Cyclo-nonapeptide with both anti-photoaging and antioxidant effects and application thereof

Abstract

This invention discloses a cyclic nonapeptide with both anti-photoaging and antioxidant effects. The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine). The cyclic nonapeptide of this invention can effectively reduce deep photoaging caused by UVA and surface oxidative damage induced by UVB, thereby achieving comprehensive skin protection.

Description

Technical Field

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

[0002] Cyclic peptides exhibit significant advantages over traditional linear peptides in terms of bioactivity, stability, and penetration.

[0003] Due to their closed cyclic structure, cyclic peptides exhibit higher resistance to enzymatic degradation compared to linear peptides and can maintain longer bioactivity in the complex environment of the skin barrier. The high rigidity provided by their cyclic backbone not only enhances their binding affinity to targets but also strengthens the functional effects at specific sites. The lipophilicity and small molecule properties of cyclic peptides contribute to improved transdermal absorption, allowing them to more easily reach the deep dermal tissue and exert stronger antioxidant and anti-aging effects. Therefore, with the increasing consumer demand for natural and functional ingredients, cyclic peptides are widely used in skincare products, anti-aging moisturizers, and repair serums, demonstrating particularly outstanding performance in repairing collagen, inhibiting matrix metalloproteinases (MMPs), and neutralizing free radicals.

[0004] UVA (320-400 nm) radiation can penetrate the skin to reach the dermis, inducing various types of cell damage and is one of the main causes of photoaging. The specific mechanisms are as follows: UVA radiation induces the large-scale generation of reactive oxygen species (ROS) through photosensitive molecules. ROS damages biomolecules in the skin, leading to DNA damage, lipid peroxidation, and protein carbonylation. Collagen and elastin fibers are the main targets of ROS attack, and damage to them leads to skin laxity and wrinkle formation. UVA radiation activates the MAPK / ERK signaling pathway and the AP-1 transcription factor. AP-1 regulates the expression of MMP genes; these enzymes directly degrade the collagen matrix in the skin, accelerating skin aging. The inflammatory response induced by UVA can activate nuclear factor NF-κB, further promoting the secretion of inflammatory factors (such as IL-6 and TNF-α) in the skin, exacerbating the photoaging effect.

[0005] UVB (280-320 nm) primarily targets epidermal cells in the skin's surface layer. Its high-energy direct effects significantly disrupt skin cells, structural proteins, and genomic homeostasis. UVB irradiation induces redox imbalance in keratinocytes, significantly increasing ROS levels, which in turn triggers a three-tiered chain of damage to intracellular lipids, proteins, and DNA. ROS and DNA damage activate skin macrophages and dendritic cells, leading to the massive release of inflammatory mediators such as IL-1β and COX-2, promoting acute skin redness and chronic aging. ROS attacks unsaturated fatty acids in the cell membrane, generating hydrogen peroxide and aldehydes, resulting in loss of transmembrane transport and activation of apoptosis signals.

[0006] Currently, there are relatively few reports on cyclic nonapeptides that have both anti-photoaging and antioxidant effects. Summary of the Invention

[0007] The purpose of this invention is to provide a cyclic nonapeptide with both anti-photoaging and antioxidant effects, and its applications. The cyclic nonapeptide of this invention can effectively reduce deep photoaging caused by UVA and surface oxidative damage induced by UVB, thereby achieving comprehensive skin protection.

[0008] 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-asparagine-lysine-valine-lysine-phenylalanine).

[0009] The aforementioned cyclic nonapeptide with repairing and antioxidant effects has the following structural formula: .

[0010] The present invention also provides the application of the above-mentioned cyclic nonapeptide in the preparation of cosmetics, food or pharmaceuticals with anti-photoaging and / or antioxidant effects.

[0011] In the aforementioned applications, anti-photoaging refers to resistance to deep photoaging caused by UVA.

[0012] In the aforementioned applications, antioxidant refers to resistance to surface oxidative damage caused by UVB.

[0013] In the aforementioned applications, the effective concentration of the cyclic nonapeptide is not less than 40 ppm.

