Cyclopeptide capable of resisting oxidation and inflammation and inhibiting matrix metalloproteinase as well as preparation method and application of cyclopeptide

Abstract

The invention belongs to the technical field of active polypeptides and preparation, and particularly relates to an antioxidant and anti-inflammatory cyclopeptide capable of inhibiting matrix metalloproteinase as well as a preparation method and application of the antioxidant and anti-inflammatory cyclopeptide. The cyclic peptide is formed by end-to-end covalent coupling of linear peptide with the amino acid sequence of DEETGEF, has the activity of resisting oxidation, resisting inflammation and inhibiting matrix metalloproteinase, is free of cytotoxicity and skin irritation, and is suitable for being used as cosmetics for resisting skin aging, wrinkles and the like.

Description

Technical Field

[0001] This invention belongs to the field of active peptides and their preparation technology, specifically relating to a cyclic peptide that has antioxidant and anti-inflammatory properties and inhibits matrix metalloproteinases, as well as its preparation method and uses. Background Technology

[0002] Oxidative stress is a redox imbalance in the body and a significant factor contributing to tissue damage and inflammation-related diseases. Inflammatory factors can induce the overexpression of matrix metalloproteinases (MMPs), which in turn can trigger the degradation of skin elastin and other proteins, leading to decreased skin elasticity and wrinkles. Therefore, anti-oxidation (including oxidative stress caused by radiation), anti-inflammation, and MMP inhibition are key strategies for delaying skin aging and improving skin elasticity.

[0003] Nuclear factor erythrocyte-2-related factor 2 (Nrf2) is a major regulator of antioxidant gene expression. Nrf2 is regulated by Kelch-like ECH-associated protein 1 (Keap1). Previous studies have shown that the protein-protein interaction between Nrf2E and Keap1 is related to the ECH homology domain 2 (Neh2) of Nrf2E, primarily through the ETGE peptide of this domain, which binds to Kelch via hydrogen bonds, electrostatics, and van der Waals forces. Therefore, based on the ETGE peptide, researchers have prepared numerous modified linear or cyclic peptides to competitively inhibit the interaction between Nrf2 and Keap1, thereby exerting antioxidant and other activities. This includes a series of linear and cyclic peptides developed by Ortet et al. (Ortet PC, Muellers SN, Viarengo-Baker LA, et al. Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning[J]. Journal of the American Chemical Society, 2021, 143(10).) and a series of linear and cyclic peptides developed by Jessica Iegre et al. (Iegre J, Krajcovicova S, Gunnarsson A, Wissler L, H,Luchniak A, S, Narjes F, Spring DR. A cell-active cyclic peptide targeting the Nrf2 / Keap1 protein-protein interaction. Chemical Science. 2023; 14(39):10800-5.); However, the former developed cyclic peptide without disulfide bonds, only c[(D)-β-homoAla-DPETGE] showed better activity than the linear peptide; the latter developed cyclic peptide based on disulfide bond ring formation also did not show a significant improvement in activity compared with the corresponding linear peptide.

[0004] Although peptide cyclization is thought to improve enzymatic stability, for topical cosmetic peptides, the primary influencing factor is not enzymatic hydrolysis, but rather the peptide's polarity and hydrophilicity. While polar and hydrophilic peptides are more likely to bind to Keap1, they hinder transdermal absorption across cell membranes. Therefore, most developers of Nrf2-mimicking peptides have employed strategies such as reducing polar amino acids and removing or replacing amino acids unrelated to Keap1 binding to optimize linear peptides, followed by peptide cyclization. For example, replacing the 8th amino acid F (Phe) of linear peptides like Ac-LDEETGEFL-NH2 with amino acid A (Ala). However, to date, most Nrf2-mimicking peptides, whether modified peptides obtained through amino acid substitution or cyclic peptides based on optimized linear peptides, have not yielded ideal results. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties, along with its preparation method and uses. This cyclic peptide is formed by decarboxylating the linear peptide segment with the amino acid sequence shown in SEQ ID NO.1 from head to tail. Compared to other Nrf2-mimicking peptides or the linear peptide segment shown in SEQ ID NO.1, this cyclic peptide exhibits superior activity. The technical solution of the present invention is as follows:

[0006] First, this invention provides a cyclic peptide that has antioxidant and anti-inflammatory properties and inhibits matrix metalloproteinases, the structural formula of which is shown in Formula I:

[0007]

[0008] Secondly, the present invention provides a method for preparing the cyclic peptide shown in Formula I, comprising the following steps:

[0009] Step S1: Preparation of linear peptide: The linear peptide segment required for the synthesis of the cyclic peptide shown in Formula I is prepared by solid-phase synthesis. The linear peptide segment contains at least one amino acid with a side-chain protecting group.

