Anti-aging cyclic peptides with both free radical scavenging and procollagen production promoting functions and applications thereof

Abstract

The application provides a group of cyclic peptides with free radical scavenging and collagen synthesis promoting functions and applications thereof. The anti-aging cyclic peptides of the application have a core structural feature of containing a cyclic 5 peptide or a cyclic 6 peptide of a PYY, PPY or YPY tripeptide motif. The cyclic peptides contain tripeptide motifs distributed as follows: motif 1 (PYY): proline (Pro, P)-tyrosine (Tyr, Y)-tyrosine (Tyr, Y), with an amino acid sequence of P-Y-Y; motif 2 (PPY): proline (Pro, P)-proline (Pro, P)-tyrosine (Tyr, Y), with an amino acid sequence of P-P-Y; and motif 3 (YPY): tyrosine (Tyr, Y)-proline (Pro, P)-tyrosine (Tyr, Y), with an amino acid sequence of Y-P-Y. The cyclic peptides prepared by the application have excellent antioxidant efficacy, can promote the synthesis of SOD and collagen, and can reduce the destruction of metal matrix proteinase to collagen. The cyclic peptides exhibit excellent anti-aging effects.

Description

Technical Field

[0001] This invention relates to the preparation of a group of anti-aging cyclic peptides and their application in skincare products, belonging to the field of daily chemical products technology. Specifically, it relates to a group of anti-aging cyclic peptides that have both free radical scavenging and collagen-promoting properties and their application in skincare and cosmetic products. Background Technology

[0002] Skin aging is a complex result of the combined effects of the body's natural physiological processes and external environmental factors. Its core characteristics include decreased skin elasticity, wrinkles, weakened barrier function, and pigmentation, which seriously affect the appearance and physiological function of the skin. Therefore, the research and development of anti-aging skincare and related bioactive ingredients has always been a research hotspot in the fields of biomedicine and cosmetics.

[0003] The core molecular mechanisms of skin aging involve free radical damage and extracellular matrix (ECM) metabolic disorders. Firstly, reactive oxygen species (ROS) generated during oxidative metabolism, including superoxide anions and hydroxyl radicals, attack biomolecules such as lipids, proteins, and DNA in skin cells, triggering oxidative stress responses. This disrupts cell structural integrity and functional stability, accelerating cell aging and apoptosis, and is a significant initiating factor for photoaging and intrinsic aging of the skin. While existing antioxidants such as vitamin C and glutathione can scavenge free radicals to some extent, they suffer from poor stability, low transdermal absorption efficiency, and short duration of action, limiting their full anti-aging effects. Secondly, the extracellular matrix, as the main structural component of the dermis, is composed of biomacromolecules such as collagen, elastin, and hyaluronic acid. The dynamic balance between its synthesis and degradation is crucial for maintaining skin elasticity and firmness. With age and external stimuli, the proliferation and synthesis capacity of fibroblasts declines, while the activity of matrix metalloproteinases (MMPs) increases, leading to a significant loss of core components such as collagen and elastin, and a decrease in hyaluronic acid content. This ultimately manifests as skin laxity, deeper wrinkles, and decreased hydration. Existing ingredients that promote extracellular matrix production, such as retinol and peptides (e.g., signal peptides and neuropeptides), suffer from drawbacks such as insufficient bioavailability, single target of action, strong irritation (e.g., retinol), or limited efficacy, making it difficult to simultaneously achieve the synergistic anti-aging effects of "scavenging free radicals - anti-inflammation - promoting extracellular matrix production." Current anti-aging bioactive ingredients generally suffer from problems such as single function, incomplete mechanism of action, and poor stability or bioavailability, failing to simultaneously and synergistically regulate various mechanisms of skin aging, thus hindering the achievement of highly effective and safe anti-aging results. Therefore, developing a novel anti-aging ingredient that combines antioxidant and extracellular matrix-promoting functions with strong stability, good biocompatibility, and high transdermal absorption efficiency is of great significance for solving skin aging problems. Summary of the Invention

[0004] To develop a novel anti-aging ingredient that combines antioxidant and extracellular matrix-promoting functions with high stability, good biocompatibility, and high transdermal absorption efficiency, the primary objective of this invention is to provide a group of anti-aging cyclic peptides that combine free radical scavenging and collagen-promoting effects. Another objective of this invention is to provide a method for preparing a group of cyclic peptides that combine antioxidant and extracellular matrix-promoting functions, as well as their applications.

[0005] The anti-aging cyclic peptide provided by this invention is characterized by a core structural feature of a cyclic 5-peptide or a cyclic 6-peptide containing a PYY, PPY, or YPY tripeptide motif.

[0006] Furthermore, the tripeptide motifs contained in the cyclic peptide are distributed as follows: Motif 1 (PYY): Proline (Pro, P) - Tyrosine (Tyr, Y) - Tyrosine (Tyr, Y), with the amino acid sequence PYY; Motif 2 (PPY): Proline (Pro, P) - Proline (Pro, P) - Tyrosine (Tyr, Y), with the amino acid sequence PPY; Motif 3 (YPY): Tyrosine (Tyr, Y) - Proline (Pro, P) - Tyrosine (Tyr, Y), with the amino acid sequence YPY; Furthermore, the cyclic peptide is an intramolecularly cyclic polypeptide with head-to-tail cyclization, where the cyclization site is formed by the α-amino group of the N-terminal amino acid and the α-carboxyl group of the C-terminal amino acid linked by an amide bond to form a cyclic structure.

[0007] Furthermore, the cyclic 5-peptide is formed by the above-described cyclization of 5 amino acid residues, with the general structural formula: cyclo (-X1-X2-X3-X4-X5-). Among them, X1-X5 are natural L-amino acids, D-amino acids or non-natural amino acids (such as phosphorylated tyrosine, methylated proline, etc.), and X1-X5 must contain a continuous PYY, PPY or YPY tripeptide motif (the motif can be located at any three consecutive sites, such as X1-X3, X2-X4 or X3-X5). Furthermore, the cyclic 6-peptide is formed by the above-described cyclization of 6 amino acid residues, and its general structural formula is: cyclo (-X1-X2-X3-X4-X5-X6-) Among them, X1-X6 are natural L-amino acids, D-amino acids or non-natural amino acids, and X1-X6 must contain a continuous PYY, PPY or YPY tripeptide motif (the motif can be located at any three consecutive sites, such as X1-X3, X2-X4, X3-X5 or X4-X6).