[0014] In the aforementioned applications, the effective concentration of the cyclic nonapeptide is 40~120ppm.

[0015] A cosmetic composition with anti-photoaging effects, comprising the aforementioned cyclononapeptide.

[0016] A cosmetic composition with antioxidant properties, comprising the aforementioned cyclononapeptide.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The cyclic nonapeptide of this invention can effectively reduce the oxidative burden caused by both UVA and UVB by inhibiting ROS generation and capturing free radicals, thus protecting the stratum corneum of the skin, reducing the damage of sunburn, and providing comprehensive antioxidant protection for the skin.

[0019] The cyclic nonapeptide of this invention can increase the synthesis of collagen type I and III, while inhibiting the expression level of MMPs associated with the breakdown of collagen type I and III, thereby reversing UV-induced skin laxity and wrinkles, reducing pigmentation, and improving skin elasticity.

[0020] The cyclic nonapeptide of this invention targets the damage mechanisms of both UVA and UVB wavelengths, and can regulate antioxidant activity, growth factor expression, and cell proliferation at multiple targets, providing differentiated product functional claims and meeting the growing market demand.

[0021] In summary, the cyclic nonapeptide of this invention is non-toxic, non-irritating, and has good stability. It has the effects of scavenging ROS (reactive oxygen species), inhibiting MMP expression, and increasing COL1 and COL3 expression. It can effectively reduce deep photoaging caused by UVA and surface oxidative damage induced by UVB, thereby achieving comprehensive protection of the skin and slowing down skin aging. It can be applied to related products in the cosmetic field. Attached Figure Description

[0022] Figure 1 This is a simulation diagram of the docking of the cyclic nonapeptide with integrin aVβ3 in this invention.

[0023] Figure 2 This is a simulation diagram of the docking of the cyclic nonapeptide and integrin a5β1 molecules in this invention.

[0024] Figure 3 The graph shows the DCF (ROS) MFI values ​​of fluorescence intensity for each experimental group. One-way ANOVA was used in the graph. * indicates that compared with the NC group, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001.

[0025] Figure 4 The bar chart shows the effect of each experimental group on the intracellular COL3 content. In the figure, one-way ANOVA was used. * indicates that compared with the NC group, and "****" indicates that P<0.0001.

[0026] Figure 5 The bar chart shows the effect of each experimental group on the intracellular MMP3 content. In the figure, one-way ANOVA was used. * indicates that compared with the NC group, "***" indicates that P<0.001 and "****" indicates that P<0.0001.

[0027] Figure 6 The bar chart shows the effect of each experimental group on the intracellular COL1 content. In the figure, one-way ANOVA was used. * indicates that compared with the NC group, "**" indicates that P<0.01, and "****" indicates that P<0.0001.

[0028] Figure 7 The bar chart shows the effect of each experimental group on the intracellular MMP1 content; one-way ANOVA was used in the figure, and "****" indicates P<0.0001. Detailed Implementation

[0029] 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.

[0030] Example:

[0031] The cyclic nonapeptide was designed as RGDS-X1-KVK-X2, where X1 and X2 are one of 20 L-amino acids and one of 20 D-amino acids, respectively, for a total of 40*40=1600 cyclic nonapeptide candidate sequences.

[0032] Subsequently, structural simulation software was used to simulate the three-dimensional structures of the aforementioned 1600 cyclic peptides, and molecular docking software was used to calculate the energy difference between the 1600 cyclic peptides and 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 RGDSNKVKF was selected.

[0033] Table 1. Difference between the energies of cyclic nonapeptide sequences and their corresponding linear peptide sequences.

[0034] First, molecular docking simulations were performed between the RGDSNKVKF cyclic nonapeptide structure and integrin αVβ3. Based on the crystal structure (αVβ3_PDB ID: 4MMX) in the Protein Data Bank, the RGDS sequence from the cyclic peptide was docked to the RGDS sequence in the αVβ3 complex crystal structure. The specific docking structure simulation diagram is shown below. Figure 1 As shown. Figure 1 In the diagram, the red linear structures represent the positions of the RGDS peptide segments in the known 4MMX crystal structure, while the green and blue rod-shaped structures represent cyclic nonapeptides.

