An anti-aging and antioxidant bicyclic peptide compound and its preparation method and application

Abstract

The present invention discloses a kind of anti-aging and antioxidant bicyclic peptide compound and its preparation method and application, belong to the technical field of polypeptide compound synthesis, specifically to adopt solid phase synthesis to prepare and obtain fully protected peptide resin; The fully protected peptide resin is cut and monocyclicized to obtain monocyclic peptide; The monocyclic peptide is subjected to dicyclic treatment to obtain bicyclic peptide compound Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond bridged Cys & Cys), and dicyclic is cyclized under the action of iodine methanol and ascorbic acid; Or, dicyclic is cyclized in DMSO. The anti-aging and antioxidant bicyclic peptide compound prepared by the present invention has the following beneficial effects: low toxicity, good stability, can improve type I collagen expression, good antioxidant effect, good anti-inflammatory effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide compound synthesis, and particularly relates to an anti-aging and antioxidant bicyclic peptide compound and a preparation method and application thereof. Background Art

[0002] The oligopeptide GHK (tripeptide-1, glycyl-L-histidyl-L-lysine) is a natural tripeptide first isolated from human plasma by Pickart et al. in 1973. During extracellular matrix degradation, GHK is cleaved and released by the secreted protein acidic and rich in cysteine ​​(SPARC) protein, which is secreted by stromal cells. This protein helps with tissue remodeling by regulating angiogenesis. Studies have found that the average blood level of GHK in 20-year-olds is 200 ng / ml, but it drops to an average of 80 ng / ml by age 60, showing a clear downward regulation with aging.

[0003] During wound healing, GHK stimulates the proliferation and migration of fibroblasts, promotes the synthesis of collagen and elastin fibers, increases skin toughness and elasticity, and accelerates wound repair and regeneration. GHK also possesses antioxidant properties: it scavenges free radicals, reduces oxidative stress-induced cell damage, protects cell integrity and function, and delays skin aging. GHK also exhibits anti-inflammatory effects: it regulates inflammatory responses, inhibits the release of inflammatory factors, and reduces tissue damage caused by inflammation. It has a therapeutic effect on inflammatory skin diseases such as acne and eczema.

[0004] Based on these functions, GHK possesses certain anti-wrinkle and repairing properties as a cosmetic: it can activate the synthesis of the extracellular matrix, increase collagen and elastin content, reduce wrinkles, and make the skin firmer and smoother. For damaged skin, such as sunburn and sensitive skin, GHK has a good repairing effect, relieving discomfort, promoting the skin's self-repair ability, and enhancing the skin's barrier function. However, GHK has a reported half-life of only 30 minutes under physiological conditions, and its high degradation rate limits its continued effectiveness. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-aging and antioxidant bicyclic peptide compound with low toxicity, good stability, the ability to enhance type I collagen expression, good antioxidant effect and good anti-inflammatory effect, as well as a preparation method and application thereof.

[0006] Peptide cyclization and side-chain stapling are important tools in the development of peptide drugs. The cyclic structure reduces the exposure of terminal groups and the possibility of unwanted chemical reactions, thereby improving overall chemical stability. It also reduces the recognition and degradation of terminal amino acids by some proteases and enhances resistance to enzymatic degradation. Due to this increased stability and resistance to enzymatic degradation, peptides that have undergone cyclization and side-chain stapling can maintain effective concentrations in the body for longer periods, reducing the frequency of dosing.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0008] The method for preparing a monocyclic peptide comprises: mixing a solid-phase synthetic resin with an amino acid reagent, preparing a fully protected peptide resin by solid-phase synthesis, and performing a cleavage treatment and a monocyclic cyclization on the fully protected peptide resin to obtain a monocyclic peptide. The structural formula of the monocyclic peptide is as follows:

[0009] ,in,

[0010] X1 and X2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg;

[0011] Y1 and Y2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg;

[0012] R1 and R2 are independently selected from Cys or its derivatives.

[0013] Preferably,

[0014] X1 and X2 are independently selected from any one of His and Lys; or,

[0015] Y1 and Y2 are independently selected from any one of His and Lys; or,

[0016] R1 and R2 are independently selected from Cys.

[0017] The invention discloses a monocyclic peptide prepared by the method.

[0018] Preferably, the monocyclic peptide is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0019] The present invention discloses a bicyclic peptide compound, the structural formula of which is as follows:

[0020] ,in,

[0021] X1 and X2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg;

[0022] Y1 and Y2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg;

[0023] R1 and R2 are independently selected from Cys or its derivatives;

[0024] L1 is formed by bonding R1 and R2.

[0025] Preferably, R1 and R2 are independently selected from Cys, and L1 is a disulfide bond formed by the bonding of R1 and R2.

[0026] Preferably, the bicyclic peptide compound is Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bridged Cys & Cys).

[0027] The invention discloses a preparation method of a bicyclic peptide compound, comprising: the preparation method of the monocyclic peptide mentioned above.

[0028] Preferably, the monocyclic peptide is subjected to a dicyclic treatment to obtain a bicyclic peptide compound;

[0029] The dicyclization is carried out under the action of iodine methanol and ascorbic acid; or, the dicyclization is carried out in DMSO.

[0030] The present invention discloses the use of the monocyclic peptide in preparing a bicyclic peptide compound and / or a product for improving type I collagen expression and / or an antioxidant product and / or an anti-inflammatory product.

[0031] The present invention discloses use of the bicyclic peptide compound in preparing products for improving type I collagen expression and / or antioxidant products and / or anti-inflammatory products.

[0032] The present invention discloses a method for preparing a fully protected peptide resin, comprising:

[0033] After mixing Fmoc-Gly-OH, DIEA, and DCM, add CTC resin for coupling to obtain the first amino acid coupling resin; the molar amount of Fmoc-Gly-OH used is 100-300% of the reaction sites on the CTC resin, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used;

[0034] The first amino acid coupling resin is deprotected, and then the activated amino acid reagents are coupled in sequence according to the peptide sequence to obtain a fully protected peptide resin. The structure of the fully protected peptide resin is: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin.

