Full-protection polypeptide and preparation method thereof

Abstract

The invention discloses a fully-protected polypeptide and a preparation method thereof, belongs to the technical field of polypeptide compound synthesis, and particularly relates to fully-protected peptide resin prepared through solid-phase synthesis. Carrying out cutting and unitary cyclization on the full-protection peptide resin to obtain unitary cyclic peptide; carrying out binary cyclization treatment on the unitary cyclic peptide to obtain a bicyclic peptide compound Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, and bridging a disulfide bond with Cysamp; the binary cyclization comprises cyclization under the action of iodo methanol and ascorbic acid; or the binary cyclization is cyclized in DMSO (Dimethylsulfoxide). The anti-aging and anti-oxidation bicyclic peptide compound prepared by the preparation method disclosed by the invention has the following beneficial effects of low toxicity, good stability, capability of improving I-type collagen expression, good anti-oxidation effect and good anti-inflammatory effect.

Description

[0001] This invention is a divisional application of CN120289573A, the original application was filed on June 12, 2025, with application number CN202510782311.6, entitled "An Anti-aging and Antioxidant Bicyclic Peptide Compound and Its Preparation Method and Application". Technical Field

[0002] This invention belongs to the field of polypeptide compound synthesis technology, specifically relating to a fully protected polypeptide and its preparation method. Background Technology

[0003] Oligopeptide GHK (tripeptide-1, glycyl-L-histyl-L-lysine) is a natural tripeptide first isolated from human plasma by Pickart et al. in 1973. During extracellular matrix degradation, acidic proteins (Secreted Protein Acidic and Rich in Cysteine, SPARC protein) secreted by cysteine-rich stromal cells cleave and release GHK, which helps 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 decreases to an average of 80 ng / ml by age 60, showing a clear downregulation with age.

[0004] During wound healing, GHK can stimulate the proliferation and migration of fibroblasts, promote the synthesis of collagen and elastin fibers, increase skin toughness and elasticity, and accelerate wound repair and regeneration. GHK has antioxidant properties: it can scavenge free radicals, reduce oxidative stress damage to cells, protect cell integrity and function, and delay skin aging. Simultaneously, GHK also exhibits certain anti-inflammatory effects: it can regulate inflammatory responses, inhibit the release of inflammatory factors, and reduce inflammation-induced tissue damage, thus having a certain therapeutic effect on inflammatory skin diseases such as acne and eczema.

[0005] Based on these functions, GHK, as a cosmetic, possesses certain anti-wrinkle and repairing effects: it can activate the synthesis of extracellular matrix, increase the content of collagen and elastin fibers, reduce the formation of wrinkles, and make the skin firmer and smoother; for damaged skin, such as sunburned or sensitive skin, GHK has a good repairing effect, relieving skin discomfort, promoting the skin's self-repair ability, and enhancing the skin's barrier function. However, it has been reported that GHK has a half-life of only 30 minutes under physiological conditions, and its highly degradable nature affects the sustained effectiveness of its functions. Summary of the Invention

[0006] The purpose of this invention is to provide a bicyclic peptide compound with low toxicity, good stability, and the ability to enhance type I collagen expression, as well as its preparation method and application, which has good antioxidant and anti-inflammatory effects.

[0007] Peptide cyclization and side-chain binding are important techniques in peptide drug development. Cyclic structures reduce the exposure of terminal groups, decreasing the likelihood of unnecessary chemical reactions and thus improving overall chemical stability. They also reduce the recognition and degradation of terminal amino acids by some proteases, enhancing resistance to enzymatic degradation. Due to their higher stability and resistance to enzymatic degradation, cyclized and side-chain-bound peptides can maintain effective concentrations in vivo for longer periods, reducing the frequency of dosing.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for preparing a monocyclic peptide includes: mixing a solid-phase synthetic resin with an amino acid reagent, preparing a fully protected peptide resin by solid-phase synthesis, and then cleaving and cyclizing the fully protected peptide resin to obtain a monocyclic peptide. The structural formula of the monocyclic peptide is as follows: ,in, X1 and X2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; Y1 and Y2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; R1 and R2 are independently selected from Cys or its derivatives.

[0009] Preferably, X1 and X2 are independently selected from either His or Lys; or, Y1 and Y2 are independently selected from either His or Lys; or, R1 and R2 are independently selected from Cys.

[0010] This invention discloses a monocyclic peptide prepared by the above method.

[0011] Preferably, the monocyclic peptide is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0012] This invention discloses a bicyclic peptide compound with the following structural formula: ,in, X1 and X2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; Y1 and Y2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; R1 and R2 are independently selected from Cys or its derivatives; L1 is formed by the bonding of R1 and R2.

[0013] Preferably, R1 and R2 are independently selected from Cys, and L1 is a disulfide bond formed by the bonding of R1 and R2.

[0014] Preferably, the bicyclic peptide compound is Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys).

[0015] This invention discloses a method for preparing bicyclic peptide compounds, including: the method for preparing the monocyclic peptides described above.

[0016] Preferably, a monocyclic peptide is subjected to dicyclic cyclization to obtain a bicyclic peptide compound; The binary cyclization is carried out by cyclization under the action of iodine methanol and ascorbic acid; or, the binary cyclization is carried out in DMSO.

[0017] This invention discloses the use of the above-mentioned monocyclic peptide in the preparation of bicyclic peptide compounds and / or products that enhance type I collagen expression and / or antioxidant and / or anti-inflammatory products.