[0035] The docking fraction between the cyclic nonapeptide RGDSNKVKF and integrin αVβ3 was -10.2881, which is less than -7, indicating a strong binding interaction. The docking results showed that the RMSD of the RGDS sequence of the cyclic nonapeptide and the RGDS sequence in the 4MMX crystal structure was 0.505 Å, and the positions of each amino acid and the direction of side chain extension were very close, which is conducive to the binding of the cyclic nonapeptide to integrin αVβ3.

[0036] Subsequently, molecular docking simulations were performed between the RGDSNKVKF cyclic peptide structure and integrin α5β1. Based on the crystal structure (α5β1_PDB ID: 4WK2) in the Protein Data Bank, the RGDS sequence in the cyclic peptide was docked to the RGDS sequence in the α5β1 complex crystal structure. The specific docking structure simulation diagram is shown below. Figure 2 As shown. Figure 2 In the diagram, the red linear structures represent the position of the RGDS peptide in the known crystal structure 4WK2, while the green and blue rod-shaped structures represent cyclic nonapeptides.

[0037] The docking fraction between the cyclic nonapeptide RGDSNKVKF and integrin α5β1 was -10.1263, which is less than -7, indicating a strong binding interaction. The docking results showed that the RMSD of the RGDS sequence of the cyclic nonapeptide and the RGDS sequence in the crystal structure 4WK2 was 1.072 Å, and the positions of the amino acids and the direction of the side chain extension were very close, which is conducive to the binding of the cyclic nonapeptide to integrin α5β1.

[0038] 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 modulating inflammation, the cyclic nonapeptide can reduce ROS production, thus exerting an antioxidant effect.

[0039] The amino acid sequence of the cyclic nonapeptide of the present invention is: cyclic (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine), i.e. Cyclo(Arg-Gly-Asp-Ser-Asn-Lys-Val-Lys-Phe), and the SMILES formula is O=C(NCC(N[C@@H](CC(O)=O)C(N[C@@H](CO)C(N[C@@H](CC(N)=O)C(N[C@@H](CCCCN)C(N[C@@H](C(C)C)C(N[C@@H](CCCCN)C(N[C@@H](CC1=CC=CC=C1)C2=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)[C@H](CCCNC(O2)=N)N.

[0040] The structural formula is: .

[0041] The preparation method of cyclic nonapeptide is as follows:

[0042] S1. Using natural amino acids arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine 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.

[0043] The specific steps are as follows:

[0044] 1) Swelling resin:

[0045] 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 induce swelling for 5 min.

[0046] 2) Resin deprotection:

[0047] Vacuum-dry the swollen reagent DCM, add 20% piperidine (Pip) / DMF (10 mL / g) as a deprotection agent, stir for 5 min and then vacuum dry, add 20% piperidine (Pip) / DMF (10 mL / g) again and stir for 5 min.

[0048] 3) Remove protective washing:

[0049] 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 seconds each time and then dried for 20 seconds to obtain resin-1.

[0050] 4) Deprotection detection:

[0051] 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.

[0052] 5) Condensation of the second amino acid:

[0053] Add 3 equivalents of Fmoc-Val-OH and 3 equivalents of Oxyma to resin-1, dissolve in 10 mL of DMF, add 3 equivalents of DIC, activate for 5 min, pour into a reactor, and stir to react for 1 h.

[0054] 6) Reaction washing:

[0055] 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.

[0056] 7) Reaction detection:

[0057] 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.

[0058] 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-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, until Fmoc-Phe-OH, to obtain resin-3.

[0059] 9) Deprotection:

[0060] 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) again and stir for 5 min.

[0061] 10) Remove protective wash:

[0062] 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.

[0063] 11) Deprotection detection:

[0064] 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 higher for 2 minutes, remove and observe the resin color. A darker resin color indicates a positive result, signifying successful deprotection.

[0065] 12) Washing:

[0066] The resin was washed 4-5 times with methanol (10 mL), and then vacuum dried for 10 min to complete solid-phase condensation, yielding a resin containing linear nonapeptide chains.

