Anti-aging and anti-oxidation bicyclic peptide compound as well as preparation method and application thereof

Abstract

The invention discloses an anti-aging and anti-oxidation bicyclic peptide compound as well as a preparation method and application thereof, belongs to the technical field of polypeptide compound synthesis, and particularly relates to 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

Technical Field

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

[0002] 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 degradation process of the extracellular matrix, Secreted Protein Acidic and Rich in Cysteine (SPARC protein) secreted by matrix cells rich in cysteine cleaves and releases GHK, which helps tissue remodeling by regulating the angiogenesis level. Studies have found that the average content of GHK in the blood of 20-year-old people is 200 ng / ml, but it drops to an average of 80 ng / ml at 60 years old, showing a clear down-regulation with aging.

[0003] 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 the damage of oxidative stress to cells, protect the integrity and function of cells, and delay skin aging. At the same time, GHK also shows certain anti-inflammatory effects: it can regulate the inflammatory response, inhibit the release of inflammatory factors, reduce the damage of inflammation to tissues, and has a certain therapeutic effect on skin inflammatory diseases such as acne and eczema.

[0004] Based on the above functions, GHK has certain anti-wrinkle and repair effects as a cosmetic: it can activate the synthesis of the extracellular matrix, increase the content of collagen and elastic fibers, reduce the generation of wrinkles, and make the skin more firm and smooth; for damaged skin, such as sunburned and sensitive skin, GHK has a good repair effect, can relieve skin discomfort, promote the self-repair 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 extremely easy-to-degrade property affects the continuous exertion of its functions. Summary of the Invention

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

[0006] Peptide cyclization and side-chain binding are important means in the development of peptide drugs. The cyclic structure reduces the exposure of terminal groups, lowers the possibility of unnecessary chemical reactions, thereby enhancing the overall chemical stability. At the same time, it reduces the recognition and decomposition of terminal amino acids by some proteases, enhancing the anti-enzyme digestion ability. Due to the higher stability and anti-enzyme digestion ability, the cyclized and side-chain-bound peptides can maintain a longer effective concentration in vivo and reduce the dosing frequency.

[0007] The technical solution adopted by the present invention to achieve the above object is as follows: A method for preparing a monocyclic peptide, comprising: mixing a solid-phase synthesis resin with an amino acid reagent, preparing a fully protected peptide resin by solid-phase synthesis, subjecting the fully protected peptide resin to cleavage treatment and monocyclization to obtain a monocyclic peptide, and the structural formula of the monocyclic peptide is as follows: , wherein, X1 and X2 independently selected from any one of His, Lys, Gly, Trp, Phe, and Arg; Y1 and Y2 independently selected from any one of His, Lys, Gly, Trp, Phe, and Arg; R1 and R2 independently selected from Cys or its derivatives.

[0008] Preferably, X1 and X2 independently selected from any one of His and Lys; or, Y1 and Y2 independently selected from any one of His and Lys; or, R1 and R2 independently selected from Cys.

[0009] The present invention discloses a monocyclic peptide prepared by the above method.

[0010] Preferably, the monocyclic peptide is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0011] The present invention discloses a bicyclic peptide compound, and the structural formula is as follows: , wherein, X1 and X2 independently selected from any one of His, Lys, Gly, Trp, Phe, and Arg; Y1 and Y2 independently selected from any one of His, Lys, Gly, Trp, Phe, and Arg; R1 and R2 independently selected from Cys or its derivatives; L1 is formed by bonding R1 and R2.

[0012] Preferably, R1 and R2 independently selected from Cys, and L1 is a disulfide bond formed by bonding R1 and R2.

[0013] Preferably, the bicyclic peptide compound is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with disulfide bridges between Cys&Cys).

[0014] The present invention discloses a preparation method of the bicyclic peptide compound, including: the preparation method of the above-mentioned monocyclic peptide.

[0015] Preferably, the monocyclic peptide is subjected to bicyclization treatment to obtain the bicyclic peptide compound; The bicyclization is carried out by the action of iodomethanol and ascorbic acid; or, the bicyclization is carried out in DMSO.

[0016] The present invention discloses the use of the above-mentioned monocyclic peptide in the preparation of bicyclic peptide compounds and / or products for enhancing the expression of type I collagen and / or antioxidant products and / or anti-inflammatory products.

[0017] The present invention discloses the use of the above-mentioned bicyclic peptide compound in the preparation of products for enhancing the expression of type I collagen and / or antioxidant products and / or anti-inflammatory products.

