A cyclic polypeptide and its application in cosmetics

Abstract

This invention provides a cyclic polypeptide and its application in cosmetics, belonging to the field of active molecule development technology. The cyclic polypeptide provided by this invention has a stable cyclic structure, which can significantly promote fibroblast proliferation, increase the content of elastin and type III collagen in the skin, and effectively scavenge DPPH free radicals and reduce reactive oxygen species levels. This cyclic octapeptide can be widely used in the preparation of cosmetics or topical skin preparations 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 cyclic 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. Therefore, developing RGD-related peptides with better structural stability and higher receptor-binding activity remains of significant research value and market potential. Summary of the Invention

[0004] The purpose of this invention is to provide a cyclic polypeptide that solves the problems of insufficient conformational stability, easy degradation, and low binding efficiency to integrin receptors of existing polypeptides containing RGD motifs in topical skin applications.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] The present invention provides a cyclic polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1, and the N-terminal amino group of the polypeptide forms an amide bond with the C-terminal carboxyl group.

[0007] The present invention also provides a cosmetic composition comprising the above-mentioned cyclic polypeptide.

[0008] Preferably, the cosmetic composition further comprises at least one of a moisturizer, emulsifier, preservative, thickener, antioxidant, pH adjuster, or solvent.

[0009] This invention also provides the application of the above-mentioned cyclic polypeptide in the preparation of topical skin preparations with anti-aging effects.

[0010] This invention also provides the application of the above-mentioned cyclic polypeptide in the preparation of topical skin formulations with antioxidant effects.

[0011] The present invention also provides the application of the above-mentioned cyclic polypeptide in the preparation of topical skin formulations for increasing the content of elastin in the skin.

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

[0013] The present invention also provides the use of the above-mentioned cyclic polypeptide in the preparation of topical skin formulations for reducing the level of reactive oxygen species in skin cells.

[0014] The present invention also provides the use of the above-mentioned cyclic polypeptide in the preparation of topical skin formulations for promoting fibroblast proliferation.

[0015] The present invention also provides an anti-aging topical product, using the above-mentioned cyclic polypeptide as an active ingredient; the dosage form of the topical product includes solution, gel, cream, lotion, serum, facial mask liquid or lyophilized powder.

[0016] The beneficial effects of this invention are: The cyclic polypeptide provided by this invention has a stable cyclic structure, which can significantly promote fibroblast proliferation, increase the content of elastin and type III collagen in the skin, and effectively scavenge DPPH free radicals and reduce reactive oxygen species levels. This cyclic octapeptide can be widely used in the preparation of cosmetics or topical skin preparations with anti-aging, antioxidant, and skin repair functions. It has the characteristics of high activity, good stability, and excellent safety, making it suitable as an active ingredient in functional skin care products. Attached Figure Description

[0017] Figure 1 These are liquid chromatography (LC) and mass spectrometry (MS) chromatograms of the cyclic polypeptide of the present invention; Figures 2-1 to 2-12 This is a report on fibroblast proliferation testing (report number: SHA01-25081184-JC-04), which includes: Figure 2-1 For the report cover; Figure 2-2 This is the report's basic information page; Figure 2-3For testing purposes and test project pages; Figure 2-4 For test materials pages; 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 Figure showing cytotoxicity results (c(RGDKTTKS)); Figure 2-9 This is a graph showing the cytotoxicity results (RGD). Figure 2-10 This is a graph showing the cytotoxicity results (KTTKS). Figure 2-11 The results of the cell proliferation test are presented in a table and bar chart. Figure 2-12 Page 13 contains the test results and statement. Figures 3-1 to 3-13 This is a test report on the content of elastin and type III collagen (report number: SHA01-25081184-JC-16), 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 test materials pages; 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 Figure showing cytotoxicity results (c(RGDKTTKS)); Figure 3-9 This is a graph showing the cytotoxicity results (RGD). Figure 3-10 This is a graph showing the cytotoxicity results (KTTKS). 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 Page 14 contains the test results and statement. Figures 4-1 to 4-8 This is a DPPH free radical scavenging rate test report (test report number: SHA01-25081190-JC-04), 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 5-13This is a report on the detection of reactive oxygen species content in UVA-stimulated fibroblasts (report number: SHA01-25081190-JC-16), 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 The results table for the cytotoxicity test (c(RGDKTTKS)); Figure 5-7 For the cytotoxicity test results (RGD); Figure 5-8 For the results of the cytotoxicity test (KTTKS); Figure 5-9 Cell viability plot (c(RGDKTTKS), RGD, KTTKS); Figure 5-10 Fluorescence spectrum of ROS content (blank control, negative control, positive control, c(RGDKTTKS)-1ppm); Figure 5-11 Fluorescence diagrams of ROS content (RGD-1ppm, RGD-10ppm, KTTKS-1ppm, KTTKS-10ppm). Figure 5-12 This is a summary table of ROS data analysis. Figure 5-13 This is the conclusion and statement page; Figure 6 This is a diagram of the cyclic polypeptide structure of the present invention. Detailed Implementation

[0018] 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.

