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

Abstract

The invention discloses a cyclic nonapeptide with the effects of resisting light aging and resisting oxidation. The amino acid sequence of the cyclic nonapeptide is as follows: cyclic (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine). The cyclic nonapeptide disclosed by the invention can be used for effectively relieving deep photoaging caused by UVA and surface oxidative damage induced by UVB, so that comprehensive protection on skin is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a cyclic peptide, in particular a cyclic nonapeptide with both anti-photoaging and antioxidant effects and its application. BACKGROUND

[0002] Compared with traditional linear peptides, cyclic peptides exhibit significant advantages in biological activity, stability, and penetration.

[0003] Due to its closed cyclic structure, cyclic peptides have higher resistance to enzymatic degradation than linear peptides and can maintain longer biological activity in complex skin barrier environments. The high rigidity provided by the cyclic backbone of cyclic peptides not only enhances their binding affinity to targets but also enhances the functional effects at specific sites. The lipophilicity and small molecule characteristics of cyclic peptides help improve their transdermal absorption performance, making them more easily reach the dermis tissue in the deep layer of the skin and exert stronger antioxidant and anti-aging effects. Therefore, with the growing demand for natural and functional ingredients among consumers, cyclic peptides are widely used in skin care products, anti-aging moisturizing creams, and repair serums, and cyclic peptides are particularly outstanding in repairing collagen, inhibiting matrix metalloproteinases (MMPs), and neutralizing free radicals.

[0004] UVA (320-400 nm) radiation can penetrate the skin to the dermis layer and induce various cell damage, which is one of the main causes of photoaging. The specific mechanism is as follows: UVA radiation induces the generation of a large amount of reactive oxygen species (ROS) through photosensitive molecules. ROS can damage biological macromolecules in the skin, leading to DNA damage, lipid peroxidation, and protein carbonylation. Among them, collagen and elastic fibers are the main targets of ROS attack, and after damage, they can cause skin relaxation and wrinkle formation. UVA radiation activates the MAPK / ERK signaling pathway and the AP-1 transcription factor. AP-1 regulates the expression of MMPs genes, and these enzymes directly degrade the collagen matrix in the skin, accelerating skin aging. The inflammatory response caused by UVA can activate the nuclear factor NF-κB, further promoting the secretion of inflammatory factors (such as IL-6, TNF-α) in the skin, exacerbating the effects of photoaging.

[0005] UVB (280-320 nm) mainly acts on the epidermal cells on the surface layer of the skin, and its high-energy direct effect has significant destructive effects on subcutaneous cells, structural proteins, and genomic homeostasis. UVB irradiation can induce oxidation-reduction imbalance in keratinocytes, significantly increasing ROS levels, and then triggering a three-level cascade damage of intracellular lipids, proteins, and DNA. ROS and DNA damage can activate skin macrophages and dendritic cells, leading to the release of a large amount of inflammatory mediators such as IL-1β and COX-2, and triggering acute skin redness and chronic aging. ROS attacks unsaturated fatty acids in the cell membrane, generating hydrogen peroxide and aldehyde products, leading to loss of cell membrane transport function and activation of apoptosis signals.

[0006] At present, there are still few reports on cyclo-nonapeptides with both anti-photoaging and antioxidant effects. SUMMARY

[0007] The present application aims to provide a cyclo-nonapeptide with both anti-photoaging and antioxidant effects and its application. The cyclo-nonapeptide of the present application can effectively reduce deep photoaging induced by UVA and surface oxidative damage induced by UVB, thereby achieving comprehensive protection of the skin.

[0008] The technical solution of the present application is a cyclo-nonapeptide with repair and antioxidant effects, wherein the amino acid sequence of the cyclo-nonapeptide is cyclo(arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine).

[0009] In the aforementioned cyclo-nonapeptide with repair and antioxidant effects, the structural formula of the cyclo-nonapeptide is: .

[0010] The present application also provides the application of the aforementioned cyclo-nonapeptide in the preparation of cosmetics or food or drugs with anti-photoaging and / or antioxidant effects.

[0011] In the aforementioned application, the anti-photoaging is against deep photoaging caused by UVA.

[0012] In the aforementioned application, the antioxidant is against surface oxidative damage caused by UVB.

[0013] In the aforementioned application, the effective concentration of the cyclo-nonapeptide is not less than 40 ppm.

[0014] In the aforementioned application, the effective concentration of the cyclo-nonapeptide is 40-120 ppm.