[0010] Step S2 Cycling of linear peptides: Using a condensing agent and an organic base, the linear peptides are cyclized by decarboxylation of the amino and carboxyl groups at the beginning and end of the linear peptide segment.

[0011] Preferably, in step S1, the solid resin used in the solid-phase synthesis method is 2-CTC Resin resin.

[0012] Preferably, in step S1, the protected amino acids used in the solid-phase synthesis method are: Fmoc-Phe-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Glu(Otbu)-OH, and Fmoc-Asp(Otbu)-OH.

[0013] Preferably, in step S2, the condensing agent and the organic base are benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate and N,N-diisopropylethylamine, respectively.

[0014] In a preferred embodiment, the method includes the following steps:

[0015] Step S1: Preparation of linear peptide: 2-CTC resin was added to a solid-phase column, and the 2-CTC resin was swollen, washed, and dried. Fmoc-Phe-OH, Fmoc-Glu(Otbu), Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Glu(Otbu)-OH, and Fmoc-Asp(Otbu)-OH were added sequentially using a solid-phase synthesis method to gradually condense and remove Fmoc. The resulting linear peptide was cleaved from the resin to prepare a linear peptide with side-chain protecting groups.

[0016] Cycling of S2 linear peptide: A linear peptide with a side-chain protecting group was condensed into a cyclization using the condensing agent benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate and the organic base N,N-diisopropylethylamine.

[0017] S3 Removal of side-chain protecting groups: The side-chain protecting groups are cleaved with trifluoroacetic acid solution to obtain the cyclic peptide shown in Formula I.

[0018] Furthermore, the present invention provides the use of the cyclic peptide shown in Formula I in the preparation of topical cosmetics. The cyclic peptide can exert beneficial effects on the skin, such as anti-aging, inhibition of skin elasticity reduction, and anti-wrinkle effects, through antioxidant, anti-inflammatory, and matrix metalloproteinase inhibition effects.

[0019] The technology of the present invention has the following beneficial effects

[0020] (1) The cyclic peptide of the present invention has a small molecular weight and only 7 amino acids, which helps to reduce the difficulty of preparing polypeptides.

[0021] (2) The cyclic peptides of the present invention do not rely on disulfide bonds to form cyclization, which helps to avoid structural instability caused by the destruction of disulfide bonds.

[0022] (3) The cyclic peptides of the present invention do not undergo complex side chain modifications such as amino acid substitution or fatty acid linkage to their linear parent peptides, but their activity is still unexpectedly improved.

[0023] (4) The cyclic peptides of the present invention have not shown cytotoxicity or skin irritation and have good safety. Attached Figure Description

[0024] Figure 1 The HPLC chromatogram of the purified cyclic 7-peptide-1 product is shown below.

[0025] Figure 2 This is the mass spectrum of cyclic 7-peptide-1;

[0026] Figure 3 The image shows the NMR spectrum of cyclic 7-peptide-1.

[0027] Figure 4 The results are from the cytotoxicity test of cyclic 7-peptide-1;

[0028] Figure 5 The results show the regulatory effect of cyclic 7-peptide-1 on HO1.

[0029] Figure 6 The results show the regulatory effect of cyclic 7-peptide-1 on NQO1.

[0030] Figure 7 The results are from the anti-inflammatory activity test of cyclic 7-peptide-1;

[0031] Figure 8 The results are from the MMP inhibitory activity assay of cyclic 7-peptide-1. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are merely illustrative examples and should not be considered as limiting the scope of protection of the present invention.

[0033] In the following examples, 2-CTC refers to 2-chlorotriphenylmethyl chloride resin; DCM refers to dichloromethane; DMF refers to N,N-dimethylformamide; DIPEA refers to ,N-diisopropylethylamine; TFA refers to trifluoroacetic acid; HOBT refers to 1-hydroxybenzotriazole; DIC refers to N,N-diisopropylcarbodiimide; and PyBop refers to benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate. All reagents and raw materials used in the following examples are commercially available conventional reagents and raw materials.

[0034] In the following examples, "wt%" is an abbreviation for weight percentage, used to indicate the mass percentage of a component in a mixture.

[0035] Example 1: Preparation of a cyclic peptide

[0036] This embodiment provides a solid-phase synthesis method for the cyclic peptide shown in Formula I. The cyclic peptide is hereinafter named "Cyclic heptapeptide-1". The synthetic route is as follows: using 2-CTC Resin resin, Fmoc-Phe-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Glu(Otbu)-OH, and Fmoc-Asp(Otbu)-OH are coupled sequentially. After amino acid coupling, the Fmoc protecting group is removed, and the peptide chain is cleaved to obtain linear heptapeptide-1. Then, a condensing agent is used to condense the peptide chain into a ring. Finally, the side chain protecting group is cleaved with trifluoroacetic acid solution to obtain crude cyclic heptapeptide-1, which is then purified to obtain a refined cyclic heptapeptide-1.

