CN118126127A

CN118126127A - Repair cyclic peptide and preparation method and application thereof

Info

Publication number: CN118126127A
Application number: CN202410344720.3A
Authority: CN
Inventors: 姬胜利; 郭凯; 董玉香; 张文杰
Original assignee: Reali Tide Biological Technology Weihai Co ltd
Current assignee: Reali Tide Biological Technology Weihai Co ltd
Priority date: 2024-03-25
Filing date: 2024-03-25
Publication date: 2024-06-04

Abstract

The invention belongs to the technical field of biological medicines, and relates to a repair cyclic peptide and a preparation method and application thereof. The cyclic peptide structure is shown in any one of structures I-IV,

Description

Repair cyclic peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a repair cyclic peptide and a preparation method and application thereof.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Tylotoin (amino acid sequence is KCVRQNNKRVCK, see SEQ ID NO.1, dithio cyclic peptide derived from the skin of the salamander), tiger17 (amino acid sequence is WCKPKPKPRCH-NH ₂, see SEQ ID NO.2, dithio cyclic peptide derived from the skin of the rana japonica) and the like have the capability of promoting wound healing, and can be applied to anti-aging, wrinkle removal, scar removal, freckle removal and skin brightening products. However, the inventor researches and discovers that the natural amino acid polypeptide Tylotoin, tiger and the like have the defects of easy enzyme hydrolysis, poor biological stability and the like when being applied to human bodies, and limit the application of the natural amino acid polypeptide to medical products and cosmetics.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide the repair cyclopeptide, and the preparation method and the application thereof. In order to achieve the above object, the present invention provides the following technical solutions:

in a first aspect, a repair cyclic peptide having a cyclic peptide structure as shown in any one of structures I-IV,

The structural I-II cyclic peptide in the repair polypeptide provided by the invention takes Tylotoin as an improved cyclic peptide to

Val-Arg-Gln-Asn-Asn-Orn-Arg-Val fragment is taken as a core, the cyclic peptide with structures III-IV is formed by taking Tiger17 as an improved cyclic peptide and Lys-Pro-Lys-Hyp-Lys-Pro-Arg fragment as a core, and disulfide bonds and 1,2, 3-triazole are respectively connected to form a cyclic peptide structure. According to the research of the invention, the repair cyclic peptide can enhance proliferation and migration of keratinocytes and fibroblasts, has remarkable repair effect, higher plasma stability, strong biological stability, lower cytotoxicity and high biological safety, and is more suitable for preparing medical products and cosmetics.

On the other hand, the preparation method of the repair cyclic peptide comprises the steps of adopting an Fmoc-protected solid-phase polypeptide synthesis method to synthesize linear peptide, and then cyclizing by utilizing the linear peptide to obtain the Fmoc-protected solid-phase polypeptide;

Wherein the structure of the linear peptide is as follows:

In some embodiments, the solid phase carrier employed in the preparation of the cyclic peptides of structures I-II is a CTC resin.

In some embodiments, a solid support amidatable RINK AMIDE AM resin is used in the preparation of the cyclic peptides of structures III-IV.

In some embodiments, the linear peptide is solubilized, the pH is adjusted to be alkaline, oxidation is performed with air, disulfide bonds are generated, and the cyclization reaction is achieved. Specifically, the pH is adjusted to 7.2 to 7.8.

In some embodiments, the linear peptide is synthesized by click chemistry to form a 1,2, 3-triazole group, effecting a cyclization reaction. Specifically, the click chemistry synthesis process is to dissolve the linear peptide, and add copper salt, sodium ascorbate and N, N-diisopropylethylamine to react.

In a third aspect, a composition comprises the repair cyclopeptide and a carrier. The carrier mainly represents auxiliary materials necessary for skin external products and mainly comprises an oil phase, a water phase, an excipient and the like.

In some embodiments, the oil phase comprises animal oil, vegetable oil, fat, higher fatty acid, hydrocarbon, ester, wax, higher alcohol, and the like. Such as mink oil, wheat oil, corn oil, stearic acid, oleic acid, stearyl alcohol, lanolin alcohol, paraffin wax, microcrystalline wax, and the like.

In some embodiments, the excipient comprises a binder, filler, disintegrant, preservative, chelating agent, thickener, gelling agent, emollient, emulsifier, antioxidant, penetration enhancer, stabilizer, and the like.

In some embodiments, the composition is formulated in the form of a solution, suspension, gel, emulsion, cream, paste, wipe, plaster, ointment, patch, foam, plaster, lyophilized powder, or the like.

In a fourth aspect, the use of a repair cyclic peptide or composition as described above for the preparation of a medical product and/or cosmetic.

The cosmetic product of the present invention is, for example, a skin care product.

In particular, the medical product and/or cosmetic of the invention is used for anti-aging, wrinkle removal, scar removal, freckle removal and/or skin lightening.