[0015] A cosmetic composition with anti-photoaging effect comprises the aforementioned cyclo-nonapeptide.

[0016] A cosmetic composition with antioxidant effect comprises the aforementioned cyclo-nonapeptide.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The cyclo-nonapeptide of the present application can effectively reduce the oxidative burden caused by UVA and UVB together by inhibiting ROS generation and capturing free radicals, protect the stratum corneum of the skin, reduce the harm of sunburn, and provide comprehensive antioxidant protection for the skin.

[0019] The cyclic nonapeptide of the present application can improve the synthesis of collagen type I and type III, while inhibiting the expression level of MMPs related to the decomposition of collagen type I and type III, thereby reversing UV-induced skin relaxation and wrinkles, reducing pigmentation, and improving skin elasticity.

[0020] The cyclic nonapeptide of the present application can regulate antioxidant, growth factor expression and cell proliferation through multi-targets against the damage mechanism of both UVA and UVB wavelengths, thereby providing differentiated product functional appeals and meeting the growing market demand.

[0021] In summary, the cyclic nonapeptide of the present application is non-toxic, non-irritating, and stable, and has the effects of scavenging ROS (reactive oxygen species), inhibiting the expression of MMPs, and increasing the expression of COL1 and COL3, and can effectively reduce the deep photoaging induced by UVA and the surface oxidative damage induced by UVB, thereby achieving comprehensive protection of the skin and slowing down skin aging, and can be applied to related products in the field of cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a molecular docking simulation diagram of the cyclic nonapeptide and integrin aVβ3 in the present application.

[0023] Figure 2 is a molecular docking simulation diagram of the cyclic nonapeptide and integrin a5β1 in the present application.

[0024] Figure 3 is a column chart of the fluorescence intensity DCF (ROS) MFI values of each experimental group; in the figure, single factor variance analysis is used, * indicates comparison with the NC group, “*” indicates P<0.05, “**” indicates P<0.01, “***” indicates P<0.001, and “****” indicates P<0.0001.

[0025] Figure 4 is a column chart of the influence of each experimental group on the content of COL3 in cells; in the figure, single factor variance analysis is used, * indicates comparison with the NC group, and “****” indicates P<0.0001.

[0026] Figure 5 is a column chart of the influence of each experimental group on the content of MMP3 in cells; in the figure, single factor variance analysis is used, * indicates comparison with the NC group, “***” indicates P<0.001, and “****” indicates P<0.0001.

[0027] Figure 6 is a column chart of the influence of each experimental group on the content of COL1 in cells; in the figure, single factor variance analysis is used, * indicates comparison with the NC group, “**” indicates P<0.01, and “****” indicates P<0.0001.

[0028] Figure 7 Figure 7 is a column chart showing the effect of each experimental group on the MMP1 content in cells; in the figure, single factor variance analysis was used, and “****” indicates P<0.0001. DETAILED DESCRIPTION

[0029] The application will be further described below in conjunction with examples, but it is not used as a basis for limiting the application.

[0030] Example:

[0031] The cyclic nonapeptide is designed as RGDS-X1-KVK-X2, wherein X1 and X2 are each one of 20 L-amino acids and 20 D-amino acids, and there are a total of 40*40=1600 candidate sequences of the cyclic nonapeptide.

[0032] Then, the three-dimensional structures of the above 1600 cyclic peptides are simulated using structure simulation software, and the energy difference between the 1600 cyclic peptides and linear peptide sequences is calculated by molecular docking software, wherein a structure simulation score Total_score<0 represents that the spatial steric hindrance between the amino acid residues of the candidate cyclic peptide structure is small, which is suitable for forming a cyclic peptide structure and suitable for being used as a candidate cyclic peptide for experimental verification. Thus, the candidate cyclic nonapeptide RGDSNKVKF with a stable structure is screened.

[0033] Table 1 Energy difference between cyclic nonapeptide sequence and corresponding linear peptide sequence

[0034] First, the RGDSNKVKF cyclic nonapeptide structure is subjected to molecular docking simulation with integrin αVβ3. According to the crystal structure (αVβ3_PDB ID: 4MMX) in the Protein Data Bank, the RGDS sequence in the cyclic peptide is docked to the RGDS sequence in the αVβ3 complex crystal structure, and the specific docking structure simulation diagram is shown in Figure 1 . Figure 1 In the figure, the red linear structure represents the position of the RGDS peptide segment in the known crystal structure 4MMX, and the green and blue stick structures represent the cyclic nonapeptide.