[0019] Example This embodiment provides a cyclic polypeptide containing the RGD motif, wherein the N-terminal amino group and the C-terminal carboxyl group form an amide bond to form a cyclic peptide, and the amino acid sequence is Cyclo[Ser-Arg-Gly-Asp-Lys-Thr-Thr-Lys], c(RGDKTTKS), as shown in SEQ ID NO.1.

[0020] The polypeptide was synthesized based on the above sequence: (1) Solid-phase synthesis of peptide chains: 2-chlorotriphenylmethylchloro resin (2-CTC resin, loading 0.98 mmol / g) was accurately weighed and placed in a reaction tube, and dichloromethane (DCM) was added. The mixture was swollen at room temperature for 1 hour. After swelling, the solvent was removed, and a dichloromethane solution containing the first C-terminal amino acid monomer (1.5 equivalents) and N,N-diisopropylethylamine (DIEA, 5 equivalents) was added to the reaction tube. The mixture was shaken at room temperature for 1 hour, and then the resin was washed with N,N-dimethylformamide (DMF) and DCM, respectively. After the initial amino acid coupling was completed, the Fmoc protecting group was removed: 20% piperidine / DMF solution was added, and deprotection was performed twice, each time for 10 minutes. The resin was then washed with DCM and DMF, respectively. Next, the coupling reaction of the second amino acid was carried out. An activated DMF solution containing the second amino acid monomer (containing 5 equivalents of the amino acid monomer, 4.75 equivalents of the condensing agent HBTU, and 5 equivalents of DIEA) was added, and the reaction was carried out with shaking at room temperature for 1 hour. After the reaction was complete, the resin was washed sequentially with DMF and DCM. The above "deprotection-washing-coupling-washing" procedure was repeated until the coupling of the last amino acid monomer was completed. Finally, the N-terminal Fmoc protecting group was removed: a 20% piperidine / DMF solution was added, and deprotection was performed twice, 10 minutes each time. The resin was then washed sequentially with DCM, DMF, and DCM, and finally dried.

[0021] (2) Peptide cleavage: Prepare a 20% (v / v) hexafluoroisopropanol / DCM mixed solution as the cleavage reagent, add it to the dried resin, and shake and react at room temperature for 0.5 hours. Repeat this operation once. Combine the filtrates from the two reactions into a centrifuge tube, remove the solvent by rotary evaporation, and obtain the crude peptide product.

[0022] (3) Cyclic peptide synthesis: The chain peptide was dissolved in DMF at a concentration of 0.005 M. After complete dissolution, HATU (2 equivalents) and DIEA (3–6 equivalents) were added sequentially, and the reaction was carried out at room temperature for 2 hours. The cyclization reaction was monitored by LCMS. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude cyclic peptide product. Acetonitrile was added to dissolve it, and the insoluble solids were removed by centrifugation. The supernatant was collected, concentrated by rotary evaporation, and used for subsequent deprotection steps.

[0023] (4) Deprotection of cyclic peptide side chains: Prepare a deprotection solution for peptide side chains (trifluoroacetic acid / triisopropylsilane / DCM = 50:5:45), add it to the crude cyclic peptide product, and shake the reaction at room temperature for 0.5–2 hours. Monitor the deprotection process by LCMS. After the reaction is complete, remove the solvent using an air pump, purify the product by reversed-phase preparative high-performance liquid chromatography (HPLC), and finally freeze-dry to obtain pure cyclic peptide (II).

[0024] (5) Detection of peptide samples: The prepared cyclic peptide products were analyzed by LCMS. The results of the cyclic octapeptide are shown in the figure. Figure 1 .