The beneficial effects of the invention are as follows:

the repair cyclic peptide provided by the invention has obvious repair effect, can enhance proliferation and migration of keratinocytes and fibroblasts, greatly improves stability compared with natural amino acid polypeptides, and is proved to be in accordance with application requirements through safety inspection. Can be applied to the preparation of anti-aging, wrinkle-removing, scar-removing, freckle-removing and skin-brightening products.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a schematic diagram of the synthetic route of Tylotoin in comparative example 1 of the present invention;

FIG. 2 is a mass spectrum of Tylotoin in comparative example 1 of the present invention;

FIG. 3 is a schematic representation of the synthetic route of Tiger17 of comparative example 2 of the present invention;

FIG. 4 is a mass spectrum of Tiger17 in comparative example 2 of the present invention;

FIG. 5 is a schematic diagram showing the synthetic route of the cyclic peptide of structure I in example 1 of the present invention;

FIG. 6 is a mass spectrum of a cyclic peptide of structure I in example 1 of the present invention;

FIG. 7 is a schematic diagram showing the synthetic route of the cyclic peptide of structure II in example 2 of the present invention;

FIG. 8 is a mass spectrum of a cyclic peptide of structure II in example 2 of the present invention;

FIG. 9 is a schematic diagram showing the synthetic route of the cyclic peptide of structure III in example 3 of the present invention;

FIG. 10 is a mass spectrum of a cyclic peptide of structure III in example 3 of the present invention;

FIG. 11 is a schematic diagram showing the synthetic route of the cyclic peptide of structure IV in example 4 of the present invention;

FIG. 12 is a mass spectrum of a cyclic peptide of structure IV in example 4 of the present invention;

FIG. 13 is a graph showing the results of cytotoxicity experiments of the repair cyclic peptides prepared in comparative example 1 and examples 1 to 2;

FIG. 14 is a graph showing the results of cytotoxicity test of the modified cyclic peptides prepared in comparative example 2 and examples 3 to 4;

FIG. 15 is a graph showing the results of experiments on plasma stability of the prosthetic cyclic peptides prepared in comparative example 1 and examples 1 to 2;

FIG. 16 is a graph showing the results of experiments on plasma stability of the modified cyclic peptides prepared in comparative example 2 and examples 3 to 4;

FIG. 17 is a graph showing the results of experiments on the activity of the repair cyclic peptides prepared in comparative example 1 and examples 1 to 2 in promoting fibroblasts, wherein a is HaCaT cells and b is HUVEC cells;

FIG. 18 is a graph showing the results of cell mobility experiments of the repair cyclic peptides prepared in comparative example 1 and examples 1 to 2, wherein a is HaCaT cells and b is HUVEC cells;

FIG. 19 is a graph showing the results of experiments on the fibroblast activation promotion of the modified cyclic peptides prepared in comparative example 2 and examples 3 to 4, wherein a is Tiger17 added, b is modified cyclic peptide added with structure III, and c is modified cyclic peptide added with structure IV;

FIG. 20 is a graph showing the results of cell mobility experiments of the repair cyclic peptides prepared in comparative example 2 and examples 3 to 4, wherein a is Tiger17 added, b is the repair cyclic peptide added with structure III, and c is the repair cyclic peptide added with structure IV.

Detailed Description

The english abbreviations for the substances appearing in the claims and the description of the invention correspond to chinese meanings as follows:

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.

Synthesis of comparative example 1Tylotoin

Linear peptides were synthesized using conventional Fmoc-protected solid-phase polypeptide synthesis, cleaved and cyclized to give Tylotoin, as shown in FIG. 1. The CTC resin is selected as a solid phase carrier, the resin is activated first, 9g of CTC resin (with a load of 1.11 mmol/g) is weighed, the CTC resin is swelled for 1 hour with 100mL of DCM at room temperature, then the swelled liquid is filtered by suction, and the resin is washed three times with 100mL of DMF and 100mL of DCM. 30mmol Fmoc-Lys (Boc) -OH, 30mmol DIEA were dissolved in a small amount of DCM with stirring and added to the reaction flask for 2 hours. The 100mL DIEA:MeOH:DCM (1:1:4) mixed solution is measured for capping, the reaction is carried out for half an hour, the reaction solution is filtered, washed three times by DMF and filtered. 200mL of 20% PIP/DMF was added separately for 5 minutes for the first run and 10 minutes for the second run. Then, ninhydrin test was performed, the solution was dark blue, the resin was yellow, and the reaction was complete. 30mmol of Fmoc-Cys (Trt) -OH, 30mmol of HOBt and 30mmol of DIC are weighed, stirred and dissolved, added into a reaction system, ninhydrin is detected, the solution is light yellow, the resin is colorless, the reaction is complete, the reaction liquid is filtered by suction, the resin is washed three times by DMF, the last time is reserved overnight, 200mL of 20% PIP/DMF is added for Fmoc removal, and the steps are carried out twice, namely, the first time is 5 minutes and the second time is 10 minutes. The ninhydrin test, the solution was dark blue and the resin was colorless, and the resin was washed with DMF until the pH was neutral. The steps are continuously checked, washed and swelled, and condensed Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH, in turn according to the same condensing agent and equivalent weight to synthesize the product

Lys(Boc)-Cys(Trt)-Val-Arg(Pbf)-Gln(Trt)-Asn(Trt)-Asn(Trt)-Lys(Boc)-Arg(Pbf)-Val-Cys(Trt)-Ly s(Boc)-CTC. The resin was contracted with 100mL of methanol, washed three times, and then the resin was drained and dried. The dried resin was placed in a round bottom flask at 1: lysates (TFA: tis: H ₂ O: edt=95:2:2:1) were added in 10 proportions. Stirring and cracking for three hours, spin drying the cracking liquid after cracking is completed, releasing the spin drying liquid into 500mL of methyl tertiary butyl ether, stirring for half an hour to generate a large amount of white precipitate, filtering and washing a filter cake with methyl tertiary butyl ether for multiple times, freeze-drying the filter cake, and purifying by chromatography to obtain the linear peptide

H-Lys-Cys-Val-Arg-GIn-Asn-Asn-Lys-Arg-Val-Cys-Ly-OH. Dissolving linear peptide in water, adjusting the concentration of polypeptide to 1g/L, adjusting the pH value to be alkalescent by sodium hydroxide, adjusting the pH value to be 7.2-7.8, stirring air for oxidation, and judging the reaction end point by mass spectrum. After the reaction was completed, freeze-drying was performed. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 column (50 x 250mm,10 μm), mobile phase a was 0.1% trifluoroacetic acid/water and mobile phase B was 0.1% trifluoroacetic acid/acetonitrile. The ultraviolet detection wavelength is 203nm. 4.69g of pure product is obtained after concentration and freeze-drying, the purity is 97.8 percent, and the yield is 31.8 percent. Tylotoin mass spectra are shown in FIG. 2.

