Full-protection cyclic peptide and preparation method thereof

Abstract

The invention discloses a full-protection cyclic peptide and a preparation method thereof, belongs to the technical field of polypeptide compound synthesis, and particularly relates to a full-protection peptide resin prepared by 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] The application is a divisional application of CN120289573A, the original application date is June 12, 2025, the application number is CN202510782311.6, and the title is an anti-aging and anti-oxidation bicyclic peptide compound and a preparation method and application thereof. TECHNICAL FIELD

[0002] The application belongs to the technical field of polypeptide compound synthesis, and particularly relates to a fully-protected cyclic peptide and a preparation method thereof. BACKGROUND

[0003] 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 the extracellular matrix degradation process, the acidic protein (Secreted Protein Acidic and Rich in Cysteine, SPARC protein) secreted by matrix cells rich in cysteine is cleaved and releases GHK, which helps tissue remodeling by regulating angiogenesis levels. Studies have found that the average blood content of GHK in 20-year-old people is 200 ng / ml, but it decreases to an average of 80 ng / ml by the age of 60, showing a clear down-regulation with age.

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

[0005] Based on the above functions, GHK has certain anti-wrinkle and repair effects as a cosmetic: it can activate the synthesis of extracellular matrix, increase the content of collagen and elastic fibers, reduce the formation of wrinkles, and make the skin more compact and smooth; for damaged skin such as sunburn and sensitive skin, GHK has good repair effects, can relieve skin discomfort, promote the self-repairing ability of the skin, and enhance the barrier function of the skin. However, it is reported that the half-life of GHK under physiological conditions is only 30 minutes, and its easily degradable characteristics affect the sustained exertion of its functions. SUMMARY

[0006] The application aims to provide an anti-aging and anti-oxidation bicyclic peptide compound with small toxicity, good stability, which can improve the expression of type I collagen, has good anti-oxidation and anti-inflammatory effects, and a preparation method and application thereof.

[0007] Polypeptide cyclization and side chain ligation are important means for polypeptide drug development. The cyclic structure reduces the exposure of terminal groups, reduces the possibility of unnecessary chemical reactions, thereby improving the overall chemical stability, and at the same time reduces the recognition and decomposition of some proteases to the terminal amino acids, and enhances the anti-enzymolysis ability. Due to higher stability and anti-enzymolysis ability, the polypeptide after cyclization and side chain ligation can maintain a longer effective concentration in vivo, reducing the frequency of administration.

[0008] The technical scheme adopted by the present application to achieve the above-mentioned purpose is: The preparation method of the monobasic cyclic peptide comprises the following steps: mixing a solid-phase synthesis resin with an amino acid reagent, preparing a fully protected peptide resin by solid-phase synthesis, and cutting and monobasic cyclization of the fully protected peptide resin to obtain a monobasic cyclic peptide. The structural formula of the monobasic cyclic peptide is as follows: , wherein, 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 derivative.

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

[0010] The present application discloses a monobasic cyclic peptide prepared by the above method.

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

[0012] The present application discloses a bicyclic peptide compound, the structural formula of which is as follows: , wherein, 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 derivative; L1 is formed by bonding 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-, disulfide bond bridged Cys&Cys).

[0015] The application discloses a preparation method of a bicyclic peptide compound.

[0016] Preferably, the bicyclic peptide compound is obtained by performing a bicyclization treatment on the monocyclic peptide. The bicyclization is performed under the action of iodomethane and ascorbic acid, or the bicyclization is performed in DMSO.

[0017] The application discloses a use of the monocyclic peptide in preparation of a bicyclic peptide compound and / or an I-type collagen expression product and / or an antioxidant product and / or an anti-inflammatory product.

[0018] The application discloses a use of the bicyclic peptide compound in preparation of an I-type collagen expression product and / or an antioxidant product and / or an anti-inflammatory product.

[0019] The application discloses a preparation method of a fully-protected peptide resin. The Fmoc-Gly-OH, the DIEA and the DCM are mixed, and then the CTC resin is added to obtain a first amino acid coupling resin; the use mole amount of the Fmoc-Gly-OH is 100-300% of the reaction sites on the CTC resin, and the use mole amount of the DIEA is 100-500% of the use mole amount of the Fmoc-Gly-OH. The first amino acid coupling resin is subjected to a deprotection treatment, and then the coupling of the activated amino acid reagent is sequentially performed according to a peptide sequence to obtain the fully-protected peptide resin.

[0020] The application discloses a fully-protected peptide resin. 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 comprises the following steps: synthesis of the first amino acid coupling resin, activation of the amino acid reagent and synthesis of the fully-protected peptide resin.

[0022] 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.

[0023] 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.