[0018] The present invention discloses a preparation method of a fully protected peptide resin, including: After mixing Fmoc-Gly-OH, DIEA and DCM, CTC resin is added and coupled to obtain the first amino acid coupled resin; the molar amount of Fmoc-Gly-OH used is 100-300% of the reaction sites on the CTC resin, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used; The first amino acid coupled resin is subjected to deprotection treatment, and then the coupling of activated amino acid reagents is carried out in sequence according to the peptide sequence to obtain the fully protected peptide resin, and 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.

[0019] The present invention discloses a fully protected peptide resin, with the structure of: H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin.

[0020] Preferably, the preparation of the fully protected peptide resin includes: the synthesis of the first amino acid coupled resin, the activation of amino acid reagents and the synthesis of the fully protected peptide resin.

[0021] Preferably, in the synthesis of the first amino acid-coupled resin, Fmoc-Gly-OH is coupled to the CTC resin. The coupling is carried out by solid-phase synthesis. After the CTC resin is swollen with DCM, it is coupled with Fmoc-Gly-OH in a DIEA-DCM solution.

[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, under a nitrogen atmosphere, the CTC resin is swollen in DCM at 20-40 °C for 5-30 min, 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 for capping for 10-60 min, suction filtration and washing are carried out to obtain Fmoc-Gly-CTC resin, which is the first amino acid-coupled resin.

[0024] 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 reaction sites on the CTC resin.

[0025] 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 usage amounts of DIEA and DCM in the DIEA-DCM solution are 0.5-2.5 mmol / mL.

[0026] More preferably, in the synthesis of the first amino acid-coupled resin, the amount of the DIEA-DCM solution is measured by DIEA therein, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used. For methanol capping, an appropriate amount can be used.

[0027] Preferably, in the activation of the amino acid reagent, the amino acid reagent is mixed with HOBT and DIC in DMF to obtain an activated amino acid reagent.

[0028] Preferably, in the activation of the amino acid reagent, the amino acid reagent and HOBT are added to DMF, and DIC is added at 0-10 °C, and the activation is carried out for 3-20 min to obtain an activated amino acid reagent.

[0029] More preferably, in the activation of the amino acid reagent, the relationship between the usage amount of the amino acid reagent and DMF is 0.1-10 mmol / mL.

[0030] 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.

[0031] 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.

[0032] 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, Fmoc-Gly-OH.

[0033] Preferably, in the synthesis of the fully protected peptide resin, the first amino acid-coupled resin is mixed with the deprotection solution for deprotection treatment, the liquid is removed by suction filtration, the activated amino acid reagent is added, and the reaction is carried out for 10 - 60 min under a nitrogen atmosphere. After the reaction is completed, suction filtration and washing are carried out; then the coupling of the above-mentioned 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, that is, the fully protected peptide resin, is obtained.

[0034] 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.

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

[0036] More preferably, in the synthesis of the fully protected peptide resin, when repeating the coupling of the activated amino acid reagent, deprotection treatment is carried out in each case. After the coupling is completed, suction filtration and washing are carried out in each case, and the washing is carried out using DMF.

[0037] More preferably, in the synthesis of the fully protected peptide resin, the washing after the coupling is completed is carried out successively using methyl tert-butyl ether, tetrahydrofuran, and methyl tert-butyl ether.

[0038] The present invention discloses the use of the 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.

[0039] The present invention 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。

[0040] The present disclosure relates to a method for preparing a fully protected polypeptide, comprising: subjecting a fully protected peptide resin to a fully protected cleavage treatment to obtain a fully protected polypeptide.

[0041] Preferably, the fully protected cleavage treatment employs a fully protected cleavage solution, which includes a DCM solution containing TFA or a DCM solution containing HFIP.

[0042] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with the cleavage solution, treated at 20 - 40 °C for 10 - 60 min, the resin is filtered off, petroleum ether is added to the filtrate for sedimentation, the supernatant is removed by centrifugation, the residue is washed with petroleum ether by centrifugation, and then dried under vacuum to obtain the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH.

[0043] More preferably, in the preparation of a fully protected polypeptide, the cleavage solution is composed of TFA and DCM, and the content of TFA in the cleavage solution is 0.1 - 5 vol %. The fully protected peptide resin is immersed in the cleavage solution, and an appropriate amount of petroleum ether is used for sedimentation and washing.