Comparative example 2 Synthesis of Tiger17

The linear peptide Tiger17 is synthesized by adopting a traditional Fmoc-protected solid-phase polypeptide synthesis method, and cyclized after cleavage to obtain the cyclic peptide Tiger17, as shown in figure 3. Resin selection of amidated RINK AMIDE AM resin (load of 0.45 mmol/g), first resin activation, weighing RINK AMIDE AM resin 8.9g in the reaction vessel, resin washing three times with 140mL DCM, adding 200mL DMF solution at room temperature under anhydrous condition for swelling 30min. The resin was checked with ninhydrin and if the solution was colorless, the next reaction was performed. 140mL of 20% PIP/DMF was added separately for 5 minutes first and 10 minutes second to remove Fmoc. Then, ninhydrin test was performed, the solution was dark blue, the resin was yellow, and Fmoc was completely removed. 12.0mmol Fmoc-His (Trt) -OH, 12.0mmol HOBt and 12.0mmol DIC were weighed out and dissolved in a stirring manner, and added to the reaction system. If the solution is light yellow and the resin is colorless, the reaction is complete as determined by ninhydrin. Then condensing Fmoc-Cys(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Trp(Boc)-OH, and coupling according to the same condensing agent and equivalent weight to obtain RINK AMIDE

AM-His (Trt) -Cys (Trt) -Arg (Pbf) -Pro-Lys (Boc) -Pro-Lys (Boc) -Pro-Lys (Boc) -Cys (Trt) -Trp (Boc) -NH ₂. The resin was contracted with 80mL of methanol, washed three times, and then the resin was drained and dried. The dried resin was placed in a round bottom flask at 1: lysates (TFA: tis: H ₂ O: edt=95:2:2:1) were added in 10 proportions. Stirring and cracking for three hours, spin drying the cracking liquid after cracking is completed, releasing the spin drying liquid into 300mL of methyl tertiary butyl ether, stirring for half an hour to generate a large amount of white precipitate, filtering and washing a filter cake with methyl tertiary butyl ether for multiple times, freeze-drying the filter cake, and purifying by chromatography to obtain the linear peptide

H-Trp-Cys-Lys-Pro-Lys-Pro-Lys-Pro-Arg-Cys-His-NH ₂. Dissolving linear peptide in water, adjusting the concentration of polypeptide to 1g/L, adjusting the pH value to be alkalescent by sodium hydroxide, adjusting the pH value to be 7.2-7.8, stirring air for oxidation, and judging the reaction end point by mass spectrum. After the reaction was completed, freeze-drying was performed. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 column (50 x 250mm,10 μm), mobile phase a was 0.1% trifluoroacetic acid/water and mobile phase B was 0.1% trifluoroacetic acid/acetonitrile. The ultraviolet detection wavelength is 203nm. After concentration and freeze-drying, 1.79g of pure product is obtained, the purity is 98.3 percent, and the yield is 32.4 percent. Tiger17 mass spectrum is shown in FIG. 4.

EXAMPLE 1 Synthesis of Structure I cyclic peptides

A linear peptide is synthesized by adopting a traditional Fmoc-protected solid-phase polypeptide synthesis method, and cyclized after cleavage to obtain a structure I cyclic peptide, as shown in figure 5. The CTC resin is selected as a solid phase carrier, the resin is activated first, 9g of CTC resin (with a load of 1.11 mmol/g) is weighed, the CTC resin is swelled for 1 hour with 100mL of DCM at room temperature, then the swelled liquid is filtered by suction, and the resin is washed three times with 100mL of DMF and 100mL of DCM. 30mmol Fmoc-Lys (Boc) -OH, 30mmol DIEA were dissolved in a small amount of DCM with stirring and added to the reaction flask for 2 hours. The 100mL DIEA:MeOH:DCM (1:1:4) mixed solution is measured for capping, the reaction is carried out for half an hour, the reaction solution is filtered, washed three times by DMF and filtered. 200mL of 20% PIP/DMF was added separately for 5 minutes for the first run and 10 minutes for the second run. Then, ninhydrin test was performed, the solution was dark blue, the resin was yellow, and the reaction was complete. 30mmol of Fmoc-Cys (Trt) -OH, 30mmol of HOBt and 30mmol of DIC are weighed, stirred and dissolved, added into a reaction system, ninhydrin is detected, the solution is light yellow, the resin is colorless, the reaction is complete, the reaction liquid is filtered by suction, the resin is washed three times by DMF, the last time is reserved overnight, 200mL of 20% PIP/DMF is added for Fmoc removal, and the steps are carried out twice, namely, the first time is 5 minutes and the second time is 10 minutes. The ninhydrin test, the solution was dark blue and the resin was colorless, and the resin was washed with DMF until the pH was neutral. The steps are continuously checked, washed and swelled, and condensed Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Orn(Boc)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH, in turn according to the same condensing agent and equivalent weight to synthesize the product