[0037]

[0038] The synthesis method of cyclic heptapeptide is as follows:

[0039] 1. Peptide resin treatment: Add 2-CTC resin to the solid-phase column, swell with DCM, remove the solvent, wash with DMF, and remove dry for later use;

[0040] Synthesis of 2Fmoc-Phe-O-2-CTC

[0041] 2.1 Add DMF containing Fmoc-Phe-OH (3.0 equivalents) and DIPEA (3.0 equivalents) to the swollen and washed resin (DMF solution volume is approximately 3 times the weight of the dry resin added, volume-to-mass ratio, mL:g). After bubbling under nitrogen for about 2 hours, drain the reaction solution and wash twice with DMF. Then add methanol containing 10% DIPEA (10% DIPEA methanol solution volume is 3 times the weight of the dry resin added, volume-to-mass ratio, mL:g). Bubble under nitrogen for about 30 minutes, drain the solvent, and wash twice with DMF.

[0042] 2.2 Add DMF containing 20% ​​piperidine to the product from step 2.1 (DMF solution volume is approximately 3 times the weight of the dry resin feed, volume-to-mass ratio, mL:g), bubble under nitrogen for 5–10 min, and then dry under vacuum. Add DMF containing 20% ​​piperidine again (DMF solution volume is approximately 3 times the weight of the dry resin feed, volume-to-mass ratio, mL:g), bubble under nitrogen for 10–15 min, and then dry under vacuum. Wash with DMF 6 times, and then dry under vacuum for later use. Confirm the completion of Fmoc deprotection using Kaiser colorimetry.

[0043] 3. Synthesis of Fmoc-Glu(Otbu)-Phe-O-2-CTC

[0044] 3.1 Add DMF containing Fmoc-Glu(Otbu)-OH (2.0 equivalents), HOBT (2.0 equivalents), and DIC (2.0 equivalents) to the product from step 2.2 (the volume of the DMF solution is approximately 3 times the weight of the dry resin added, volume-to-mass ratio, mL:g). Bubble the mixture under nitrogen for approximately 1 hour. Confirm the reaction is complete using a Kaiser colorimetric test. Drain the reaction solution and wash twice with DMF.

[0045] 3.2 Add DMF containing 20% ​​piperidine (DMF solution volume is approximately 3 times the weight of the dry resin feed, volume-to-mass ratio, mL:g) to the product from step 3.1, bubble under nitrogen for 5–10 min, dry under vacuum, add DMF containing 20% ​​piperidine again (DMF solution volume is approximately 3 times the weight of the dry resin feed, volume-to-mass ratio, mL:g), bubble under nitrogen for 10–15 min, dry under vacuum, wash with DMF 6 times, and dry under vacuum for later use. Confirm the completion of Fmoc deprotection using Kaiser colorimetry.

[0046] 4. Synthesis of linear peptides with side-chain protecting groups

[0047] Following steps 1 to 3 described above, Fmoc Gly-OH (3.0 equivalents), Fmoc Thr-OH (3.0 equivalents), Fmoc Glu(Otbu)-OH (3.0 equivalents), Fmoc Glu(Otbu)-OH (3.0 equivalents), and Fmoc Asp(Otbu)-OH (3.0 equivalents) were sequentially coupled to obtain a 7-peptide with a protecting group. The Fmoc protecting group was then removed. After washing with resin, the product was dried under reduced pressure at approximately 37°C.

[0048] 5. Cleavage of linear peptides with side-chain protecting groups

[0049] The dried linear peptide resin with side-chain protecting groups was cut with dichloromethane containing 1.5% TFA (the volume of the dichloromethane solution was 10 times the weight of the linear peptide resin, volume-to-mass ratio, mL:g) at room temperature for about 1 hour. The solution was filtered, concentrated, and the concentrate was added dropwise to water to precipitate a solid. The solution was kept at 0-10℃ for about 1 hour, filtered, and the filter cake was dried under reduced pressure at 37℃ to obtain crude linear 7-peptide-1 with side-chain protecting groups.

[0050] 6. Preparation of Cyclic Peptides

[0051] The product from step 5 was condensed into a cyclization using the condensing agent PyBop (3.0 equivalents) and the organic base DIPEA (3.0 equivalents) at 10–30°C for 6–24 hours (this temperature and reaction time range is acceptable; in this specific example, the reaction was carried out at 25°C for 24 hours). Finally, the side-chain protecting groups were cleaved with trifluoroacetic acid solution to obtain crude cyclic 7-peptide-1, which was then purified to obtain the refined cyclic 7-peptide-1.