[0067] 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.

[0068] The specific steps are as follows:

[0069] 13) Resin-protected pyrolysis:

[0070] The resin containing the linear nonapeptide chain 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.

[0071] S3. The fully protected polypeptide solid to be cyclized is mixed with polypeptide coupling agent, activator, etc., and then cyclized to obtain a cyclic nonapeptide containing a protecting group.

[0072] The specific steps are as follows:

[0073] 14) Modification cyclization reaction:

[0074] Weigh out the fully protected polypeptide solid to be cyclic, dissolve it in AR grade DMF to obtain a polypeptide solution with a concentration of 1 mM; weigh out 2 eq PyBOP using an electronic balance, and transfer 4 eq DIEA to add to the polypeptide solution. Stir the reaction at room temperature for 10 h to obtain a cyclic nonapeptide containing a protecting group.

[0075] S4. The cyclic nonapeptide containing the protecting group was purified by preparative HPLC to obtain the cyclic nonapeptide.

[0076] The specific steps are as follows:

[0077] 15) Rotary freeze-drying:

[0078] The cyclic nonapeptide containing the protecting group was concentrated using a rotary evaporator. After concentration, it was dissolved in 30% acetonitrile / water solution, then freeze-dried on the wall, and finally purified by preparative HPLC to obtain the cyclic (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine) with the sequence shown in SEQ ID NO.1.

[0079] Experimental example:

[0080] 1. Antioxidant experiment:

[0081] 1.1 Experimental grouping: A blank control group, a negative control group, a positive control group, and a sample group were set up, with at least 3 replicates in each group.

[0082] 1.2 Experimental Methods: Human immortalized keratinocytes in good growth condition were used at a density of 4 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and incubated at 37°C in a 5% CO2 incubator. Prior to irradiation, cells were administered the drug to the groups according to the design scheme in Table 2 for 4 hours. The negative control group, positive control group, and sample group received a dose of 20 mJ / cm² after 4 hours of incubation. 2 UVB irradiation stimulation was performed. After irradiation, drugs were administered according to the groupings in Table 2, and the cells were cultured for another 24 hours. Subsequently, flow cytometry analysis was conducted according to the specific operating procedures of the reactive oxygen species detection kit (Beyotime). Data analysis software was used to obtain the fluorescence intensity values ​​DCF (ROS) MFI (DM) for each group. The ROS scavenging rate was calculated using the following formula: ROS scavenging rate (%) = (Negative group DM - Sample group DM) / (Negative group DM - Blank control DM) × 100%.

[0083] The specific design scheme is shown in Table 2.

[0084] Table 2. Antioxidant efficacy design scheme

[0085]

[0086] 1.3 Experimental Results:

[0087] The antioxidant experimental results of cyclic nonapeptide are shown in Table 3 and Figure 3 As shown.

[0088] Table 3. Effect of cyclic nonapeptide on ROS content generated by UVB-induced HaCaT

[0089]

[0090] The research results show that, compared with the blank control group, the average fluorescence intensity of the negative control group was significantly increased, indicating successful modeling. Compared with the negative control group, the average fluorescence intensity of both the positive control group and the sample group was significantly decreased. Specifically, the ROS scavenging rate of the 20 ppm positive control group was 66.14%, the ROS scavenging rate of the 40 ppm cyclic nonapeptide was 32.19%, the ROS scavenging rate of the 80 ppm cyclic nonapeptide was 38.24%, and the ROS scavenging rate of the 120 ppm cyclic nonapeptide was 91.83%, demonstrating antioxidant activity. Therefore, the cyclic nonapeptide RGDSNKVKF of this invention possesses the ability to scavenge ROS under the above experimental conditions, thereby achieving antioxidant effects, and is a cyclic nonapeptide with antioxidant properties.

[0091] 2. Anti-photoaging efficacy experiment:

[0092] 2.1 Experimental grouping: A blank control group, a negative control group, and a sample group (40ppm, 80ppm, and 120ppm of cyclic nonapeptide) were set up.