Lys(Boc)-Cys(Trt)-Val-Arg(Pbf)-Gln(Trt)-Asn(Trt)-Asn(Trt)-Orn(Boc)-Arg(Pbf)-Val-Cys(Trt)-Ly s(Boc)-CTC. The resin was contracted with 100mL of methanol, washed three times, and then the resin was drained and dried. The dried resin was placed in a round bottom flask at 1: lysates (TFA: tis: H ₂ O: edt=95:2:2:1) were added in 10 proportions. Stirring and cracking for three hours, spin drying the cracking liquid after cracking is completed, releasing the spin drying liquid into 500mL of methyl tertiary butyl ether, stirring for half an hour to generate a large amount of white precipitate, filtering and washing a filter cake with methyl tertiary butyl ether for multiple times, freeze-drying the filter cake, and purifying by chromatography to obtain the linear peptide

H-Lys-Cys-Val-Arg-Gln-Asn-Asn-Orn-Arg-Val-Cys-Lys-OH. Dissolving linear peptide in water, adjusting the concentration of polypeptide to 1g/L, adjusting the pH value to be alkalescent by sodium hydroxide, adjusting the pH value to be 7.2-7.8, stirring air for oxidation, and judging the reaction end point by mass spectrum. After the reaction was completed, freeze-drying was performed. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 column (50 x 250mm,10 μm), mobile phase a was 0.1% trifluoroacetic acid/water and mobile phase B was 0.1% trifluoroacetic acid/acetonitrile. The ultraviolet detection wavelength is 203nm. 4.73g of pure product is obtained after concentration and freeze-drying, the purity is 98.6 percent, and the yield is 32.4 percent. The mass spectrum of the structure I cyclic peptide is shown in figure 6.

EXAMPLE 2 Synthesis of Structure II cyclic peptides

A linear peptide was synthesized using a conventional Fmoc-protected solid-phase polypeptide synthesis, cleaved and cyclized to give the compound of structure II, as shown in FIG. 7. The CTC resin is selected as a solid phase carrier, the resin is activated first, 9g of CTC resin (with a load of 1.11 mmol/g) is weighed, the CTC resin is swelled for 1 hour with 100mL of DCM at room temperature, then the swelled liquid is filtered by suction, and the resin is washed three times with 100mL of DMF and 100mL of DCM. 30mmol Fmoc-Lys (Boc) -OH, 30mmol DIEA were dissolved in a small amount of DCM with stirring and added to the reaction flask for 2 hours. The 100mL DIEA:MeOH:DCM (1:1:4) mixed solution is measured for capping, the reaction is carried out for half an hour, the reaction solution is filtered, washed three times by DMF and filtered. 200mL of 20% PIP/DMF was added separately for 5 minutes for the first run and 10 minutes for the second run. Then, ninhydrin test was performed, the solution was dark blue, the resin was yellow, and the reaction was complete. 30mmol Fmoc-L-DAP (N ₃) -OH, 30mmol HOBt and 30mmol DIC are weighed, stirred and dissolved, added into a reaction system, ninhydrin is detected, the solution is light yellow, the resin is colorless, the reaction is complete, the reaction solution is filtered by suction, the resin is washed three times by DMF, the last time is reserved overnight, 200mL of 20% PIP/DMF is added for Fmoc removal, and the steps are carried out twice, namely, the first time is 5 minutes and the second time is 10 minutes. The ninhydrin test, the solution was dark blue and the resin was colorless, and the resin was washed with DMF until the pH was neutral. The above steps are continuously checked, washed and swelled, and condensed Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Orn(Boc)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-PRA-OH、Fmoc-Lys(Boc)-OH, in turn to synthesize Lys(Boc)-PRA-Val-Arg(Pbf)-Gln(Trt)-Asn(Trt)-Asn(Trt)-Orn(Boc)-Arg(Pbf)-Val-DAP(N₃)-Lys(Boc)-CTC., the resin is shrunk with 100mL of methanol, washed three times, and then the resin is pumped down and dried. The dried resin was placed in a round bottom flask at 1: lysates (TFA: tis: H ₂ O: edt=95:2:2:1) were added in 10 proportions. Stirring and cracking for three hours, spin drying the cracking liquid after cracking is completed, releasing the spin drying liquid into 500mL of methyl tertiary butyl ether, stirring for half an hour to generate a large amount of white precipitate, filtering and washing a filter cake with methyl tertiary butyl ether for multiple times, freeze-drying the filter cake, and purifying by chromatography to obtain the linear peptide

H-Lys-PRA-Val-Arg-GIn-Asn-Asn-Orn-Arg-Val-DAP (N ₃) -Lys-OH. The linear peptide was dissolved in water at a polypeptide concentration of 1g/L, 10.0mmol copper (II) sulfate pentahydrate, 30.0mmol sodium ascorbate and 80.0mmol N, N-diisopropylethylamine were added, and stirred until the mass spectrometry detection reaction was completed. After the reaction was completed, freeze-drying was performed. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 column (50 x 250mm,10 μm), mobile phase a was 0.1% trifluoroacetic acid/water and mobile phase B was 0.1% trifluoroacetic acid/acetonitrile. The ultraviolet detection wavelength is 203nm. Concentrating and freeze-drying to obtain 5.14g of pure product with purity of 98.9% and yield of 35.1%. The mass spectrum of the structure II cyclic peptide is shown in figure 8.