[0052] 7. Cyclic Peptide Purification Process

[0053] The crude cyclic heptapeptide-1 was dissolved in water containing 10% acetonitrile (20 mL per g of crude product) and filtered through a 0.45 μm filter membrane. The filtrate was purified and separated using a C18 column (10 μm, 250*30 mm, 100A). The detection wavelength was 210 nm, and the sample was loaded at a flow rate of 20 mL / min. Gradient elution was used: phase A was 0.1% acetic acid solution, and phase B was acetonitrile. The elution program is shown in Table 1.

[0054] Table 1. Gradient elution procedure for cyclic 7-peptide-1 purification.

[0055]

[0056]

[0057] Elution was performed under the gradient conditions described above. Fractions from the 10th to 20th minute were collected in segments. Fractions that passed liquid phase monitoring (the qualified standard is tentatively defined as having a purity greater than 95%) were combined and freeze-dried. Low-purity fractions (purity 60% to 95%) were recovered. Fractions with a purity lower than 60% were directly poured into the waste liquid collection tank.

[0058] The purified peptide of cyclic 7-peptide-1 obtained in step 6 was analyzed by liquid chromatography (purity > 95%). The results are as follows: Figure 1 As shown, the retention time of cyclic 7-peptide-1 was 18.348 min, and the sample purity was 97.556% based on the percentage of chromatographic peak area.

[0059] Liquid phase conditions:

[0060] Chromatographic column: Syncronis aQ 250*4.6mm 5um; column temperature: 30℃; flow rate: 1ml / min; detection wavelength: 220nm; mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution; elution program is shown in Table 2.

[0061] Table 2. HPLC gradient elution procedure for the detection of cyclic 7-peptide-1

[0062] Time (min) Mobile phase A (%) Mobile phase B (%) 0 92 8 25 67 33 25.1 20 80 27 20 80 27.1 92 8 32 92 8

[0063] Cyclic 7-peptide-1 has the molecular formula C 34 H 45 N7O 16 The theoretical molecular weight is 807.29; its mass spectrometry data are as follows: Figure 2 As shown, the NMR spectrum is as follows Figure 3 As shown. The structural confirmation data for cyclic 7-peptide-1 are as follows:

[0064] LC-MS (m / z): 808.2982 [M] + ;

[0065] 1H-NMR(DMSO): 1.01(11H,d,J=6.20Hz), 1.57(3H,d,J=14.00Hz), 1.57(4H,t,J=14.08Hz), 1.69(2H,d,J=6.3 5Hz),1.86(4H,d,J=7.16Hz),1.89(3H,d,J=5.08Hz),1.90(5H,d,J=14.17Hz),1.93(4H,d,J=7.71Hz),1.98( 9H,d,J=19.17Hz), 1.98(5H,d,J=7.01Hz), 2.01(3H,d,J=2.41Hz), 2.04(1H,d,J=3.05Hz), 2.23(7H,d,J=7. 27Hz), 2.27 (5H, d, J = 17.98Hz), 2.30 (4H, d, J = 15.60Hz), 2.33 (1H, d, J = 3.33Hz), 2.58 (3H, d, J = 16.17Hz), 2. 60(3H,d,J=16.30Hz), 2.89(4H,d,J=13.70Hz), 2.91(3H,d,J=13.72Hz), 3.05(4H,d,J=13.64Hz), 3.36(4H, d,J=4.44Hz), 3.40(3H,d,J=4.26Hz), 3.92(2H,d,J=6.82Hz), 3.96(2H,d,J=7.08Hz), 3.99(3H,d,J=3.16Hz) ,4.00(4H,d,J=3.01Hz), 4.04(5H,d,J=9.37Hz), 4.07(5H,d,J=6.72Hz), 4.11(6H,d,J=5.81Hz), 4.23(3H,d, J=5.97Hz), 7.11(11H,d,J=7.80Hz), 7.18(7H,d,J=7.23Hz), 7.55(3H,d,J=6.24Hz), 8.16(3H,d,J=4.16Hz).

[0066] Example 2 Cytotoxicity test of cyclic 7-peptide-1

[0067] 1. Sample and preparation: The purified product of cyclic 7-peptide-1 was prepared according to the method in Example 1.

[0068] 2. Test method: Human immortalized epidermal cells HaCaT (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu64) were used to perform cytotoxicity tests using the CCK-8 assay.