[0093] 2.2 Experimental Methods: Human primary fibroblasts (HDF) were seeded in 6-well plates at a density of 2.5 × 10⁻⁶ cells / well. 5 When the cell density reached 40%–60% and the cell plate formation rate was 40%–60%, the cells were grouped and administered drugs according to the design scheme in Table 4. 2 mL of culture medium was added to each well of the blank control group and negative control group, while 2 mL of culture medium containing 40 ppm, 80 ppm, and 120 ppm cyclic nonapeptide was added to each well of the sample group. After culturing in a CO2 incubator for 24 h, the well plates were removed, the culture medium was discarded, and an appropriate amount of PBS was added to cover the cell surface. The negative control group and sample group were exposed to UVA irradiation at a dose of 10 J. After irradiation, the cells were cultured in a CO2 incubator for another 24 h. Total RNA was extracted from each group of cells, and the expression levels of COL1, COL3, MMP1, and MMP3 genes in the cells were detected according to the instructions for real-time quantitative PCR.

[0094] The specific design scheme is shown in Table 4.

[0095] Table 4. Anti-photoaging design scheme

[0096]

[0097] Experimental results:

[0098] The experimental results of the anti-photoaging efficacy of cyclic nonapeptide are shown in Tables 5 and 6. Figure 4-7 As shown.

[0099] Table 5. Effects of cyclic nonapeptide on intracellular COL3 & MMP3 in HDF cells

[0100]

[0101] The results showed that, compared with the blank control group, COL3 mRNA expression decreased significantly and MMP3 mRNA expression increased significantly after UVA irradiation, indicating successful model establishment. Compared with the negative control group, the relative expression levels of COL3 mRNA and MMP3 mRNA significantly increased and decreased respectively in different doses of cyclic nonapeptide groups, indicating that the cyclic nonapeptide of the present invention has anti-aging and anti-photoaging effects at concentrations of 40ppm, 80ppm, and 120ppm, and can reverse UV-induced skin laxity and wrinkles, and improve skin elasticity.

[0102] Table 6. Effects of cyclic nonapeptide on intracellular COL1 & MMP1 in HDF cells

[0103]

[0104] The results showed that, compared with the blank control group, COL1 mRNA expression decreased significantly and MMP1 mRNA expression increased significantly after UVA irradiation, indicating successful model establishment. Compared with the negative control group, the relative expression level of COL1 mRNA in the 120 ppm cyclic nonapeptide group increased significantly and the relative expression level of MMP1 mRNA decreased significantly, indicating an anti-aging effect.

[0105] Based on the above research results, the cyclic nonapeptide RGDSNKVKF, under the aforementioned experimental conditions, can reduce the expression of MMPs and increase the expression of COL1 and COL3, thereby achieving an anti-photoaging effect. Therefore, it is a cyclic nonapeptide with anti-photoaging properties.

[0106] 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 both anti-photoaging and antioxidant effects, characterized in that: The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine).

2. The cyclic nonapeptide with both anti-photoaging and antioxidant effects according to claim 1, characterized in that: The structural formula of the cyclic nonapeptide is: .

3. The use of the cyclic nonapeptide according to any one of claims 1-2 in the preparation of cosmetics for improving skin aging caused by ultraviolet radiation.

4. The application according to claim 3, characterized in that: Skin aging caused by ultraviolet radiation includes deep skin aging caused by UVA radiation.

5. The application according to claim 3, characterized in that: Skin aging caused by ultraviolet radiation includes aging caused by oxidative damage to the skin surface due to UVB radiation.

6. The application according to claim 3, characterized in that: The effective concentration of the cyclic nonapeptide is not less than 40 ppm.

7. The application according to claim 3, characterized in that: The effective concentration of the cyclic nonapeptide is 40~120ppm.

8. A cosmetic composition with anti-photoaging effects, characterized in that: It includes the cyclic nonapeptide as described in any one of claims 1-2.

9. A cosmetic composition with antioxidant properties, characterized in that: It includes the cyclic nonapeptide as described in any one of claims 1-2.

Images