[0024] Preferably, in the synthesis of the first amino acid coupled resin, the CTC resin is swelled in DCM at 20-40°C for 5-30 min under a nitrogen atmosphere, the DCM is removed by suction filtration, Fmoc-Gly-OH is added, then a DIEA-DCM solution is added at 10-20°C, and the reaction is carried out at 20-30°C for 2-5 h. After the reaction is completed, methanol is added to cap the ends for 10-60 min, suction filtration is carried out, and washing is performed to obtain the Fmoc-Gly-CTC resin, i.e., the first amino acid coupled resin.

[0025] More preferably, in the synthesis of the first amino acid coupled resin, the molar amount of Fmoc-Gly-OH used is 100-300% of the reactive sites on the CTC resin.

[0026] More preferably, in the synthesis of the first amino acid coupled resin, the DIEA-DCM solution is prepared by mixing DIEA and DCM, and the amounts of DIEA and DCM used in the DIEA-DCM solution are 0.5-2.5 mmol / mL.

[0027] More preferably, in the synthesis of the first amino acid coupled resin, the amount of the DIEA-DCM solution is measured in terms of the amount of DIEA therein, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used. An appropriate amount of methanol is used for capping.

[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°C, and the activation is carried out for 3-20 min to obtain the activated amino acid reagent.

[0030] More preferably, in the activation of the amino acid reagent, the amounts of the amino acid reagent and DMF used are in a ratio 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 comprises Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH.

[0034] Preferably, in the synthesis of the fully-protected peptide resin, the first amino acid coupled resin is mixed with a deprotection solution for deprotection treatment, liquid is removed by suction filtration, the activated amino acid reagent is added, and the reaction is carried out under a nitrogen atmosphere for 10-60 min, after the reaction is completed, suction filtration is carried out, and then washing is carried out; then the above-mentioned coupling of the activated amino acid reagent is repeated, after the coupling is completed, washing and drying are carried out, and finally H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e. the fully-protected peptide resin, is obtained.

[0035] More preferably, in the synthesis of the fully-protected peptide resin, the coupling order of the activated 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 the fully-protected peptide resin, in the deprotection treatment, the resin to which the amino acid reagent is coupled is first immersed in a 20vol% Pip / DMF solution, and deprotection treatment is carried out at 20-30°C for 10-60 min.

[0037] More preferably, in the synthesis of the fully-protected peptide resin, when the coupling of the activated amino acid reagent is repeated, deprotection treatment is carried out, after the coupling is completed, suction filtration and washing are carried out, and DMF is used for washing.

[0038] More preferably, in the synthesis of the fully-protected peptide resin, after the coupling is completed, washing is carried out in turn using methyl tert-butyl ether, tetrahydrofuran and methyl tert-butyl ether.

[0039] The application discloses the use of a fully-protected peptide resin in the preparation of a fully-protected polypeptide, and the structure of the fully-protected polypeptide is H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0040] The application discloses a fully-protected polypeptide, and the structure is: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0041] The application discloses a preparation method of a fully-protected polypeptide, comprising: subjecting a fully-protected peptide resin to a full-protection cleavage treatment to obtain a fully-protected polypeptide.

[0042] Preferably, the full-protection cleavage treatment adopts a full-protection cleavage solution, and the full-protection cleavage solution comprises a TFA-containing DCM solution or a HFIP-containing DCM solution.

[0043] Preferably, in the preparation of the fully-protected polypeptide, the fully-protected peptide resin is mixed with the cleavage solution, and is treated at 20-40 DEG 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 by centrifugation; and vacuum drying is performed 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 the fully-protected polypeptide, the cleavage solution is formed by mixing TFA and DCM, and the content of TFA in the cleavage solution is 0.1-5 vol %; the cleavage solution is used to immerse the fully-protected peptide resin; and the sedimentation and washing of petroleum ether are appropriately used.

[0045] Preferably, in the preparation of the fully-protected polypeptide, the fully-protected peptide resin is mixed with the cleavage solution, and is treated at 20-40 DEG C for 10-60 min; the resin is removed by filtration; the filtrate is concentrated and rotary 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 the fully-protected polypeptide, the cleavage solution is formed by mixing HFIP and DCM, and the content of HFIP in the cleavage solution is 20-40 vol %; and the cleavage solution is used to immerse the fully-protected peptide resin.

[0047] The application discloses a use of a fully-protected polypeptide in preparation of a fully-protected cyclic peptide, and 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] The application discloses a fully-protected cyclic peptide, and the structure is: Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0049] The application discloses a preparation method of a fully-protected cyclic peptide.

[0050] Preferably, the fully-protected polypeptide is subjected to the fully-protected cyclization by using a HATU-DIEA solution in the fully-protected cyclization treatment; or the fully-protected cyclization is performed by using a DMF solution containing HOBT, DIC and NMM in the fully-protected cyclization treatment.