[0018] This invention discloses the use of the above-mentioned bicyclic peptide compound in the preparation of products that enhance type I collagen expression and / or antioxidant and / or anti-inflammatory products.

[0019] This invention discloses a method for preparing a fully protected peptide resin, comprising: Fmoc-Gly-OH, DIEA, and DCM were mixed and added to CTC resin to obtain the first amino acid coupling resin. The molar amount of Fmoc-Gly-OH used was 100-300% of the reaction sites on the CTC resin, and the molar amount of DIEA used was 100-500% of the molar amount of Fmoc-Gly-OH used. The first amino acid coupling resin was deprotected, and then the activated amino acid reagents were coupled sequentially 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.

[0020] This invention discloses a fully protected peptide resin with the following structure: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin.

[0021] Preferably, the preparation of the fully protected peptide resin includes: the synthesis of the first amino acid coupling resin, the activation of the amino acid reagent, and the synthesis of the fully protected peptide resin.

[0022] Preferably, in the synthesis of the first amino acid coupling resin, Fmoc-Gly-OH is coupled to CTC resin. The coupling is performed using a solid-phase synthesis method. After swelling treatment with DCM, the CTC resin is coupled with Fmoc-Gly-OH in a DIEA-DCM solution.

[0023] Preferably, in the synthesis of the first amino acid coupling resin, Fmoc-Gly-OH is coupled to the CTC resin in a DCM solution containing DIEA.

[0024] Preferably, in the synthesis of the first amino acid coupling resin, under a nitrogen atmosphere, CTC resin is swollen in DCM at 20-40℃ for 5-30 min, DCM is removed by filtration, Fmoc-Gly-OH is added, and then DIEA-DCM solution is added at 10-20℃. The reaction is carried out at 20-30℃ for 2-5 h. After the reaction is completed, methanol is added to end-cap for 10-60 min, and the mixture is filtered and washed to obtain Fmoc-Gly-CTC resin, which is the first amino acid coupling resin.

[0025] 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 reaction sites on the CTC resin.

[0026] More preferably, in the synthesis of the first amino acid coupling resin, the DIEA-DCM solution is a mixture of DIEA and DCM, and the amount of DIEA and DCM used in the DIEA-DCM solution is 0.5-2.5 mmol / mL.

[0027] More preferably, in the synthesis of the first amino acid coupling resin, the amount of DIEA-DCM solution used is measured by DIEA, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used. Methanol is used for end-capping, and an appropriate amount is sufficient.

[0028] Preferably, in the activation of the amino acid reagent, the amino acid reagent is mixed with HOBT and DIC in DMF to obtain the activated amino acid reagent.

[0029] Preferably, in the activation of the amino acid reagent, the amino acid reagent and HOBT are added to DMF, DIC is added at 0-10℃, and activation is carried out for 3-20 minutes to obtain the activated amino acid reagent.

[0030] More preferably, in the activation of the amino acid reagent, the amount of amino acid reagent used is related to the amount of DMF used in the range of 0.1-10 mmol / mL.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 activating amino acid reagent is added, and the reaction is carried out under a nitrogen atmosphere for 10-60 min. After the reaction is completed, the mixture is filtered and washed. Then, the coupling with the activating amino acid reagent is repeated. After the coupling is completed, the mixture is washed and dried to finally obtain H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, which is the fully protected peptide resin.

[0035] More preferably, in the synthesis of the fully protected peptide resin, the coupling sequence of the activating amino acid reagent 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.

[0036] More preferably, in the synthesis of 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 then deprotected at 20-30°C for 10-60 min.

[0037] More preferably, in the synthesis of fully protected peptide resins, each time the amino acid activating reagent is repeatedly coupled, it undergoes deprotection treatment. After coupling, it is filtered and washed using DMF.

[0038] More preferably, in the synthesis of the fully protected peptide resin, the washing after coupling is performed sequentially using methyl tert-ether, tetrahydrofuran, and methyl tert-ether.

[0039] This invention discloses the use of fully protected peptide resin in the preparation of fully protected polypeptides, the structure of which is: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0040] This invention discloses a fully protected polypeptide with the following structure: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0041] This invention discloses a method for preparing a fully protected peptide, comprising: subjecting a fully protected peptide resin to a fully protected cleavage process to obtain a fully protected peptide.

[0042] Preferably, the fully protective cutting process uses a fully protective cutting fluid, which includes a DCM solution containing TFA or a DCM solution containing HFIP.

[0043] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with the cleavage fluid and treated at 20-40℃ for 10-60 min. The resin is removed by filtration, petroleum ether is added to the filtrate for precipitation, the supernatant is removed by centrifugation, the polypeptide is washed with petroleum ether and centrifuged again, and then vacuum dried to obtain the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0044] More preferably, in the preparation of a fully protected peptide, the cutting fluid is a mixture of TFA and DCM, wherein the content of TFA in the cutting fluid is 0.1-5 vol%. The cutting fluid is used to immerse the fully protected peptide resin, and petroleum ether is used for sedimentation and washing in appropriate amounts.

[0045] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with the cleavage solution and 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.

[0046] More preferably, in the preparation of a fully protected peptide, the cutting fluid is a mixture of HFIP and DCM, wherein the content of HFIP in the cutting fluid is 20-40 vol%. The cutting fluid immerses the fully protected peptide resin.