[0069] After HaCaT cell resuscitation, cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Sigma-Aldrich, catalog number 102443039) and 1% penicillin-streptomycin (Gibco, catalog number 15140122) until the exponential growth phase. Cells were collected, counted, and seeded into 96-well plates and incubated at 37°C for 24 h at a seeding density of 5 × 10⁶ cells / well. 4 cell / well (200 μl / well).

[0070] Different concentrations (wt%) of cyclic 7-peptide-1 were added to each well. No test substance was added to the control group.

[0071] Follow the kit instructions and add 10% CCK-8 solution at a volume ratio of 10:1 (medium-to-volume). Incubate at 37°C for 45 minutes, then measure the OD450 of each well using a microplate reader.

[0072] The formula for calculating cell viability (survival rate) for each group of samples is as follows:

[0073] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100

[0074] As = Absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8, and the analyte compound)

[0075] Ab = Absorbance of blank wells (absorbance of wells containing culture medium and CCK-8)

[0076] Ac = Absorbance of control wells (absorbance of wells containing cells, culture medium, and CCK-8)

[0077] 3. Test Results: The test results are as follows Figure 4 As shown, cyclic 7-peptide-1 is not cytotoxic to HaCaT cells at concentrations below 0.5 wt%.

[0078] Example 3: Stimulation test of cyclic 7-peptide

[0079] I. Materials and Methods

[0080] 1. Sample and Preparation

[0081] Sample group: Microemulsion containing cyclic 7-peptide-1 (a purified product of cyclic 7-peptide-1 prepared according to the method of Example 1). Blank control group: Blank excipient of microemulsion without cyclic 7-peptide-1. The microemulsion formulations are shown in Table 3, and all contents in Table 3 are mass percentages.

[0082] Table 3 Microemulsion Formulations

[0083]

[0084] 2. Subjects: A total of 30 subjects, including 11 males and 19 females, who met the subject voluntary inclusion criteria.

[0085] 3. Skin patch test method: Using qualified patch testing equipment, an occlusive patch test method was employed. Approximately 0.020g–0.025g of the test substance was placed in the patch testing device, and a hypoallergenic adhesive tape was applied to the back of the subject. After 24 hours, the test substance was removed, and skin reactions were observed at 0.5, 24, and 48 hours after removal. The results were recorded according to the skin reaction grading standards in the "Cosmetic Safety Technical Specifications" (2015 edition). The results showed that no adverse skin reactions were observed at any test time point when the sample and blank control were used on 30 subjects.

[0086] Example 4: Antioxidant Activity Test of Cyclic 7-Peptide-1

[0087] 1. Samples and Preparation: The purified cyclic 7-peptide-1 and the linear peptide with the amino acid sequence shown in SEQ ID NO. 1 were prepared according to the method of Example 1. Hereinafter referred to as the linear peptide, the preparation method differs from the example in that it does not involve a cyclization step. The purification method is as follows:

[0088] The crude linear peptide was dissolved in water containing 10% acetonitrile (20 mL per gram of crude product) and filtered through a 0.45 μm filter membrane. The filtrate was purified and separated using a C18 column (10 μm, 250*30 mm, 100A). The detection wavelength was 210 nm, and the sample was loaded at a flow rate of 20 mL / min. Gradient elution was used: phase A was 0.1% acetic acid solution, and phase B was acetonitrile. The elution program is shown in the table below.

[0089] Table 4 Gradient elution procedure for linear peptide purification

[0090] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 10 89 11 30 68 32 33 40 60 35 95 5 43 95 5

[0091] Elution was performed under the gradient conditions described above. Fractions from 15 to 21 minutes were collected in segments. Fractions that passed liquid phase monitoring (the qualified standard is tentatively defined as having a purity greater than 95%) were combined and freeze-dried. Low-purity fractions (purity 60% to 95%) were recovered. Fractions with a purity lower than 60% were directly poured into the waste liquid collection tank.

[0092] The HPLC detection conditions for linear peptides are as follows:

[0093] Chromatographic column: Syncronis aQ 250*4.6mm 5um; Column temperature: 30℃; Flow rate: 1ml / min;

[0094] Detection wavelength: 220 nm; Mobile phase A: 0.1% trifluoroacetic acid aqueous solution; Mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution.

[0095] Table 5. HPLC gradient elution procedure for linear peptide detection.