[0044] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with the cleavage solution, treated at 20 - 40 °C for 10 - 60 min, the resin is filtered off, and after the filtrate is concentrated by rotary evaporation to dryness, the fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH is obtained.

[0045] More preferably, in the preparation of a fully protected polypeptide, the cleavage solution is composed of HFIP and DCM, and the content of HFIP in the cleavage solution is 20 - 40 vol %. The fully protected peptide resin is immersed in the cleavage solution.

[0046] The present invention discloses the use of a fully protected polypeptide in the 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-).

[0047] The present invention discloses a fully protected cyclic peptide, the structure of which is: Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-).

[0048] The present invention discloses a method for preparing a fully protected cyclic peptide, which includes: subjecting a fully protected polypeptide to a fully protected cyclization treatment to obtain a fully protected cyclic peptide.

[0049] Preferably, in the fully protected cyclization treatment, a HATU-DIEA solution is used to perform the fully protected cyclization on the fully protected polypeptide; or, in the fully protected cyclization treatment, a DMF solution containing HOBT, DIC, and NMM is used for the fully protected cyclization.

[0050] Preferably, in the preparation of the fully protected cyclic peptide, a 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, and the temperature of the HATU-DIEA solution is controlled at 20 - 30 °C. Then, the fully protected polypeptide 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, and 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, 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-).

[0051] 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-to-volume ratio of 1 g : 5 - 20 mL.

[0052] 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-to-volume ratio of 1 g : 3 - 10 mL : 0.1 - 5 mL.

[0053] More preferably, in the preparation of the fully protected cyclic peptide, based on the fully protected polypeptide as the measurement benchmark for the amount of the fully protected polypeptide DMF solution, and based on HATU as the measurement benchmark for the HATU-DIEA solution, the usage amount of HATU is 40 - 60 wt% of the fully protected polypeptide.

[0054] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide DMF solution; then HOBT is added, the temperature of the reaction solution is controlled to be 0-10 °C, NMM and DIC are added, and then the reaction is stirred at 20-40 °C for 8-24 h. 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, and then washed successively 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-).

[0055] 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: 0.2-2 L.

[0056] More preferably, in the preparation of the fully protected cyclic peptide, the usage amount of HOBT is 10-30 wt% of the fully protected polypeptide.

[0057] More preferably, in the preparation of the fully protected cyclic peptide, the usage amount of NMM is 10-40 wt% of the fully protected polypeptide.

[0058] More preferably, in the preparation of the fully protected cyclic peptide, the usage amount of DIC is 5-30 wt% of the fully protected polypeptide.

[0059] More preferably, in the preparation of the fully protected cyclic peptide, the water added when precipitating the solid is used in an appropriate amount, the ethyl acetate for dissolving the solid is used in an appropriate amount, and the saturated sodium bicarbonate solution, water and saturated sodium chloride solution used in the washing are all used in an appropriate amount.

[0060] The present invention discloses the use of the fully protected cyclic peptide in the preparation of a monocyclic peptide, and the structure of the monocyclic peptide is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0061] The present invention discloses a monocyclic peptide with the structure: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-), and the structural formula is: .

[0062] The present invention discloses a preparation method of a monocyclic peptide, including: Solid-phase synthesis is used to prepare H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, that is, the fully protected peptide resin; The fully protected peptide resin is cleaved and monocyclized to obtain a monocyclic peptide, and the structure of the monocyclic peptide is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0063] 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.

[0064] Preferably, the cleavage solution for the cleavage treatment is composed 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 composed of HFIP and DCM, and the content of HFIP in the cleavage solution is 20-40 vol%; or, the monocyclization is carried out by cyclizing and deprotecting H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH obtained by the cleavage treatment.

[0065] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the cleavage solution for 1-4 h, concentrated, precipitated with ice-cold diethyl ether, washed, centrifuged, and dried by rotation to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0066] More preferably, in the preparation of the monocyclic peptide, the cleavage solution is a mixed solution of TFA, DCM, and EDT, and TFA, DCM, and EDT in the cleavage solution are mixed at a volume ratio of 1:0.4-1.6:0.05-0.5.

[0067] More preferably, in the preparation of the monocyclic peptide, the usage relationship between the fully protected cyclic peptide and the cleavage solution is 1 g:5-20 mL. Ice-cold diethyl ether is used in an appropriate amount during precipitation.

