Cyclic nonapeptide with antioxidant and soothing anti-inflammatory efficacy and use thereof

Abstract

The application discloses a cyclic nonapeptide with antioxidant and soothing anti-inflammatory effects, and the amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-glutamine-lysine-valine-lysine-arginine). The application finds a new cyclic nonapeptide which can effectively scavenge ROS, inhibit secretion of inflammatory factors, and has antioxidant effect and soothing anti-inflammatory capacity.

Description

Technical Field

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

[0002] Ultraviolet B (UVB) radiation, with a wavelength of 290-320 nm, also known as medium-wave erythema-inducing ultraviolet radiation, can reach the epidermis. UVB exposure can have a variety of harmful effects on human skin, including sunburn, tanning, immunosuppression, photoaging, and skin cancer. In addition, UVB exposure can induce the production of excessive and highly destructive reactive oxygen species (ROS), disrupting cellular oxygen balance and causing oxidative stress, resulting in significant oxidative damage to proteins, lipids, and DNA in the skin.

[0003] In addition, UVB irradiation-induced reactive oxygen species (ROS) activate inflammatory signaling pathways, generating inflammatory responses and accelerating skin inflammation. The pathogenesis of inflammation is a complex process, with inflammatory responses being a major contributing factor. After UVB irradiation, key inflammatory factors observed in the skin include IL-6, IL-1α, and TNF-α. IL-6, produced by HaCaT cells after UVB irradiation, participates in the acute phase of inflammation and influences the proliferation, differentiation, and apoptosis of keratinocytes, acting as a trigger for the inflammatory response. IL-1α acts as an "initiator" of the inflammatory response; once released extracellularly, IL-1α immediately binds to IL-1 receptors on its own cells or neighboring cells, rapidly initiating downstream signaling pathways such as NF-κB, thereby inducing the expression and release of other inflammatory factors (such as IL-6, IL-8, and TNF-α).

[0004] To combat skin oxidative damage and suppress inflammation, various antioxidant and anti-inflammatory products have emerged on the market. Common antioxidant and anti-inflammatory ingredients include vitamin E, vitamin C, and coenzyme Q10. However, these ingredients have limitations such as easy oxidation and inactivation, and the need to build up skin tolerance during use.

[0005] There are currently no publicly available research reports on cyclic nonapeptides, which have antioxidant and soothing anti-inflammatory effects. Summary of the Invention

[0006] The purpose of this invention is to provide a cyclic nonapeptide with antioxidant and soothing anti-inflammatory effects and its applications. This invention discovers a novel cyclic nonapeptide that can effectively scavenge ROS, inhibit the secretion of inflammatory factors, and possesses antioxidant and soothing anti-inflammatory capabilities.

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

[0008] The aforementioned cyclic nonapeptide with antioxidant and soothing anti-inflammatory effects has the following structure: .

[0009] The present invention also provides the application of the above-mentioned cyclic nonapeptide in the preparation of cosmetics, food or pharmaceuticals with antioxidant and / or soothing and anti-inflammatory effects.

[0010] In the aforementioned applications, the antioxidant refers to the protection against skin oxidation caused by UVB radiation.

[0011] In the aforementioned applications, the soothing and anti-inflammatory effect refers to relieving skin inflammation caused by UVB.

[0012] In the aforementioned applications, the effective concentration of the cyclic nonapeptide is 10~100 μg / mL.

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

[0014] A cosmetic composition having soothing and anti-inflammatory effects, comprising the aforementioned cyclononapeptide.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The amino acid sequence of the cyclic nonapeptide of this invention is: cyclic (arginine-glycine-aspartic acid-serine-glutamine-lysine-valine-lysine-arginine). This structure facilitates the binding of integrin αVβ3 and α5β1, thereby alleviating inflammation by inhibiting the MAPK pathway of integrin signaling. By regulating inflammation, the cyclic nonapeptide can reduce ROS production, thus exerting antioxidant and soothing anti-inflammatory effects.

[0017] Antioxidant and anti-inflammatory experiments verified that the cyclic nonapeptide of the present invention can significantly inhibit the production of ROS in HaCaT cells and reduce the secretion of inflammatory factors IL-6 and IL-1α at concentrations of 10 μg / mL and 100 μg / mL, thereby achieving the purpose of antioxidation and soothing anti-inflammation.

[0018] The cyclic nonapeptide of this invention has the characteristics of small molecular weight, good skin permeability, and high biocompatibility. It is also not easily oxidized and deactivated, does not require the establishment of skin tolerance, and has good and unrestricted effects. Attached Figure Description

[0019] Figure 1 This is a simulation diagram of the docking of the cyclic nonapeptide with integrin aVβ3 in this invention.

[0020] Figure 2 This is a simulation diagram of the docking of the cyclic nonapeptide and integrin a5β1 molecules in this invention.