[0096] Time (min) Mobile phase A (%) Mobile phase B (%) 0 92 8 25 67 33 25.1 20 80 27 20 80 27.1 92 8 32 92 8

[0097] LC-MS (m / z) of linear peptide: 825.5 [M] + ; 1 H-NMR(DMSO): 1.06(1H,d,J=5.82Hz), 1.16(1H,d,J=10.68Hz), 1.69(1H,d,J=8.88Hz), 1.77(1H,d ,J=8.34Hz),1.79(1H,d,J=9.24Hz),1.85(1H,d,J=8.94Hz),1.96(1H,d,J=7.74Hz),1.98(1H,d,J =7.20Hz), 2.16(1H,d,J=10.92Hz), 2.17(1H,d,J=9.06Hz), 2.19(1H,d,J=6.24Hz), 2.24(1H,d,J= 6.78Hz), 2.28 (1H, d, J = 5.22Hz), 2.59 (1H, d, J = 3.48Hz), 2.65 (1H, d, J = 5.88Hz), 2.67 (1H, d, J = 6. 30Hz), 2.89 (1H, d, J = 13.50Hz), 2.90 (1H, d, J = 13.26Hz), 3.03 (1H, d, J = 4.32Hz), 3.05 (1H, d, J = 4. 62Hz), 3.66 (1H, d, J = 4.92Hz), 3.69 (1H, d, J = 4.74Hz), 3.72 (1H, d, J = 5.22Hz), 3.75 (1H, d, J = 5.64 Hz), 4.14 (1H, d, J = 6.12Hz), 4.26 (1H, d, J = 6.66Hz), 4.36 (1H, d, J = 6.48Hz), 7.18 (1H, d, J = 7.02Hz ), 7.24 (1H, d, J = 7.20Hz), 7.87 (1H, d, J = 8.22Hz), 8.13 (1H, d, J = 6.42Hz), 8.17 (1H, d, J = 7.56Hz).

[0098] 2. Real-time quantitative PCR detection

[0099] 2.1 After HaCaT cells were resuscitated, they were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin until the exponential growth phase. Cells were collected, counted, and seeded into 6-well plates. The cells were incubated at 37°C for 24 h at a seeding density of 1 × 10⁻⁶ cells / well. 6 cell / well (2mL / well).

[0100] 2.2 Cell grouping: Using the optimal concentration screened in Example 2, 24-well plates were used for cell seeding and active ingredient treatment. A total of 4 groups were set up: control group, H2O2 (200 μM) model group, cyclic 7-peptide-1 + H2O2 group, and linear peptide + H2O2 group, with 3 replicates in each group;

[0101] Table 6. Treatments and concentrations of different HaCaT cells

[0102] Grouping Handling method NC group (normal control) No modeling or addition of test material Model group <![CDATA[200μM H2O2 model establishment]]> Cyclic 7-peptide-1 group <![CDATA[0.5%(g / 100mL)+H2O2(200μM) <!-- 8 -->]]> Linear peptidome <![CDATA[0.5%(g / 100mL)+H2O2(200μM)]]>

[0103] 2.3 Total RNA extraction: Total RNA was isolated from the cells to be tested using the SteadyPure Rapid RNA Extraction Kit (Aikerui, catalog number AG21023). The specific steps are as follows:

[0104] (1) Collect approximately 1×10 6 Cells were lysed thoroughly with lysis buffer RL; the lysis buffer was transferred to an RNA purification column and centrifuged at 12,000×g for 1 minute; the purification column was washed sequentially with buffers RW1 and RW2.

[0105] Total RNA was eluted with RNase-free water, and its concentration and purity were determined (an A260 / A280 ratio of 1.9-2.1 is considered acceptable).

[0106] (2) cDNA synthesis

[0107] RNA was reverse transcribed into cDNA using a reverse transcription kit (Akerui, catalog number AG11728):

[0108] Take 1 μg of total RNA, add 4 μL of 5×RT Mix and 2 μL of RT Enzyme Mix, and add RNase-free water to a final volume of 20 μL. Incubate at 42℃ for 15 min, then heat at 85℃ for 5 s to terminate the reaction. Store the resulting cDNA at -20℃ for later use.

[0109] (3) Real-time quantitative PCR amplification

[0110] Reaction system: Prepared using SYBR GREEN premixed reagent (Akerui, catalog number AG11740) according to the following proportions (Table 7):

[0111] Table 7. Reaction system for real-time quantitative PCR amplification

[0112] reaction system Added amount 2×SYBRqPCRMix 10μL Forward / reverse primers (10 μM) 0.4 μL each cDNA template 2μL <![CDATA[ddH2O]]> Make up to 20 μL

[0113] Reaction Procedure: Run the following procedure on the Bio-Rad real-time quantitative PCR system (Table 8):

[0114] Table 8. Reaction Procedure for Real-Time Quantitative PCR

[0115] 95℃ pre-denaturation 30s denaturation at 95℃ 5s Annealing / Extension at 60℃ 30 seconds (40 cycles total) Melting curve analysis 65℃~95℃ From 65℃ to 95℃, increment by 5 seconds for every 0.5℃ increment.

[0116] (4) Data analysis: The relative expression level of the target gene mRNA was calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene.