[0068] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the cleavage solution for 1-4 h, and after the cleavage is completed, it is precipitated with ice-cold diethyl ether, washed, centrifuged, and dried by rotation to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

[0069] More preferably, in the preparation of the monocyclic peptide, the cleavage solution is a mixed solution of TFA, Tis, and H2O, and TFA, Tis, and H2O in the cleavage solution are mixed at a volume ratio of 1:0.01-0.05:0.01-0.05, and the usage relationship between the fully protected cyclic peptide and the cleavage solution is 1 g:5-20 mL. Ice-cold diethyl ether is used in an appropriate amount during precipitation.

[0070] The present invention discloses the use of a monocyclic peptide in the preparation of a bicyclic peptide compound, and the structure of the bicyclic peptide compound is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with a disulfide bond bridging Cys&Cys).

[0071] The present invention discloses a bicyclic peptide compound, and the structure is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with a disulfide bond bridging Cys&Cys).

[0072] The bicyclic peptide compound exhibits the functions of inducing extracellular matrix generation, anti-inflammatory and antioxidant. In cell function tests, the compound has excellent efficacy.

[0073] The structural formula of the bicyclic peptide compound is: 。

[0074] The present invention discloses a preparation method of a bicyclic peptide compound, including: the preparation method of the above-mentioned monocyclic peptide.

[0075] Preferably, the monocyclic peptide is subjected to bicyclic cyclization to obtain the bicyclic peptide compound, and the bicyclic cyclization is carried out by the action of iodomethanol and ascorbic acid; or, the bicyclic cyclization is carried out in DMSO. The monocyclic peptide is dissolved in an organic solvent and oxidized so that two cysteines are connected by forming a disulfide bond, and finally the bicyclic peptide compound is obtained.

[0076] Preferably, in the preparation of the bicyclic peptide, the monocyclic peptide is mixed with an acetic acid solution, and insoluble substances are filtered off. The filtrate is diluted with purified water, and an iodomethanol solution is added with stirring at 20-40 °C. Stir until the reaction solution turns yellow and does not fade, and judge that the reaction is complete; then an ascorbic acid solution is added with stirring for reduction until the reaction solution changes back from yellow to the original milky white, and 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 μm filter membrane to obtain a crude solution of the bicyclic peptide, which is purified by reverse-phase C18 chromatography and freeze-dried to obtain the bicyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with a disulfide bond bridging Cys&Cys).

[0077] More preferably, in the preparation of the bicyclic peptide, the acetic acid solution is prepared by mixing acetic acid and pure water in a volume ratio of 1:0.2-5, and the usage amount of the monocyclic peptide is 3-15 wt% of the acetic acid solution.

[0078] More preferably, in the preparation of the bicyclic peptide, the usage amount of purified water for dilution is 500-1500 wt% of the acetic acid solution.

[0079] More preferably, in the preparation of the bicyclic 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.

[0080] Preferably, in the preparation of the bicyclic peptide, the monocyclic peptide is mixed with DMSO, the insoluble matter is filtered off, and the reaction is stirred at 20 - 40 °C for 4 - 14 d. The reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45 μm filter membrane to obtain a crude solution of the bicyclic peptide. The crude solution is purified by reverse-phase C18 chromatography and freeze-dried to obtain the bicyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, with a disulfide bond bridging Cys&Cys).

[0081] More preferably, in the preparation of the bicyclic peptide, the usage amount of the monocyclic peptide is 5 - 20 wt% of DMSO.

[0082] The present invention discloses the use of the bicyclic peptide compound in the preparation of products for increasing the expression of type I collagen and / or antioxidant products and / or anti-inflammatory products.

[0083] Preferably, the antioxidant product includes the use in products for increasing the expression of the antioxidant key enzyme SOD and / or products for increasing the expression of GSH.

[0084] Preferably, the anti-inflammatory product includes products for inhibiting TNF-α and / or products for inhibiting IL-6 and / or products for inhibiting IL-8.

[0085] The present invention also discloses a preparation method of the above-mentioned bicyclic peptide compound, including: synthesis of the linear polypeptide, cyclization of the linear polypeptide, and secondary cyclization of the cyclized polypeptide.

[0086] Preferably, the preparation method of the cyclic peptide compound specifically includes: Using the polypeptide coupling synthesis method to prepare a fully protected peptide resin including a pair of cysteines in the sequence; performing full protection cleavage to obtain a chain-like fully protected polypeptide, i.e., a linear polypeptide; dissolving the fully protected polypeptide in an organic solvent and stirring the reaction under the condition of a catalytic system to form a fully protected cyclic peptide; taking the fully protected cyclic peptide for cleavage to obtain a cyclic peptide; dissolving the cyclic peptide in an organic solvent and performing oxidation so that the two cysteines are connected by forming a disulfide bond, and finally obtaining the bicyclic peptide compound.