[0051] 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 be 20-30 DEG C, then the fully-protected polypeptide DMF solution is added dropwise into the HATU-DIEA solution, and the reaction is performed for 20-120 min, the reaction is monitored by HPLC, after the reaction is completed, water and ethyl acetate are added for extraction, the water phase is extracted once again by using ethyl acetate, then the organic phases are combined, and then the combined organic phase is sequentially washed by using a saturated sodium bicarbonate solution, water and a saturated sodium chloride solution, dried by using 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 polypeptide and DMF in the fully-protected polypeptide DMF solution are mixed at a mass-volume ratio of 1 g:5-20 mL.

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

[0054] More preferably, in the preparation of the fully-protected cyclic peptide, the amount of the fully-protected polypeptide DMF solution is measured by taking the fully-protected polypeptide as a measurement reference, and the amount of HATU in the HATU-DIEA solution is measured by taking HATU as a measurement reference, and the use amount of HATU is 40-60 wt% of the fully-protected polypeptide.

[0055] Preferably, in the preparation of the fully-protected cyclic peptide, the fully-protected polypeptide is mixed with DMF to obtain a DMF solution of the fully-protected polypeptide; then HOBT is added, the temperature of the reaction solution is controlled at 0-10 DEG C, NMM and DIC are added, then the reaction is stirred at 20-40 DEG C for 8-24 hours, the reaction is monitored by HPLC, after the reaction is completed, water is added to precipitate the solid, the solid is dissolved in ethyl acetate, then the solution is sequentially washed with saturated sodium bicarbonate solution, water and 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-).

[0056] More preferably, in the preparation of the fully-protected cyclic peptide, the fully-protected polypeptide in the DMF solution of the fully-protected polypeptide is mixed with DMF 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 10-30wt% of the fully-protected polypeptide.

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

[0059] More preferably, in the preparation of the fully-protected cyclic peptide, the amount of DIC used is 5-30wt% of the fully-protected polypeptide.

[0060] More preferably, in the preparation of the fully-protected cyclic peptide, the amount of water added when the solid is precipitated is appropriate, the amount of ethyl acetate used for dissolving the solid is appropriate, and the amounts of the saturated sodium bicarbonate solution, water and saturated sodium chloride solution used for washing are appropriate.

[0061] The application discloses the use of a fully-protected cyclic peptide in the preparation of a monomeric cyclic peptide, and the monomeric cyclic peptide has the structure Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0062] The application discloses a monomeric cyclic peptide, which has the structure Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-) and the structural formula: .

[0063] The application discloses a preparation method of a monomeric cyclic peptide, which comprises the following steps: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e., a fully-protected peptide resin, is prepared by solid-phase synthesis; The full-protected peptide resin is cleaved and monomeric cyclized to obtain a monomeric cyclic peptide, and the structure of the monomeric cyclic peptide is 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 cleavage solution for the cleavage treatment is a mixture of TFA and DCM, and the content of TFA in the cleavage solution is 0.1-5 vol %; or, the cleavage solution for the cleavage treatment is a mixture of HFIP and DCM, and the content of HFIP in the cleavage solution is 20-40 vol %; or, the monomeric cyclization is cyclization and deprotection of H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH obtained by the cleavage treatment.

[0066] Preferably, in the preparation of the monomeric cyclic peptide, the full-protected cyclic peptide is mixed with the cleavage solution for 1-4 h, and then concentrated, precipitated with ice ethyl ether, washed, centrifuged, and dried to obtain the monomeric cyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0067] More preferably, in the preparation of the monomeric cyclic peptide, the cleavage solution is a mixture of TFA, Tis and H2O, and the cleavage solution is mixed in a volume ratio of 1:0.01-0.05:0.01-0.05.

[0068] More preferably, in the preparation of the monomeric cyclic peptide, the full-protected cyclic peptide is mixed with the cleavage solution in a ratio of 1 g:5-20 mL, and ice ethyl ether is used in an appropriate amount in the precipitation.

[0069] Preferably, in the preparation of the monomeric cyclic peptide, the full-protected cyclic peptide is mixed with the cleavage solution for 1-4 h, and then concentrated, precipitated with ice ethyl ether, washed, centrifuged, and dried to obtain the monomeric cyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0070] More preferably, in the preparation of the monomeric cyclic peptide, the cleavage solution is a mixture of TFA, Tis and H2O, and the cleavage solution is mixed in a volume ratio of 1:0.01-0.05:0.01-0.05.

[0071] The application discloses purposes of a monomeric cyclic peptide in preparing a bicyclic peptide compound, and the bicyclic peptide compound has a structure of Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, Cys&Cys bridged by a disulfide bond).