[0117] 3. Test Results: Figure 5 , Figure 6 The effect of the active ingredient on H2O2-induced HO-1 and NQO1 transcription levels was detected by real-time quantitative PCR (qRT-PCR). * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0118] Cellular experiments showed that the expression levels of HO-1 and NQO1 decreased in a 200 μM H2O2-induced skin oxidation model.

[0119] Compared to the H2O2 group, the expression levels of heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) were significantly increased in the cyclic heptapeptide-1 group, with increases of 7.61-fold (p<0.001) and 2.37-fold (p<0.001), respectively. In the linear peptide group, the expression levels of HO-1 and NQO1 were significantly increased, with increases of 3.63-fold (p<0.01) and 1.42-fold (p<0.001), respectively. The expression levels of HO-1 and NQO1 in the cyclic heptapeptide-1 group were significantly higher than those in the linear peptide group (p<0.001).

[0120] Heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) both play important antioxidant roles. This study shows that cyclic 7-peptide-1, despite having the same sequence as the linear peptide, exhibits significantly higher antioxidant regulatory activity than the linear peptide.

[0121] Example 5: Anti-inflammatory and MMP-inhibiting activity tests of cyclic 7-peptide-1

[0122] 1. Sample: The purified product of cyclic 7-peptide-1 and the linear peptide with the amino acid sequence shown in SEQ ID NO.1 were prepared according to the methods of Examples 1 and 4.

[0123] 2. Testing method: ELISA detection method

[0124] 2.1 Sample Preparation

[0125] Cell culture and treatment: HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin until the exponential growth phase. Cells were digested with trypsin and counted at 5 × 10⁻⁶ cells / year. 4Cells / well were seeded at a density of 200 μL / well in 96-well plates and incubated at 37°C and 5% CO2 for 24 h. The supernatant was discarded, the cells were washed once with PBS, and the analyte was added and the cells were incubated for another 24 h. The supernatant was collected by centrifugation (1,000 × g, 10 min) and stored at -80°C for later use.

[0126] 2.2 ELISA Detection Procedure

[0127] Reagent kits: YEASEN Human IL-6 ELISA Kit (catalog number: 97068ES48) and Wuhan Huamei Biotechnology Human Matrix metalloproteinase 1, MMP-1 ELISA kit (catalog number: CSB-E04672h) were used.

[0128] 2.2.1 Preparation of Standards and Samples: IL-6 and MMP-1 standards were serially diluted according to the instructions (range: 0-500 pg / mL). Overexpression of inflammatory factors IL-6 and MMP-1 was induced by stimulation with lipopolysaccharide (LPS) (10 ug / ml). The experimental groups and cell treatment methods are shown in the table below.

[0129] Table 9. Treatments and concentrations of different HaCaT cells

[0130] Grouping Handling method NC group (normal control) No modeling or addition of test material Model group LPS (10ug / ml) modeling Cyclic 7-peptide-1 group 0.1% (g / 100mL) + LPS (10μg / ml) Linear peptidome 0.1% (g / 100mL) + LPS (10μg / ml)

[0131] 2.2.2 Addition and incubation: Add 100 μL / well of standard or sample to the pre-coated ELISA plate and incubate at 37°C for 2 h.

[0132] 2.2.3 Enzyme conjugate incubation: Discard the liquid, add the working concentration of enzyme conjugate (100 μL / well), and incubate at room temperature for 20 min.

[0133] 2.2.4 Wash the plate three times with 1× washing solution (300 μL / well), then pat dry.

[0134] 2.2.5 Enzyme conjugate reaction: Add 100 μL / well of HRP-labeled streptavidin and incubate at 37°C in the dark for 30 min.

[0135] 2.2.6 Color development: After washing 5 times, add 100 μL / well of TMB substrate and develop at room temperature in the dark for 15 min.

[0136] 2.2.7 Termination and Detection: Add 50 μL of stop solution per well and immediately read the absorbance values ​​at 450 nm (dominant wavelength) / 630 nm (calibration wavelength) using a microplate reader.

[0137] 2.3. Data Analysis

[0138] Statistical analysis: One-way ANOVA was performed using GraphPad Prism software. Significance criteria: * for P < 0.05; ** for P < 0.01; *** for P < 0.001.

[0139] 3. Test Results: See results below. Figure 7 , Figure 8 .

[0140] Compared with the NC group (normal control group), the model group showed a significant increase in the expression of inflammatory cytokines IL-6 and MMP-1 by adding LPS (10ug / ml) (p<0.001).