[0035] This invention discloses a fully protected peptide resin having the structure:

[0036] H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin.

[0037] Preferably, the preparation of the fully protected peptide resin includes: synthesis of a first amino acid coupling resin, activation of an amino acid reagent, and synthesis of a fully protected peptide resin.

[0038] Preferably, in the synthesis of the first amino acid coupling resin, Fmoc-Gly-OH is coupled to CTC resin using a solid phase synthesis method. After swelling the CTC resin with DCM, the Fmoc-Gly-OH is coupled in a DIEA-DCM solution.

[0039] Preferably, in the synthesis of the first amino acid coupled resin, Fmoc-Gly-OH is coupled to the CTC resin in a DCM solution containing DIEA.

[0040] Preferably, in the synthesis of the first amino acid coupling resin, under a nitrogen atmosphere, the CTC resin is swelled in DCM at 20-40°C for 5-30 minutes, the DCM is removed by filtration, Fmoc-Gly-OH is added, and then a DIEA-DCM solution is added at 10-20°C, and the reaction is carried out at 20-30°C for 2-5 hours. After the reaction is completed, methanol is added for end-capping for 10-60 minutes, filtered, and washed to obtain Fmoc-Gly-CTC resin, i.e., the first amino acid coupling resin.

[0041] More preferably, in the synthesis of the first amino acid coupling resin, the molar amount of Fmoc-Gly-OH used is 100-300% of the reactive sites on the CTC resin.

[0042] More preferably, in the synthesis of the first amino acid coupling resin, the DIEA-DCM solution is prepared by mixing DIEA and DCM, and the amount of DIEA and DCM used in the DIEA-DCM solution is 0.5-2.5 mmol / mL.

[0043] More preferably, in the synthesis of the first amino acid coupling resin, the amount of DIEA in the DIEA-DCM solution is measured by measuring the DIEA therein, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used. Methanol end-capping can be used in an appropriate amount.

[0044] Preferably, in the activation of the amino acid reagent, the amino acid reagent is mixed with HOBT and DIC in DMF to obtain an activated amino acid reagent.

[0045] Preferably, in the activation of the amino acid reagent, the amino acid reagent and HOBT are added to DMF, and DIC is added at 0-10° C. and activated for 3-20 min to obtain the activated amino acid reagent.

[0046] More preferably, in the activation of the amino acid reagent, the usage ratio of the amino acid reagent to DMF is 0.1-10 mmol / mL.

[0047] More preferably, in the activation of the amino acid reagent, the molar amount of HOBT used is 50-200% of the molar amount of the amino acid reagent used.

[0048] More preferably, in the activation of the amino acid reagent, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used.

[0049] More preferably, in the activation of the amino acid reagent, the amino acid reagent includes Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

[0050] Preferably, in the synthesis of the fully protected peptide resin, the first amino acid coupling resin is mixed with the deprotection solution for deprotection treatment, the liquid is removed by filtration, an activated amino acid reagent is added, and the reaction is carried out under a nitrogen atmosphere for 10-60 minutes. After the reaction is completed, the resin is filtered and washed; then the coupling of the activated amino acid reagent is repeated. After the coupling is completed, the resin is washed and dried to finally obtain H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e., the fully protected peptide resin.

[0051] More preferably, in the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagents is: Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH.

[0052] More preferably, in the synthesis of the fully protected peptide resin, during the deprotection treatment, the resin coupled with the amino acid reagent is first immersed in a 20 vol% Pip / DMF solution and subjected to a deprotection treatment at 20-30° C. for 10-60 min.

[0053] More preferably, in the synthesis of the fully protected peptide resin, when the coupling of the activated amino acid reagent is repeated, the resin is subjected to deprotection treatment. After the coupling is completed, the resin is filtered and washed with DMF.

[0054] More preferably, in the synthesis of the fully protected peptide resin, the washing after coupling is carried out in sequence using tert-methyl ether, tetrahydrofuran and tert-methyl ether.

[0055] The invention discloses use of a fully protected peptide resin in preparing a fully protected polypeptide. The fully protected polypeptide has the structure of H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0056] The present invention discloses a fully protected polypeptide having the structure:

[0057] H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0058] The present invention discloses a method for preparing a fully protected polypeptide, comprising: subjecting a fully protected peptide resin to a fully protected cleavage treatment to obtain the fully protected polypeptide.

[0059] Preferably, the full protective cutting process uses a full protective cutting solution, which includes a DCM solution containing TFA or a DCM solution containing HFIP.

[0060] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with a cutting solution, treated at 20-40° C. for 10-60 min, the resin is removed by filtration, petroleum ether is added to the filtrate for sedimentation, the supernatant is removed by centrifugation, the petroleum ether is washed and centrifuged, and vacuum dried to obtain the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0061] More preferably, in the preparation of a fully protected peptide, the cutting solution is a mixture of TFA and DCM, and the TFA content in the cutting solution is 0.1-5 vol%. The cutting solution is immersed in the fully protected peptide resin, and petroleum ether is used in an appropriate amount for precipitation and washing.

[0062] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with a cutting solution, treated at 20-40° C. for 10-60 min, the resin is removed by filtration, and the filtrate is concentrated and evaporated to dryness to obtain the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0063] More preferably, in the preparation of a fully protected peptide, the cutting solution is prepared by mixing HFIP and DCM, and the HFIP content in the cutting solution is 20-40 vol%. The cutting solution immerses the fully protected peptide resin.

[0064] The invention discloses use of a fully protected polypeptide in preparing a fully protected cyclic peptide. The structure of the fully protected cyclic peptide is: Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0065] The present invention discloses a fully protected cyclic peptide having the structure:

[0066] Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0067] The invention discloses a method for preparing a fully protected cyclic peptide, which comprises: subjecting a fully protected polypeptide to a fully protected cyclization treatment to obtain the fully protected cyclic peptide.

[0068] Preferably, the fully protected cyclization treatment is performed using a HATU-DIEA solution to perform fully protected cyclization on the fully protected polypeptide; or, the fully protected cyclization treatment is performed using a DMF solution containing HOBT, DIC, and NMM.