EXAMPLE 3 Synthesis of Structure III cyclic peptides

A linear peptide is synthesized by adopting a traditional Fmoc-protected solid-phase polypeptide synthesis method, and cyclized after cleavage to obtain a structure I cyclic peptide, as shown in figure 9. Resin selection of amidated RINK AMIDE AM resin (load of 0.45 mmol/g), first resin activation, weighing RINK AMIDE AM resin 8.9g in the reaction vessel, resin washing three times with 140mL DCM, adding 200mL DMF solution at room temperature under anhydrous condition for swelling 30min. The resin was checked with ninhydrin and if the solution was colorless, the next reaction was performed. 140mL of 20% PIP/DMF was added separately for 5 minutes first and 10 minutes second to remove Fmoc. Then, ninhydrin test was performed, the solution was dark blue, the resin was yellow, and Fmoc was completely removed. 12.0mmol Fmoc-His (Trt) -OH, 12.0mmol HOBt and 12.0mmol DIC were weighed out and dissolved in a stirring manner, and added to the reaction system. If the solution is light yellow and the resin is colorless, the reaction is complete as determined by ninhydrin. Then condensing Fmoc-Cys(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Hyp(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Trp(Boc)-OH, and coupling according to the same condensing agent and equivalent weight in turn to obtain Rink AmideAM-His(Trt)-Cys(Trt)-Arg(Pbf)-Pro-Lys(Boc)-Hyp(OtBu)-Lys(Boc)-Pro-Lys(Boc)-Cys(Trt)-Trp(Boc)-NH₂., shrinking the resin with 80mL of methanol, washing the resin three times, and then pumping the resin and drying the resin. The dried resin was placed in a round bottom flask at 1: lysates (TFA: tis: H2O: edt=95:2:2:1) were added in 10 proportions. Stirring and cracking for three hours, spin drying the cracking liquid after cracking is completed, releasing the spin drying liquid into 300mL of methyl tertiary butyl ether, stirring for half an hour to generate a large amount of white precipitate, filtering and washing a filter cake with methyl tertiary butyl ether for multiple times, freeze-drying the filter cake, and purifying by chromatography to obtain the linear peptide

H-Trp-Cys-Lys-Pro-Lys-Hyp-Lys-Pro-Arg-Cys-His-NH ₂. Dissolving linear peptide in water, adjusting the concentration of polypeptide to 1g/L, adjusting the pH value to be alkalescent by sodium hydroxide, adjusting the pH value to be 7.2-7.8, stirring air for oxidation, and judging the reaction end point by mass spectrum. After the reaction was completed, freeze-drying was performed. The crude peptide was purified by reverse phase high performance liquid chromatography using a C18 column (50 x 250mm,10 μm), mobile phase a was 0.1% trifluoroacetic acid/water and mobile phase B was 0.1% trifluoroacetic acid/acetonitrile. The ultraviolet detection wavelength is 203nm. After concentration and freeze-drying, 1.66g of pure product is obtained, the purity is 97.9%, and the yield is 29.7%. The mass spectrum of the cyclic peptide of the structure III is shown in figure 10.

EXAMPLE 4 Synthesis of Structure IV cyclic peptides

A linear peptide was synthesized using a conventional Fmoc-protected solid-phase polypeptide synthesis, cleaved and cyclized to give a cyclic peptide of structure II, as shown in FIG. 11. Resin selection of amidated RINK AMIDE AM resin (load of 0.45 mmol/g), first resin activation, weighing RINK AMIDE AM resin 8.9g in the reaction vessel, resin washing three times with 140mL DCM, adding 200mL DMF solution at room temperature under anhydrous condition for swelling 30min. The resin was checked with ninhydrin and if the solution was colorless, the next reaction was performed. 140mL of 20% PIP/DMF was added separately for 5 minutes first and 10 minutes second to remove Fmoc. Then, ninhydrin test was performed, the solution was dark blue, the resin was yellow, and Fmoc was completely removed. 12.0mmol Fmoc-His (Trt) -OH, 12.0mmol HOBt and 12.0mmol DIC were weighed out and dissolved in a stirring manner, and added to the reaction system. If the solution is light yellow and the resin is colorless, the reaction is complete as determined by ninhydrin. Then condensing Fmoc-L-DAP(N₃)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-Hyp(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Pro-OH、Fmoc-Lys(Boc)-OH、Fmoc-PRA-OH、Fmoc-Trp(Boc)-OH, and coupling according to the same condensing agent and equivalent weight to obtain RINK AMIDE

AM-His(Trt)-DAP(N₃)-Arg(Pbf)-Pro-Lys(Boc)-Hyp(Boc)-Lys(Boc)-Pro-Lys(Boc)-PRA-Trp(Boc)-NH₂. The resin was contracted with 80mL of methanol, washed three times, and then the resin was drained and dried. The dried resin was placed in a round bottom flask at 1: lysates (TFA: tis: H ₂ O: edt=95:2:2:1) were added in 10 proportions. Stirring and cracking for three hours, spin drying the cracking liquid after cracking is completed, releasing the spin drying liquid into 300mL of methyl tertiary butyl ether, stirring for half an hour to generate a large amount of white precipitate, filtering and washing a filter cake with methyl tertiary butyl ether for multiple times, freeze-drying the filter cake, and purifying by chromatography to obtain the linear peptide

After completion of the reaction, the crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparative column (50X 250mm,10 μm), mobile phase A was 0.1% trifluoroacetic acid/water solution, mobile phase B was 0.1% trifluoroacetic acid/acetonitrile solution, UV detection wavelength 203nm was concentrated and lyophilized to give 1.94g of pure product, 98.9% purity, 34.8% yield, structure IV cyclic peptide mass spectrum as shown in FIG. 12.