[0141] (1) Compared with the model group, 0.1% cyclic heptapeptide-1 reduced IL-6 expression by 51.3%, and 0.1% linear peptide reduced IL-6 expression by 39.2%. The inhibitory effect of cyclic heptapeptide-1 on IL-6 was significantly higher than that of linear peptide at the same concentration (p<0.01).

[0142] IL-6 belongs to the interleukin family of inflammatory factors and is mainly secreted by immune cells (such as T cells and macrophages), keratinocytes, fibroblasts, and endothelial cells. Various factors can induce elevated IL-6 levels, such as enhanced inflammatory responses, photoaging and oxidative stress, and environmental damage.

[0143] This experiment induced an inflammation model in skin cells using LPS, and found that LPS significantly increased IL-6 expression in HaCaT cells. The addition of cyclic heptapeptide-1 reduced IL-6 expression, indicating that cyclic heptapeptide-1 has an anti-inflammatory effect.

[0144] (2) Compared with the model group, 0.1% cyclic 7-peptide-1 reduced the expression of MMP-1 by 43.2%, and 0.1% linear peptide reduced the expression of MMP-1 by 30.4%. The inhibitory effect of cyclic 7-peptide-1 on MMP-1 was significantly higher than that of linear peptide at the same concentration (p<0.01).

[0145] One of the main causes of skin aging is the alteration of the dermal structure. Many factors contribute to this structural change, among which excessive expression of matrix metalloproteinases (MMPs) leads to the over-degradation of collagen and elastin, which support the skin's structure, resulting in wrinkles, decreased elasticity, and other signs of aging. MMP-1 is also a major factor in the age-associated secretory phenotype (SASP).

[0146] This experiment used LPS to create an inflammation model in skin cells and found that LPS significantly increased the secretion of MMP-1 by the cells. The addition of cyclic heptapeptide-1 reduced MMP-1 expression, indicating that cyclic heptapeptide-1 has an inhibitory effect on skin aging.

Claims

1. A cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties, characterized in that, The structural formula of the cyclic peptide is shown in Formula I:

2. The method for preparing a cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties as described in claim 1, characterized in that, The preparation method includes the following steps: Preparation of S1 linear peptide: The linear peptide segment required for the synthesis of the cyclic peptide shown in Formula I was prepared by solid-phase synthesis, wherein the linear peptide segment contains at least one amino acid with a side-chain protecting group. Cycling of S2 linear peptides: Using condensing agents and organic bases, linear peptides are cyclized by decarboxylation of the amino and carboxyl groups at the beginning and end of the linear peptide segment.

3. The method for preparing a cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties according to claim 2, characterized in that, In step S1, the solid resin used in the solid-phase synthesis method is 2-CTC Resin resin.

4. The method for preparing a cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties according to claim 2, characterized in that, In step S1, the protected amino acids used in the solid-phase synthesis method are: Fmoc-Phe-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Glu(Otbu)-OH, and Fmoc-Asp(Otbu)-OH.

5. The method for preparing a cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties according to claim 2, characterized in that, In step S2, the condensing agent and the organic base are benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate and N,N-diisopropylethylamine, respectively.

6. The method for preparing a cyclic peptide with antioxidant, anti-inflammatory, and matrix metalloproteinase-inhibiting properties according to claim 2, characterized in that, The method includes the following steps: Preparation of S1 linear peptide: 2-CTC resin was added to a solid-phase column, and the 2-CTC resin was swollen, washed, and dried. Fmoc-Phe-OH, Fmoc-Glu(Otbu), Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Glu(Otbu)-OH, and Fmoc-Asp(Otbu)-OH were added sequentially by solid-phase synthesis to gradually condense and remove Fmoc. The resulting linear peptide was cleaved from the resin to prepare a linear peptide with side-chain protecting groups. Cycling of S2 linear peptide: A linear peptide with a side-chain protecting group was condensed into a cyclization using the condensing agent benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate and the organic base N,N-diisopropylethylamine. S3 Removal of side-chain protecting groups: The side-chain protecting groups are cleaved with trifluoroacetic acid solution to obtain the cyclic peptide shown in Formula I.

7. Use of the cyclic peptide of claim 1 or the cyclic peptide prepared by any one of claims 2 to 6 in the preparation of topical cosmetics for antioxidant purposes.

8. Use of the cyclic peptide of claim 1 or the cyclic peptide prepared by any one of claims 2 to 6 in the preparation of anti-inflammatory topical cosmetics.

9. Use of the cyclic peptide of claim 1 or the cyclic peptide prepared by any one of claims 2 to 6 in the preparation of a topical cosmetic for inhibiting matrix metalloproteinases.

10. A topical cosmetic product, characterized in that, It contains at least the cyclic peptide of claim 1 or the cyclic peptide prepared by the preparation method of any one of claims 2 to 6.

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