[0008] Further, the cyclo5 peptide is selected from Cyclo(PYYAY), Cyclo(YPPYP), Cyclo(PPYAP), Cyclo(YPYYA), Cyclo(TYPYY), Cyclo(YPYRY), Cyclo( GPYYY), Cyclo(RTYPY), Cyclo(AAPYY), Cyclo(YPYVA), Cyclo(PYPYY), Cyclo(YPYGG), Cyclo(PYYGY), Cyclo(PPYGR); Furthermore, the cyclic 6-peptide is selected from Cyclo(PYPYGA), Cyclo(PYYAPP), Cyclo(PYYGPG), Cyclo(AGYPYR), Cyclo(YPYGGY), Cyclo(PPYAYA), Cyclo(YGYPYY), Cyclo(YPYVPP), Cyclo(YPYPVP), Cyclo(GPPYYV), and Cyclo(YAPPYP).

[0009] In addition, the present invention also discloses a method for preparing the above-mentioned cyclic peptide, comprising the following steps: Step 1: Linear peptide synthesis: 2-Chlorotriphenylmethyl chloride (2-CTC resin, 0.98 mmol / g loading) was weighed into a reaction tube and swollen in dichloromethane (DCM) at room temperature for 1 hour. After the solvent was removed, a dichloromethane solution of the first amino acid monomer at the C-terminus (1.5 equiv) and N,N-diisopropylethylamine (DIEA, 5 equiv) was added. The mixture was shaken at room temperature for 1 hour, and the resin was washed three times sequentially with N,N-dimethylformamide (DMF) and DCM, and then dried. Deprotection with 20% piperidine / DMF solution was then performed twice, 10 min each time, followed by washing the resin three times sequentially with DCM and DMF. After drying, the second amino acid was coupled by adding a DMF solution containing the activated second amino acid monomer (5 equiv of amino acid monomer, 4.75 equiv of condensing agent HBTU, and 5 equiv of DIEA). The mixture was reacted with shaking at room temperature for 1 h. The resin was then washed three times sequentially with DMF and DCM, and dried. This process of de-Fmoc protection, washing, coupling, and washing was repeated until the last amino acid monomer was obtained. The cleavage reagent hexafluoroisopropanol (HFIP) / DCM (20%) was added to the dried resin, and the mixture was reacted with shaking at room temperature for 0.5 h. This process was repeated once. The filtrates were combined and collected in centrifuge tubes. The solvent was removed by rotary evaporation to obtain the crude product.

[0010] Step 2, Cycloning: The crude cyclic peptide was dissolved in DMF (0.005 M). After complete dissolution, HATU (2 equiv) and DIEA (3-6 equiv) were added sequentially, and the reaction was carried out at room temperature for 2 h. The cyclization process was monitored by LCMS. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude cyclic peptide. The product was dissolved in acetonitrile, and the insoluble solids were removed by centrifugation. The filtrate was collected and evaporated to dryness for further deprotection.

[0011] Step 3: Remove protection: Prepare a deprotection solution of TFA / triisopropylsilane (TIPS) / DCM = 50:5:45 or TFA / TIPS / 1,2-ethylenedithiol (EDT) / DCM = 50:2.5:2.5:45, and add it to the crude cyclic peptide product. Incubate at room temperature with shaking for 0.5–2 h, and monitor the deprotection process using LCMS. After complete deprotection, dry the solvent with an air pump and purify the product using a column chromatography system.

[0012] In addition, the above-mentioned anti-aging cyclic peptides can be used in skin care and cosmetics. The cyclic peptides can be pre-dispersed in an aqueous solution containing polyols and then directly added to the prepared cosmetics at room temperature.

[0013] Based on the above technical solution, the beneficial effects of the present invention are as follows: 1) Multi-mechanism coordinated anti-aging: The prepared cyclic peptide has both antioxidant and extracellular matrix-promoting functions; 2) Excellent safety performance: Non-cytotoxic and will not cause skin sensitivity; 3) The cyclic peptides prepared by this invention have excellent antioxidant effects, can promote the synthesis of SOD and collagen, and can reduce the damage of collagen to matrix metalloproteinases; the cyclic peptides exhibit excellent anti-aging effects. Attached Figure Description

[0014] Figure 1 The image shows the cyclic peptide structure and LCMS spectrum of Example 2.

[0015] Figure 2 The image shows the cyclic peptide structure and LCMS spectrum of Example 3.

[0016] Figure 3 The image shows the cyclic peptide structure and LCMS spectrum of Example 5.

[0017] Figure 4 The image shows the cyclic peptide structure and LCMS spectrum of Example 16.

[0018] Figure 5 The image shows the cyclic peptide structure and LCMS spectrum of Example 20.

[0019] Figure 6 This is a graph for assessing the cytotoxicity of cyclic peptides.

[0020] Figure 7 This is a diagram showing the scavenging of intracellular reactive oxygen species by cyclic peptides.

[0021] Figure 8 The cyclic peptide represents the intracellular reactive oxygen species scavenging rate.

[0022] Figure 9 The SOD enzyme antagonistic inhibition rate.

[0023] Figure 10 This refers to the SOD enzyme content.

[0024] Figure 11 This represents the amount of type I collagen promoted by the cyclic peptide.

[0025] Figure 12 This represents the amount of type III collagen promoted by the cyclic peptide.

[0026] Figure 13 This is the inhibition of matrix metalloproteinase MMP-1 by cyclic peptides.

[0027] Figure 14 This is the inhibition of matrix metalloproteinase MMP-3 by cyclic peptides.

[0028] Figure 15SA-β-gal staining for cyclic peptides. Detailed Implementation

[0029] To better illustrate the present invention, the following detailed description is provided in conjunction with specific embodiments. However, these specific embodiments are merely for illustrative purposes and are not intended to limit the scope of the invention.

[0030] This invention provides a method for preparing a group of cyclic peptides with both antioxidant and extracellular matrix-promoting functions, and their application in cosmetics. The core structural feature of these anti-aging cyclic peptides is a cyclic 5-peptide or cyclic 6-peptide containing a PYY, PPY, or YPY tripeptide motif.

[0031] The tripeptide motifs contained in the cyclic peptide are distributed as follows: Motif 1 (PYY): Proline (Pro, P) - Tyrosine (Tyr, Y) - Tyrosine (Tyr, Y), with the amino acid sequence PYY; Motif 2 (PPY): Proline (Pro, P) - Proline (Pro, P) - Tyrosine (Tyr, Y), with the amino acid sequence PPY; Motif 3 (YPY): Tyrosine (Tyr, Y) - Proline (Pro, P) - Tyrosine (Tyr, Y), with the amino acid sequence YPY; The cyclic peptide is an intramolecularly cyclic polypeptide with head-to-tail cyclization, where the cyclization site is formed by the α-amino group of the N-terminal amino acid and the α-carboxyl group of the C-terminal amino acid linked by an amide bond to form a cyclic structure.