[0069] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide DMF solution; HATU, DIEA and DMF are mixed to obtain a HATU-DIEA solution, the temperature of the HATU-DIEA solution is controlled to 20-30°C, and then the fully protected polypeptide DMF solution is dropwise added to the HATU-DIEA solution, the reaction is carried out for 20-120 minutes, and the reaction is monitored by HPLC. After the reaction is completed, water and ethyl acetate are added for extraction, the aqueous phase is extracted again with ethyl acetate, the organic phases are combined, and then washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0070] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF are mixed in a DMF solution of the fully protected peptide at a mass volume ratio of 1 g: 5-20 mL.

[0071] More preferably, in the preparation of the fully protected cyclic peptide, HATU, DIEA and DMF are mixed in the HATU-DIEA solution at a mass volume ratio of 1 g: 3-10 mL: 0.1-5 mL.

[0072] More preferably, in the preparation of the fully protected cyclic peptide, the amount of the fully protected polypeptide in the DMF solution is based on the fully protected polypeptide, and the amount of HATU in the HATU-DIEA solution is based on HATU, and the amount of HATU used is 40-60 wt % of the fully protected polypeptide.

[0073] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide DMF solution; then HOBT is added, the reaction solution temperature is controlled to 0-10°C, NMM and DIC are added, and then the reaction is stirred at 20-40°C for 8-24 hours, and the reaction is monitored by HPLC. After the reaction is completed, water is added to precipitate a solid, and the solid is dissolved with ethyl acetate, and then washed with a saturated sodium bicarbonate solution, water, and a saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0074] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF are mixed in a DMF solution of the fully protected peptide at a mass volume ratio of 1 g:0.2-2 L.

[0075] More preferably, in the preparation of the fully protected cyclic peptide, the amount of HOBT used is 10-30 wt % of the fully protected peptide.

[0076] More preferably, in the preparation of the fully protected cyclic peptide, the amount of NMM used is 10-40 wt % of the fully protected polypeptide.

[0077] More preferably, in the preparation of the fully protected cyclic peptide, the amount of DIC used is 5-30 wt % of the fully protected peptide.

[0078] More preferably, in the preparation of the fully protected cyclic peptide, an appropriate amount of water is added when the solid is precipitated, an appropriate amount of ethyl acetate is used to dissolve the solid, and an appropriate amount of saturated sodium bicarbonate solution, water and saturated sodium chloride solution are used in washing.

[0079] The invention discloses use of a fully protected cyclic peptide in preparing a monocyclic peptide. The monocyclic peptide has the structure of Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0080] The present invention discloses a monocyclic peptide having a structure of Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-) and a structural formula of: .

[0081] The present invention discloses a method for preparing a monocyclic peptide, comprising:

[0082] H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e., fully protected peptide resin, was prepared by solid phase synthesis;

[0083] The fully protected peptide resin is cleaved and cyclized to obtain a monocyclic peptide having the structure of Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0084] Preferably, the amino acid reagents for solid phase synthesis include Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH and Fmoc-Gly-OH.

[0085] Preferably, the cleavage treatment solution is a mixture of TFA and DCM, and the TFA content in the cleavage solution is 0.1-5 vol%; or, the cleavage treatment solution is a mixture of HFIP and DCM, and the HFIP content in the cleavage solution is 20-40 vol%; or, the monocyclic cyclization is performed by cyclizing and deprotecting H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH obtained by the cleavage treatment.

[0086] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the cutting solution for 1-4 hours, concentrated, precipitated with glacial ether, washed, centrifuged, and dried to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0087] More preferably, in the preparation of the monocyclic peptide, the cutting solution is a mixture of TFA, DCM and EDT, and the volume ratio of TFA, DCM and EDT in the cutting solution is 1:0.4-1.6:0.05-0.5.

[0088] More preferably, in the preparation of the monocyclic peptide, the ratio of the amount of the fully protected cyclic peptide to the amount of the cutting solution is 1 g: 5-20 mL. During the precipitation, an appropriate amount of glacial ether is used.

[0089] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the cutting solution for 1-4 hours. After unsuccessful cutting, the peptide is precipitated with glacial ether, washed, centrifuged, and dried to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0090] More preferably, in the preparation of the monocyclic peptide, the cutting solution is a mixture of TFA, Tis, and H2O, wherein the TFA, Tis, and H2O are mixed in a volume ratio of 1:0.01-0.05:0.01-0.05, and the amount of the fully protected cyclic peptide to the cutting solution is 1 g: 5-20 mL. During the precipitation, glacial ether is used in an appropriate amount.

[0091] The invention discloses use of a monocyclic peptide in preparing a bicyclic peptide compound. The bicyclic peptide compound has the structure of Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bond bridging Cys and Cys).

[0092] The invention discloses a bicyclic peptide compound having the structure of Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bond bridging Cys and Cys).

[0093] The bicyclic peptide compound exhibits the functions of inducing extracellular matrix production, anti-inflammatory and antioxidant effects, and has excellent efficacy in cell function tests.

[0094] The structural formula of the bicyclic peptide compound is: .

[0095] The invention discloses a preparation method of a bicyclic peptide compound, comprising: a preparation method of the monocyclic peptide.

[0096] Preferably, the monocyclic peptide is subjected to a dicyclic cyclization treatment to obtain a bicyclic peptide compound, wherein the dicyclic cyclization is carried out under the action of iodine methanol and ascorbic acid; or, the dicyclic cyclization is carried out in DMSO. The monocyclic peptide is dissolved in an organic solvent and oxidized to form a disulfide bond between the two cysteines, thereby obtaining a bicyclic peptide compound.

[0097] Preferably, in the preparation of the binary cyclic peptide, the monocyclic peptide is mixed with an acetic acid solution, the insoluble matter is removed by filtration, the filtrate is diluted with purified water, and an iodine-methanol solution is added at 20-40° C. with stirring, and the reaction solution is stirred until it turns yellow and does not fade, and the reaction is judged to be complete; then, an ascorbic acid solution is added with stirring for reduction until the reaction solution turns from yellow back to the original milky white, and stirred until the color does not change, and the reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45 μm filter membrane to obtain a crude binary cyclic peptide solution, which is purified by reverse phase C18 chromatography and lyophilized to obtain the binary cyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond bridged Cys & Cys).

