Cyclic nonapeptide with repairing and anti-oxidation effects and application of cyclic nonapeptide

Abstract

The invention discloses a cyclic nonapeptide with repairing and anti-oxidation effects and application of the cyclic nonapeptide, the amino acid sequence of the cyclic nonapeptide is cyclic (arginine-glycine-aspartic acid-serine-arginine-lysine-valine-lysine-serine), the prepared cyclic nonapeptide has the repairing and anti-oxidation effects, and the cyclic nonapeptide has the repairing and anti-oxidation effects. The composition can be applied to preparation of cosmetics with repairing and / or anti-oxidation effects.

Description

Technical Field

[0001] This invention relates to a cyclic peptide, particularly a cyclic nonapeptide with repairing and antioxidant effects and its applications. Background Technology

[0002] As living standards improve, people are paying more attention to their appearance, and the condition of one's skin is crucial to their overall look. Scientific research shows that environmental factors and life stress can lead to deteriorating skin condition, resulting in problems such as skin oxidation and barrier damage. To meet market demand, there is a growing number of skincare products with antioxidant and repairing effects, and consequently, the requirements for antioxidant and repairing ingredients are increasing year by year.

[0003] There are many types of antioxidants and repair products on the market today. Peptide ingredients have become a research hotspot in the skin care field in recent years due to their high activity and high targeting properties. Cyclic peptides, due to their unique structure, provide a new direction for upgrading raw materials with antioxidant and repair effects.

[0004] RGDS peptides can alleviate lipopolysaccharide-induced inflammation by inhibiting the MAPK pathway of integrin signaling. Inflammatory responses are typically accompanied by the production of reactive oxygen species (ROS) and increased oxidative stress; by regulating inflammation, RGDS peptides can reduce ROS production, thereby exerting an antioxidant effect. KVK peptides, by mimicking the function of TSP-1, trigger the release of active TGF-β. TGF-β can induce keratinocyte proliferation and migration, promoting cell repair.

[0005] However, there are currently no literature reports on the cyclic nonapeptide structure formed by the amino acid linkage between RGDS and KVK, which gives the cyclic nonapeptide both repair and antioxidant effects. Summary of the Invention

[0006] The purpose of this invention is to provide a cyclic nonapeptide with repairing and antioxidant effects, and its applications. The cyclic nonapeptide of this invention has repairing and antioxidant effects.

[0007] The technical solution of the present invention is: a cyclic nonapeptide with repair and antioxidant effects, wherein the amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-arginine-lysine-valine-lysine-serine).

[0008] The aforementioned cyclic nonapeptide with repairing and antioxidant effects has the following structure: .

[0009] The present invention also provides the application of the above-mentioned cyclic nonapeptide in the preparation of cosmetics with repair and / or antioxidant effects.

[0010] In the aforementioned applications, the effective concentration of the cyclic nonapeptide is 70 μg / mL.

[0011] In the aforementioned applications, the effective concentration of the cyclic nonapeptide is 140 μg / mL.

[0012] A cosmetic composition with repairing effects, comprising the aforementioned cyclic nonapeptide.

[0013] A cosmetic composition with antioxidant properties, comprising the aforementioned cyclononapeptide.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The structure of the cyclic nonapeptide of this invention facilitates binding to integrins αVβ3 and α5β1, thereby alleviating lipopolysaccharide-induced inflammation by inhibiting the MAPK pathway of integrin signaling. By regulating inflammation, it can reduce ROS production, thus exerting an antioxidant effect. Simultaneously, the KVK peptide within it mimics the function of TSP-1, triggering the release of active TGF-β. TGF-β can induce keratinocyte proliferation and migration, promoting cell repair.

[0015] Antioxidant and repair experiments verified that the cyclic nonapeptide of the present invention can significantly inhibit ROS production in HaCaT cells and promote HaCaT cell healing at concentrations of 70 μg / mL and 140 μg / mL, thereby achieving the purpose of antioxidation and repair.

[0016] Therefore, the cyclic nonapeptide designed in this invention has the characteristics of good solubility, structural stability, and small molecular weight, and also has antioxidant and repair effects, making it safe and effective to use. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the docking of the cyclic nonapeptide with integrin aVβ3 in this invention.