[0047] This invention discloses the use of fully protected polypeptides in the preparation of fully protected cyclic peptides. The structure of the fully protected cyclic peptide is: Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0048] This invention discloses a fully protected cyclic peptide with the following structure: Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0049] This invention discloses a method for preparing a fully protected cyclic peptide, comprising: subjecting a fully protected polypeptide to a fully protected cyclic peptide by a fully protected cyclization process to obtain a fully protected cyclic peptide.

[0050] Preferably, the fully protected cyclization treatment uses HATU-DIEA solution to perform fully protected cyclization of the peptide; or, the fully protected cyclization treatment uses DMF solution containing HOBT, DIC, and NMM to perform fully protected cyclization.

[0051] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide is mixed with DMF to obtain a fully protected peptide DMF solution; HATU, DIEA and DMF are mixed to obtain a HATU-DIEA solution, and the temperature of the HATU-DIEA solution is controlled at 20-30℃. Then, the fully protected peptide DMF solution is added dropwise to the HATU-DIEA solution, and the reaction is carried out for 20-120 min. 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 successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The mixture is dried with 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-).

[0052] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF in the fully protected peptide DMF solution are mixed at a mass-volume ratio of 1g:5-20mL.

[0053] More preferably, in the preparation of the fully protected cyclic peptide, HATU, DIEA and DMF in the HATU-DIEA solution are mixed in a mass-volume ratio of 1g:3-10mL:0.1-5mL.

[0054] More preferably, in the preparation of the fully protected cyclic peptide, the amount of the fully protected peptide DMF solution is based on the fully protected peptide, and the amount of HATU in the HATU-DIEA solution is based on HATU, with the amount of HATU being 40-60 wt% of the fully protected peptide.

[0055] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide is mixed with DMF to obtain a fully protected peptide DMF solution; then HOBT is added, and NMM and DIC are added while controlling the reaction temperature at 0-10℃. The reaction is then stirred at 20-40℃ for 8-24 hours, and the reaction is monitored by HPLC. After the reaction is complete, water is added to precipitate the solid, which is dissolved in ethyl acetate and then washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The solid is dried with 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-).

[0056] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF in the fully protected peptide DMF solution are mixed at a mass-volume ratio of 1g:0.2-2L.

[0057] 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.

[0058] More preferably, in the preparation of the fully protected cyclic peptide, the amount of NMM used is 10-40 wt% of the fully protected peptide.

[0059] 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.

[0060] More preferably, in the preparation of the fully protected cyclic peptide, an appropriate amount of water is used when precipitating the solid, 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 the washing process.

[0061] This invention discloses the use of fully protected cyclic peptides in the preparation of monocyclic peptides, the structure of which is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0062] This invention discloses a monocyclic peptide with the structure: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-), and the structural formula is: .

[0063] This invention discloses a method for preparing a monocyclic peptide, comprising: 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. The fully protected peptide resin was cleaved and cyclized to obtain a cyclic peptide with the structure: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0064] 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.

[0065] Preferably, the cutting fluid for the cutting process is a mixture of TFA and DCM, with the TFA content in the cutting fluid being 0.1-5 vol%; or, the cutting fluid for the cutting process is a mixture of HFIP and DCM, with the HFIP content in the cutting fluid being 20-40 vol%; or, the cyclization is performed by cyclizing and deprotecting H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH obtained from the cutting process.

[0066] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the cleavage solution for 1-4 hours, concentrated, precipitated with ice-cold ether, washed, centrifuged, and evaporated to dryness to obtain the monocyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0067] More preferably, in the preparation of the monocyclic peptide, the cleavage 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.

[0068] More preferably, in the preparation of the monocyclic peptide, the ratio of the amount of fully protected cyclic peptide to the cleavage solution used is 1g: 5-20mL. An appropriate amount of icy diethyl ether is used during sedimentation.

[0069] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the cleavage solution for 1-4 hours. After cleavage without formation, the peptide is settled with ice-cold ether, washed, centrifuged, and evaporated to dryness to obtain the monocyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0070] More preferably, in the preparation of the monocyclic peptide, the cleavage solution is a mixture of TFA, Tis, and H2O, wherein TFA, Tis, and H2O are mixed in a volume ratio of 1:0.01-0.05:0.01-0.05, and the ratio of the amount of fully protected cyclic peptide to the amount of cleavage solution used is 1g:5-20mL. An appropriate amount of ice-cold ether is used during sedimentation.

[0071] This invention discloses the use of a monocyclic peptide in the preparation of a bicyclic peptide compound, the structure of which is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys).

[0072] This invention discloses a bicyclic peptide compound with the structure: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys).

[0073] The bicyclic peptide compound exhibited functions such as inducing extracellular matrix production, anti-inflammatory and antioxidant effects, and demonstrated excellent efficacy in cell function tests.

[0074] The structural formula of the bicyclic peptide compound is: .

[0075] This invention discloses a method for preparing bicyclic peptide compounds, including: the method for preparing the above-mentioned monocyclic peptide.

[0076] Preferably, the monocyclic peptide undergoes dicyclic cyclization to obtain a bicyclic peptide compound, wherein the dicyclic cyclization is performed by iodine methanol and ascorbic acid; or, the dicyclic cyclization is performed in DMSO. The monocyclic peptide is dissolved in an organic solvent and oxidized, causing the two cysteine ​​residues to be linked by disulfide bonds, ultimately yielding the bicyclic peptide compound.