[0072] The application discloses a bicyclic peptide compound, which has a structure of Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, Cys&Cys bridged by a disulfide bond).

[0073] The bicyclic peptide compound has the functions of inducing extracellular matrix production and anti-inflammation and anti-oxidation, and has excellent efficacy in cell function tests.

[0074] The bicyclic peptide compound has a structural formula of Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, Cys&Cys bridged by a disulfide bond). .

[0075] The application discloses a preparation method of the bicyclic peptide compound, and the preparation method comprises the preparation method of the monomeric cyclic peptide.

[0076] Preferably, the monomeric cyclic peptide is subjected to a bicyclization treatment to obtain the bicyclic peptide compound, the bicyclization is performed under the action of iodomethane and ascorbic acid; or the bicyclization is performed in DMSO. The monomeric cyclic peptide is dissolved in an organic solvent, is subjected to oxidation, is connected by forming a disulfide bond between two cysteines, and finally the bicyclic peptide compound is obtained.

[0077] Preferably, in the preparation of the bicyclic peptide, the monomeric cyclic peptide is mixed with an acetic acid solution, insoluble substances are removed by filtration, the filtrate is diluted by adding purified water, an iodomethane solution is added under stirring at 20-40 DEG C until the reaction solution turns yellow and does not fade, the reaction is judged to be complete; then an ascorbic acid solution is added under stirring for reduction until the reaction solution turns from yellow to the original milky white color, and the stirring is continued until the color does not change, the reaction is monitored by HPLC, after the reaction is completed, the reaction solution is filtered through a 0.45 mu m filter membrane to obtain a crude bicyclic peptide solution, the solution is purified by reverse phase C18 chromatography, and is freeze-dried to obtain the bicyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, Cys&Cys bridged by a disulfide bond).

[0078] More preferably, in the preparation of the bicyclic peptide, the acetic acid solution is prepared by mixing acetic acid and purified water at a volume ratio of 1:0.2-5, and the monomeric cyclic peptide is used in an amount of 3-15 wt% of the acetic acid solution.

[0079] More preferably, in the preparation of the bicyclic peptide, the purified water is used in an amount of 500-1500 wt% of the acetic acid solution.

[0080] More preferably, in the preparation of the binary cyclic peptide, the content of iodomethanol in the iodomethanol solution is 0.01-1 mol / L, the content of ascorbic acid in the ascorbic acid solution is 0.05-5 wt%, and the iodomethanol solution and the ascorbic acid solution are used in appropriate amounts.

[0081] Preferably, in the preparation of the binary cyclic peptide, the monomeric cyclic peptide is mixed with DMSO, insoluble substances are removed by filtration, and the reaction is stirred at 20-40℃ for 4-14d, 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 binary cyclic peptide solution, which is purified by reverse phase C18 chromatography and freeze-dried to obtain the binary cyclic 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 monomeric cyclic peptide is used in an amount of 5-20wt% of DMSO.

[0083] The application discloses a binary cyclic peptide compound in the preparation of a product for improving expression of collagen type I and / or an antioxidant product and / or an anti-inflammatory product.

[0084] Preferably, the antioxidant product includes the use in a product for improving expression of the key antioxidant enzyme SOD and / or a product for improving expression of GSH.

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

[0086] The application also discloses a preparation method of the binary cyclic peptide compound.

[0087] Preferably, the preparation method of the cyclic peptide compound specifically comprises: The polypeptide coupling method is used to prepare a fully-protected peptide resin including a pair of cysteines in the sequence; the fully-protected cutting is performed to obtain a chain-shaped fully-protected polypeptide, i.e., a straight-chain polypeptide; the fully-protected polypeptide is dissolved in an organic solvent, and is stirred under the condition of a catalytic system to form a fully-protected cyclic peptide; the fully-protected cyclic peptide is cut to obtain a cyclic peptide; and the cyclic peptide is dissolved in an organic solvent, and is oxidized to connect two cysteines through a disulfide bond, so that the binary cyclic peptide compound is finally obtained.