[0035] The docking score of the cyclic nonapeptide RGDSNKVKF and integrin αVβ3 is -10.2881, and the docking score < -7 is a strong binding. The docking result shows that the RMSD of the RGDS of the cyclic nonapeptide and the RGDS sequence in the crystal structure 4MMX is 0.505 Å, and the positions of the amino acids and the directions of the side chains are very close, which is conducive to the combination of the cyclic nonapeptide and integrin αVβ3.

[0036] Subsequently, the RGDSNKVKF cyclic peptide structure is simulated by molecular docking with integrin α5β1. According to the crystal structure (α5β1_PDB ID: 4WK2) in the Protein Data Bank, the RGDS sequence in the cyclic peptide is docked to the RGDS sequence in the α5β1 complex crystal structure, and the specific docking structure simulation diagram is as shown in Figure 2 . Figure 2 In the figure, the red linear structure represents the position of the RGDS peptide segment in the known crystal structure 4WK2, and the green and blue stick structures represent the cyclic nonapeptide.

[0037] The docking score of the cyclic nonapeptide RGDSNKVKF and integrin α5β1 is -10.1263, and the docking score < -7 is a strong binding. The docking result shows that the RGDS of the cyclic nonapeptide and the RGDS sequence in the crystal structure 4WK2 have an RMSD of 1.072 Å, and the positions of each amino acid and the direction of the side chain extension are very close, which is conducive to the binding of the cyclic nonapeptide and integrin α5β1.

[0038] The above molecular docking simulation results show that the structure of the cyclic nonapeptide is conducive to the binding of integrin αVβ3 and α5β1, and reduces the inflammation induced by lipopolysaccharide by inhibiting the MAPK pathway of integrin signal transduction. By regulating inflammation, the cyclic nonapeptide can reduce the generation of ROS, thereby playing an antioxidant role.

[0039] The amino acid sequence of the cyclic nonapeptide of the present application is: cyclic (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine), that is, 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)[C@H](CCCNC(O2)=N)N.

[0040] The structural formula is: .

[0041] The preparation method of the cyclic nonapeptide is:

[0042] S1, with natural amino acid arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine as starting material, using fluorenylmethyloxycarbonyl (Fmoc) N-terminal protection strategy, sequentially connecting the corresponding amino acids according to the resin solid phase synthesis method, and sequentially removing the Fmoc-protecting group during the process, to obtain a resin containing a linear nonapeptide chain.

[0043] The specific steps are as follows:

[0044] 1) Swelling the resin:

[0045] 0.6g of 2-Cl Trt-Lys resin (substitution degree SD=0.39 mmol / g) was added to the reactor, and DCM (10mL / g) was added as a swelling agent for swelling for 5min.

[0046] 2) Resin deprotection:

[0047] The swelling agent DCM was vacuumed dry, and 20% piperidine (Pip) / DMF (10mL / g) was added as a deprotection agent, and after stirring for 5min, it was vacuumed dry, and 20% piperidine (Pip) / DMF (10mL / g) was added again and stirred for 5min.

[0048] 3) Deprotection washing:

[0049] The deprotection agent Pip / DMF was vacuumed dry, and DMF (10mL / g) was washed for 5 times, each time stirring for 20~30s, and vacuuming for 20s, to obtain resin-1.

[0050] 4) Deprotection detection:

[0051] About 20 particles of resin-1 were taken into a detection test tube, 1mL of ninhydrin detection reagent was added to the detection test tube, and then the detection test tube was placed in a metal bath pot above 120°C for 2min, and the resin color was observed after taking it out. The resin color becomes dark, which is positive, indicating that the deprotection is successful.

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

[0053] 3 equivalents of Fmoc-Val-OH and 3 equivalents of Oxyma were added to the resin-1, 10mL of DMF was added for dissolution, and then 3 equivalents of DIC was added, and after activation for 5min, it was poured into the reactor, and stirred for 1h.

[0054] 6) Reaction washing:

[0055] The reaction reagent DMF was vacuumed dry, and DMF (10mL / g) was washed for 5 times, each time stirring for 20~30s, and vacuuming for 20s, to obtain resin-2.

[0056] 7) Reaction detection:

[0057] Take about 20 resin-2 into the test tube, add 1 mL of ninhydrin test reagent to the test tube, then put the test tube into a metal bath pot above 120°C for 2 min, take it out and observe the color of the resin, and there is no obvious change in the color of the resin, indicating that the reaction condensation is successful.