[0032] The cyclic 5-peptide is formed by cyclizing 5 amino acid residues as described above, and has the general structural formula: cyclo (-X1-X2-X3-X4-X5-). Among them, X1-X5 are natural L-amino acids, D-amino acids or non-natural amino acids (such as phosphorylated tyrosine, methylated proline, etc.), and X1-X5 must contain a continuous PYY, PPY or YPY tripeptide motif (the motif can be located at any three consecutive sites, such as X1-X3, X2-X4 or X3-X5). The cyclo5 peptide is preferably selected from Cyclo(PYYAY), Cyclo(YPPYP), Cyclo(PPYAP), Cyclo(YPYYA), Cyclo(TYPYY), Cyclo(YPYRY), Cyclo(GPY YY), Cyclo(RTYPY), Cyclo(AAPYY), Cyclo(YPYVA), Cyclo(PYPYY), Cyclo(YPYGG), Cyclo(PYYGY), Cyclo(PPYGR). That is, Examples 1-14.

[0033] The cyclic 6-peptide is formed from 6 amino acid residues through the above-described cyclization process, and its general structural formula is: cyclo (-X1-X2-X3-X4-X5-X6-); Among them, X1-X6 are natural L-amino acids, D-amino acids or non-natural amino acids, and X1-X6 must contain a continuous PYY, PPY or YPY tripeptide motif (the motif can be located at any three consecutive sites, such as X1-X3, X2-X4, X3-X5 or X4-X6). The cyclic 6-peptide is preferably derived from Cyclo(PYPYGA), Cyclo(PYYAPP), Cyclo(PYYGPG), Cyclo(AGYPYR), Cyclo(YPYGGY), Cyclo(PPYAYA), Cyclo(YGYPYY), Cyclo(YPYVPP), Cyclo(YPYPVP), Cyclo(GPPYYV), and Cyclo(YAPPYP). See Examples 15-25.

[0034] The cyclic peptides prepared in this invention possess excellent antioxidant properties, promoting SOD synthesis and collagen synthesis while reducing the damage to collagen caused by matrix metalloproteinases. These cyclic peptides exhibit superior anti-aging effects.

[0035] In addition, the present invention also discloses a method for preparing the above-mentioned cyclic peptide, comprising the following steps: Step 1: Linear peptide synthesis: 2-Chlorotriphenylmethyl chloride (2-CTC resin, 0.98 mmol / g loading) was weighed into a reaction tube and swollen in dichloromethane (DCM) at room temperature for 1 hour. After the solvent was removed, a dichloromethane solution of the first amino acid monomer at the C-terminus (1.5 equiv) and N,N-diisopropylethylamine (DIEA, 5 equiv) was added. The mixture was shaken at room temperature for 1 hour, and the resin was washed three times sequentially with N,N-dimethylformamide (DMF) and DCM, and then dried. Deprotection with 20% piperidine / DMF solution was then performed twice, 10 min each time, followed by washing the resin three times sequentially with DCM and DMF. After drying, the second amino acid was coupled by adding a DMF solution containing the activated second amino acid monomer (5 equiv of amino acid monomer, 4.75 equiv of condensing agent HBTU, and 5 equiv of DIEA). The mixture was reacted with shaking at room temperature for 1 h. The resin was then washed three times sequentially with DMF and DCM, and dried. This process of de-Fmoc protection, washing, coupling, and washing was repeated until the last amino acid monomer was obtained. The cleavage reagent hexafluoroisopropanol (HFIP) / DCM (20%) was added to the dried resin, and the mixture was reacted with shaking at room temperature for 0.5 h. This process was repeated once. The filtrates were combined and collected in centrifuge tubes. The solvent was removed by rotary evaporation to obtain the crude product.

[0036] Step 2, Cycloning: The crude cyclic peptide product was dissolved in DMF (0.005 M). After complete dissolution, HATU (2 equiv) and DIEA (3-6 equiv) were added sequentially, and the reaction was carried out at room temperature for 2 h. The cyclization process 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 the product, and the insoluble solids were removed by centrifugation. The filtrate was collected and evaporated to dryness for the next step of deprotection.

[0037] Step 3: Remove protection: Prepare a deprotection solution of TFA / triisopropylsilane (TIPS) / DCM = 50:5:45 or TFA / TIPS / 1,2-ethylenedithiol (EDT) / DCM = 50:2.5:2.5:45, and add it to the crude cyclic peptide product. Incubate at room temperature with shaking for 0.5–2 h, and monitor the deprotection process using LCMS. After complete deprotection, dry the solvent with an air pump and purify the product using a column chromatography system.

[0038] In addition, the above-mentioned cyclic peptides can be used in skin care and cosmetic products. The cyclic peptides can be pre-dispersed in an aqueous solution containing polyols and then directly added to the prepared skin care and cosmetic products at room temperature.

[0039] The following are relevant contents of efficacy test examples of the present invention.

[0040] Efficacy Test Example 1: LCMS Identification Method for Anti-aging Cyclic Peptides LCMS test conditions: Shimadzu LCMS 2050; column Shimazu shim-pack GISTC18 (2 μm, 100 mm x 2.1 mm); flow rate 0.3 mL / min; mobile phase A (H2O); mobile phase B (acetonitrile, containing 0.01% HCOOH); Test method: Wavelength 204 nm. Elution gradient: 0-0.5 min, maintain 5% B; 0.5-5 min, increase B proportion to 95%; 5-8 min, maintain B proportion at 95%; then decrease to 5% within 0.5 min and maintain for 1.5 min to equilibrate the column.

[0041] The structures and LCMS spectra of Examples 2, 3, 5, 16, and 20 are as follows: Figures 1-5 As shown.

[0042] Efficacy Test Example 2: Cytotoxicity Test of Anti-aging Cyclic Peptides This invention employs the CCK8 (Cell Counting Kit-8) method to detect the toxicity of a target cyclic peptide to human skin fibroblasts (HSF). The core principle is as follows: the water-soluble tetrazolium salt (WST-8) in the CCK8 reagent is reduced to orange-yellow formazan by intracellular dehydrogenases. The absorbance (OD value) of formazan is positively correlated with the number of viable cells. The biocompatibility of the cyclic peptide is assessed by calculating cell viability. The specific method is as follows: (1) Cell resuscitation and culture After thawing the frozen HSF cells, they were seeded into culture flasks, DMEM complete medium was added, and they were cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, they were passaged using 0.25% trypsin, and cells in the logarithmic growth phase of the 3rd-5th generation were used for experiments.

[0043] (2) Cell seeding and adhesion HSF cells in logarithmic growth phase were digested with trypsin, resuspended in complete culture medium, and the cell concentration was adjusted to 5 × 10³ cells / well. They were then seeded into 96-well cell culture plates, with 100 μL of cell suspension added to each well. The culture plates were incubated at 37°C in a 5% CO2 incubator for 24 h to allow the cells to adhere completely.