[0088] The catalytic system for synthesizing the binary cyclic peptide includes DIC, HOBt, HATU and 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 Figure 2 is a graph of IL-8 expression levels. DETAILED DESCRIPTION

[0102] The technical solutions in the embodiments of the present application will be apparently and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0103] The concepts involved in the present application will be described below with reference to the drawings. It should be noted that the following descriptions of the concepts are only for the purpose of making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application. Meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0104] Embodiment 1: A preparation method of a fully protected peptide resin Synthesis of the first amino acid coupling resin: under a nitrogen atmosphere, the CTC resin was treated with DCM at 25°C for 10 min, and then filtered to remove the DCM. Fmoc-Gly-OH was added, followed by the addition of a DIEA-DCM solution at 10°C. The reaction was carried out at 25°C for 2.5 h. After the reaction was completed, methanol was added for capping for 30 min. The mixture was filtered and washed to obtain the Fmoc-Gly-CTC resin, i.e., the first amino acid coupling resin. The use amount of Fmoc-Gly-OH was 200% of the reaction sites on the CTC resin. The DIEA-DCM solution was prepared by mixing DIEA and DCM. The use amount of DIEA and DCM in the DIEA-DCM solution was 1.25 mmol / mL. The use amount of the DIEA-DCM solution was measured based on the amount of DIEA. The use amount of DIEA was 250% of the use amount of Fmoc-Gly-OH. The amount of methanol used for capping was appropriate. The reaction sites of the CTC resin were 125 mmol.

[0105] Activation of the amino acid reagent: the amino acid reagent and HOBT were added to DMF, and DIC was added at 5°C. The mixture was activated for 5 min to obtain the activated amino acid reagent. The use amount of the amino acid reagent and DMF was 1 mmol / mL. The use amount of HOBT was 100% of the use amount of the amino acid reagent. The use amount of DIC was 100% of the use amount of the amino acid reagent. The amino acid reagent included Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

[0106] Synthesis of the fully protected peptide resin: the first amino acid coupled resin was mixed with the deprotection solution for deprotection treatment, and the liquid was removed by suction filtration. The activated amino acid reagent was added, and the reaction was carried out under a nitrogen atmosphere for 40 min. After the reaction was completed, suction filtration and washing were carried out. Then the above-mentioned coupling of the activated amino acid reagent was repeated. After the coupling was completed, washing and drying were carried out, and finally H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, i.e. the fully protected peptide resin, was obtained. The coupling order of the activated 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 treatment, the resin coupled with the amino acid reagent was first immersed in a 20vol% Pip / DMF solution for deprotection treatment at 25°C for 30 min. When the coupling of the activated amino acid reagent was repeated, deprotection treatment was carried out. After the coupling was completed, suction filtration and washing were carried out, and washing was carried out using DMF. Washing after the coupling was completed was carried out using methyl tert-butyl ether, tetrahydrofuran and methyl tert-butyl ether in sequence.

[0107] Example 2: A method for preparing a fully protected polypeptide Preparation of the fully protected polypeptide: the fully protected peptide resin was mixed with the cleavage solution, and was treated at 30°C for 30 min. The resin was removed by filtration, and petroleum ether was added to the filtrate for sedimentation. The supernatant was removed by centrifugation, and the petroleum ether was washed by centrifugation. Vacuum drying was carried out, and the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH was obtained. The cleavage solution was obtained by mixing TFA and DCM, and the content of TFA in the cleavage solution was 1vol%. The fully protected peptide resin was immersed in the cleavage solution, and the sedimentation and washing of the petroleum ether were carried out using 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%.

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

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

[0110] Example 5: A preparation method of a fully protected cyclic peptide Preparation of the full-protected cyclic peptide: the full-protected polypeptide was mixed with DMF to obtain a full-protected polypeptide DMF solution; then HOBT was added, and the temperature of the reaction solution was controlled at 0°C, NMM and DIC were added, and then the reaction was stirred at 25°C for 16h, the reaction was monitored by HPLC, and after the reaction was completed, water was added to precipitate the solid, the solid was dissolved with ethyl acetate, and then sequentially washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and rotary evaporated to obtain the full-protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-). The full-protected polypeptide DMF solution was prepared by mixing the full-protected polypeptide with DMF at a mass-volume ratio of 1g:1L, the amount of HOBT used was 18.8wt% of the full-protected polypeptide, the amount of NMM used was 23.2wt% of the full-protected polypeptide, and the amount of DIC used was 14.5wt% of the full-protected polypeptide. The amount of water added when the solid was precipitated was appropriately used, the amount of ethyl acetate used for dissolving the solid was appropriately used, and the amounts of saturated sodium bicarbonate solution, water and saturated sodium chloride solution used for washing were all appropriately used. The full-protected polypeptide was prepared by the preparation method of Example 3. The yield of the full-protected cyclic peptide was 75.73%, and the purity was 77.86%.

[0111] Example 6: Preparation method of the monocyclic peptide Preparation of the monocyclic peptide: the full-protected cyclic peptide was mixed with a cleavage solution for 2h, concentrated, precipitated with ice ethyl ether, washed, centrifuged, and rotary evaporated to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cleavage solution was a mixture of TFA, DCM and EDT, the volume ratio of TFA, DCM and EDT in the cleavage solution was 1:0.8:0.2, and the amount of the full-protected cyclic peptide used was 1g:10mL. The amount of ice ethyl ether used for precipitation was appropriately used. The full-protected cyclic peptide was prepared by the preparation method of 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 reverse phase C18 liquid chromatography, and after freeze-drying, the purity of the pure product was 96%, and the number was PR2495B. The chromatography of the reverse phase C18 chromatography in Example 6 in the present application is shown in Figure 1 .