[0098] More preferably, in the preparation of the binary cyclic peptide, the acetic acid solution is prepared by mixing acetic acid and pure water in a volume ratio of 1:0.2-5, and the amount of the monocyclic peptide used is 3-15 wt % of the acetic acid solution.

[0099] More preferably, in the preparation of the binary cyclic peptide, the amount of purified water used for dilution is 500-1500 wt % of the acetic acid solution.

[0100] More preferably, in the preparation of the binary cyclic peptide, the content of iodine methanol in the iodine methanol solution is 0.01-1 mol / L, the content of ascorbic acid in the ascorbic acid solution is 0.05-5 wt %, and the iodine methanol solution and ascorbic acid solution are used in appropriate amounts.

[0101] Preferably, in the preparation of the binary cyclic peptide, the monocyclic peptide is mixed with DMSO, the insoluble matter is removed by filtration, the reaction is stirred at 20-40° C. for 4-14 days, and the reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45 μm filter membrane to obtain a crude binary cyclic peptide solution, which is purified by reverse phase C18 chromatography and lyophilized to obtain the binary cyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond bridged Cys & Cys).

[0102] More preferably, in the preparation of the binary cyclic peptide, the amount of the monocyclic peptide used is 5-20 wt % of DMSO.

[0103] The invention discloses a bicyclic peptide compound for preparing a product for improving type I collagen expression and / or an antioxidant product and / or an anti-inflammatory product.

[0104] Preferably, the antioxidant product includes a product for increasing the expression of the key antioxidant enzyme SOD and / or a product for increasing the expression of GSH.

[0105] Preferably, the anti-inflammatory product comprises a TNF-α inhibiting product and / or an IL-6 inhibiting product and / or an IL-8 inhibiting product.

[0106] The present invention also discloses a method for preparing the bicyclic peptide compound, which comprises: synthesis of a linear polypeptide, cyclization of the linear polypeptide, and secondary cyclization of the cyclized polypeptide.

[0107] Preferably, the preparation method of the cyclic peptide compound specifically comprises:

[0108] A fully protected peptide resin including a pair of cysteines in its sequence is prepared by a polypeptide coupling synthesis method; fully protected cleavage is performed to obtain a chain-like fully protected polypeptide, i.e., a linear polypeptide; the fully protected polypeptide is dissolved in an organic solvent, and stirred under catalytic system conditions to form a fully protected cyclic peptide; the fully protected cyclic peptide is cleaved to obtain a cyclic peptide; the cyclic peptide is dissolved in an organic solvent and oxidized to form a disulfide bond between the two cysteines, thereby finally obtaining a bicyclic peptide compound.

[0109] The catalytic system for synthesizing the binary cyclic peptide comprises DIC, HOBt, HATU, DIEA and NMM.

[0110] The present invention adopts solid phase synthesis to prepare H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e., peptide resin; the peptide resin is cut and cyclized to obtain a monocyclic peptide, and the monocyclic peptide structure is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly -); The monocyclic peptide is subjected to dicyclic treatment to obtain a dicyclic peptide compound Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond bridged Cys & Cys), and the dicyclic is cyclized under the action of iodine methanol and ascorbic acid; or, the dicyclic is cyclized in DMSO, thereby having the following beneficial effects: low toxicity, good stability, can enhance the expression of type I collagen, good antioxidant effect, good anti-inflammatory effect. Therefore, the present invention is a dicyclic peptide compound with low toxicity, good stability, can enhance the expression of type I collagen, good antioxidant effect, good anti-inflammatory effect, and its preparation method and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 This is the liquid chromatogram of a single-ring peptide compound.

[0112] Figure 2 This is the mass spectrum of a single-ring peptide compound.

[0113] Figure 3 This is the liquid chromatogram of the bicyclic peptide compound.

[0114] Figure 4 This is the mass spectrum of the bicyclic peptide compound.

[0115] Figure 5 This is a diagram showing the cytotoxicity test results of the bicyclic peptide compound.

[0116] Figure 6 This is the result of serum stability test of bicyclic peptide compound.

[0117] Figure 7 This is a graph showing the results of the COL1A expression test.

[0118] Figure 8 This is the SOD expression level test result diagram.

[0119] Figure 9 This is a graph showing the GSH level test results.

[0120] Figure 10 Figure 2 is a graph of TNF-α expression levels.

[0121] Figure 11 Graph showing IL-6 expression levels.

[0122] Figure 12 Graph showing IL-8 expression levels. DETAILED DESCRIPTION

[0123] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0124] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0125] Example 1: Preparation of a fully protected peptide resin

[0126] Synthesis of the first amino acid coupling resin: Under a nitrogen atmosphere, CTC resin was swollen in DCM at 25°C for 10 minutes. The DCM was removed by filtration, and Fmoc-Gly-OH was added. DIEA-DCM solution was then added at 10°C and allowed to react for 2.5 hours at 25°C. After completion, methanol was added for end-capping for 30 minutes. The resin was filtered and washed to obtain Fmoc-Gly-CTC resin, the first amino acid coupling resin. The molar amount of Fmoc-Gly-OH was 200% of the reactive sites on the CTC resin. The DIEA-DCM solution was prepared by mixing DIEA and DCM at a combined amount of 1.25 mmol / mL. The amount of DIEA in the DIEA-DCM solution was calculated based on the DIEA content, with the molar amount of DIEA being 250% of the molar amount of Fmoc-Gly-OH. Methanol was used as the end-capping agent, and the amount of CTC resin was 125 mmol.