[0087] The catalytic system for synthesizing the bicyclic peptide of the present invention includes DIC, HOBt, HATU, DIEA, and NMM.

[0088] In the present invention, the peptide resin, i.e., H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-CTC resin, is prepared by solid-phase synthesis. The peptide resin is cleaved and mono-cyclized to obtain a mono-cyclic peptide, and the structure of the mono-cyclic peptide is: Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The mono-cyclic peptide is subjected to a dual-cyclization treatment to obtain a bicyclic peptide compound Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bridge connecting Cys&Cys), and the dual-cyclization is carried out by cyclization under the action of iodomethanol and ascorbic acid; or, the dual-cyclization is carried out in DMSO. Therefore, the present invention has the following beneficial effects: low toxicity, good stability, can enhance the expression of type I collagen, good antioxidant effect, and good anti-inflammatory effect. Accordingly, the present invention relates to a bicyclic peptide compound with anti-aging and antioxidant properties, which has low toxicity, good stability, can enhance the expression of type I collagen, good antioxidant effect, and good anti-inflammatory effect, and its preparation method and application. Description of the Drawings

[0089] Figure 1 It is the liquid chromatography diagram of the mono-cyclic peptide compound.

[0090] Figure 2 It is the mass spectrometry diagram of the mono-cyclic peptide compound.

[0091] Figure 3 It is the liquid chromatography diagram of the bicyclic peptide compound.

[0092] Figure 4 It is the mass spectrometry diagram of the bicyclic peptide compound.

[0093] Figure 5 It is the test result diagram of the cytotoxicity of the bicyclic peptide compound.

[0094] Figure 6 It is the test result diagram of the serum stability of the bicyclic peptide compound.

[0095] Figure 7 It is the test result diagram of the COL1A expression.

[0096] Figure 8 It is the test result diagram of the SOD expression level.

[0097] Figure 9 It is the test result diagram of the GSH level.

[0098] Figure 10 It is the TNF-α expression level diagram.

[0099] Figure 11 It is the IL-6 expression level diagram.

[0100] Figure 12 It is a graph of IL-8 expression level. Detailed implementation manners

[0101] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0102] First, the concepts involved in the present application will be described in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments and the features in the embodiments in the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0103] Example 1: A preparation method of a fully protected peptide resin Synthesis of the first amino acid-coupled resin: Under a nitrogen atmosphere, the CTC resin was swollen in DCM at 25°C for 10 minutes, and the DCM was removed by suction filtration. Fmoc-Gly-OH was added, and then a DIEA-DCM solution was added at 10°C, and the reaction was carried out at 25°C for 2.5 hours. After the reaction was completed, methanol was added for capping for 30 minutes, and suction filtration and washing were carried out to obtain Fmoc-Gly-CTC resin, that is, the first amino acid-coupled resin. The molar amount of Fmoc-Gly-OH used was 200% of the reaction sites on the CTC resin. The DIEA-DCM solution was composed of DIEA and DCM. The usage amounts of DIEA and DCM in the DIEA-DCM solution were 1.25 mmol / mL. The usage amount of the DIEA-DCM solution was measured by DIEA therein, and the molar amount of DIEA used was 250% of the molar amount of Fmoc-Gly-OH used. For methanol capping, an appropriate amount can be used. The reaction sites of the CTC resin were 125 mmol.

[0104] Activation of the amino acid reagent: The amino acid reagent and HOBT were added to DMF, and DIC was added at 5°C, and activation was carried out for 5 minutes to obtain an activated amino acid reagent. The usage amount relationship between the amino acid reagent and DMF was 1 mmol / mL. The molar amount of HOBT used was 100% of the molar amount of the amino acid reagent used, and the molar amount of DIC used was 100% of the molar amount of the amino acid reagent used. The amino acid reagent included Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

[0105] Synthesis of fully protected peptide resin: The first amino acid-coupled resin was mixed with the deprotection solution for deprotection treatment. The liquid was removed by suction filtration, and the activated amino acid reagent was added. The reaction was carried out for 40 min under a nitrogen atmosphere. After the reaction was completed, suction filtration was performed and washing was carried out. Then, the coupling of the above-mentioned 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, that is, 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. During the deprotection treatment, the resin coupled with the amino acid reagent was first immersed in a 20 vol% Pip / DMF solution, and deprotection treatment was carried out at 25 °C for 30 min. When repeating the coupling of the activated amino acid reagent, deprotection treatment was carried out in all cases. After the coupling was completed, suction filtration and washing were carried out in all cases, and the washing was carried out with DMF. After the coupling was completed, washing was carried out successively with methyl tert-butyl ether, tetrahydrofuran, and methyl tert-butyl ether.