[0058] 8) Repeat steps 2) - 7), and according to the sequence of the polypeptide, condense the subsequent amino acids in the sequence from right to left, respectively 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 is finished, to get resin-3.

[0059] 9) Deprotection:

[0060] Add 20% Pip / DMF (10 mL / g) as deprotection reagent to resin-3, stir for 5 min, then dry, and add 20% Pip / DMF (10 mL / g) again, stir for 5 min.

[0061] 10) Deprotection washing:

[0062] Vacuum dry the deprotection reagent Pip / DMF, and then wash with DMF (10 mL / g) for 5 times, each time stirring for 20-30 s, and drying for 20 s, to get resin-4.

[0063] 11) Deprotection detection:

[0064] Take about 20 resin-4 into the test tube, add 1 mL of ninhydrin test reagent, and put the test tube into a metal bath pot above 120°C for 2 min, take it out and observe the color of the resin, and the resin color becomes darker, which is positive, indicating that the deprotection is successful.

[0065] 12) Washing:

[0066] Wash resin-4 with methanol (10 mL) for 5 times, and vacuum dry for 10 min, to complete the solid-phase condensation, and obtain a resin containing a linear nonapeptide chain.

[0067] S2, cleave the resin containing the linear nonapeptide chain, cut the linear nonapeptide chain from the resin, and remove the remaining protecting groups of the peptide chain, collect the cleavage liquid containing the crude linear nonapeptide chain, and obtain a full-protected polypeptide solid to be cycled.

[0068] The specific steps are as follows:

[0069] 13) Resin full-protection cleavage:

[0070] The resin containing the linear nonapeptide chain is loaded into a boat-shaped reactor, 10 mL / g of cleavage solution (volume ratio of TFE:DCM is 30:70) is added, and cleavage is carried out at room temperature for 3 times, 1 h each time, the reaction solution is filtered out, and the solvent is concentrated and evaporated using a rotary evaporator. After evaporation, a 30% acetonitrile / water solution is added to dissolve the sample, and then freeze-drying is performed to obtain a full-protected polypeptide solid to be ringed.

[0071] S3, the full-protected polypeptide solid to be ringed is mixed with a polypeptide coupling agent, an activator, etc., and then ringed to obtain a protected ring nonapeptide.

[0072] The specific steps are as follows:

[0073] 14) Modification of the ring-opening reaction:

[0074] The full-protected polypeptide solid to be ringed is weighed and dissolved in AR-grade DMF to obtain a polypeptide solution with a concentration of 1 mM. 2 eq of PyBOP and 4 eq of DIEA are weighed and transferred to the polypeptide solution, and the mixture is stirred at room temperature for 10 h to obtain a protected ring nonapeptide.

[0075] S4, the protected ring nonapeptide is purified by preparative HPLC to obtain a ring nonapeptide.

[0076] The specific steps are as follows:

[0077] 15) Rotary evaporation and freeze-drying:

[0078] The protected ring nonapeptide is concentrated using a rotary evaporator. After concentration, a 30% acetonitrile / water solution is added to dissolve the sample, and then wall-hanging freeze-drying is performed. Finally, preparative HPLC purification is performed to obtain a ring (arginine-glycine-aspartic acid-serine-asparagine-lysine-valine-lysine-phenylalanine), the sequence of which is shown in SEQ ID NO. 1.

[0079] Experimental example:

[0080] 1. Antioxidant experiment:

[0081] 1.1 Experimental grouping: blank control group, negative control group, positive control group, sample group, at least 3 replicates per group.

[0082] 1.2 Experimental method: well-grown human immortalized keratinocytes were inoculated in a 6-well plate at a density of 4×10 5 cells per well, and cultured in a 37℃, 5% CO2 incubator. Before irradiation, the cells were grouped and dosed according to the design in Table 2 for 4 h. The negative control group, the positive control group and the sample group received a dose of 20 mJ / cm 2UVB irradiation stimulation. After irradiation, the grouping according to Table 2 was administered, and the culture was continued for 24 h. Subsequently, the flow cytometer was detected according to the specific operation steps of the active oxygen detection kit (Bi Yun Tian). Data analysis software was used for data analysis, and the fluorescence intensity value DCF (ROS) MFI (referred to as DM) of each group was obtained. The ROS clearance rate calculation formula is as follows: ROS clearance rate (%) = (negative group DM-sample group DM) / (negative group DM-blank control DM) x 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 experiment results of the cyclic nonapeptide are shown in Table 3 and Figure 3 .