[0113] The mass spectrum of the monocyclic peptide purified by reverse phase C18 chromatography in Example 6 in the present application is shown in Figure 2 .

[0114] Example 7: Preparation method of the monocyclic peptide Preparation of the mono-cyclic peptide: the fully protected cyclic peptide was mixed with the cleavage solution for 2 h, and then precipitated with ice-ethyl ether, washed, centrifuged, and dried to obtain the mono-cyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cleavage solution was a mixture of TFA, Tis and H2O, and the TFA, Tis and H2O in the cleavage solution were mixed at a volume ratio of 1:0.026:0.026. The amount of the fully protected cyclic peptide used was 1 g per 10 mL of the cleavage solution. The ice-ethyl ether used in the precipitation was used in an appropriate amount. The fully protected cyclic peptide was prepared by the preparation method of Example 4. The purity of the mono-cyclic peptide was 32.26%, and the yield was 126.74%.

[0115] Example 8: Preparation method of the di-cyclic peptide Preparation of the di-cyclic peptide: the mono-cyclic peptide was mixed with an acetic acid solution, and insoluble substances were removed by filtration. The filtrate was diluted with purified water, and an iodomethanol solution was added under stirring at 25°C until the reaction solution turned yellow and no fading occurred, indicating that the reaction was complete. Then, an ascorbic acid solution was added under stirring for reduction until the reaction solution turned from yellow to milky white, and the color did not change upon stirring. 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 di-cyclic peptide solution, which was purified by reverse phase C18 chromatography and freeze-dried to obtain the di-cyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide-bridged Cys&Cys), i.e., the di-cyclic peptide compound. The acetic acid solution was prepared by mixing acetic acid and purified water at a volume ratio of 1:1. The amount of the mono-cyclic peptide used was 6.8 wt% of the acetic acid solution. The amount of the purified water used for dilution was 1000 wt% of the acetic acid solution. The iodomethanol solution contained 0.1 mol / L of iodomethanol. The ascorbic acid solution contained 1 wt% of ascorbic acid. The iodomethanol solution and the ascorbic acid solution were used in appropriate amounts. The mono-cyclic peptide was prepared by the preparation method of Example 6 and was not purified by reverse phase C18 liquid chromatography. The purity of the di-cyclic peptide was 98.33%.

[0116] Example 9: Preparation method of the di-cyclic peptide Preparation of the di-cyclic peptide: the mono-cyclic peptide was mixed with DMSO, and insoluble substances were removed by filtration. The reaction was stirred at 25°C for 7 d, 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 di-cyclic peptide solution, which was purified by reverse phase C18 chromatography and freeze-dried to obtain the di-cyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide-bridged Cys&Cys), i.e., the di-cyclic peptide compound. The amount of the mono-cyclic peptide used was 10 wt% of the DMSO. The mono-cyclic peptide was prepared by the preparation method of Example 6. The purity of the di-cyclic peptide was 98.21%. The di-cyclic peptide compound prepared in Example 9 was numbered PR2495.

[0117] The chromatogram of PR2495 purified by reversed-phase C18 liquid chromatography in Example 9 of the present application is shown as follows. Figure 3

[0118] The mass spectrum of the bicyclic peptide compound obtained in Example 9 of the present application is shown as follows. Figure 4

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

[0120] 1. Cell toxicity test The present application evaluated the potential toxicity of PR2495 and PR2495B to cells and determined the possible safe concentration range thereof.

[0121] In the cell toxicity test of the present application, HFF-1 cells were inoculated into a 96-well plate, 200 μL of culture medium was added to each well, containing about 10,000 cells / well, to ensure uniform cell density. The cells were cultured overnight at 37°C in a 5% CO2 incubator to allow them to adhere and grow. After 24 hours, the cell culture medium was aspirated and 200 μL of DMEM medium containing different concentrations of test samples was added. The 96-well plate was returned to the incubator for continued culture. After 24 hours, the absorbance value was measured at 490 nm wavelength by MTT method using a microplate reader 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 with only medium without cells was designed as the blank control group. The cell viability was calculated as follows: cell viability = (test group absorbance - blank control group absorbance / negative control group absorbance - blank control group absorbance) x 100%.