[0021] Figure 3 This is a graph showing the toxic effects of different concentrations of cyclic nonapeptide on HaCaT cells.

[0022] Figure 4 This is a bar chart of the average ROS fluorescence intensity values ​​for each experimental group. In the figure, analysis of variance was used. # indicates a significant difference compared to group BC, where "#" indicates P-value < 0.05, "##" indicates P-value < 0.01, and "###" indicates P-value < 0.001. * indicates a significant difference compared to group NC, where "*" indicates P-value < 0.05, "**" indicates P-value < 0.01, and "***" indicates P-value < 0.001.

[0023] Figure 5 The figures show the effects of each experimental group on the levels of intracellular inflammatory factors. In the figures, Figure A is a bar chart of IL-6 levels; Figure B is a bar chart of IL-1α levels. Analysis of variance was used. # indicates a significant difference compared to group BC, "#" indicates P-value < 0.05, "##" indicates P-value < 0.01, and "###" indicates P-value < 0.001. * indicates a significant difference compared to group NC, "*" indicates P-value < 0.05, "**" indicates P-value < 0.01, and "***" indicates P-value < 0.001. Detailed Implementation

[0024] 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.

[0025] Example:

[0026] 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.

[0027] 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 (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 RGDSQKVKR was selected.

[0028] Table 1. Difference between the energies of cyclic nonapeptide sequences and their corresponding linear peptide sequences.

[0029]

[0030] First, molecular docking simulations were performed between the RGDSQKVKR 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.

[0031] The docking fraction between the cyclic nonapeptide RGDSQKVKR and integrin αVβ3 was -10.5057, 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 1.243 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 αVβ3.

[0032] Subsequently, molecular docking simulations were performed between the RGDSQKVKR cyclic nonapeptide 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.

[0033] The docking fraction between the cyclic nonapeptide RGDSQKVKR and integrin α5β1 was -11.7136, 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 1.558 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.

[0034] The above molecular docking simulation results indicate that the structure of the cyclic nonapeptide is conducive to binding integrin αVβ3 and α5β1.

[0035] The amino acid sequence of the cyclic nonapeptide of the present invention is: cyclic (arginine-glycine-aspartic acid-serine-glutamine-lysine-valine-lysine-arginine), i.e., Cyclo(Arg-Gly-Asp-Ser-Gln-Lys-Val-Lys-Arg), and the cyclic sequence is shown in SEQ ID NO.1. The structural formula is: .

[0036] The preparation method of cyclic nonapeptide is as follows:

[0037] S1. Using natural amino acids arginine-glycine-aspartic acid-serine-glutamine-lysine-valine-lysine-arginine 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.

[0038] The specific steps are as follows:

[0039] 1) Swelling resin:

[0040] 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.

[0041] 2) Resin deprotection:

[0042] 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.

[0043] 3) Remove protective washing:

[0044] 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.

[0045] 4) Deprotection detection:

[0046] 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.

[0047] 5) Condensation of the second amino acid:

[0048] 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.

[0049] 6) Reaction washing:

[0050] 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.

[0051] 7) Reaction detection:

[0052] 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.

[0053] 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-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, until Fmoc-Arg(Pbf)-OH ends, to obtain resin-3.

[0054] 9) Deprotection:

[0055] 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.

[0056] 10) Remove protective wash:

[0057] 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.

[0058] 11) Deprotection detection:

[0059] 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.

[0060] 12) Washing:

[0061] 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.

[0062] 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.

[0063] The specific steps are as follows:

[0064] 13) Resin-protected pyrolysis:

[0065] 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.

[0066] 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.

[0067] The specific steps are as follows:

[0068] 14) Modification cyclization reaction:

[0069] 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.

[0070] S4. The cyclic nonapeptide containing the protecting group was purified by preparative HPLC to obtain the cyclic nonapeptide.

[0071] The specific steps are as follows:

[0072] 15) Rotary freeze-drying:

[0073] 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-glutamine-lysine-valine-lysine-arginine) with a relative molecular weight of 1055.2100.

[0074] Experimental example:

[0075] 1. Cytotoxicity test:

[0076] 1.1 Experimental groups: blank control group (BC), zeroing group, and sample group.

[0077] 1.2 Experimental Methods: HaCaT cells were incubated at 37℃ at a density of 1×10⁻⁶ cells per well.4 Cells were seeded at a concentration of [missing information] into 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. The supernatant was discarded, and the cells were washed once with 200 μL of PBS. The blank control group and the zeroing group were then treated with complete culture medium for 24 hours, while the sample groups were treated with different concentrations of cyclic nonapeptide for 24 hours. Cell viability was measured using the CCK-8 colorimetric assay kit (Beyotime) according to the manufacturer's instructions. The effect of cyclic nonapeptide on HaCaT cell viability was calculated.

[0078] The specific design scheme is shown in Table 2.