[0127] Activation of amino acid reagents: Add the amino acid reagent and HOBT to DMF, then add DIC at 5°C for 5 minutes to obtain the activated amino acid reagent. The amount of amino acid reagent to DMF used is 1 mmol / mL. The molar amount of HOBT is 100% of the molar amount of the amino acid reagent, and the molar amount of DIC is 100% of the molar amount of the amino acid reagent. Amino acid reagents include Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

[0128] Synthesis of fully protected peptide resin: The first amino acid coupling resin is mixed with the deprotection solution for deprotection. The liquid is removed by filtration, and the activated amino acid reagent is added. The reaction is carried out under a nitrogen atmosphere for 40 minutes. After completion of the reaction, the mixture is filtered and washed. The coupling with the activated amino acid reagent is then repeated. After completion of the coupling, the mixture is washed and dried to obtain H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e., the fully protected peptide resin. The coupling order of the activated amino acid reagents is: Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-His(Trt)-OH. During the deprotection treatment, the resin coupled with the amino acid reagent was immersed in a 20 vol% Pip / DMF solution and deprotected at 25°C for 30 minutes. Repeated coupling of the activated amino acid reagent was followed by deprotection. After coupling, the resin was filtered and washed with DMF. Washing after coupling was sequentially performed with tertiary methyl ether, tetrahydrofuran, and finally tertiary methyl ether.

[0129] Example 2: A method for preparing a fully protected polypeptide

[0130] Preparation of fully protected polypeptide: The fully protected peptide resin was mixed with a cutting solution, treated at 30°C for 30 minutes, the resin was removed by filtration, petroleum ether was added to the filtrate for sedimentation, the supernatant was removed by centrifugation, the petroleum ether was washed and centrifuged, and vacuum dried to obtain the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH. The cutting solution was prepared by a mixture of TFA and DCM, and the TFA content in the cutting solution was 1 vol%. The cutting solution was used to immerse the fully protected peptide resin, and petroleum ether was used for sedimentation and washing in an appropriate amount. The fully protected peptide resin was prepared by the preparation method of Example 1. The yield of the fully protected polypeptide was 104.17%, and the purity was 92.20%.

[0131] Example 3: A method for preparing a fully protected polypeptide

[0132] Preparation of a fully protected peptide: A fully protected peptide resin was mixed with a cutting solution and treated at 30°C for 30 minutes. The resin was removed by filtration, and the filtrate was concentrated and evaporated to dryness to obtain the fully protected peptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH. The cutting solution was a mixture of HFIP and DCM, with an HFIP content of 33 vol%. The fully protected peptide resin was immersed in the cutting solution. The fully protected peptide resin was prepared according to the preparation method of Example 1. The yield of the fully protected peptide was 117.27%, and the purity was 91.24%.

[0133] Example 4: Preparation of a fully protected cyclic peptide

[0134] Preparation of fully protected cyclic peptide: take a fully protected polypeptide and mix it with DMF to obtain a fully protected polypeptide DMF solution; mix HATU, DIEA and DMF to obtain a HATU-DIEA solution, control the temperature of the HATU-DIEA solution to 25°C, then add the fully protected polypeptide DMF solution dropwise to the HATU-DIEA solution, react for 60 minutes, monitor the reaction by HPLC, and after the reaction is completed, add water and ethyl acetate for extraction, extract the aqueous phase again with ethyl acetate, combine the organic phases, and then wash with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, dry over anhydrous sodium sulfate, filter, and rotary evaporate to obtain the fully protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-). The fully protected peptide was mixed with DMF at a mass-volume ratio of 1 g:10 mL in a DMF solution. HATU, DIEA, and DMF were mixed at a mass-volume ratio of 1 g:5.88 mL:0.90 mL in a HATU-DIEA solution. The DMF solution was measured based on the fully protected peptide, while the HATU-DIEA solution was measured based on HATU, with the amount of HATU used being 51 wt % of the fully protected peptide. The fully protected peptide was prepared according to the method of Example 3. The yield of the fully protected cyclic peptide was 84.50%, and the purity was 94.26%.

[0135] Example 5: Preparation of a fully protected cyclic peptide

[0136] Preparation of a fully protected cyclic peptide: A fully protected peptide was mixed with DMF to obtain a fully protected peptide DMF solution; HOBT was then added, and the reaction solution temperature was controlled at 0°C before NMM and DIC were added. The reaction was then stirred at 25°C for 16 hours and monitored by HPLC. After completion of the reaction, water was added to precipitate a solid, which was dissolved in ethyl acetate and then washed sequentially with a saturated sodium bicarbonate solution, water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-). In the fully protected peptide DMF solution, the fully protected peptide and DMF were mixed at a mass volume ratio of 1 g:1 L. The amount of HOBT, the amount of NMM, and the amount of DIC were 18.8 wt % of the fully protected peptide, 23.2 wt % of the fully protected peptide, and 14.5 wt % of the fully protected peptide. The amount of water added to precipitate the solid was appropriate, as was the amount of ethyl acetate used to dissolve the solid. The saturated sodium bicarbonate solution, water, and saturated sodium chloride solution used in washing were all appropriate. A fully protected peptide was prepared using the method described in Example 3. The yield of the fully protected cyclic peptide was 75.73%, and the purity was 77.86%.

[0137] Example 6: Preparation of a Monocyclic Peptide

[0138] Preparation of a monocyclic peptide: A fully protected cyclic peptide was mixed with a cutting solution for 2 hours. After concentration, the mixture was precipitated with glacial ether, washed, centrifuged, and spin-dried to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cutting solution was a mixture of TFA, DCM, and EDT in a volume ratio of 1:0.8:0.2. The amount of fully protected cyclic peptide to cutting solution was 1 g:10 mL. Glacial ether was used in an appropriate amount during the precipitation. The fully protected cyclic peptide was prepared according to the preparation method of Example 4. The monocyclic peptide had a purity of 60.71% and a yield of 98.61%.

[0139] The monocyclic peptide prepared in Example 6 was purified by reverse phase C18 liquid chromatography and lyophilized to obtain a pure product with a purity of 96%, numbered PR2495B. Figure 1 shown.

[0140] The mass spectrum of the monocyclic peptide obtained by reverse phase C18 chromatography purification in Example 6 of the present invention is as follows: Figure 2 shown.