[0122] The results of the cell toxicity experiment are shown as follows. Figure 5 The cell viability of epithelial cells treated with different concentrations of PR2495 and PR2495B did not show a significant decrease in cell viability, and the cell viability remained at about 96% relative to the control group at a concentration of 500 μM, confirming that PR2495 and PR2495B both had very low cytotoxicity.

[0123] 2. Serum stability test The present application evaluated the decomposition of PR2495 and PR2495B in a serum environment to predict their metabolism in vivo.

[0124] ​​Test sample lyophilized powder was directly dissolved in 10 vol% fetal bovine serum to a final concentration of 1 mg / mL. Lyophilized oligopeptide GHK was also dissolved under the same conditions as a control group. The above prepared solution was sterile filtered through a 0.22 μM filter and then placed in a sterile container and incubated in a 37°C incubator. Sampling was performed at the set time points of 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. The residual amount of the polypeptide was determined using analytical high-performance liquid chromatography, with the 0 time content taken as 100%, and 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 0 time peak area) x 100%. The residual percentage versus time curve was plotted, and the half-life was calculated. The test sample was PR2495 or PR2495B.

[0125] The serum stability experiment results are shown in Table 1. Figure 6 As shown in Table 1, the GHK control group was almost completely degraded in 10 vol% fetal bovine serum in 4 hours, and the degradation half-life was calculated to be about 0.7 hours according to the degradation curve. Under the same conditions, PR2495 was nearly completely degraded in 48 hours, and the half-life was calculated to be about 13 hours according to the degradation curve. Under the same conditions, the half-life of PR2495B was about 9 hours, indicating that the intramolecular secondary cyclization produced by the disulfide bond can enhance the stability of the polypeptide.

[0126] 3. Test of type I collagen expression promotion rate The present application studies the effect of PR2495 and PR2495B on the expression level of COL1A in human fibroblasts HFF-1.

[0127] Logarithmic growth phase fibroblasts HFF-1 were inoculated into a 6-well plate at 50,000 cells / well. 2 mL of culture medium was added to each well, and the cells were allowed to adhere and grow for 24 hours in an incubator. The cells were divided into groups and DMEM medium containing test samples was added. A DMEM blank control group and 100 ng / mL TGF-β1 as a positive control group were also set up. The test samples were GHK or PR2495 or PR2495B, and the concentration of the test sample was 10, 20, and 40 μM.

[0128] After 24 hours of treatment, the medium was removed, and the RNA of the cells in each well was extracted. 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 according to the formula: RNA relative expression level = 2ΔΔC(t).

[0129] t-test statistical analysis was used for comparison between groups. P < 0.05 was considered to be significantly different, marked with * in the figure; P < 0.01 was considered to be extremely significantly different, marked with ** in the figure.

[0130] The up-regulation rate was calculated according to the formula: up-regulation rate = (test group-blank control group) / blank control group x 100%.

[0131] The experimental results of collagen type I expression are shown in Table 2. Figure 7 As shown in Table 2, compared with the blank control group, the expression of COL1A was significantly increased after TGF-β1 treatment in the positive control group, proving that the cell model and qPCR system were working normally. Compared with the blank control group, PR2495B treatment significantly positively regulated the expression of COL1A, with an up-regulation rate of about 32% at 10 μM, about 42% at 10 μM, and about 49% at 40 μM. Compared with the blank control group, PR2495 treatment significantly positively regulated the expression of COL1A, with an up-regulation rate of about 37% at 10 μM, about 58% at 10 μM, and about 69% at 40 μM. Both PR2495 and PR2495B can promote the production of collagen type I, and the up-regulation ability of PR2495 is slightly stronger than that of PR2495B.

[0132] 4. Anti-oxidation effect detection Superoxide dismutase (SOD) is an important member of the antioxidant enzyme system in biological systems, which is a terminal compound that effectively removes superoxide anion free radicals to avoid excessive damage to cells. Glutathione (GSH) is continuously oxidized and reduced in cells, and is oxidized to glutathione disulfide (GSSG) by the action of glutathione peroxidase (GPx). GSSG is rapidly reduced to GSH by glutathione reductase, maintaining the level of reduced glutathione in cells and reducing oxidative stress.

[0133] The present application studies whether PR2495 and PR2495B can improve the antioxidant capacity of cells, whether they affect the expression level of antioxidant-related enzymes, and the ultimate antioxidant capacity.

[0134] Logarithmic growth period fibroblast HFF-1 cells were inoculated into a 6-well plate at 50,000 cells per well. 2 mL of culture medium was added to each well, and the cells were allowed to adhere and grow for 24 hours in an incubator. The cells were divided into groups and treated with DMEM medium containing test samples. A blank control group was set up without adding polypeptides and oxidative stress. A negative control group was set up without adding 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 2The cell supernatant was collected after 24 hours, and the SOD and GSH levels were detected according to the SOD detection kit and GSH quantitative kit instructions. The test samples were GHK or PR2495 or PR2495B, and the concentration of the test sample was 10 μM.