[0044] (3) Cyclic peptide gradient treatment Discard the old culture medium from the 96-well plate, add complete culture medium containing different concentrations of cyclic peptides, and set up the following groups: Blank control group: only 100 μL of complete culture medium (cell-free, cyclic peptide-free) was added. Cell control group: Add 100 μL of complete culture medium (with cells, without cyclic peptides); Cyclic peptide experimental group: 100 μL of complete culture medium containing cyclic peptides was added, and the final concentration of cyclic peptides was set to 1 ppm, 2 ppm, 5 ppm, 10 ppm, 20 ppm, and 50 ppm. Each group should have 3-5 parallel duplicate wells to avoid experimental errors.

[0045] The inoculated 96-well plates were placed in a 37°C, 5% CO2 incubator and incubated for 24 h, 48 h, and 72 h (multiple time points were used to comprehensively evaluate the time-dependent toxicity of the cyclic peptide).

[0046] (4) CCK8 staining and absorbance detection After incubation, add 10 μL of CCK8 reagent to each well (the reagent to culture medium volume ratio is 1:10), gently shake the culture plate to mix the reagent evenly, and avoid generating air bubbles; place the culture plate back in a 37℃, 5% CO2 incubator and incubate in the dark for 2 h; after incubation, use a microplate reader to measure the OD value of each well at a wavelength of 450 nm (main absorption peak) and 630 nm (corrected background absorption), and record the data.

[0047] (5) Data processing and repeatability verification Experimental data were analyzed using GraphPad Prism 8.0 software. The OD values ​​of the cell control group and each experimental group were first corrected using the OD value of the blank control group (subtracting background uptake from the culture medium and CCK8 reagent). Cell viability was then calculated using the following formula: Cell viability (%) = (Corrected OD value of experimental group / Corrected OD value of control group) × 100% Each cyclic peptide sample was subjected to at least three independent replicate experiments, and the mean ± standard deviation (x ± s) was taken as the final result.

[0048] The cytotoxicity test results for Examples 2, 3, 5, 16, and 20 are shown in Table 1 and... Figure 6 As shown in Table 1 and Figure 6 It can be seen that when the concentration of cyclic peptide is ≤50ppm, the cell survival rate is ≥90%, indicating extremely high safety.

[0049] Table 1. Cytotoxicity test of cyclic peptides (%)

[0050] Efficacy test example 3: Molecular docking between cyclic peptides and target sites: Molecular docking technology, as a core tool in computer-aided drug design, can theoretically predict and quantitatively evaluate the bioactivity and mechanism of action of designed cyclic peptides at the molecular level by simulating the interaction patterns between peptides and protein targets, providing direct theoretical support for the functional targeting of cyclic peptides. This invention targets the core anti-aging effects of cyclic peptides—"antioxidant and promotion of extracellular matrix production"—and selects two key pathway target proteins for molecular docking verification: KEAP1 (Kelch-like ECH-associated protein 1), a key target protein of the oxidative stress pathway, can regulate the activation of the Nrf2 antioxidant pathway by binding to cyclic peptides, thereby playing a role in scavenging free radicals. TGFβ (transforming growth factor β), a key target protein in the extracellular matrix synthesis pathway, can promote fibroblast proliferation and the synthesis of collagen and elastin by specifically binding to cyclic peptides. Tables 2 and 3 list the molecular docking binding energies of the preferred cyclic 5-peptide and cyclic 6-peptide (containing PYY / PPY / YPY core motifs) of the present invention with KEAP1 and TGFβ target proteins. The larger the absolute value of the binding energy, the stronger the binding affinity between the cyclic peptide and the target protein, providing a theoretical basis for the structure-function correlation of the cyclic peptide's synergistic anti-aging effects.

[0051] Table 2. Molecular docking binding energy data of the preferred cyclic 5-peptide with KEAP1 and TGFβ target proteins.

[0052] Table 3. Molecular docking binding energy data of the preferred cyclic hexapeptide with KEAP1 and TGFβ target proteins.

[0053] The binding energy in molecular docking analysis is expressed as free energy (ΔG), with units of kcal / mol. The core principle is: the more negative the ΔG value (i.e., the larger the absolute value of the binding energy), the stronger the binding affinity between the cyclic peptide and the target protein, the more stable the intermolecular interaction, and theoretically, the more significant the biological activity. A binding energy absolute value < 6 kcal / mol, i.e., ΔG > -6 kcal / mol, indicates weak binding, meaning the active ingredient has a weak affinity for the target protein, belonging to a "non-specific / unstable binding mode"; a binding energy absolute value of 6~8 kcal / mol, i.e., ΔG ∈ [-8, -6) kcal / mol, indicates moderate binding, meaning the active ingredient forms a specific and effective binding with the target protein, belonging to a "moderate affinity binding mode"; a binding energy absolute value ≥ 8 kcal / mol, i.e., ΔG ≤ -8 kcal / mol, indicates strong binding, meaning the active ingredient forms a highly specific and stable binding with the target protein, belonging to a "strong affinity binding mode".

[0054] As can be seen from Tables 2 and 3, the cyclic pentapeptide and cyclic hexapeptide of the present invention exhibit a "strong affinity binding mode" with KEAP1 and TGFβ. This means that when the cyclic pentapeptide and cyclic hexapeptide of the present invention strongly bind to KEAP1, they can efficiently relieve the inhibition of Nrf2 by KEAP1 and significantly activate the antioxidant pathway; when they strongly bind to TGFβ, they can effectively promote the phosphorylation of TGFβ receptor and accelerate the synthesis of collagen by fibroblasts.

[0055] Efficacy Test Example 4: Antioxidant Performance Test of Anti-aging Cyclic Peptides The antioxidant properties of cyclic peptides were assessed using the DCFH-DA probe to detect intracellular ROS levels. DCFH-DA (2',7'-dichlorofluorescein diacetate) is a non-fluorescent, lipid-soluble ROS-specific probe that can penetrate the cell membrane and enter the cell. Intracellular esterases hydrolyze it into non-fluorescent DCFH (2',7'-dichlorofluorescein). When reactive oxygen species (ROS) are present in the cell (such as superoxide anions and hydroxyl radicals), DCFH is oxidized to highly fluorescent DCF (2',7'-dichlorofluorescein). The fluorescence intensity of DCF, observed and quantified using a fluorescence microscope, indirectly reflects the relative content of intracellular ROS—higher fluorescence intensity indicates a higher intracellular ROS level; conversely, lower fluorescence intensity indicates a more significant ROS scavenging effect. The specific operating steps are as follows: Step 1: Sample Processing (1) Place a sterile coverslip in a 6-well cell culture plate and sterilize it by ultraviolet irradiation for 30 min; (2) HSF cells in the logarithmic growth phase were digested with trypsin, resuspended in complete culture medium, and the cell concentration was adjusted to 2×10⁻⁶. 4 Inoculate each well with 2 mL of complete culture medium into a 6-well plate containing a coverslip; (3) Incubate in a 37℃, 5% CO2 incubator for 24 h to allow the cells to adhere completely to the coverslip; (4) Discard the old culture medium, add 2 mL of complete culture medium containing the corresponding reagents according to the following groups, and continue incubation for 24 h (consistent with the time for cell action by cyclic peptides to ensure full manifestation of ROS scavenging effect): Blank control group (ROS basic group) (NC): Complete culture medium (with cells, without cyclic peptides, without H2O2); Positive induction group (ROS positive control) (H2O2): Complete culture medium containing 100 μM H2O2 (with cells, no cyclic peptides, to induce ROS generation). Cyclic peptide experimental group: complete culture medium containing 10 ppm cyclic peptide + 100 μM H2O2; Three parallel holes are set in each group to ensure data repeatability.