[0106] Example 2: A preparation method of fully protected polypeptide Preparation of fully protected polypeptide: 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. Petroleum ether was added to the filtrate for sedimentation, and the supernatant was removed by centrifugation. Washing with petroleum ether and centrifugation were carried out, and vacuum drying was carried out to obtain fully protected polypeptide H-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-OH. The cleavage solution was composed of TFA and DCM, and the content of TFA in the cleavage solution was 1 vol%. The fully protected peptide resin was immersed in the cleavage solution, and the sedimentation and washing with petroleum ether were used appropriately. 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%.

[0107] Example 3: A preparation method of fully protected polypeptide Preparation of fully protected polypeptide: 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. After the filtrate was concentrated and rotary evaporated to dryness, 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 composed of HFIP and DCM, and the content of HFIP in the cleavage solution was 33 vol%. The cleavage solution submerged the fully protected peptide resin. The fully protected peptide 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%.

[0108] Example 4: A preparation method of a fully protected cyclic peptide Preparation of 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, and then the fully protected polypeptide DMF solution was dropped into the HATU-DIEA solution and reacted for 60 min. The reaction was monitored by HPLC. After the reaction was completed, water and ethyl acetate were added for extraction, and 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, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-). In the fully protected polypeptide DMF solution, the fully protected polypeptide 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 dosage of the fully protected polypeptide DMF solution was based on the fully protected polypeptide therein, and the HATU-DIEA solution was based on HATU. The usage amount of HATU 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%.

[0109] Example 5: A preparation method of a fully protected cyclic peptide Preparation of fully protected cyclic peptide: Mix the fully protected polypeptide with DMF to obtain a fully protected polypeptide DMF solution; then add HOBT, control the temperature of the reaction solution at 0 °C, add NMM and DIC, and then stir and react at 25 °C for 16 h. Monitor the reaction by HPLC. After the reaction is completed, add water to precipitate a solid. Dissolve the solid in ethyl acetate, and then wash it successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. Dry it over anhydrous sodium sulfate, filter, and rotary evaporate to obtain the fully protected cyclic peptide Cyclo(His(Trt)-Cys(Trt)-Lys(Boc)-Gly-His(Trt)-Cys(Trt)-Lys(Boc)-Gly-). In the fully protected polypeptide DMF solution, the fully protected polypeptide and DMF are mixed at a mass-to-volume ratio of 1 g:1 L. The usage amount of HOBT is 18.8 wt% of the fully protected polypeptide, the usage amount of NMM is 23.2 wt% of the fully protected polypeptide, and the usage amount of DIC is 14.5 wt% of the fully protected polypeptide. The amount of water added for precipitation is used appropriately, the ethyl acetate for dissolving the solid is used appropriately, and the saturated sodium bicarbonate solution, water, and saturated sodium chloride solution used in the washing are all used appropriately. The fully protected polypeptide is prepared by the preparation method of Example 3. The yield of the fully protected cyclic peptide is 75.73%, and the purity is 77.86%.

[0110] Example 6: Preparation method of monocyclic peptide Preparation of monocyclic peptide: Mix the fully protected cyclic peptide with the cleavage solution for 2 h, concentrate, precipitate with ice-cold diethyl ether, wash and centrifuge, and rotary evaporate to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cleavage solution is a mixed solution of TFA, DCM, and EDT, and TFA, DCM, and EDT in the cleavage solution are mixed at a volume ratio of 1:0.8:0.2. The usage amount relationship between the fully protected cyclic peptide and the cleavage solution is 1 g:10 mL. The ice-cold diethyl ether is used appropriately during precipitation. The fully protected cyclic peptide is prepared by the preparation method of Example 4. The purity of the monocyclic peptide is 60.71%, and the yield is 98.61%.