Example 5 cytotoxicity assay

HaCaT cells were selected and cultured in high-sugar DMEM containing 10% calf serum and 1% green-streptomycin diabody. Cells were transferred to an incubator containing 5% carbon dioxide at 37℃overnight for culture. After 24 hours the cell growth state was observed and liquid change or passaging treatment was performed. After the end of the culture, the cell suspension was added to a 96-well cell culture plate, and the cell count per well was 1×10 ⁴. Tylotoin, tiger17, structure I, structure II, structure III and structure IV cyclic peptides (final concentration 1-128 μg/mL) were added to the cell culture at different concentrations after dilution of the double ratio and incubated for 24 hours. After the incubation, 10. Mu.l MTT solution was added to each well and incubation was continued for 4 hours at 37 ℃. The supernatant in the wells was removed, 150. Mu.l DMSO was added, and the mixture was shaken for 10 minutes to dissolve the purple crystals. The light absorption (OD) of each well was measured on a microplate reader at a wavelength of 490nm, and the cell viability was calculated as follows.

Cell viability% = (absorbance of experimental group/absorbance of control group) ×100%

As can be seen from FIG. 13, the cell viability was over 70% at concentrations of the structure I, structure II and Tylotoin cyclic peptides below 64. Mu.g/mL, indicating that the structure I, structure II and Tylotoin cyclic peptides have high biocompatibility for mammalian cells. Wherein, the cytotoxicity of the cyclic peptide with structure I is the lowest, and the biocompatibility with mammals is the highest; when the concentration is 128 mug/mL, the survival rate of the structure I cyclic peptide and the structure II cyclic peptide to cells is still more than 70%, and at the moment, the survival rate of the corresponding cells of Tylotoin cyclic peptides is lower than 70%. This demonstrates that the cyclic peptides of Structure I and Structure II of examples 1 and 2 of the present invention are relatively non-toxic to cells and relatively safe at concentrations below 128 μg/mL. The structure I cyclic peptide and the structure II cyclic peptide have high biocompatibility for mammals at the concentration below 128 mug/mL, and the safety and the biocompatibility are better than Tylotoin.

From fig. 14, it can be seen that both the structure III and structure IV cyclic peptides have higher cell viability than Tiger17 prior to the non-engineering. When the concentrations of Tiger17, the structure III cyclic peptide and the structure IV cyclic peptide are lower than 64 mug/mL, the cell survival rate is above 70%, which shows that the three cyclic peptides have high biocompatibility on mammalian cells. Among them, the structure IV cyclic peptide has the lowest cytotoxicity and highest biocompatibility with mammals. This demonstrates that structure III and structure IV cyclic peptides are relatively non-toxic to cells and relatively safe at concentrations below 64 μg/mL; when the concentration reaches 128 mug/mL, the cytotoxicity is high, and the risk is high. The structure III cyclic peptide and the structure IV cyclic peptide have high biocompatibility to mammals at the concentration of below 64 mug/mL, and the safety and the biocompatibility are improved compared with Tiger17.

Example 6 evaluation of skin safety

Skin closed patch test: ages between 18-50 years, 100 females, 100 males, and a total of 200 people. The male and female animals were divided into 2 groups of 100 men and 100 women. The subjects are all good in skin, free of sensitive skin and free of skin allergy history, no antihistamine is used in the last week or no immunosuppressant is used in the last month, no anti-inflammatory drug is applied to the tested parts in the last two months, and the volunteer selection standard of the subjects is met.

The experimental method comprises the following steps: and respectively diluting the solutions of the structure I cyclic peptide, the structure II cyclic peptide, the structure III cyclic peptide and the structure IV cyclic peptide to 50 mug/mL, dripping the solutions into a qualified patch device, dripping 0.02-0.03 mL, completely pasting the back of a subject, taking notes by a marker pen, and pasting for 48 hours.

According to the plaque test result interpretation and recording method recommended by the international contact dermatitis study group, after 48 hours, the skin of 200 subjects was negative, and no adverse reaction was found. This demonstrates that structure I cyclic peptide, structure II cyclic peptide, structure III cyclic peptide, and structure IV cyclic peptide are safe for skin evaluation.

EXAMPLE 8 plasma stability experiment

Series of experiments were performed on plasma stability of 6 cyclic peptides, respectively. The plasma of mice containing Tylotoin, tiger, structure I, structure II, structure III and structure IV cyclic peptides was diluted (8-week-old male ICR mice, heparin sodium as anticoagulant), and incubated at 37℃with 100. Mu.L samples every 12 hours, and then 36 hours. The sample was precipitated by adding 4% phosphoric acid, vortexed for 3 minutes and centrifuged at 12000rpm for 10 minutes at 4 ℃. The supernatant was taken and assayed in LC-MS/MS.

As can be seen from FIG. 15, the stability of the cyclic peptides of structure I and structure II in plasma was significantly improved compared to Tylotoin. The cyclic peptides of the structure I and the cyclic peptides of the structure II are incubated for 12 hours without obvious degradation, after incubation for 24 hours, the residual amounts of the cyclic peptides of the structure I and the cyclic peptides of the structure II are more than 90%, and the residual amount of Tylotoin is reduced to below 90%; after 36 hours of incubation, tylotoin residual amounts had been reduced to about 76%, while the residual amounts of both structure I and structure II cyclic peptides were above 80%; after 48 hours of incubation, the residual amounts of the polypeptides of the cyclic peptides of structure I and the cyclic peptides of structure II are still more than 74%, and at this time, the residual amount of the polypeptide Tylotoin is reduced to less than 65%. The plasma stability of the cyclic peptides of structure I and structure II is superior to Tylotoin. The structural I cyclic peptide and the structural II cyclic peptide are improved in enzymolysis resistance stability of plasma greatly after structural transformation.