[0056] Step 2: DCFH-DA probe loading and incubation (1) After the cyclic peptide incubation is completed, the culture medium of each well is aspirated and the cells are gently washed twice (5 min each time) with PBS buffer preheated to 37°C to remove residual culture medium, unbound cyclic peptides and H2O2. (2) Preparation of DCFH-DA working solution: Dilute the DCFH-DA stock solution (10 mM, dissolved in DMSO, stored at -20℃ in the dark) with serum-free DMEM medium to a final concentration of 10 μM (final DMSO concentration ≤0.1%, to avoid cytotoxicity); (3) Add 1 mL of 10 μM DCFH-DA working solution to each well to ensure complete coverage of the cells on the coverslip. Incubate in a 37°C, 5% CO2 incubator for 20-30 min in the dark (avoid probe photolysis; gently shake the culture plate once every 10 min during this period to ensure even probe loading).

[0057] Step 3: Fluorescence Microscopy Observation and Image Acquisition (1) After the probe incubation is complete, the DCFH-DA working solution is discarded, and the cells are washed three times (5 min each time) with pre-warmed PBS buffer to completely remove the free probes that have not entered the cells (to reduce background fluorescence interference). (2) Add 2 mL of PBS buffer to each well (to keep the cells moist and avoid drying which would affect fluorescence stability); (3) Carefully remove the coverslip from the 6-well plate and attach it to the slide with the cell side facing up (you can add 1 drop of anti-fluorescence quenching mounting medium to prolong the fluorescence preservation time). (4) Fluorescence microscope parameter settings: excitation wavelength 488 nm, emission wavelength 525 nm, objective magnification 20× (or 40×, adjusted according to cell density), exposure time uniformly set to 100 ms (all groups should be kept consistent to avoid quantitative error); (5) Five non-overlapping fields of view are randomly selected from each group to collect fluorescence images (including bright field images and fluorescence images, which are used to locate cells).

[0058] Step 4: Quantitative analysis of fluorescence intensity Fluorescence intensity was quantified using ImageJ software, and the relative inhibition rate of ROS was calculated using the following formula: ROS relative inhibition rate (%) = [1 - average fluorescence intensity of experimental group / average fluorescence intensity of positive induction group] × 100% Antioxidant test results as follows Figure 7 As shown, the H2O2 group exhibited high fluorescence intensity, while the blank control and the examples showed very low fluorescence intensity, indicating that the peptides in the examples possessed excellent ROS scavenging effects. Specific fluorescence intensities are shown in Table 4. The relative inhibition rates of reactive oxygen species are as follows: Figure 8 As shown, the scavenging rate of reactive oxygen species is much higher than 50%, indicating that the cyclic peptides synthesized on this surface have strong antioxidant activity.

[0059] Table 4. Average fluorescence intensity (au)

[0060] Efficacy Test Example 5: Antioxidant Enzyme Activity Test of Anti-aging Cyclic Peptides The antioxidant enzyme activity of the anti-aging cyclic peptides was detected using a total SOD activity assay kit (WST-8 method), with the xanthine-xanthine oxidase system as the superoxide anion free radical (O2) assay. 2- ) generator, continuously generating O 2- It can undergo a redox reaction with the water-soluble tetrazolium salt substrate WST-8 in the kit to generate a water-soluble formazan dye with a characteristic absorption peak at 450 nm. Meanwhile, the superoxide dismutase (SOD) in the sample will specifically remove O from the system. 2- This inhibits the formation of formazan dye, and the inhibition rate is positively correlated with SOD activity. Finally, the absorbance value of each reaction well is detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the total SOD activity in the sample can be calculated by combining the standard curve plotted with SOD standards.

[0061] The specific experimental steps include: Step 1: Sample Preparation (1) Preparation of cell samples: For adherent cells, aspirate the cell culture medium, wash once with PBS or physiological saline pre-cooled at 4°C or in an ice bath, add SOD sample preparation solution at a ratio of 100-200 μL per 1 million cells, and pipette appropriately to fully lyse the cells; (2) Preparation of WST-8 / enzyme working solution: Prepare an appropriate amount of WST-8 / enzyme working solution according to a volume of 160µl for each reaction. Mix 151µl of SOD detection buffer, 8µl of WST-8 and 1µl of enzyme solution evenly; (3) Preparation of reaction start working solution: Dissolve the reaction start solution (40X) and mix well. Dilute it by adding 39µl of SOD detection buffer to every 1 µl of reaction start solution (40X). After mixing well, the reaction start working solution is obtained.

[0062] Step 2: Sample Testing (1) Refer to the table below to set up the sample wells and various blank control wells using a 96-well plate. Add the test samples and other solutions in the order shown in Table 5 below. After adding the reaction start-up working solution, mix thoroughly.

[0063] Table 5. Amount of reaction solution added

[0064] (2) After incubating at 37°C for 30 minutes, the absorbance was measured at 450 nm.

[0065] (3) The inhibition percentage is calculated using the formula: (A blank control 1 - A sample) / (A blank control 1 - A blank control 2) × 100% The results of the absorbance inhibition rate test are shown in Table 6. Figure 9 and Figure 10 As shown, H2O2 treatment reduced the inhibition rate of formazan dye formation, indicating successful modeling and a decrease in SOD enzyme content in the model. Compared to the H2O2-treated model group, the inhibition rate of formazan dye formation in the example sample group increased from 26.76% in the model group to 47.1%~56.69%, indicating an increase in intracellular SOD enzyme content in the examples.

[0066] Table 6. Inhibition rate of formazan dye formation (%)

[0067] Efficacy Test Example 6: Extracellular Matrix Synthesis Test of Anti-aging Cyclic Peptides Extracellular matrix synthesis assays were performed using two types of kits: the Human Collagen Type I ELISA Kit and the Human Collagen Type III ELISA Kit. Specific procedures included: Step 1: Preliminary Preparations (1) Reagent preparation Complete culture medium: Take DMEM high glucose medium, add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (final concentration of 100 U / mL penicillin and 100 μg / mL streptomycin), mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃ for later use. Washing solution: Dilute the concentrated washing solution in the ELISA Kit with sterile ultrapure water at a ratio of 1:20, mix thoroughly and set aside. 10ppm cyclic peptide stock solution: Accurately weigh 1 mg of cyclic peptide sample and place it in a 100 mL sterile volumetric flask. Add a small amount of DMSO to dissolve it (final DMSO concentration ≤0.1% to avoid cell toxicity). Dilute to the mark with complete culture medium and mix thoroughly to obtain 100 ppm cyclic peptide stock solution. Take another 10 mL of the 100 ppm stock solution and dilute to 100 mL with complete culture medium. Mix thoroughly to obtain 10 ppm cyclic peptide working solution. Prepare and use immediately. Standard dilution: Take the Human Collagen Type I standard from the ELISA Kit and dilute it serially with standard diluent (or complete culture medium) to prepare a series of standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, and 32 ng / mL according to the kit instructions. Keep on ice for later use.