[0077] Preferably, in the preparation of the dicyclic peptide, the monocyclic peptide is mixed with acetic acid solution, filtered to remove insoluble matter, the filtrate is diluted with purified water, and iodine methanol solution is added under stirring at 20-40℃ until the reaction solution turns yellow and does not fade, indicating that the reaction is complete; then ascorbic acid solution is added under stirring for reduction until the reaction solution changes from yellow back to the original milky white, and the color does not change, the reaction is monitored by HPLC, and after the reaction is completed, the reaction solution is filtered through a 0.45μm filter membrane to obtain a crude dicyclic peptide solution, which is purified by reversed-phase C18 chromatography and lyophilized to obtain the dicyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond-bridged Cys&Cys).

[0078] More preferably, in the preparation of the binary cyclic peptide, the acetic acid solution is prepared by mixing acetic acid and pure water at 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.

[0079] 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.

[0080] 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.

[0081] Preferably, in the preparation of the dicyclic peptide, the monocyclic peptide is mixed with DMSO, the insoluble matter is removed by filtration, and the mixture is stirred at 20-40℃ for 4-14 days. The reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45μm filter membrane to obtain a crude dicyclic peptide solution. The solution is purified by reversed-phase C18 chromatography and lyophilized to obtain the dicyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond-bridged Cys & Cys).

[0082] More preferably, in the preparation of the binary cyclic peptide, the amount of the monocyclic peptide used is 5-20 wt% of DMSO.

[0083] This invention discloses the use of bicyclic peptide compounds in the preparation of products that enhance type I collagen expression and / or antioxidant and / or anti-inflammatory products.

[0084] Preferably, the antioxidant product includes the use of products that enhance the expression of the key antioxidant enzyme SOD and / or products that enhance the expression of GSH.

[0085] Preferably, the anti-inflammatory product includes a TNF-α inhibitor and / or an IL-6 inhibitor and / or an IL-8 inhibitor.

[0086] The present invention also discloses a method for preparing the above-mentioned bicyclic peptide compound, comprising: synthesis of a linear polypeptide, cyclization of the linear polypeptide, and secondary cyclization of the cyclized polypeptide.

[0087] Preferably, the method for preparing cyclic peptide compounds specifically includes: A fully protected peptide resin containing a pair of cysteine ​​residues in its sequence was prepared by peptide coupling synthesis. Fully protected cleavage was performed to obtain a linear fully protected peptide. The fully protected peptide was dissolved in an organic solvent and reacted under catalytic conditions to form a fully protected cyclic peptide. The fully protected cyclic peptide was then cleaved to obtain a cyclic peptide. The cyclic peptide was dissolved in an organic solvent and oxidized, allowing the two cysteine ​​residues to be linked by disulfide bonds, ultimately yielding a bicyclic peptide compound.

[0088] The catalytic system for synthesizing binary cyclic peptides in this invention includes DIC, HOBt, HATU, DIEA, and NMM.

[0089] This invention utilizes 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 then cleaved and cyclized to obtain a monocyclic peptide with the structure: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly). -); A monocyclic peptide undergoes dicyclic cyclization to obtain the bicyclic peptide compound Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys). The dicyclic cyclization is performed under the action of iodine methanol and ascorbic acid; or, the dicyclic cyclization is performed in DMSO. Therefore, it has the following beneficial effects: low toxicity, good stability, ability to enhance type I collagen expression, good antioxidant effect, and good anti-inflammatory effect. Therefore, this invention relates to a bicyclic peptide compound with low toxicity, good stability, ability to enhance type I collagen expression, good antioxidant effect, and good anti-inflammatory effect, as well as its preparation method and applications. Attached Figure Description

[0090] Figure 1 This is a liquid chromatogram of a monocyclic peptide compound.

[0091] Figure 2 This is the mass spectrum of a monocyclic peptide compound.

[0092] Figure 3 This is a liquid chromatogram of a bicyclic peptide compound.

[0093] Figure 4 This is the mass spectrum of the bicyclic peptide compound.

[0094] Figure 5 This is a graph showing the cytotoxicity test results of the bicyclic peptide compound.

[0095] Figure 6 The image shows the serum stability test results of the bicyclic peptide compound.

[0096] Figure 7 The graph shows the results of the COL1A expression test.

[0097] Figure 8 This is a graph showing the results of the SOD expression level test.

[0098] Figure 9 This is a graph showing the results of the GSH level test.

[0099] Figure 10 This is a graph showing the expression level of TNF-α.

[0100] Figure 11 A graph showing IL-6 expression levels.

[0101] Figure 12 A graph showing IL-8 expression levels. Detailed Implementation

[0102] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0103] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0104] Example 1: A method for preparing a fully protected peptide resin Synthesis of the first amino acid coupling resin: Under a nitrogen atmosphere, CTC resin was swollen in DCM at 25°C for 10 min. DCM was removed by filtration. Fmoc-Gly-OH was added, followed by DIEA-DCM solution at 10°C. The reaction was carried out at 25°C for 2.5 h. After the reaction was complete, methanol was added for end-capping for 30 min. The mixture was then filtered and washed to obtain Fmoc-Gly-CTC resin, the first amino acid coupling resin. The molar amount of Fmoc-Gly-OH used was 200% of the reaction sites on the CTC resin. The DIEA-DCM solution was a mixture of DIEA and DCM, with a combined DIEA and DCM concentration of 1.25 mmol / mL. The amount of DIEA in the DIEA-DCM solution was measured based on the amount of DIEA, and the molar amount of DIEA was 250% of the molar amount of Fmoc-Gly-OH. Methanol end-capping was used in appropriate amounts. The reaction sites of the CTC resin were 125 mmol.