From the graph 16, the stability of the cyclic peptides of structure III and IV in plasma is greatly improved compared with Tiger 17. The cyclic peptides of the structure III and the cyclic peptides of the structure IV are incubated for 12 hours without obvious degradation, after incubation for 24 hours, the residual amounts of the cyclic peptides of the structure III and the cyclic peptides of the structure IV are more than 90%, and the residual amount of Tiger17 is reduced to below 85%; after 36 hours of incubation, the residual amount of Tiger17 had fallen to about 72%, while the residual amounts of both structure III and structure IV cyclic peptides were above 84%. The structural III cyclic peptide and the structural IV cyclic peptide of the invention have greatly improved stability against enzymolysis in blood plasma after structural transformation.

Example 8 promotion of fibroblast proliferation Activity

Tylotin, structure I cyclic peptide and Structure II cyclic peptide

HaCaT cells and HUVEC cells were first cultured in high-sugar DMEM containing 10% calf serum and 1% neo-streptomycin diabody, respectively. Cells were transferred to an incubator containing 5% carbon dioxide at 37℃overnight for culture. The following day the cell growth state was observed and liquid change or passaging treatment was performed. After the end of the culture, the cell suspension was added to a 96-well cell culture plate, and the cell count per well was 1×10 ⁴.

The cells in logarithmic growth phase were taken and the diluted Tylotoin, structure I and structure II cyclic peptides (final concentrations 10, 20, 30, 40 and 50. Mu.g/mL) were added to the cell cultures of the experimental group for 24 hours. The MTT assay detects the proliferation rate of HaCaT cells and HUVEC cells.

Taking cells in logarithmic phase, scratching by a sterile gun, and washing with PBS until the scratches are clear and tidy. Control group was not added Tylotoin, structure I and structure II cyclic peptides, experimental group was added structure I and structure II cyclic peptides (20. Mu.g/mL) to cell culture, and the recordings were observed every 12 hours, and ended after 48 hours. The mobility of HaCaT cells and HSF cells is calculated, and the migration capacity of each cell is analyzed and compared.

From fig. 17, it is clear that the proliferation activity of these two cells is related to the concentration of the structure. Compared with Tylotoin, at the concentration of 50 mug/mL and the time of 24 hours, the proliferation of the structure I cyclic peptide and the structure II cyclic peptide to HaCaT cells is respectively improved to 126 percent and 128 percent, which are both superior to Tylotoin (123 percent); the proliferation of HUVEC cells was increased to 120% and 122%, respectively, both of which were superior to Tylotoin (117%). The cyclic peptides of the structure I and the cyclic peptides of the structure II can promote fibroblast proliferation to different degrees, and the activity is obviously improved after structural transformation.

As can be seen from FIG. 18, the scratch repair effect of the cyclic peptides of structure I and II is better than Tylotoin, and the scratch repair effect of the cyclic peptide of structure II is the best. The repair rates of the structural I cyclic peptide and the structural II cyclic peptide on HaCaT cells are 64 percent and 66 percent respectively at the concentration of 50 mug/mL and the time of 24 hours, which are both superior to Tylotoin (61 percent); after 48 hours, the repair rates of the structural I cyclic peptide and the structural II cyclic peptide on HaCaT cells are respectively 76% and 80% which are better than Tylotoin (70%); the repair rates of the structure I cyclic peptide and the structure II cyclic peptide on HUVEC cells in 24 hours are 67 percent and 68 percent respectively, which are better than Tylotoin (65 percent); after 48 hours, the repair rates of the structure I cyclic peptide and the structure II cyclic peptide on HUVEC cells for 48 hours are 76% and 82%, respectively, which are superior to Tylotoin (74%). The scratch experiment results of the structure I cyclic peptide and the structure II cyclic peptide show that the structure I cyclic peptide and the structure II cyclic peptide can promote migration of HaCaT cells and HUVEC cells to different degrees, and the activity of the structure I cyclic peptide and the structure II cyclic peptide is improved compared with Tylotoin after structural transformation.

Tiger17, structural III cyclic peptide and structural IV cyclic peptide

HaCaT cells and HSF cells were first cultured in high-sugar DMEM containing 10% calf serum and 1% green-streptomycin diabody, respectively. Cells were transferred to an incubator containing 5% carbon dioxide at 37℃overnight for culture. The following day the cell growth state was observed and liquid change or passaging treatment was performed. After the end of the culture, the cell suspension was added to a 96-well cell culture plate, and the cell count per well was 1×10 ⁴.

The log phase cells were taken and the diluted Tiger17, structure III and Structure IV cyclic peptides (final concentrations 2.5, 5, 10 and 20. Mu.g/mL) were added to the experimental cell cultures for 24 hours. MTT assay detects proliferation rates of HaCaT cells and HSF cells.

Taking cells in logarithmic phase, scratching by a sterile gun, and washing with PBS until the scratches are clear and tidy. Control group without Tiger17, structure III and Structure IV cyclopeptides, experimental group added Structure III and Structure IV cyclopeptides (20 μg/mL) to cell cultures, observations were recorded every 12 hours, and ended after 48 hours. The mobility of HaCaT cells and HSF cells is calculated, and the migration capacity of each cell is analyzed and compared.