[0068] Hydrogen peroxide working solution: Take 30% hydrogen peroxide solution and dilute it with sterile PBS to a final concentration of 200 μM. Prepare and use immediately. After preparation, store on ice in the dark to avoid decomposition.

[0069] (2) Cell pretreatment After thawing cryopreserved human fibroblasts (HSF cells), they were seeded into 15 mL culture flasks, and 5 mL of complete culture medium was added. The flasks were then incubated in a CO2 incubator at 37°C, 5% CO2, and 95% humidity. When cell confluence reached 80%-90%, the cells were digested with trypsin-EDTA digestion solution, collected by centrifugation, resuspended in complete culture medium, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 4 Quantity / mL, for later use.

[0070] Step 2: Cell Seeding and Drug Intervention (1) Take a 96-well cell culture plate, add 100 μL of cell suspension with adjusted concentration to each well, and incubate in a 37℃, 5% CO2 incubator for 24 h to allow the cells to adhere completely to the plate. (2) After the cells adhered to the wall, the old culture medium was discarded and the cells were divided into groups: 100 μL of complete culture medium was added to the normal control group; 100 μL of 200 μM hydrogen peroxide working solution was added to the model control group and the cyclic peptide experimental group, and they were incubated in a 37℃, 5% CO2 incubator for 2 h to establish an oxidative stress injury model. (3) After the modeling is completed, discard the hydrogen peroxide working solution in each modeling group well, and wash gently twice with sterile PBS. After each wash, let stand for 30 seconds and then discard the washing solution. (4) After washing, 100 μL of complete culture medium containing 0.1% DMSO (with the same DMSO concentration as the experimental group) was added to both the normal control group and the model control group; 100 μL of 10 ppm cyclic peptide working solution was added to the experimental group; 6 replicates were set up for each group; (5) The 96-well plate was placed in a 37°C, 5% CO2 incubator for 48 hours for cyclic peptide intervention.

[0071] Step 3: Collection and analysis of cell supernatant (1) After 48 hours of drug intervention, the 96-well cell culture plate was placed in a low-speed centrifuge and centrifuged at 1000 r / min for 5 min. The cell supernatant of each well was carefully aspirated and transferred to the corresponding 1.5 mL sterile centrifuge tube. The tube was centrifuged again at 12000 r / min for 10 min to remove cell debris from the supernatant. The supernatant was collected and stored in a -80℃ freezer for later use in ELISA detection.

[0072] (2) Strictly follow the instructions for Human Collagen Type I ELISA Kit and Human Collagen Type III ELISA Kit.

[0073] (3) Place the microplate into the microplate reader, use 450nm as the detection wavelength and 630nm as the reference wavelength, measure the absorbance (OD value) of each well, and record the experimental data.

[0074] (4) Use ELISACalc software to perform calculations, select a four-parameter regression curve, and then calculate the sample concentration based on the sample absorbance. The data are summarized in Tables 7 and 8. Figure 11 and Figure 12 As shown in Table 7 and Figure 11 It can be seen that the type I collagen content in the H2O2 model group was 111.93 ng / ml, while the type I collagen content in the examples was higher than 170 ng / ml, and in Example 2 it even reached 365.21 ng / ml. This indicates that the prepared cyclic peptide has a good promoting effect on type I collagen production.

[0075] Table 7. Collagen Col1 Content (ng / ml)

[0076] From Table 8 and Figure 12 It can be seen that the type III collagen content in the H2O2 model group was 0.59 ng / ml, while the type III collagen content in the examples was higher than 1.88 ng / ml, and in Example 2 it even reached 2.3 ng / ml. This indicates that the prepared cyclic peptide has a good promoting effect on type III collagen production.

[0077] Table 8. Collagen Col3 Content (ng / ml)

[0078] Efficacy Test Example 7: Extracellular Matrix Degradation Inhibition Test of Anti-aging Cyclic Peptides Extracellular matrix synthesis assays were performed using the Human MMP-1 ELISA Kit and the Human MMP-3 ELISA Kit. Two types of reagent kits are used for detection. Specific operating procedures include: Step 1: Preliminary Preparations (1) Reagent preparation Complete culture medium: Take DMEM high glucose medium, add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (final concentration of 100 U / mL penicillin and 100 μg / mL streptomycin), mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃ for later use. Washing solution: Dilute the concentrated washing solution in the ELISA Kit with sterile ultrapure water at a ratio of 1:20, mix thoroughly and set aside. 10ppm cyclic peptide stock solution: Accurately weigh 1 mg of cyclic peptide sample and place it in a 100 mL sterile volumetric flask. Add a small amount of DMSO to dissolve it (final DMSO concentration ≤0.1% to avoid cell toxicity). Dilute to the mark with complete culture medium and mix thoroughly to obtain 100 ppm cyclic peptide stock solution. Take another 10 mL of the 100 ppm stock solution and dilute to 100 mL with complete culture medium. Mix thoroughly to obtain 10 ppm cyclic peptide working solution. Prepare and use immediately. Standard dilution: Take the Human MMP-1 ELISA Kit and Human MMP-3 ELISA Kit standards from the ELISA Kit and, according to the kit instructions, serially dilute them with standard diluent (or complete culture medium) to obtain a series of standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, and 32 ng / mL. Keep them on ice for later use.

[0079] Hydrogen peroxide working solution: Take 30% hydrogen peroxide solution and dilute it with sterile PBS to a final concentration of 200 μM. Prepare and use immediately. After preparation, store on ice in the dark to avoid decomposition.

[0080] (2) Cell pretreatment After thawing cryopreserved human fibroblasts (HSF cells), they were seeded into 15 mL culture flasks, and 5 mL of complete culture medium was added. The flasks were then incubated in a CO2 incubator at 37°C, 5% CO2, and 95% humidity. When cell confluence reached 80%-90%, the cells were digested with trypsin-EDTA digestion solution, collected by centrifugation, resuspended in complete culture medium, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 4 Quantity / mL, for later use.