[0105] Activation of amino acid reagents: Add amino acid reagents and HOBT to DMF, add DIC at 5℃, and activate for 5 min to obtain activated amino acid reagents. The ratio of amino acid reagent to DMF usage is 1 mmol / mL. The molar amount of HOBT and DIC used is 100% of the molar amount of amino acid reagents used. Amino acid reagents include Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

[0106] Synthesis of fully protected peptide resin: The first amino acid coupling resin was mixed with a deprotection solution for deprotection treatment. The liquid was removed by filtration, and an activating amino acid reagent was added. The reaction was carried out under a nitrogen atmosphere for 40 min. After the reaction was completed, the mixture was filtered and washed. The coupling process with the activating amino acid reagent was then repeated. After coupling was completed, the mixture was 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 sequence of the activating amino acid reagent was: 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. In the deprotection process, the resin coupled with the amino acid reagent was first immersed in a 20 vol% Pip / DMF solution and deprotected at 25°C for 30 min. During repeated coupling with the activated amino acid reagent, deprotection was performed in each case. After coupling, the resin was filtered and washed with DMF. The washing after coupling was performed sequentially with methyl tert-ether, tetrahydrofuran, and methyl tert-ether.

[0107] Example 2: A method for preparing a fully protected polypeptide Preparation of the fully protected peptide: The fully protected peptide resin was mixed with the cleavage fluid and treated at 30°C for 30 min. The resin was removed by filtration, and petroleum ether was added to the filtrate for precipitation. The supernatant was removed by centrifugation, followed by washing with petroleum ether and centrifugation. The peptide was then vacuum dried to obtain the fully protected peptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH. The cleavage fluid was a mixture of TFA and DCM, with TFA content of 1 vol%. The fully protected peptide resin was immersed in the cleavage fluid, and the amount of petroleum ether used for precipitation and washing was appropriate. The fully protected peptide resin was prepared by the method described in Example 1. The yield of the fully protected peptide was 104.17%, and the purity was 92.20%.

[0108] Example 3: A method for preparing a fully protected polypeptide Preparation of the fully protected peptide: The fully protected peptide resin was mixed with the cleavage solution and treated at 30°C for 30 min. 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 cleavage solution was a mixture of HFIP and DCM, with HFIP content of 33 vol%. The cleavage solution was used to immerse the fully protected peptide resin. The fully protected peptide resin was prepared by the method described in Example 1. The yield of the fully protected peptide was 117.27%, and the purity was 91.24%.

[0109] Example 4: A method for preparing a fully protected cyclic peptide Preparation of fully protected cyclic peptide: The fully protected peptide was mixed with DMF to obtain a fully protected peptide DMF solution; HATU, DIEA and DMF were mixed to obtain a HATU-DIEA solution, and the temperature of the HATU-DIEA solution was controlled at 25℃. Then, the fully protected peptide DMF solution was added dropwise to the HATU-DIEA solution, and the reaction was carried out for 60 min. The reaction was monitored by HPLC. After the reaction was completed, water and ethyl acetate were added for extraction. The aqueous phase was extracted again with ethyl acetate. The organic phases were combined and then washed successively with saturated sodium bicarbonate solution, water and saturated sodium chloride solution. The mixture was dried with 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-to-volume ratio of 1 g:10 mL. In the HATU-DIEA solution, HATU, DIEA, and DMF were mixed at a mass-to-volume ratio of 1 g:5.88 mL:0.90 mL. The amount of the fully protected peptide in the DMF solution was based on the fully protected peptide, and the amount of HATU in the HATU-DIEA solution was based on HATU, with HATU used at 51 wt% of the fully protected peptide. The fully protected peptide was prepared by the method described in Example 3. The yield of the fully protected cyclic peptide was 84.50%, and the purity was 94.26%.

[0110] Example 5: A method for preparing a fully protected cyclic peptide Preparation of fully protected cyclic peptide: The fully protected peptide was mixed with DMF to obtain a fully protected peptide DMF solution; then HOBT was added, and NMM and DIC were added while controlling the reaction temperature at 0℃. The reaction was then stirred at 25℃ for 16 h, and the reaction was monitored by HPLC. After the reaction was completed, water was added to precipitate the solid, which was dissolved in ethyl acetate and then washed successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The solid was 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-). The fully protected peptide and DMF in the fully protected peptide DMF solution were mixed at a mass-to-volume ratio of 1 g:1 L. The amount of HOBT used was 18.8 wt% of the fully protected peptide, the amount of NMM used was 23.2 wt% of the fully protected peptide, and the amount of DIC used was 14.5 wt% of the fully protected peptide. Appropriate amounts of water and ethyl acetate were added during the precipitation of the solid. Appropriate amounts of saturated sodium bicarbonate solution, water, and saturated sodium chloride solution were also used in the washing process. The fully protected polypeptide 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%.

[0111] Example 6: Preparation method of monocyclic peptide Preparation of the monocyclic peptide: The fully protected cyclic peptide was mixed with the cleavage buffer for 2 hours, concentrated, and then precipitated with ice-cold diethyl ether. After washing and centrifugation, the mixture was evaporated to dryness to obtain the monocyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cleavage buffer was a mixture of TFA, DCM, and EDT, with a volume ratio of 1:0.8:0.2. The ratio of the fully protected cyclic peptide to the cleavage buffer was 1 g:10 mL. An appropriate amount of ice-cold diethyl ether was used during precipitation. The fully protected cyclic peptide was prepared according to the method described in Example 4. The purity of the monocyclic peptide was 60.71%, and the yield was 98.61%.