From fig. 19, it is understood that the proliferation activity of these two cells is related to the concentration of the structure. Compared with a control group, at the concentration of 20 mug/mL and the time of 24 hours, the proliferation of the structure III cyclic peptide and the structure IV cyclic peptide on HaCaT cells is respectively improved by 215 percent and 220 percent, which are both superior to Tiger17 (200 percent); the proliferation of HSF cells is respectively improved by 100% and 105%, and is superior to Tiger17 (95%). The cyclic peptides of the structure III and the cyclic peptides of the structure IV can promote fibroblast proliferation to different degrees, and the activity after structural modification is obviously improved compared with that of unmodified Tiger 17.

As can be seen from fig. 20, the scratch repair effect of the structure III cyclic peptide and the structure IV cyclic peptide is better than Tiger17, and the scratch repair effect of the structure IV cyclic peptide is the best. The repair rates at the concentration of 20 mug/mL and the time of 48 hours are respectively 90% and 92%, which are superior to Tiger17 (88%). The scratch experiment results of the structure III cyclic peptide and the structure IV cyclic peptide show that the structure III cyclic peptide and the structure IV cyclic peptide can promote the migration of HaCaT cells to different degrees, and the activity is obviously improved after the structure transformation.

Example 9 evaluation of cosmetic Effect

At room temperature, butylene glycol with the mass fraction of 5% and PE9010 with the mass fraction of 0.5% are respectively added into sterile water to serve as matrixes, the structure I cyclic peptide and the structure II cyclic peptide are respectively added according to 128 mug/mL to prepare essence, and the structure III cyclic peptide and the structure IV cyclic peptide are respectively added according to 50 mug/mL to prepare essence.

A population of subjects comprising a cyclic peptide of structure I or a cyclic peptide of structure II: between ages 18-50, 42 females and 18 males, 60 total, were divided into 2 groups, half of each male and female. A population of subjects containing a cyclic peptide of structure III and a cyclic peptide of structure IV: between ages 18-50, women 38, men 22, 60 total, are divided into 2 groups. All subjects had freckle, dark yellow and light facial color, wrinkles and skin with postoperative scars, acne pits and other unsightly factors. The testing method comprises the following steps: after the skin of a subject is cleaned, essence containing the cyclic peptides of the structure I, the cyclic peptides of the structure II, the cyclic peptides of the structure III and the cyclic peptides of the structure IV is uniformly smeared on the surface of the skin of the subject, and the using effect is observed and felt. The evaluation and feedback results of the 8-week test are shown in tables 1 to 4.

Table 1 summary of feedback for trial of cyclic peptide essence

Table 2 summary of feedback for trial of cyclic peptide essence II

Table 3 feedback summary table for trial of structure III cyclopeptide serum

Table 4 summary of trial feedback of structure IV cyclopeptide serum

The subjects indicate that the moisture content of the skin can be improved, the moisture retention and the skin elasticity of the skin can be enhanced, and the moisture and the luster of the skin can be improved. The product has effects of whitening skin, removing wrinkle, removing scar and removing mottle. The subject did not develop allergy.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A repair cyclic peptide is characterized in that the cyclic peptide structure is shown in any one of structures I-IV,

2. A method for preparing the repair cyclic peptide according to claim 1, characterized in that the linear peptide is synthesized by adopting Fmoc-protected solid-phase polypeptide synthesis method, and then cyclized by utilizing the linear peptide to obtain the repair cyclic peptide;

Wherein the structure of the linear peptide is as follows:

3. the method for preparing a repair cyclic peptide according to claim 2, wherein in the process of preparing the cyclic peptides of structures I to II, the solid phase carrier used is CTC resin;

Or, in the process of preparing the cyclic peptides of structures III-IV, a solid phase carrier is adopted to amidate RINK AMIDE AM resin.

4. The method for producing a repair cyclic peptide according to claim 2, wherein the linear peptide is dissolved, the pH is adjusted to be alkaline, oxidation is performed by air to generate disulfide bonds, and cyclization reaction is performed; preferably, the pH is adjusted to 7.2-7.8;

Or, the linear peptide is synthesized by click chemistry to form a1, 2, 3-triazole group, so as to realize cyclization reaction; preferably, the click chemistry synthesis is performed by dissolving the linear peptide, adding copper salt, sodium ascorbate and N, N-diisopropylethylamine for reaction.

5. A composition comprising the repair cyclic peptide of claim 1 and a carrier.

6. The composition of claim 5, wherein the carrier comprises an oil phase, an aqueous phase, and/or an excipient.

7. The composition of claim 6, wherein the oil phase comprises one or more of animal oil, vegetable oil, fat, higher fatty acid, hydrocarbon, ester, wax, higher alcohol;

Or, the excipient comprises one or more of binder, filler, disintegrating agent, preservative, chelating agent, thickener, gelatinizer, emollient, emulsifier, antioxidant, penetration enhancer, and stabilizer.

8. The composition of claim 5, wherein the composition is in the form of a solution, suspension, gel, emulsion, cream, paste, liniment, plaster, ointment, patch, foam, plaster, or lyophilized powder.

9. Use of a repair cyclic peptide according to claim 1 or a composition according to any one of claims 5 to 8 for the preparation of a medical product and/or a cosmetic.

10. The use according to claim 9, wherein the medical product and/or cosmetic is for anti-aging, wrinkle-removing, scar-removing, freckle-removing and/or skin lightening.