[0018] Figure 2 This is a schematic diagram of the docking of the cyclic nonapeptide with integrin a5β1 in this invention.

[0019] Figure 3 The bar chart shows the average ROS fluorescence intensity values ​​for each experimental group. Analysis of variance was used in the chart. Significance compared to the BC group is indicated by # (# indicates P-value < 0.05, ## indicates P-value < 0.01, ### indicates P-value < 0.001); significance compared to the NC group is indicated by * (* indicates P-value < 0.05, ** indicates P-value < 0.01, *** indicates P-value < 0.001).

[0020] Figure 4The figure shows the HaCaT cell healing rate of each experimental group. In the figure, analysis of variance was used. Compared with the BC group, the significance is indicated by *, where * indicates P-value < 0.05, ** indicates P-value < 0.01, and *** indicates P-value < 0.001. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0022] Example: Constructing cyclic nonapeptides of RGDS and KVK requires screening for suitable amino acids to link the two peptides. The cyclic nonapeptide was designed as RGDS-X1-KVK-X2, where X1 and X2 are one of 20 L-amino acids and 20 D-amino acids, respectively, resulting in a total of 40*40=1600 candidate cyclic nonapeptide sequences.

[0023] Subsequently, structural simulation software was used to simulate the three-dimensional structures of the aforementioned 1600 cyclic peptides, and molecular simulation software was used to calculate the energy difference between the 1600 cyclic peptides and their linear peptide sequences. A structural simulation score of Total_score < 0 indicates that the steric hindrance between the amino acid residues of the candidate cyclic peptide structure is small, suitable for forming a cyclic peptide structure, and suitable for experimental verification as a candidate cyclic peptide. Thus, the structurally stable candidate cyclic nonapeptide RGDSRKVKS was selected.

[0024] Table 1. Difference between the energy of cyclic peptide sequences and the corresponding linear peptide sequences. Molecular docking simulations were performed between the designed cyclic nonapeptide and integrin αVβ3. Based on the crystal structure in the Protein Data Bank (αVβ3_PDB ID: 4MMX), the RGDS sequence in the cyclic nonapeptide was docked to the RGDS sequence in the crystal structure of the αVβ3 complex. The specific docking structure simulation diagram is shown below. Figure 1 As shown. Figure 1 The thin, linear structure is the RGDS sequence that binds to integrin αVβ3 in the crystal structure, while the stick-like structure is a cyclic nonapeptide structure. The spatial differences in the amino acid residues between the two are as follows: Figure 1 As shown.

[0025] The docking results showed that the RGDS of the cyclic nonapeptide had an RMSD of 0.407 Å with the RGDS sequence in the 4MMX crystal structure. The positions of each amino acid and the direction of side chain extension were very similar, which is conducive to the binding of the cyclic nonapeptide to integrin αVβ3.

[0026] Molecular docking simulations were performed between the designed cyclic nonapeptide and integrin α5β1. Based on the crystal structure in the Protein Data Bank (α5β1_PDB ID: 7NWL), the RGDS sequence in the cyclic nonapeptide was docked to the RGDS sequence in the crystal structure of the α5β1 complex. The specific docking structure simulation diagram is shown below. Figure 2 As shown. Figure 2 The thin, linear structure is the RGDS sequence that binds to integrin α5β1 in the crystal structure, while the stick-like structure is a cyclic nonapeptide structure. The spatial differences in the amino acid residues between the two are as follows: Figure 2 As shown.

[0027] The docking results showed that the RGDS of the cyclic nonapeptide had an RMSD of 0.589 Å with the RGDS sequence in the crystal structure 7NWL. The positions of each amino acid and the direction of side chain extension were very similar, which is conducive to the binding of the cyclic nonapeptide to integrin α5β1.