[0112] The monocyclic peptide prepared in Example 6 was purified by reversed-phase C18 liquid chromatography, and after lyophilization, a pure product with a purity of 96% was obtained, designated PR2495B. The chromatogram of the reversed-phase C18 chromatography purification in Example 6 of this invention is shown below. Figure 1 As shown.

[0113] The mass spectra of the monocyclic peptide obtained by reverse-phase C18 chromatography in Example 6 of this invention are as follows: Figure 2 As shown.

[0114] Example 7: Preparation method of monocyclic peptide Preparation of the monocyclic peptide: The fully protected cyclic peptide was mixed with the cleavage buffer for 2 hours. After cleavage without formation, the peptide was precipitated with ice-cold diethyl ether, washed, centrifuged, and evaporated to dryness to obtain the monocyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cleavage buffer was a mixture of TFA, Tis, and H2O, with a volume ratio of 1:0.026:0.026. The ratio of the fully protected cyclic peptide to the cleavage buffer was 1 g:10 mL. An appropriate amount of ice-cold diethyl ether was used during precipitation. The fully protected cyclic peptide was prepared according to the method described in Example 4. The purity of the monocyclic peptide was 32.26%, and the yield was 126.74%.

[0115] Example 8: Preparation method of binary cyclic peptide Preparation of the bicyclic peptide: The monocyclic peptide was mixed with acetic acid solution, filtered to remove insoluble matter, and the filtrate was diluted with purified water. Iodine methanol solution was added and stirred at 25°C until the reaction solution turned yellow and did not fade, indicating that the reaction was complete. Then, ascorbic acid solution was added and reduced while stirring until the reaction solution changed from yellow to milky white and the color did not change. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was filtered through a 0.45 μm filter membrane to obtain a crude bicyclic peptide solution. The crude solution was purified by reversed-phase C18 chromatography and lyophilized to obtain the bicyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys), i.e., the bicyclic peptide compound. The acetic acid solution was prepared by mixing acetic acid and pure water in a 1:1 volume ratio. The amount of the monocyclic peptide used was 6.8 wt% of the acetic acid solution, and the amount of purified water used for dilution was 1000 wt% of the acetic acid solution. The iodine methanol solution contained 0.1 mol / L of iodine methanol, and the ascorbic acid solution contained 1 wt% of ascorbic acid. The iodine methanol solution and ascorbic acid solution were used in appropriate amounts. The monocyclic peptide was prepared by the method described in Example 6 without purification by reversed-phase C18 liquid chromatography. The purity of the binary cyclic peptide was 98.33%.

[0116] Example 9: Preparation method of binary cyclic peptide Preparation of the bicyclic peptide: A monocyclic peptide was mixed with DMSO, and insoluble matter was removed by filtration. The mixture was stirred at 25°C for 7 days, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was filtered through a 0.45 μm filter membrane to obtain a crude bicyclic peptide solution. This solution was purified by reversed-phase C18 chromatography and lyophilized to obtain the bicyclic peptide Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys), i.e., the bicyclic peptide compound. The amount of monocyclic peptide used was 10 wt% of DMSO. The monocyclic peptide was prepared by the method described in Example 6. The purity of the bicyclic peptide was 98.21%. The bicyclic peptide compound prepared in Example 9 was designated PR2495.

[0117] In Example 9 of this invention, the chromatogram of PR2495 purified by reversed-phase C18 liquid chromatography is as follows: Figure 3 As shown.

[0118] The mass spectra of the bicyclic peptide compound obtained in Example 9 of this invention are as follows: Figure 4 As shown.

[0119] Experimental example: To verify the physicochemical properties and physiological functions of PR2495 and PR2495B of the present invention, the following tests were conducted. The culture medium or cell culture medium used in the tests of the present invention was DMEM medium.

[0120] 1. Cytotoxicity test This invention evaluated the potential toxicity of PR2495 and PR2495B to cells and determined their possible safe concentration ranges.

[0121] In the cytotoxicity test of this invention, HFF-1 cells were seeded into 96-well plates, with 200 μL of culture medium added to each well, containing approximately 10,000 cells / well to ensure a uniform cell density. The plates were incubated overnight at 37°C with 5% CO2 to allow cell adhesion and growth. After 24 hours, the cell culture medium was aspirated, and 200 μL of DMEM culture medium containing different concentrations of test samples was added. The 96-well plates were then returned to the incubator for further incubation. After 24 hours, the absorbance was measured at 490 nm using the MTT assay with a microplate reader to determine whether the test sample exhibited cytotoxicity and its safe concentration range. The test samples were PR2495 or PR2495B, and the different concentrations included 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM. 0 μM was used as the negative control group. Another group was designed with only culture medium and no cells 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%.

[0122] Cytotoxicity test results as follows Figure 5 As shown, epithelial cells treated with different concentrations of PR2495 and PR2495B did not show a significant decrease in cell viability. At a concentration of 500 μM, the cell viability remained at approximately 96% compared to the control group, confirming that both PR2495 and PR2495B have very low cytotoxicity.

[0123] 2. Serum stability test This invention evaluates the degradation of PR2495 and PR2495B in the serum environment to predict their metabolism in vivo.