[0135] The t-test statistical analysis was used for comparison between groups. P<0.05 was considered to have significant difference, marked * in the figure; P<0.01 was considered to have extremely significant difference, marked ** in the figure.

[0136] According to the formula: promotion rate = (test group - positive control group) / positive control group x 100%, the SOD and GSH promotion levels of each group were calculated.

[0137] The results of the antioxidant experiment are shown in Table 1. Figures 8-9 As shown in Table 1, compared with the blank control group, the SOD and GSH levels of the negative control group were significantly down-regulated, and the levels of the positive control group were significantly up-regulated, with an SOD level increase of about 53.7% and a GSH level increase of about 119.7%, proving that the oxidative stress stimulation was effective and the antioxidant positive control was working normally. GHK treatment showed certain antioxidant capacity, with an SOD level increase of about 36.5% and a GSH level increase of about 61.0% at 10 μM compared with the negative control. Compared with the negative control, the SOD level of PR2495B increased by about 88.5%, and the GSH level increased by about 124%. Compared with the negative control, the SOD level of PR2495 increased by about 74.7%, and the GSH level increased by about 120%. PR2495 and PR2495B can both increase the expression level of the antioxidant key enzyme SOD and ultimately increase the expression amount of the antioxidant key substance GSH.

[0138] 5. Anti-inflammatory effect detection In the inflammatory response, cytokines such as TNF-α, IL-6 and IL-8 are important regulatory molecules. They interact through a complex network, together participating 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 immune response, and IL-8 enhances the infiltration and activation of immune cells. There is a complex synergistic relationship between the three factors, which together constitute the core network of 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 and PR2495 or PR2495B to inhibit the production of inflammatory factors by macrophages under the condition of inflammatory stress induced by lipopolysaccharide LPS.

[0139] The macrophage cell line RAW264.7 was seeded into a 6-well plate at 50,000 cells per well and cultured for 24 hours. The cells were divided into groups and treated with DMEM medium containing the test sample. A blank control group was set up, and a negative control group was set up without adding the polypeptide. 0.01% dexamethasone was used as an anti-inflammatory positive control group. After 2 hours of treatment, 200 μL of LPS working solution was added to each well of the remaining groups except the blank control group, and the cells were incubated in the incubator for 22 hours. The cell culture supernatant was collected, and TNF-α, IL-6, and IL-8 were detected and analyzed according to the instructions of the ELISA kit. The test sample was GHK or PR2495 or PR2495B, and the concentration of the test sample was 10 μM. The t-test statistical analysis was used for comparison between groups. P < 0.05 was considered to be significantly different, marked with * in the figure; P < 0.01 was considered to be extremely significantly different, marked with ** in the figure.

[0140] The inhibition rate was calculated according to the formula: inhibition rate% = (negative control group-test group) / negative control group x 100%.

[0141] The results of the anti-inflammatory experiment are shown in Table 1. Figures 10-12 As shown in Table 1, compared with the blank control group, the content of TNF-α, IL-6, and IL-8 in the negative control group increased significantly, indicating that the test stimulation conditions were effective. Compared with the negative control group, the content of TNF-α, IL-6, and IL-8 in the positive control group decreased significantly, and the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with the positive control group were about 34.7%, 31.8%, and 48.0%, respectively, indicating that the test positive control was effective. Compared with the negative control group, the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with 10 μM GHK were about 15.1%, 34.2%, and 19.4%, respectively; the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with 10 μM PR2495B were about 30.0%, 55.4%, and 33.0%, respectively; and the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with 10 μM PR2495 were about 30.6%, 55.7%, and 36.7%, respectively. PR2495 and PR2495B both showed stronger effects than GHK at the same concentration and both showed significant anti-inflammatory ability.

[0142] The above examples and / or embodiments are only used to illustrate the preferred examples and / or embodiments of the present technology, and do not limit the embodiments of the present technology in any form. Any person skilled in the art can make some changes or modifications to other equivalent examples without departing from the scope of the technology disclosed in the present disclosure, but should be considered as substantially the same technology or embodiment as the present disclosure.

[0143] The principles and implementation manners of the present application are described herein by using specific examples, and the above example descriptions are only used to help understand the method of the present application and its core idea. The above is only a preferred implementation manner of the present application. It should be noted that, due to the limited nature of the language expression, there are objectively infinite specific structures, and for ordinary skilled persons in the technical field, some improvements, refinements or changes can be made without departing from the principles of the present application, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, shall be regarded as the protection scope of the present 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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