[0079] Table 2. Cytotoxicity Design Scheme

[0080]

[0081] 1.2 Experimental Results:

[0082] The results of the cytotoxicity assay for cyclic nonapeptide are shown in Table 3 and Figure 3 As shown.

[0083] Table 3. Effects of cyclic nonapeptide on keratinocyte toxicity

[0084] Based on the toxicity test results, as shown in Table 3 and Figure 3 As shown, when cyclic nonapeptide was used to treat keratinocytes, compared with the blank control group, the cell viability reached more than 90% when the concentration was ≤200μg / mL, and it was non-toxic to keratinocytes.

[0085] 2. Antioxidant experiment:

[0086] 2.1 Experimental groups: blank control group (BC), negative control group (NC), and sample group (CP: 10, 100 μg / mL cyclic nonapeptide).

[0087] 2.2 Experimental Methods: Cells were cultured at 2.0 × 10⁻⁶. 5 Inoculate 100 μg / well of each sample into a 6-well plate and incubate overnight (37°C, 5% CO2). After overnight incubation, add 100 μg / mL of cyclic nonapeptide to the sample groups, while replacing the blank control and negative control groups with complete culture medium. Continue incubation for 24 hours, discard the supernatant, add 500 μL of PBS, and use 200 mJ / cm² water. 2UVB 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 was repeated twice, and the fluorescence value was detected using a fluorescence microplate reader to obtain the fluorescence intensity value (DCF (ROS) MFI) of each group. The effect of cyclic nonapeptide on ROS production in the sample group was observed and compared.

[0088] Table 4. Antioxidant Design Scheme

[0089]

[0090] 2.3 Experimental Results:

[0091] The antioxidant experimental results of cyclic nonapeptide are shown in Table 5 and Figure 4 As shown.

[0092] Table 5. Effect of cyclic nonapeptide on ROS content generated by UVB-induced HaCaT

[0093]

[0094] Based on the research results, the cyclic nonapeptide RGDSQKVKR possesses the ability to scavenge ROS under the aforementioned experimental conditions, thereby achieving an antioxidant effect. Therefore, it is a cyclic nonapeptide with antioxidant properties.

[0095] 3. Soothing and anti-inflammatory effects experiment:

[0096] 3.1 Experimental grouping: Blank control group (BC), model control group (NC), and sample group (CP: 10, 100 μg / mL cyclic nonapeptide).

[0097] 3.2 Experimental Methods: HaCaT cells were cultured at 2.0 × 10⁻⁶ cells / mL. 5 Inoculate 100 μg / well of each sample into a 6-well plate and incubate overnight (37°C, 5% CO2). After overnight incubation, add 100 μg / mL of cyclic nonapeptide to the sample groups, while replacing the blank control and negative control groups with complete culture medium. Continue incubation for 24 hours, discard the supernatant, add 500 μL of PBS, and use 200 mJ / cm² water. 2 After UVB irradiation stimulation, PBS was discarded, 2 mL of complete culture medium was added, and the mixture was placed in an incubator overnight. After incubation for 24 hours, the supernatant was collected for the determination of IL-6 and IL-1α kits.

[0098] The specific design scheme is shown in Table 6.

[0099] Table 6. Soothing and Anti-inflammatory Design Scheme

[0100] 3.3 Experimental Results:

[0101] Experimental results of the soothing and anti-inflammatory effects of cyclic nonapeptide are shown in Tables 7-8 and 8. Figure 5 As shown.

[0102] Table 7. Effects of cyclic nonapeptide on intracellular IL-6 in HaCaT cells

[0103]

[0104] Table 8. Effects of cyclic nonapeptide on intracellular IL-1α in HaCaT cells

[0105]

[0106] The research results indicate that the cyclic nonapeptide RGDSQKVKR, at the aforementioned concentrations, reduces the secretion of inflammatory factors IL-6 and IL-1α, thereby achieving a soothing and anti-inflammatory effect. Therefore, it is a cyclic nonapeptide with soothing and anti-inflammatory properties.

[0107] 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. The application of a cyclic nonapeptide with antioxidant and soothing anti-inflammatory effects in the preparation of cosmetics with antioxidant and / or soothing anti-inflammatory effects, characterized in that: The amino acid sequence of the cyclic nonapeptide is: cyclic (arginine-glycine-aspartic acid-serine-glutamine-lysine-valine-lysine-arginine).

2. The application according to claim 1, characterized in that: The structure of the cyclic nonapeptide is as follows: 。 3. The application according to claim 1, characterized in that: The antioxidant mentioned refers to the protection against skin oxidation caused by UVB radiation.

4. The application according to claim 1, characterized in that: The soothing and anti-inflammatory properties are intended to soothe skin inflammation caused by UVB.

5. The application according to claim 1, characterized in that: The effective concentration of the cyclic nonapeptide is 10~100μg / mL.

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