[0111] The monocyclic peptide prepared in Example 6 was purified by reversed-phase C18 liquid chromatography, and the purity of the pure product after freeze-drying was 96%, numbered PR2495B. The chromatography of the reversed-phase C18 chromatography purification in Example 6 of the present invention is as Figure 1 shown.

[0112] The mass spectrum of the monocyclic peptide purified by reversed-phase C18 chromatography in Example 6 of the present invention is as Figure 2 shown.

[0113] Example 7: Preparation method of monocyclic peptide Preparation of monocyclic peptide: The fully protected cyclic peptide was mixed with the cleavage solution for 2 h. After cleavage, it was precipitated with ice-cold diethyl ether, washed, centrifuged, and dried by rotation to obtain the monocyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-). The cleavage solution was a mixed solution of TFA, Tis, and H2O, and TFA, Tis, and H2O in the cleavage solution were mixed at a volume ratio of 1:0.026:0.026. The usage relationship between the fully protected cyclic peptide and the cleavage solution was 1 g:10 mL. An appropriate amount of ice-cold diethyl ether was used for precipitation. The fully protected cyclic peptide was prepared by the preparation method of Example 4. The purity of the monocyclic peptide was 32.26%, and the yield was 126.74%.

[0114] Example 8: Preparation method of bicyclic peptide Preparation of bicyclic peptide: The monocyclic peptide was mixed with acetic acid solution, and the insoluble matter was filtered off. The filtrate was diluted with purified water. An iodine-methanol solution was added with stirring at 25 °C, and stirring was continued until the reaction solution turned yellow and did not fade, indicating that the reaction was complete; then an ascorbic acid solution was added with stirring for reduction until the reaction solution changed 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 solution of the bicyclic peptide. After purification by reverse-phase C18 chromatography and lyophilization, the bicyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide-bridged Cys&Cys), that is, the bicyclic peptide compound, was obtained. The acetic acid solution was prepared by mixing acetic acid and pure water at a volume ratio of 1:1. The usage amount of the monocyclic peptide was 6.8 wt% of the acetic acid solution. The usage amount of purified water for dilution was 1000 wt% of the acetic acid solution. The content of iodine-methanol in the iodine-methanol solution was 0.1 mol / L, and the content of ascorbic acid in the ascorbic acid solution was 1 wt%. The iodine-methanol solution and the ascorbic acid solution were used in appropriate amounts. The monocyclic peptide was prepared by the preparation method of Example 6 and was not purified by reverse-phase C18 liquid chromatography. The purity of the bicyclic peptide was 98.33%.

[0115] Example 9: Preparation method of bicyclic peptide Preparation of bicyclic peptide: The monocyclic peptide was mixed with DMSO, and the insoluble matter was filtered off. The reaction was stirred at 25 °C for 7 d. 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 solution of the bicyclic peptide. After purification by reverse-phase C18 chromatography and lyophilization, the bicyclic peptide Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide-bridged Cys&Cys), that is, the bicyclic peptide compound, was obtained. The usage amount of the monocyclic peptide was 10 wt% of DMSO. The monocyclic peptide was prepared by the preparation method of Example 6. The purity of the bicyclic peptide was 98.21%. The bicyclic peptide compound prepared in Example 9 was numbered PR2495.

[0116] In Example 9 of the present invention, the chromatogram of the purification of PR2495 by reverse-phase C18 liquid chromatography is as Figure 3 shown.

[0117] The mass spectrum of the bicyclic peptide compound obtained in Example 9 of the present invention is as Figure 4 shown.

[0118] Test Example: To verify the physical and chemical properties and physiological functions of PR2495 and PR2495B of the present invention, the following tests were carried out. The culture medium or cell culture medium used in the tests of the present invention was DMEM medium.

[0119] 1. Cytotoxicity test The present invention evaluated the potential toxicity of PR2495 and PR2495B to cells and determined their possible safe concentration ranges.

[0120] In the cytotoxicity test of the present invention, HFF-1 cells were inoculated into a 96-well plate, and 200 μL of culture medium containing about 10,000 cells / well was added to each well to ensure consistent cell density. The cells were cultured overnight in an incubator at 37 °C and 5% CO2 to allow the cells to adhere and grow. After 24 hours, the cell culture medium was aspirated, and 200 μL of DMEM medium containing test samples at different concentrations was added. The 96-well plate was returned to the incubator for continued culture. After 24 hours, the absorbance value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader by the MTT method to determine whether the test samples had cytotoxicity and their safe concentration ranges. The test samples were PR2495 or PR2495B, and the different concentrations of the test samples included 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM. 0 μM was the negative control group, and another group containing only the culture medium without cells was designed as the blank control group. The cell viability was calculated as follows: Cell viability = ((Absorbance of the test group - Absorbance of the blank control group) / (Absorbance of the negative control group - Absorbance of the blank control group)) × 100%.