[0088] Table 3. Influence of cyclic nonapeptide on ROS content induced by UVB in HaCaT

[0089]

[0090] According to the research results, compared with the blank control group, the average fluorescence intensity of the negative control group was significantly up-regulated, indicating that the modeling was successful. Compared with the negative control group, the average fluorescence intensity of the positive control group and the sample group was significantly down-regulated, wherein the ROS clearance rate of the 20ppm positive control group was 66.14%, the ROS clearance rate of the 40ppm cyclic nonapeptide was 32.19%, the ROS clearance rate of the 80ppm cyclic nonapeptide was 38.24%, and the ROS clearance rate of the 120ppm cyclic nonapeptide was 91.83%, which had an antioxidant effect. Therefore, the cyclic nonapeptide RGDSNKVKF of the present application has the ability to remove ROS under the above experimental conditions, and further achieves the antioxidant effect, and is a cyclic nonapeptide with antioxidant effect.

[0091] 2. Anti-photoaging efficacy experiment:

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

[0093] 2.2 Experimental method: human primary fibroblasts HDF were inoculated in a 6-well plate with a density of 2.5x10 5When the concentration of the cell suspension reaches 1 x 106cells / mL and the cell plating rate reaches 40-60%, the cells are grouped and dosed according to the design in Table 4. The blank control group and the negative control group are each added with 2 mL of culture solution, and the sample group is each added with 2 mL of culture solution containing 40 ppm, 80 ppm, and 120 ppm of the cyclic nonapeptide. After 24 hours of culture in a CO2incubator, the well plate is removed, the culture solution is removed, and an appropriate amount of PBS is added to cover the cell surface. The negative control group and the sample group are exposed to UVA irradiation at a dose of 10 J. After the irradiation is completed, the CO2incubator is continued to be used for 24 hours of culture, total RNA of the cells in each group is extracted, and then the expression amounts of COL1, COL3, MMP1, and MMP3 genes in the cells are detected according to the real-time fluorescent quantitative PCR operation instruction.

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

[0095] Table 4. Anti-photoaging design

[0096]

[0097] Experimental results:

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

[0099] Table 5. Influence of the cyclic nonapeptide on COL3 and MMP3 in HDF cells

[0100]

[0101] The results show that, compared with the blank control group, the expression of COL3 mRNA is significantly decreased and the expression of MMP3 mRNA is significantly increased after UVA irradiation, which indicates that the modeling is successful. Compared with the negative control group, the relative expression amount of COL3 mRNA in the cyclic nonapeptide groups at different dosages is significantly increased, and the relative expression amount of MMP3 mRNA is significantly decreased, which indicates that the cyclic nonapeptide has an anti-aging and anti-photoaging effect at concentrations of 40 ppm, 80 ppm, and 120 ppm, and can reverse UV-induced skin relaxation and wrinkles and improve skin elasticity.

[0102] Table 6. Influence of the cyclic nonapeptide on COL1 and MMP1 in HDF cells

[0103]

[0104] The results show that, compared with the blank control group, the expression of COL1 mRNA is significantly decreased and the expression of MMP1 mRNA is significantly increased after UVA irradiation, which indicates that the modeling is successful. Compared with the negative control group, the relative expression amount of COL1 mRNA in the cyclic nonapeptide group at 120 ppm is significantly increased, and the relative expression amount of MMP1 mRNA is significantly decreased, which has an anti-aging effect.​

[0105] According to the above research results, it is shown that the cyclic nonapeptide RGDSNKVKF has the effects of reducing the expression of MMPs, increasing the expression of COL1 and COL3, and achieving the effect of anti-photoaging under the above experimental conditions. Therefore, it is a cyclic nonapeptide with the effect of anti-photoaging.

[0106] It should be understood that the above examples are only used to illustrate the technical solutions of the present application, but not limit it. For those skilled in the art, the technical solutions recorded in the above examples can be modified, or some technical features can be replaced by equivalent ones. All these modifications and replacements shall belong to the protection scope of the appended claims of the present application.

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 cyclo-nonapeptide according to claim 1, having both anti-photoaging and antioxidant effects, characterized by: The structural formula 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 anti-photoaging and / or antioxidant efficacy.

4. Use according to claim 3, characterized in that: Anti-photoaging is anti-deep photoaging caused by UVA.

5. Use according to claim 3, characterized in that: Antioxidant is anti-superficial oxidative damage caused by UVB.

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

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

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

9. 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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