[0028] The molecular docking simulation results indicate that the structure of the cyclic nonapeptide is conducive to binding to integrin αVβ3 and α5β1, thereby alleviating lipopolysaccharide-induced inflammation by inhibiting the MAPK pathway of integrin signaling. By regulating inflammation, the cyclic nonapeptide can reduce ROS production, thus exerting an antioxidant effect. Simultaneously, the cyclic nonapeptide, through its KVK peptide mimicking the function of TSP-1, triggers the release of active TGF-β, which can induce keratinocyte proliferation and migration, promoting cell repair.

[0029] The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-arginine-lysine-valine-lysine-serine), namely Cyclo(Arg-Gly-Asp-Ser-Arg-Lys-Val-Lys-Ser) or cyclic (RGDS-R-KVK-S).

[0030] The structure of the cyclic nonapeptide is as follows: .

[0031] The preparation method of cyclic nonapeptide is as follows: S1. Using natural amino acids arginine-glycine-aspartic acid-serine-arginine-lysine-valine-lysine-serine as starting materials, a fluorene methoxycarbonyl (Fmoc) N-terminal protection strategy is adopted, and the corresponding amino acids are sequentially linked according to the resin solid-phase synthesis method, during which the Fmoc- protecting group is sequentially removed to obtain a resin containing a linear nonapeptide chain.

[0032] The specific steps are as follows: 1) Swelling resin: 0.6 g of 2-Cl Trt-Lys resin (degree of substitution SD = 0.39 mmol / g) was added to the reactor, and DCM (10 mL / g) was added as a swelling agent to allow swelling for 5 min.

[0033] 2) Resin deprotection: Vacuum-dry the swollen reagent DCM, add 20% piperidine (Pip) / DMF (10 mL / g) as a deprotection agent, stir for 5 min, dry under vacuum, and then add 20% piperidine (Pip) / DMF (10 mL / g) and stir for 5 min.

[0034] 3) Remove protective washing: The protective reagent Pip / DMF was removed by vacuum drying, and the resin was washed 5 times with DMF (10 mL / g), stirring for 20-30 s each time, and then dried for 20 s to obtain resin-1.

[0035] 4) Deprotection detection: Take about 20 resin-1 particles and put them into a test tube. Add 1 mL of ninhydrin detection reagent to the test tube, and then put the test tube into a metal bath at 120°C or above for 2 minutes. Take it out and observe the color of the resin. If the resin color becomes darker, it is a positive result, indicating that the deprotection was successful.

[0036] 5) Condensation of the second amino acid: Add 3 equivalents of Fmoc-Val-OH and 3 equivalents of Oxyma to resin 1, add 10 mL of DMF to dissolve, then add 3 equivalents of DIC, activate for 5 min, pour into the reactor, and stir to react for 1 h.

[0037] 6) Reaction washing: The reaction reagent DMF was dried under vacuum, and the mixture was washed 5 times with DMF (10 mL / g), stirring for 20-30 seconds each time and then dried under vacuum for 20 seconds to obtain resin-2.

[0038] 7) Reaction detection: Take about 20 resin-2 particles and put them into a test tube. Add 1 mL of ninhydrin test reagent to the test tube, and then put the test tube into a metal bath at 120°C or above for 2 minutes. Take it out and observe the color of the resin. If there is no obvious change in the color of the resin, it indicates that the reaction condensation is successful.

[0039] 8) Repeat steps 2)-7), condensing the subsequent amino acids in the sequence from right to left according to the polypeptide sequence, namely Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH until Fmoc-Arg(Pbf)-OH, to obtain resin-3.

[0040] 9) Deprotection: Add 20% Pip / DMF (10 mL / g) as a deprotection agent to resin-3, stir for 5 min and then dry it. Add 20% Pip / DMF (10 mL / g) and stir for 5 min.

[0041] 10) Remove protective wash: The protective reagent Pip / DMF was removed by vacuum drying, and then washed 5 times with DMF (10 mL / g), stirring for 20-30 s each time and drying for 20 s to obtain resin-4.

[0042] 11) Deprotection detection: Place approximately 20 resin-4 particles into a test tube, add 1 mL of ninhydrin detection reagent, place the test tube in a metal bath at 120°C or above for 2 minutes, remove and observe the resin color. A darker resin color indicates a positive result, signifying successful deprotection.