[0081] Step 2: Cell Seeding and Drug Intervention (1) Take a 96-well cell culture plate, add 100 μL of cell suspension with adjusted concentration to each well, and incubate in a 37℃, 5% CO2 incubator for 24 h to allow the cells to adhere completely to the plate. (2) After the cells adhered to the wall, the old culture medium was discarded and the cells were divided into groups: 100 μL of complete culture medium was added to the normal control group; 100 μL of 200 μM hydrogen peroxide working solution was added to the model control group and the cyclic peptide experimental group, and they were incubated in a 37℃, 5% CO2 incubator for 2 h to establish an oxidative stress injury model. (3) After the modeling is completed, discard the hydrogen peroxide working solution in each modeling group well, and wash gently twice with sterile PBS. After each wash, let stand for 30 seconds and then discard the washing solution. (4) After washing, 100 μL of complete culture medium containing 0.1% DMSO (with the same DMSO concentration as the experimental group) was added to both the normal control group and the model control group; 100 μL of 10 ppm cyclic peptide working solution was added to the experimental group; 6 replicates were set up for each group; (5) The 96-well plate was placed in a 37°C, 5% CO2 incubator for 48 hours for cyclic peptide intervention.

[0082] Step 3: Collection and analysis of cell supernatant (1) After 48 hours of drug intervention, the 96-well cell culture plate was placed in a low-speed centrifuge and centrifuged at 1000 r / min for 5 min. The cell supernatant of each well was carefully aspirated and transferred to the corresponding 1.5 mL sterile centrifuge tube. The tube was centrifuged again at 12000 r / min for 10 min to remove cell debris from the supernatant. The supernatant was collected and stored in a -80℃ freezer for later use in ELISA detection.

[0083] (2) Strictly follow the instructions for Human MMP-1 ELISA Kit and Human MMP-3 ELISA Kit.

[0084] (3) Place the microplate into the microplate reader, use 450nm as the detection wavelength and 630nm as the reference wavelength, measure the absorbance (OD value) of each well, and record the experimental data.

[0085] (4) Use ELISACalc software to perform calculations, select a four-parameter regression curve, and then calculate the sample concentration based on the sample absorbance. The data are summarized in Tables 9 and 10. Figure 13 and Figure 14 As shown in Table 9 and Figure 13It can be seen that the content of matrix metalloproteinase MMP-1 in the H2O2 model group was 924.37 ng / ml, while the MMP-1 content in the examples was lower than 306.12 ng / ml, and in Example 2 it was even lower than 180.7 ng / ml. This indicates that the prepared cyclic peptide can effectively inhibit the damage of collagen by matrix metalloproteinase MMP-1.

[0086] Table 9. Content of matrix metalloproteinase MMP-1 (ng / ml)

[0087] From Table 10 and Figure 14 It can be seen that the content of matrix metalloproteinase MMP-3 in the H2O2 model group is 1114.69 ng / ml, while the content of matrix metalloproteinase MMP-3 in the examples is lower than 296.13 ng / ml, and in Example 20 it is even lower than 213.95 ng / ml. This indicates that the prepared cyclic peptide can effectively inhibit the damage of collagen by matrix metalloproteinase MMP-3.

[0088] Table 10. Content of matrix metalloproteinase MMP-3 (ng / ml)

[0089] Efficacy Test Example 8: Evaluation of the Anti-aging Ability of Anti-aging Cyclic Peptides This experiment evaluated the anti-aging effect of cyclic peptides at a final concentration of 10 ppm using the SA-β-gal staining method. The specific steps are as follows: Step 1: Cell Culture and Senescence Model Construction (1) Cell resuscitation and passage: Human dermal fibroblasts (HDFa, ATCC PCS-201-012) were taken out of liquid nitrogen, rapidly revived in a 37°C water bath, and then transferred to a culture flask containing complete culture medium (DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin double antibiotic) and placed in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were digested with 0.25% trypsin-EDTA and passaged. Cells from the 3rd to 5th generations were selected for subsequent experiments.

[0090] (2) Cell seeding: Cells were seeded simultaneously in 6-well and 96-well plates. The 6-well plates were used for morphological observation and positive cell counting. 5 × 10⁶ cells were seeded in each well. 4 For quantitative absorbance analysis, seed 1 × 10⁶ cells per well with 2 mL of complete culture medium; use 96-well plates for absorbance quantification, seeding each well with 1 × 10⁶ cells. 4 Add 100 μL of complete culture medium to each cell; after inoculation, place both culture plates in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere completely.

[0091] (3) Senescence induction: The old culture medium in both culture plates was discarded, and culture medium containing senescence inducer was added to both. The senescence inducer was H2O2 (final concentration 200 μM) or D-galactose (final concentration 50 mM). After adding the inducer, the plates were cultured in a 37℃, 5% CO2 incubator for 48 h to construct a cell senescence model. The criterion for successful model determination was that the positive rate of SA-β-gal after induction was ≥60% as verified in the preliminary experiment.

[0092] Step 2: Sample Processing (1) Preparation of cyclic peptide stock solution: Accurately weigh the target cyclic peptide with a purity ≥95%, dissolve it in sterile DMSO as solvent, and prepare a cyclic peptide stock solution with a concentration of 10 mg / mL; filter the stock solution with a 0.22 μm filter membrane to remove bacteria, and store it in the dark and cold for later use.

[0093] (2) Experimental grouping and sample addition: Cells in 6-well and 96-well plates after senescence induction were divided into 5 groups, with 3 parallel wells in each group. The specific grouping and processing methods are as follows: Blank control group: No senescence induction was performed, only complete culture medium was added; Aging control group: After aging induction, a complete culture medium (containing 0.1% DMSO) without cyclic peptides was added. Cyclic peptide experimental group: After aging induction, a complete culture medium containing cyclic peptides was added to achieve a final concentration of 10 ppm for cyclic peptides, with a final concentration of DMSO ≤0.1%; (3) Incubation: The culture plates after the above grouping treatment were placed in a 37℃, 5% CO2 incubator for 72h to ensure that the cyclic peptides could fully exert their effects.

[0094] Step 3: SA-β-gal staining and observation (1) Cell fixation: Discard the culture medium in each well, wash the cells gently twice with PBS buffer at pH 7.4, and then add 4% paraformaldehyde solution to each well and fix for 15 min at room temperature.

[0095] (2) Washing: Discard the fixative and wash the cells three times with PBS buffer at pH 7.4 for 5 minutes each time to completely remove residual formaldehyde.

[0096] (3) Preparation of staining solution: Prepare SA-β-gal staining solution according to the following ratio. Each 1 mL of staining solution contains: 100 μL of citrate-sodium phosphate buffer (pH 6.0), 50 μL of X-Gal stock solution (20 mg / mL), 10 μL of 50 mM potassium ferricyanide solution, 10 μL of 50 mM potassium ferrocyanide solution, 2 μL of 1 M MgCl2 solution, and 828 μL of sterile deionized water. Mix well and set aside.