[0141] Example 7: Preparation of a monocyclic peptide

[0142] Preparation of a monocyclic peptide: A fully protected cyclic peptide was mixed with a cutting solution for 2 hours. If cleavage was unsuccessful, the peptide was precipitated with glacial ether, washed, centrifuged, and spin-dried to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cutting solution was a mixture of TFA, Tis, and H₂O in a volume ratio of 1:0.026:0.026. The amount of fully protected cyclic peptide to cutting solution was 1 g:10 mL. Glacial ether was used in an appropriate amount during the precipitation. The fully protected cyclic peptide was prepared according to the preparation method of Example 4. The monocyclic peptide had a purity of 32.26% and a yield of 126.74%.

[0143] Example 8: Preparation of a binary cyclic peptide

[0144] Preparation of the binary cyclic peptide: a monocyclic peptide is mixed with an acetic acid solution, insoluble matter is removed by filtration, the filtrate is diluted with purified water, and an iodine-methanol solution is added at 25° C. with stirring, and the reaction solution is stirred until it turns yellow and does not fade, which indicates that the reaction is complete; then, an ascorbic acid solution is added with stirring for reduction, until the reaction solution changes from yellow to milky white, and the color is stirred until it does not change. The reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45 μm filter membrane to obtain a crude binary cyclic peptide solution, which is purified by reverse-phase C18 chromatography and lyophilized to obtain the binary cyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond-bridged Cys & Cys), i.e., a bicyclic peptide compound. The acetic acid solution is prepared by mixing acetic acid and pure water in a volume ratio of 1:1. The amount of the monocyclic peptide used is 6.8wt% of the acetic acid solution, the amount of purified water used for dilution is 1000wt% of the acetic acid solution, the content of iodine methanol in the iodine methanol solution is 0.1mol / L, the content of ascorbic acid in the ascorbic acid solution is 1wt%, and the iodine methanol solution and ascorbic acid solution are used in appropriate amounts. The monocyclic peptide was prepared by the preparation method of Example 6 without reverse-phase C18 liquid chromatography purification. The purity of the binary cyclic peptide is 98.33%.

[0145] Example 9: Preparation of a binary cyclic peptide

[0146] Preparation of a dicyclic peptide: A monocyclic peptide was mixed with DMSO, insoluble matter was removed by filtration, and the reaction was stirred at 25°C for 7 days. The reaction was monitored by HPLC. After completion of the reaction, the reaction solution was filtered through a 0.45 μm filter membrane to obtain a crude dicyclic peptide solution. The crude solution was purified by reverse-phase C18 chromatography and lyophilized to obtain the dicyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond bridged Cys & Cys), i.e., a dicyclic peptide compound. The monocyclic peptide was used in an amount of 10 wt % based on DMSO. The monocyclic peptide was prepared by the preparation method of Example 6. The dicyclic peptide had a purity of 98.21%. The dicyclic peptide compound prepared in Example 9 was numbered PR2495.

[0147] The chromatographic results of the purification of PR2495 by reverse phase C18 liquid chromatography in Example 9 of the present invention are as follows: Figure 3 shown.

[0148] The mass spectrum of the bicyclic peptide compound obtained in Example 9 of the present invention is as follows Figure 4 shown.

[0149] Test example:

[0150] To verify the physicochemical properties and physiological functions of PR2495 and PR2495B of the present invention, the following tests were performed. The culture medium or cell culture medium used in the tests of the present invention was DMEM medium.

[0151] 1. Cytotoxicity Test

[0152] The present invention evaluates the potential toxicity of PR2495 and PR2495B to cells and determines their possible safe concentration ranges.

[0153] In the cytotoxicity test of the present invention, HFF-1 cells were seeded into 96-well plates, and 200 μL of culture medium was added to each well, containing about 10,000 cells / well, to ensure consistent cell density. Cultured overnight in an incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall and grow. After 24 hours, the cell culture medium was aspirated, 200 μL of DMEM culture medium containing test samples at different concentrations was added, and the 96-well plates were returned to the incubator for continued culture. After 24 hours, the absorbance value was measured at a wavelength of 490 nm using an enzyme reader by the MTT method to determine whether the test sample had cytotoxicity and its safe concentration range. The test sample was PR2495 or PR2495B, and the different concentrations of the test sample included 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM. 0 μM was the negative control group, and another group containing only culture medium but no cells was designed as the blank control group. Cell viability was calculated as follows: cell viability = (absorbance of test group - absorbance of blank control group / absorbance of negative control group - absorbance of blank control group) × 100%.

[0154] The results of cytotoxicity experiments were as follows Figure 5 As shown, the cell viability of epithelial cells treated with different concentrations of PR2495 and PR2495B did not show a significant decrease. When treated with a concentration of 500 μM, the cell viability remained at about 96% relative to the control group, confirming that both PR2495 and PR2495B have very low cytotoxicity.

[0155] 2. Serum stability test

[0156] The present invention evaluates the decomposition of PR2495 and PR2495B in a serum environment to predict their metabolism in vivo.

[0157] The lyophilized test sample was directly dissolved in 10 vol% fetal bovine serum to a final concentration of 1 mg / mL. Lyophilized oligopeptide GHK was dissolved under the same conditions and served as a control. The prepared solution was sterile-filtered through a 0.22 μM filter, placed into sterile containers, and incubated in a 37°C incubator. Samples were collected at designated time points: 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. The residual peptide content was determined using analytical HPLC. The degradation percentage was calculated by comparing the peak areas at different time points, with the content at time 0 being considered 100%. The calculation formula was: Relative content = (test sample peak area / test sample peak area at time 0) × 100%. The residual percentage was plotted against time to calculate the half-life. The test samples were PR2495 or PR2495B.

[0158] Serum stability test results Figure 6 As shown, the GHK control group was almost completely degraded within 4 hours under 10 vol% fetal bovine serum, with a half-life of approximately 0.7 hours calculated from the degradation curve. Under the same conditions, PR2495 was nearly completely degraded within 48 hours, with a half-life of approximately 13 hours calculated from the degradation curve. Under the same conditions, PR2495B had a half-life of approximately 9 hours, demonstrating that intramolecular secondary cyclization via disulfide bonds can enhance peptide stability.