[0043] 12) Washing: The resin was washed 45 times with methanol (10 mL) and vacuum dried for 10 min to complete solid-phase condensation, yielding a resin containing linear nonapeptide chains.

[0044] S2. Cut the resin containing the linear nonapeptide chain, cut the linear nonapeptide chain off the resin, remove the remaining protecting groups of the peptide chain, collect the cutting fluid containing the crude linear nonapeptide chain, and obtain a fully protected polypeptide solid to be cyclic.

[0045] The specific steps are as follows: 13) Resin-protected pyrolysis: The resin containing the linear peptide was loaded into a boat-shaped reactor, and 10 mL / g lysis buffer (TFE:DCM volume ratio of 30:70) was added. The mixture was lysed three times at room temperature for 1 hour each time. The reaction solution was filtered out, and the solvent was concentrated and evaporated using a rotary evaporator. After evaporation, the sample was dissolved in 30% acetonitrile / water solution and lyophilized to obtain a fully protected peptide solid.

[0046] S3. The fully protected polypeptide solid to be cyclized is mixed with polypeptide coupling agent, activator, etc., and then cyclized to obtain a cyclic peptide containing a protecting group.

[0047] The specific steps are as follows: 14) Modification cyclization reaction: Weigh out the fully protected polypeptide solid to be cyclic, dissolve it in AR grade DMF to obtain a polypeptide solution with a polypeptide concentration of 1 mM; after dissolution, weigh out 2 eq PyBOP and transfer 4 eq DIEA using an electronic balance, add them to the polypeptide solution, and stir at room temperature for 10 h to obtain a cyclic peptide containing a protecting group.

[0048] S4. The cyclic peptide containing the protecting group was purified by preparative HPLC to obtain cyclic nonapeptide.

[0049] The specific steps are as follows: 15) Rotary freeze-drying: After the reaction was complete, the reaction solution was concentrated using a rotary evaporator. After concentration, 30% acetonitrile / water solution was added to dissolve it, and then the solution was freeze-dried on the wall. Finally, the solution was purified by preparative HPLC to obtain the cyclic (arginine-glycine-aspartic acid-serine-arginine-lysine-valine-lysine-serine).

[0050] Experimental example: Biosafety cabinet (Nuaire, NU-543-600s), CO2 incubator (Panasonic, MCO-18AIC), centrifuge (Eppendorf, Centrifuge 5804R), mini cryogenic centrifuge (Sigma, 1-14K), analytical balance (Sartorius, BCE224-1CCN), microscope (Zeiss, Axio Observer3), microplate reader (TECAN, SPARK), ultraviolet irradiator (Genefos, UV-621111801), flow cytometer (Beckman, Cytoflex S).

[0051] Experimental reagents: Phosphate-buffered saline (PBS, Biosharp), high-glucose DMEM medium (Gibco, 11965092), 0.25% trypsin solution (Gibco, 25200056), penicillin-streptomycin (Gibco, 15070063), fetal bovine serum (Beyotime, C0234), and ROS assay kit (Beyotime, S0033M).

[0052] 1. Antioxidant experiment: 1.1 Experimental grouping: A blank control group, a negative control group, and a sample group were set up, with at least 3 replicates in each group.

[0053] 1.2 Experimental Methods: Cells were cultured at 4.0 × 10⁻⁶. 5Inoculate 12-well plates evenly with 1 inoculum sample per well and incubate overnight (37°C, 5% CO2). After overnight incubation, add 35, 70, and 140 μg / mL of cyclic nonapeptide to the sample groups, respectively. Replace the blank and negative control groups with complete culture medium. After culturing for another 24 hours, discard the supernatant and add 500 μL of PBS, using 1000 mJ / cm² water. 2 UVB irradiation stimulation was performed. After irradiation, the probe was incubated according to the specific operating procedures of the reactive oxygen species detection kit (Beyotime). After loading the probe, trypsin digestion was performed, digestion was terminated with complete culture medium, centrifuged at 1200 r / min, the supernatant was discarded, and HBS was added for washing. This process was repeated twice, and flow cytometry was performed to obtain the fluorescence intensity values ​​(DCF (ROS) MFI) of each group. The ROS scavenging rate was calculated using the following formula: ROS clearance rate (%) = (negative group DM - sample group DM) / (negative group DM - blank control DM) × 100%.