[0124] The lyophilized sample powder was directly dissolved in 10 vol% fetal bovine serum to a final concentration of 1 mg / mL. The lyophilized oligopeptide GHK was dissolved under the same conditions as a control group. The prepared solutions were aseptically filtered through a 0.22 μM filter, placed in sterile containers, and incubated at 37°C. Samples were taken at set time points: 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. The residual amount of the peptide was determined using analytical high-performance liquid chromatography (HPLC), with the content at time 0 as 100%. The decomposition ratio was calculated by comparing the peak areas at different time points. The calculation formula was: Relative content = (Test sample peak area / Test sample peak area at time 0) × 100%. A curve of residual percentage versus time was plotted, and the half-life was calculated. The test sample was PR2495 or PR2495B.

[0125] Serum stability test results are as follows Figure 6 As shown, the control group's GHK was almost completely degraded in 10 vol% fetal bovine serum for 4 hours, with a calculated degradation half-life of approximately 0.7 hours based on the degradation curve. Under the same conditions, PR2495 was nearly completely degraded in 48 hours, with a calculated half-life of approximately 13 hours based on the degradation curve. Under the same conditions, PR2495B had a half-life of approximately 9 hours, indicating that intramolecular secondary cyclization due to disulfide bonds can enhance the stability of the peptide.

[0126] 3. Test on the increase rate of type I collagen expression This invention investigated the effects of PR2495 and PR2495B on the expression level of COL1A in human fibroblast HFF-1 cells.

[0127] HFF-1 fibroblasts in logarithmic growth phase were seeded at 50,000 cells / well in 6-well plates. 2 mL of culture medium was added to each well, and the plates were incubated overnight to allow cells to adhere and grow for 24 hours. Cells were then divided into groups, with DMEM medium containing the test sample added. A DMEM blank control group and a 100 ng / mL LTF-β1 positive control group were also included. The test samples were GHK, PR2495, or PR2495B at concentrations of 10, 20, and 40 μM.

[0128] After 24 hours of treatment, the culture medium was removed, and RNA was extracted from cells in each well. The Ct value was detected by qRT-PCR to determine the expression level of type I collagen mRNA in HFF-1 cells at different concentrations. The expression level was calculated using the formula: relative RNA expression level = 2ΔΔC(t).

[0129] The t-test was used for comparisons between groups. P < 0.05 was considered statistically significant and marked with * in the figure; P < 0.01 was considered highly significant and marked with ** in the figure.

[0130] The upregulation rate is calculated using the formula: Upregulation rate = (Test group - Blank control group) / Blank control group × 100%.

[0131] The experimental results of type I collagen expression are as follows: Figure 7 As shown, compared with the blank control group, the expression level of COL1A in the positive control group was significantly increased after TGF-β1 treatment, proving that the cell model and qPCR system functioned normally. Compared with the blank control group, PR2495B treatment significantly positively regulated COL1A expression, with an upregulation rate of approximately 32% at 10 μM, approximately 42% at 10 μM, and approximately 49% at 40 μM. Compared with the blank control group, PR2495 treatment significantly positively regulated COL1A expression, with an upregulation rate of approximately 37% at 10 μM, approximately 58% at 10 μM, and approximately 69% at 40 μM. Both PR2495 and PR2495B can promote type I collagen production, and PR2495 has a slightly stronger ability to induce upregulation of expression than PR2495B.

[0132] 4. Antioxidant activity detection Superoxide dismutase (SOD) is a crucial component of the antioxidant enzyme system in biological systems. As a terminal compound, it effectively scavenge superoxide anion free radicals, preventing excessive damage to cells. Glutathione (GSH) undergoes continuous oxidation-reduction within cells. Glutathione peroxidase (GPx) reduces GSH to water and oxygen molecules, while glutathione itself is oxidized to glutathione disulfide (GSSG). GSSG is then rapidly reduced back to GSH by glutathione reductase, maintaining intracellular reduced glutathione levels and mitigating oxidative stress.

[0133] This invention investigates whether PR2495 and PR2495B can enhance cellular antioxidant capacity, whether they affect the expression levels of antioxidant-related enzymes, and ultimately, antioxidant capacity.

[0134] HFF-1 fibroblasts in logarithmic growth phase were seeded at 50,000 cells / well in 6-well plates. 2 mL of culture medium was added to each well, and the plates were incubated overnight to allow cells to adhere and grow for 24 hours. Cells were then divided into groups: a blank control group (no peptides and no oxidative stress) and a negative control group (no peptides). 7 μg / mL vitamin E was used as the antioxidant positive control. Oxidative stress was applied by UVA irradiation, while the blank control group received no irradiation. The UVA irradiation concentration was 30 J / cm². 2Cell supernatant was collected 24 hours later, and SOD and GSH levels were measured according to the instructions of the SOD detection kit and GSH quantification kit. The test samples were GHK, PR2495, or PR2495B, with a concentration of 10 μM.

[0135] The t-test was used for comparisons between groups. P < 0.05 was considered statistically significant and marked with * in the figure; P < 0.01 was considered highly significant and marked with ** in the figure.

[0136] The improvement rate is calculated using the formula: Improvement rate = (Test group - Positive control group) / Positive control group × 100%. The improvement level of SOD and GSH in each group is calculated.

[0137] Antioxidant experiment results are as follows Figure 8-9 As shown, compared with the blank control group, the levels of SOD and GSH in the negative control group were significantly downregulated, while the levels in the positive control group were significantly restored, with SOD levels increasing by approximately 53.7% and GSH levels increasing by approximately 119.7%, demonstrating the effectiveness of oxidative stress stimulation and the normal function of the antioxidant positive control. GHK treatment showed certain antioxidant capacity, with SOD levels increasing by approximately 36.5% and GSH levels increasing by approximately 61.0% at 10 μM compared with the negative control. Compared with the negative control, PR2495B showed an increase of approximately 88.5% in SOD levels and approximately 124% in GSH levels. Compared with the negative control, PR2495 showed an increase of approximately 74.7% in SOD levels and approximately 120% in GSH levels. Both PR2495 and PR2495B can increase the expression level of the key antioxidant enzyme SOD, ultimately increasing the expression level of the key antioxidant substance GSH.