[0097] (4) Staining incubation: Add 1 mL of staining solution to each well of a 6-well plate and 100 μL of staining solution to each well of a 96-well plate, ensuring that the staining solution completely covers the cell surface; place the culture plate in a 37°C CO2-free incubator and incubate in the dark for 12-16 h.

[0098] (5) Termination and washing: Discard the staining solution, wash the cells twice with PBS buffer at pH 7.4, add a small amount of 50% glycerol to the 6-well plate for mounting, and leave a small amount of PBS buffer in the 96-well plate to prevent the cells from drying out.

[0099] (6) Morphological observation.

[0100] like Figure 15 The figure shows the SA-β-gal staining results in the blank group, aging control group, and sample group. The blue-stained cells in the aging control group are SA-β-gal-positive senescent cells, while the number of blue-stained cells in the sample with added cyclic peptides is significantly reduced. This indicates that the cyclic peptides of this invention have a good anti-aging effect.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An anti-aging cyclic peptide, characterized in that: Its core structural feature is a cyclic 5-peptide or cyclic 6-peptide containing a PYY, PPY, or YPY tripeptide motif. The tripeptide motifs contained in the cyclic peptide are distributed as follows: Motif 1 (PYY): Proline (Pro, P) - Tyrosine (Tyr, Y) - Tyrosine (Tyr, Y), with the amino acid sequence PYY; Motif 2 (PPY): Proline (Pro, P) - Proline (Pro, P) - Tyrosine (Tyr, Y), with the amino acid sequence PPY; Motif 3 (YPY): Tyrosine (Tyr, Y) - Proline (Pro, P) - Tyrosine (Tyr, Y), with the amino acid sequence YPY.

2. The anti-aging cyclic peptide as described in claim 1, characterized in that: The cyclic peptide is an intramolecularly cyclic polypeptide with head-to-tail cyclization, where the cyclization site is formed by the α-amino group of the N-terminal amino acid and the α-carboxyl group of the C-terminal amino acid linked by an amide bond to form a cyclic structure.

3. The anti-aging cyclic peptide as described in claim 2, characterized in that: The cyclic 5-peptide is formed by cyclization of 5 amino acid residues as described above, and has the general structural formula: cyclo (-X1-X2-X3-X4-X5-). Among them, X1-X5 are natural L-amino acids, D-amino acids or non-natural amino acids, and X1-X5 must contain a continuous PYY, PPY or YPY tripeptide motif. The cyclic 6-peptide is formed from 6 amino acid residues through the above-described cyclization process, and its general structural formula is: cyclo (-X1-X2-X3-X4-X5-X6-); Among them, X1-X6 are natural L-amino acids, D-amino acids or non-natural amino acids, and X1-X6 must contain consecutive PYY, PPY or YPY.

4. The anti-aging cyclic peptide as described in claim 3, characterized in that: The non-natural amino acids are selected from phosphorylated tyrosine and methylated proline; the tripeptide motif of the cyclic 5-peptide is located at any three consecutive sites, such as X1-X3, X2-X4 or X3-X5; the tripeptide motif of the cyclic 6-peptide is located at any three consecutive sites, such as X1-X3, X2-X4, X3-X5 or X4-X6.

5. The anti-aging cyclic peptide as described in claim 4, characterized in that: The cyclic 5 peptide is selected from Cyclo(PYYAY), Cyclo(YPPYP), Cyclo(PPYAP), Cyclo(YPYYA), Cyclo(TYPYY), Cyclo(YPYRY), Cyclo(GPY YY), Cyclo(RTYPY), Cyclo(AAPYY), Cyclo(YPYVA), Cyclo(PYPYY), Cyclo(YPYGG), Cyclo(PYYGY), Cyclo(PPYGR); The cyclic 6 peptide is selected from Cyclo(PYPYGA), Cyclo(PYYAPP), Cyclo(PYYGPG), Cyclo(AGYPYR), Cyclo(YPYGGY), Cyclo(PPYAYA), Cyclo(YGYPYY), Cyclo(YPYVPP), Cyclo(YPYPVP), Cyclo(GPPYYV), Cyclo(YAPPYP).

6. The method for preparing the anti-aging cyclic peptide according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Linear peptide synthesis: 2-Chlorotriphenylmethyl chloride (2-CTC resin, 0.98 mmol / g loading) was weighed into a reaction tube, and dichloromethane (DCM) was added to swell the resin at room temperature for 1 hour. After the solvent was removed, a dichloromethane solution of the first amino acid monomer at the C-terminus (1.5 equiv) and N,N-diisopropylethylamine (DIEA, 5 equiv) was added. The mixture was shaken at room temperature for 1 hour, and then the resin was washed three times with N,N-dimethylformamide (DMF) and DCM, and dried. Subsequently, Fmoc protection was removed by adding 20% ​​piperidine / DMF solution twice, 10 min each time. Then, the resin was washed three times with DCM and DMF in sequence. After drying, the second amino acid was coupled by adding the activated second amino acid monomer DMF solution and reacting with shaking at room temperature for 1 h. The resin was then washed three times with DMF and DCM in sequence and dried. The above steps of Fmoc protection removal, washing, coupling, and washing were repeated until the last amino acid monomer was reached. Add the cleavage reagent hexafluoroisopropanol (HFIP) / DCM (20%) to the dry resin, shake at room temperature for 0.5 h, repeat once, combine the filtrates and collect them in a centrifuge tube, remove the solvent by rotary evaporation to obtain the crude product; Step 2, Cycloning: The crude cyclic peptide product was dissolved in DMF (0.005 M). After complete dissolution, HATU (2 equiv) and DIEA (3-6 equiv) were added sequentially, and the reaction was carried out at room temperature for 2 h. The cyclization process 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 the product, and the insoluble solids were removed by centrifugation. The filtrate was collected and evaporated to dryness for the next step of deprotection. Step 3: Remove protection: Prepare a deprotection solution of TFA / triisopropylsilane (TIPS) / DCM = 50:5:45 or TFA / TIPS / 1,2-ethylenedithiol (EDT) / DCM = 50:2.5:2.5:45, and add it to the crude cyclic peptide product. Incubate at room temperature with shaking for 0.5–2 h, and monitor the deprotection process using LCMS. After complete deprotection, dry the solvent with an air pump and purify the product using a column chromatography system.

7. The method for preparing the anti-aging cyclic peptide as described in claim 6, characterized in that: The second amino acid monomer DMF solution comprises 5 equiv of amino acid monomer, 4.75 equiv of condensing agent HBTU, and 5 equiv of DIEA.

8. The use of the anti-aging cyclic peptide according to any one of claims 1-7 in the preparation of skin care products.

9. The use of the anti-aging cyclic peptide according to any one of claims 1-7 in the preparation of skin care products that scavenge free radicals and / or promote collagen production.

10. The use of the anti-aging cyclic peptide according to any one of claims 1-7 in the preparation of cosmetics.

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