[0159] 3. Test on the increase rate of type I collagen expression

[0160] The present invention studies the effects of PR2495 and PR2495B on the expression level of COL1A in human fibroblast HFF-1.

[0161] HFF-1 fibroblasts in the logarithmic growth phase were seeded at 50,000 cells / well in a 6-well plate. 2 mL of culture medium was added to each well and the cells were incubated overnight in an incubator to allow the cells to adhere and grow for 24 hours. The cells were divided into groups and treated with DMEM medium containing the test sample. A separate DMEM blank control group and 100 ng / mL TGF-β1 served as a positive control. The test samples were GHK, PR2495, or PR2495B at concentrations of 10, 20, or 40 μM.

[0162] After 24 hours of treatment, the culture medium was removed and RNA was extracted from each well. The Ct values ​​were measured by qRT-PCR to determine the expression levels of type I collagen mRNA in HFF-1 cells at different concentrations. Expression levels were calculated using the formula: relative RNA expression level = 2ΔΔC(t).

[0163] Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, marked with * in the figures; P < 0.01 was considered an extremely significant difference, marked with ** in the figures.

[0164] The expression upregulation rate was calculated according to the formula: upregulation rate = (test group - blank control group) / blank control group × 100%.

[0165] The experimental results of type I collagen expression are as follows Figure 7 As shown, compared with the blank control group, TGF-β1 treatment in the positive control group significantly increased COL1A expression, demonstrating the functional performance of the cell model and qPCR system. Compared with the blank control group, PR2495B treatment significantly positively regulated COL1A expression, with increases of approximately 32% at 10 μM, 42% at 10 μM, and 49% at 40 μM. Compared with the blank control group, PR2495 treatment significantly positively regulated COL1A expression, with increases of approximately 37% at 10 μM, 58% at 10 μM, and 69% at 40 μM. Both PR2495 and PR2495B promoted type I collagen production, with PR2495 exhibiting a slightly stronger ability to induce expression than PR2495B.

[0166] 4. Antioxidant effect detection

[0167] Superoxide dismutase (SOD) is a crucial member of the antioxidant enzyme system in biological systems. As a terminal compound, it effectively scavenges superoxide anion free radicals to prevent excessive cellular damage. Glutathione (GSH) undergoes continuous redox reactions within cells. Through the action of glutathione peroxidase (GPx), glutathione peroxidase reduces superoxide to water and oxygen molecules, while simultaneously being oxidized to glutathione disulfide (GSSG). GSSG is rapidly reduced to GSH by glutathione reductase, maintaining intracellular levels of reduced glutathione and reducing oxidative stress.

[0168] The present invention studies whether PR2495 and PR2495B can enhance the antioxidant capacity of cells, whether they affect the expression levels of antioxidant-related enzymes and the ultimate antioxidant capacity.

[0169] Fibroblasts HFF-1 in the logarithmic growth phase were seeded into 6-well plates at 50,000 cells / well. 2 mL of culture medium was added to each well and placed in an incubator overnight to allow the cells to adhere and grow for 24 hours. The cells were divided into groups and DMEM culture medium containing the test sample was added. A blank control group was also set up. The blank control group did not add polypeptides and was not subjected to oxidative stress. A negative control group was set up. The negative control group did not add polypeptides and 7 μg / mL vitamin E was used as an antioxidant positive control group. Oxidative stress was applied by ultraviolet irradiation UVA, and the blank control group was not irradiated. The ultraviolet irradiation UVA was 30 J / cm 2 After 24 hours, the cell supernatant was collected and the SOD and GSH levels were measured according to the instructions of the SOD detection kit and the GSH quantification kit. The test sample was GHK, PR2495, or PR2495B, and the test sample concentration was 10 μM.

[0170] Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, marked with * in the figures; P < 0.01 was considered an extremely significant difference, marked with ** in the figures.

[0171] According to the formula: improvement rate = (test group - positive control group) / positive control group × 100%, the improvement levels of SOD and GSH in each group were calculated.

[0172] The results of the antioxidant test were as follows Figure 8-9As shown, compared with the blank control, SOD and GSH levels in the negative control group were significantly downregulated, while those in the positive control group significantly increased, with SOD levels increasing by approximately 53.7% and GSH levels by approximately 119.7%. This demonstrates that oxidative stress stimulation is effective and that the antioxidant positive control is functioning properly. GHK treatment demonstrated some antioxidant capacity. Compared with the negative control, SOD levels increased by approximately 36.5% and GSH levels increased by approximately 61.0% at 10 μM. Compared with the negative control, PR2495B increased SOD levels by approximately 88.5% and GSH levels by approximately 124%. Compared with the negative control, PR2495 increased SOD levels by approximately 74.7% and GSH levels by approximately 120%. Both PR2495 and PR2495B increased the expression of the key antioxidant enzyme SOD, ultimately increasing the expression of the key antioxidant substance GSH.

[0173] 5. Anti-inflammatory Effect Detection

[0174] In the inflammatory response, cytokines such as TNF-α, IL-6, and IL-8 are important regulatory molecules. They interact through a complex network and jointly participate in the regulation of immune response, tissue repair, and pathological damage. TNF-α is mainly responsible for activating immune cells, IL-6 promotes the persistence of the immune response, and IL-8 enhances the infiltration and activation of immune cells. There is a complex synergistic relationship between these three factors, which together constitute the core network of the inflammatory response. Under pathological conditions, such as autoimmune diseases or infections, their overexpression may lead to tissue damage and organ dysfunction. This part of the study will compare the ability of GHK and PR2495 or PR2495B to inhibit the production of inflammatory factors in macrophages under inflammatory pressure caused by lipopolysaccharide (LPS) stimulation.