[0138] 5. Anti-inflammatory effect test In inflammatory responses, cytokines such as TNF-α, IL-6, and IL-8 are important regulatory molecules. Through complex network interactions, they jointly participate in the regulation of immune responses, tissue repair, and pathological damage. TNF-α is primarily responsible for activating immune cells, IL-6 promotes the persistence of the immune response, while IL-8 enhances the infiltration and activation of immune cells. These three factors have complex synergistic relationships, collectively forming the core network of the inflammatory response. Under pathological conditions, such as autoimmune diseases or infections, their overexpression can lead to tissue damage and organ dysfunction. This part of the study will compare the ability of GHK with PR2495 or PR2495B to inhibit the production of inflammatory factors in macrophages under lipopolysaccharide (LPS)-induced inflammatory stress.

[0139] RAW264.7 macrophages were seeded at 50,000 cells / well in 6-well plates and cultured for 24 hours. Treatment groups were administered DMEM medium containing the test sample, while a blank control group and a negative control group (without peptides) were also included. 0.01% dexamethasone served as the anti-inflammatory positive control. Two hours after drug administration, 200 μL / well of working solution was added to each well of all groups except the blank control group, and the cells were incubated for another 22 hours. The cell culture supernatant was collected, and TNF-α, IL-6, and IL-8 were analyzed according to the ELISA kit instructions. The test samples were GHK, PR2495, or PR2495B at a concentration of 10 μM. The t-test was used for comparisons between groups. P < 0.05 was considered statistically significant and marked with * in the figure; P < 0.01 was considered highly significant and marked with ** in the figure.

[0140] The inhibition rate is calculated using the formula: Inhibition rate % = (Negative control group - Test group) / Negative control group × 100%.

[0141] Anti-inflammatory test results as follows Figure 10-12 As shown, TNF represents TNF-α, IL-6 represents IL-6, and IL-8 represents IL-8. Compared with the blank control group, the levels of TNF-α, IL-6, and IL-8 in the negative control group were significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the negative control group, the levels of TNF-α, IL-6, and IL-8 in the positive control group were significantly decreased. After treatment, the inhibition rates of TNF-α, IL-6, and IL-8 in the positive control group 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 inhibition rates of approximately 15.1%, 34.2%, and 19.4% for TNF-α, IL-6, and IL-8, respectively; treatment with 10 μM R2495B resulted in inhibition rates of approximately 30.0%, 55.4%, and 33.0% for TNF-α, IL-6, and IL-8, respectively; and treatment with 10 μM PR2495 resulted in inhibition rates of approximately 30.6%, 55.7%, and 36.7% for TNF-α, IL-6, and IL-8, respectively. Both PR2495 and PR2495B showed stronger effects than GHK at the same concentration, demonstrating significant anti-inflammatory capabilities.

[0142] The above embodiments and / or implementation methods are merely illustrative of 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 way. Any person skilled in the art may make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the present invention, but these should still be regarded as technologies or embodiments that are substantially the same as the present invention.

[0143] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above are only preferred embodiments of this application. It should be noted that due to the limitations of textual expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, 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 situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing a monocyclic peptide, including: A fully protected peptide resin was prepared by mixing a solid-phase synthetic resin with an amino acid reagent and then by solid-phase synthesis. The fully protected peptide resin was then cleaved and cyclized to obtain a monocyclic peptide. The structural formula of the monocyclic peptide is as follows: ,in, X1 and X2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; Y1 and Y2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; R1 and R2 are independently selected from Cys or its derivatives.

2. The method for preparing a monocyclic peptide according to claim 1, characterized in that: X1 and X2 are independently selected from either His or Lys; or, Y1 and Y2 are independently selected from either His or Lys; or, R1 and R2 are independently selected from Cys.

3. The monocyclic peptide prepared by the method of claim 1.

4. The monocyclic peptide according to claim 3, characterized in that: The monocyclic peptide is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

5. A bicyclic peptide compound with the following structural formula: ,in, X1 and X2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; Y1 and Y2 are independently selected from any one of His, Lys, Gly, Trp, Phe and Arg; R1 and R2 are independently selected from Cys or its derivatives; L1 is formed by the bonding of R1 and R2.

6. The bicyclic peptide compound according to claim 5, characterized in that: The bicyclic peptide compound is Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bonds bridging Cys & Cys).

7. A method for preparing bicyclic peptide compounds, including: The method for preparing the monocyclic peptide according to claim 1.

8. The method for preparing the bicyclic peptide compound according to claim 7, characterized in that: A monocyclic peptide was subjected to dicyclic cyclization to obtain a bicyclic peptide compound. The binary cyclization is carried out by cyclization under the action of iodine methanol and ascorbic acid; or, the binary cyclization is carried out in DMSO.

9. Use of the monocyclic peptide of any one of claims 3-4 in the preparation of bicyclic peptide compounds and / or products for enhancing type I collagen expression and / or antioxidant and / or anti-inflammatory products.

10. Use of the bicyclic peptide compound of any one of claims 5-6 in the preparation of products that enhance type I collagen expression and / or antioxidant and / or anti-inflammatory products.

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