[0025] I. Fibroblast proliferation test (Test report SHA01-25081184-JC-04, 12 pages in total, corresponding to...) Figures 2-1 to 2-13 ) like Figures 2-1 to 2-12 As shown, this test was based on a human fibroblast model to detect the effect of the cyclic octapeptide c (RGDKTTKS), control RGD, and control KTTKS (as shown in SEQ ID NO.2) of the present invention on cell viability after treatment at a concentration of 1 ppm for 48 hours. The results showed that the cyclic octapeptide of the present invention could significantly promote fibroblast proliferation at a concentration of 1 ppm (p<0.01); control RGD could significantly promote proliferation at both 1 ppm and 10 ppm concentrations (p<0.01); control KTTKS significantly promoted proliferation at a concentration of 1 ppm (p<0.05) and significantly promoted proliferation at a concentration of 10 ppm (p<0.01); indicating that the cyclic octapeptide of the present invention has a firming effect. II. Determination of Elastin and Type III Collagen Content (Test Report SHA01-25081184-JC-16, 13 pages in total, corresponding to...) Figures 3-1 to 3-14 ) like Figures 3-1 to 3-13 As shown, this test, based on a UVA-stimulated human fibroblast model, examined the effects of the cyclic octapeptide c (RGDKTTKS), control RGD, and control KTTKS at concentrations of 1 ppm and 10 ppm on elastin and type III collagen content. After UVA stimulation, the negative control group showed a highly significant decrease in elastin and type III collagen content (p<0.01), indicating successful model establishment. The positive control group (VC+VE) significantly increased the content of both proteins (p<0.01). The cyclic octapeptide c of this invention at a concentration of 1 ppm significantly increased elastin and type III collagen content. The levels of elastin and type III collagen were significantly increased (p<0.01); control RGD significantly increased the levels of both proteins at concentrations of 1 ppm and 10 ppm (p<0.01); control KTTKS significantly increased the levels of elastin at 1 ppm (p<0.05) and extremely significantly increased the levels of type III collagen (p<0.01), and extremely significantly increased the levels of both proteins at 10 ppm (p<0.01); indicating that the cyclic octapeptide of the present invention promotes the synthesis of elastin and type III collagen. III. DPPH Free Radical Scavenging Rate Test (Test Report SHA01-25081190-JC-04, 8 pages in total, corresponding to...) Figures 4-1 to 4-8 ) like Figures 4-1 to 4-8As shown, the DPPH free radical scavenging method was used to detect the antioxidant activity of the cyclic octapeptide c (RGDKTTKS), control RGD, and control KTTKS at concentrations of 1 ppm, 10 ppm, and 50 ppm. The results showed that the cyclic octapeptide of the present invention had DPPH free radical scavenging ability at concentrations of 1 ppm, 10 ppm, and 50 ppm, with significant scavenging effect at 1 ppm (p<0.05) and extremely significant scavenging effect at 10 ppm and 50 ppm (p<0.01). Control RGD only showed extremely significant scavenging ability at a concentration of 50 ppm (p<0.01). Control KTTKS showed significant scavenging ability at concentrations of 10 ppm and 50 ppm (p<0.05 and p<0.01, respectively). This indicates that the cyclic octapeptide of the present invention has antioxidant activity. IV. Detection of Reactive Oxygen Species Content in UVA-Stimulated Fibroblasts (Detection Report SHA01-25081190-JC-16, 13 pages in total, corresponding to...) Figures 5-1 to 5-13 ) like Figures 5-1 to 5-13 As shown, this test was based on a UVA-stimulated human fibroblast model. The effect of the cyclic octapeptide c (RGDKTTKS), control RGD, and control KTTKS at concentrations of 1 ppm and 10 ppm on intracellular reactive oxygen species (ROS) levels was detected using a fluorescent probe method. 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 clearance rate of 32.89%. The cyclic octapeptide c of this invention at 1 ppm... At a concentration of ppm, it can significantly reduce the level of reactive oxygen species (ROS) (p<0.01), with a relative scavenging rate of 17.11%; the control RGD can significantly reduce the level of ROS at concentrations of 1 ppm and 10 ppm (p<0.01), with relative scavenging rates of 13.82% and 26.97%, respectively; the control KTTKS can significantly reduce the level of ROS at concentrations of 1 ppm and 10 ppm (p<0.01), with relative scavenging rates of 13.82% and 26.97%, respectively; indicating that the cyclic octapeptide of the present invention has antioxidant capacity.

[0026] As can be seen from the above embodiments, the present invention provides a cyclic octapeptide with amide bonds at both ends (structural diagram shown). Figure 6Fibroblast proliferation experiments showed that the cyclic octapeptide significantly improved the cell viability of human fibroblasts within a safe concentration range. Elastin and type III collagen content assays showed that the cyclic octapeptide effectively increased the levels of elastin and type III collagen in fibroblasts after UVA stimulation. Antioxidant function tests indicated that the cyclic octapeptide had DPPH free radical scavenging ability and significantly reduced UVA-induced reactive oxygen species levels in fibroblasts. In summary, the cyclic octapeptide provided by this invention possesses good anti-aging, antioxidant, and skin repair-promoting bioactivity, making it suitable for use in the preparation of functional cosmetics or topical skin formulations.

[0027] 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 cyclic polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and the N-terminal amino group of the polypeptide forms an amide bond with the C-terminal carboxyl group.

2. A cosmetic composition comprising the cyclic 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 cyclic polypeptide of claim 1 in the preparation of topical skin preparations with anti-aging effects.

5. The use of the cyclic polypeptide of claim 1 in the preparation of topical skin formulations with antioxidant effects.

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

7. The use of the cyclic 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 cyclic 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 cyclic 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 cyclic polypeptide of 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.

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