[0054] The specific design scheme is shown in Table 2.

[0055] Table 2. Antioxidant efficacy design scheme 1.3 Experimental Results: The antioxidant experimental results of cyclic nonapeptide are shown in Table 3 and Figure 3 As shown.

[0056] Table 3. Effect of cyclic nonapeptide on ROS content generated by UVB-induced HaCaT The results of the antioxidant experiment showed that the average fluorescence intensity of the negative control group was significantly increased compared with the blank control group, indicating successful model establishment. Compared with the negative control group, 70 μg / mL and 140 μg / mL of cyclic nonapeptide significantly inhibited ROS production in HaCaT cells, with clearance rates of 25.85% and 37.85%, respectively, achieving an antioxidant effect. Therefore, the cyclic nonapeptide of this invention is a polypeptide with antioxidant activity.

[0057] 2. Repair Experiment: 2.1 Experimental grouping: A blank control group (DMEM prepared with 1% FBS), a positive control group (DMEM prepared with 10% FBS), and a sample group (DMEM prepared with 1% FBS as reagent, containing 70 and 140 μg / mL cyclic nonapeptide) were set up, with at least 3 fields of view selected in each group.

[0058] 2.2 Experimental Methods: HaCaT cells were cultured at 5.0 × 10⁻⁶ cells / mL. 5Inoculate cells evenly into 12-well plates at a rate of [number] cells / well and incubate at 37°C, 5% CO2 until confluence. Using a sterile 200μL pipette tip, draw two regular cross-shaped lines on each well of the cells, perpendicular to the long axis of the well. Wash the cells 2-3 times with sterile PBS after the incision. Add the corresponding culture reagents according to the experimental groups, and continue culturing for 24 hours. Take photos and use image processing software to calculate the healing area. The healing rate is calculated using the following formula: Healing rate (%) = (Initial scratch area - Area of ​​blank area after 24 hours) / Initial scratch area * 100%.

[0059] The specific design scheme is shown in Table 4.

[0060] Table 4. Repair Efficacy Design Scheme Experimental results: The antioxidant experimental results of cyclic nonapeptide are shown in Table 5 and Figure 4 As shown.

[0061] Table 5. Effect of cyclic nonapeptide on the healing rate of HaCaT cells The results of the repair experiment showed that, compared with the blank control group, the healing rate of the positive control group was significantly increased, reaching 80.24%. Compared with the blank control group, 70 μg / mL and 140 μg / mL cyclic nonapeptide significantly promoted the healing rate of HaCaT cells, with healing rates of 67.12% and 65.81%, respectively, indicating that cyclic nonapeptide has a repairing effect. Cyclic nonapeptide has the ability to promote the healing of HaCaT cells at concentrations of 70 μg / mL and 140 μg / mL, thereby achieving the purpose of repair. Therefore, the cyclic nonapeptide of the present invention is a polypeptide with a repairing effect.

[0062] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Those skilled in the art can modify the technical solutions described in the above embodiments, or make equivalent substitutions for some of the technical features; and all such modifications and substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A cyclic nonapeptide with repairing and antioxidant effects, characterized in that: The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-arginine-lysine-valine-lysine-serine).

2. The cyclic nonapeptide with repairing and antioxidant effects according to claim 1, characterized in that: The structure of the cyclic nonapeptide is: 。 3. Use of the cyclic nonapeptide according to any one of claims 1-2 in the preparation of a cosmetic product having a repairing and / or antioxidant efficacy.

4. Use according to claim 3, characterized in that: The effective concentration of the cyclic nonapeptide is 70 μg / mL.

5. Use according to claim 3, characterized in that: The effective concentration of the cyclic nonapeptide is 140 μg / mL.

6. A cosmetic composition having a repairing effect, characterized by: A cosmetic product comprising the cyclic nonapeptide according to any one of claims 1-2.

7. A cosmetic composition having an antioxidant effect, characterized by: A cosmetic product comprising the cyclic nonapeptide according to any one of claims 1-2.

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