroblasts after UVA stimulation. Antioxidant function tests indicated that the peptide had DPPH free radical scavenging ability and significantly reduced UVA-induced reactive oxygen species levels in fibroblasts. Molecular docking results showed that the peptide had a higher binding affinity to integrin receptors α5β1 and αvβ3, exhibiting a more favorable docking score and model rescore than the basic RGD sequence, and forming more hydrogen bonds, salt bridges, and N-terminal additional contacts. In summary, the peptide provided by this invention possesses good anti-aging, antioxidant, and skin repair-promoting biological activities, making it suitable for the preparation of functional cosmetics or topical skin formulations.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide consists of the amino acid sequence shown in SEQ ID NO.1, with a free amino group at the N-terminus and a free carboxyl group at the C-terminus.

2. A cosmetic composition comprising the polypeptide of claim 1.

3. The cosmetic composition according to claim 2, characterized in that, The cosmetic composition further comprises at least one of a moisturizer, emulsifier, preservative, thickener, antioxidant, pH adjuster, or solvent.

4. The use of the polypeptide according to claim 1 in the preparation of topical skin preparations with anti-aging effects.

5. The use of the polypeptide according to claim 1 in the preparation of topical skin preparations with antioxidant effects.

6. The use of the polypeptide of claim 1 in the preparation of a topical skin formulation for increasing the elastin content in the skin.

7. The use of the polypeptide of claim 1 in the preparation of a topical skin formulation for increasing the content of type III collagen in the skin.

8. The use of the polypeptide of claim 1 in the preparation of a topical skin formulation for reducing the level of reactive oxygen species in skin cells.

9. The use of the polypeptide of claim 1 in the preparation of a topical skin formulation for promoting fibroblast proliferation.

10. An anti-aging topical product, characterized in that, The polypeptide described in claim 1 is used as the active ingredient; the dosage form of the topical product includes solution, gel, cream, lotion, serum, facial mask liquid or lyophilized powder.

Images