[0175] The macrophage cell line RAW264.7 was seeded into 6-well plates at 50,000 cells / well and cultured for 24 hours. The drugs were divided into groups, and the treatment group was added with DMEM culture medium containing the test sample. A blank control group was also set up, and a negative control group was set up. The negative control group did not add polypeptides, and 0.01% dexamethasone was used as an anti-inflammatory positive control group. After 2 hours of drug administration, 200 μL of LPS working solution was added to each well of the remaining groups except the blank control group, and the cells were placed in an incubator and cultured for 22 hours. The cell culture supernatant was collected, and TNF-α, IL-6 and IL-8 were detected and analyzed according to the operating instructions of the ELISA kit. The test sample was GHK or PR2495 or PR2495B, and the test sample concentration was 10 μM.

[0176] Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, marked with * in the figures; P < 0.01 was considered an extremely significant difference, marked with ** in the figures.

[0177] The inhibition rate was calculated according to the formula: inhibition rate % = (negative control group - test group) / negative control group × 100%.

[0178] The results of anti-inflammatory experiments were as follows Figure 10-12 As shown in the figure, TNF refers to TNF-α, IL6 refers to IL-6, and IL8 refers to IL-8. Compared with the blank control group, the TNF-α, IL-6, and IL-8 levels in the negative control group increased significantly, indicating that the stimulation conditions in this test were effective. Compared with the negative control group, the TNF-α, IL-6, and IL-8 levels in the positive control group decreased significantly. After treatment with the positive control group, the TNF-α, IL-6, and IL-8 inhibition rates were approximately 34.7%, 31.8%, and 48.0%, respectively, indicating that the positive control in this test was effective. Compared with the negative control group, treatment with 10 μM GHK resulted in approximately 15.1%, 34.2%, and 19.4% inhibition rates for TNF-α, IL-6, and IL-8, respectively. Treatment with 10 μM R2495B resulted in approximately 30.0%, 55.4%, and 33.0% inhibition rates for TNF-α, IL-6, and IL-8, respectively. Treatment with 10 μM PR2495 resulted in approximately 30.6%, 55.7%, and 36.7% inhibition rates for TNF-α, IL-6, and IL-8, respectively. At the same concentration, both PR2495 and PR2495B exhibited a stronger effect than GHK, demonstrating significant anti-inflammatory potential.

[0179] The above embodiments and / or implementation methods are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0180] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing a monocyclic peptide, comprising: The solid phase synthetic resin is mixed with an amino acid reagent, and a fully protected peptide resin is prepared by solid phase synthesis. The fully protected peptide resin is cleaved and cyclized to obtain a monocyclic peptide. The structural formula of the monocyclic peptide is as follows: ,in, X1 is selected from His and X2 is selected from Lys; Y1 is selected from Lys and Y2 is selected from His; R1 and R2 are independently selected from Cys.

2. The method for preparing a monocyclic peptide according to claim 1, wherein: In the preparation of the fully protected peptide resin, Fmoc-Gly-OH, DIEA, and DCM were mixed, and then added to CTC resin for coupling to obtain the first amino acid-coupled resin; The first amino acid coupling resin is deprotected, and then the activated amino acid reagents are coupled in sequence according to the peptide sequence to obtain a fully protected peptide resin. The structure of the fully protected peptide resin is: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin.

3. The method for preparing a monocyclic peptide according to claim 2, wherein: The molar amount of Fmoc-Gly-OH used is 100-300% of the reactive sites on the CTC resin, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used.

4. The method for preparing a monocyclic peptide according to claim 2, wherein: In the activation of the amino acid reagent, the amino acid reagent is mixed with HOBT and DIC in DMF to obtain an activated amino acid reagent; the usage ratio of the amino acid reagent and DMF is 0.1-10 mmol / mL, the molar amount of HOBT is 50-200% of the molar amount of the amino acid reagent, and the molar amount of DIC is 50-200% of the molar amount of the amino acid reagent. The amino acid reagent includes Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

5. The method for preparing a monocyclic peptide according to claim 1, wherein: The fully protected peptide resin is subjected to a fully protected cleavage treatment to obtain a fully protected polypeptide, which is H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

6. The method for preparing a monocyclic peptide according to claim 5, wherein: The fully protected cutting process uses a fully protected cutting fluid, which includes a DCM solution containing TFA, and the content of TFA in the fully protected cutting fluid is 0.1-5 vol%; or, the fully protected cutting fluid includes a DCM solution containing HFIP, and the content of HFIP in the fully protected cutting fluid is 20-40 vol%.

7. The method for preparing a monocyclic peptide according to claim 5, wherein: The fully protected polypeptide is subjected to a fully protected cyclization treatment to obtain a fully protected cyclic peptide, which is Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

8. The method for preparing a monocyclic peptide according to claim 7, wherein: In the fully protected cyclization treatment, a HATU-DIEA solution is used to perform fully protected cyclization on the fully protected polypeptide, and HATU, DIEA and DMF in the HATU-DIEA solution are mixed in a mass volume ratio of 1 g: 3-10 mL: 0.1-5 mL; or, in the fully protected cyclization treatment, a DMF solution containing HOBT, DIC and NMM is used for fully protected cyclization, and the amount of HOBT used is 10-30 wt% of the fully protected polypeptide, the amount of NMM used is 10-40 wt% of the fully protected polypeptide, and the amount of DIC used is 5-30 wt% of the fully protected polypeptide.

9. The method for preparing a monocyclic peptide according to claim 7, wherein: The fully protected cyclic peptide is cleaved with a cleavage solution to obtain a monocyclic peptide.

10. The method for preparing a monocyclic peptide according to claim 9, wherein: The cutting solution is a mixture of TFA, DCM and EDT, wherein TFA, DCM and EDT are mixed in a volume ratio of 1:0.4-1.6:0.05-0.

5.

11. The monocyclic peptide prepared by the preparation method according to any one of claims 1 to 10, wherein the monocyclic peptide is Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

12. Use of the monocyclic peptide according to claim 11 in the preparation of a bicyclic peptide compound and / or a product for increasing type I collagen expression and / or an antioxidant product and / or an anti-inflammatory product.

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