[0121] The results of the cytotoxicity experiment are as Figure 5 shown. For epithelial cells treated with different concentrations of PR2495 and PR2495B, the cell viability did not show a significant decrease. At a concentration of 500 μM, the cell viability was still maintained at about 96% relative to the control group, confirming that both PR2495 and PR2495B have very low cytotoxicity.

[0122] 2. Serum stability detection The present invention evaluated the degradation of PR2495 and PR2495B in a serum environment to predict their metabolism in vivo.

[0123] The freeze-dried powder of the test sample was directly dissolved in 10 vol% fetal bovine serum at a final concentration of 1 mg / mL. The freeze-dried oligopeptide GHK was dissolved under the same conditions as a control group. The solution prepared above was aseptically filtered through a 0.22 μM filter and filled into a sterile container, and then incubated in an incubator at 37 °C. Sampling was carried out 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 by analytical high performance liquid chromatography. Taking the content at 0 time as 100%, the decomposition ratio was calculated by comparing the peak areas at different time points. Calculation formula: relative content = (peak area of the test sample / peak area of the test sample at 0 time) × 100%. A curve of residual percentage against time was plotted and the half-life was calculated. The test sample was PR2495 or PR2495B.

[0124] The results of the serum stability experiment are as Figure 6 shown. GHK in the control group was almost completely degraded in 10 vol% fetal bovine serum in 4 hours, and its 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 its 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 caused by disulfide bonds can enhance the stability of the polypeptide.

[0125] 3. Test on the promotion rate of type I collagen expression The present invention studied the effects of PR2495 and PR2495B on the expression level of COL1A in human fibroblasts HFF-1.

[0126] The fibroblasts HFF-1 in the logarithmic growth phase were seeded into a 6-well plate at 50,000 cells / well. 2 mL of medium was added to each well and incubated overnight in an incubator to allow the cells to adhere and grow for 24 hours. Grouping was carried out, and DMEM medium containing the test sample was added. In addition, a DMEM blank control group and 100 ng / mL TGF-β1 as a positive control group were established. The test samples were GHK or PR2495 or PR2495B, and the concentrations of the test samples were 10, 20, and 40 μM.

[0127] After treatment for 24 hours, the medium was removed, the RNA of the cells in each well was extracted, and the Ct value was detected by qRT-PCR to judge the expression level of type I collagen mRNA in HFF-1 cells at different concentrations. The expression level was calculated according to the formula: relative RNA expression level = 2ΔΔC(t).

[0128] The t-test statistical analysis was used for comparison between groups. P < 0.05 was considered to have a significant difference, marked * in the figure; P < 0.01 was considered to have a highly significant difference, marked ** in the figure.

[0129] According to the calculation formula: up-regulation rate = (test group - blank control group) / blank control group × 100%, calculate the expression up-regulation rate.

[0130] The experimental results of type I collagen expression are as Figure 7 shown. Compared with the blank control group, after treatment with TGF-β1 in the positive control group, the expression level of COL1A increased significantly, indicating that the cell model and qPCR system were working properly. Compared with the blank control group, treatment with PR2495B had a significant positive regulation on the expression of COL1A. The up-regulation rate was approximately 32% at 10 μM, approximately 42% at 10 μM, and approximately 49% at 40 μM. Compared with the blank control group, treatment with PR2495 had a significant positive regulation on the expression of COL1A. The up-regulation rate was approximately 37% at 10 μM, approximately 58% at 10 μM, and approximately 69% at 40 μM. Both PR2495 and PR2495B could promote the production of type I collagen, and the ability of PR2495 to induce up-regulation of expression was slightly stronger than that of PR2495B.

[0131] 4. Detection of antioxidant effect Superoxide dismutase (SOD) is an important component of the antioxidant enzyme system in biological systems. As a terminal compound, it effectively scavenges superoxide anion radicals to avoid excessive damage to cells. Glutathione (GSH) is continuously oxidized and reduced in cells. Through the action of glutathione peroxidase (GPx), peroxides are reduced to water and oxygen molecules, while GSH itself is oxidized to glutathione disulfide (GSSG). GSSG is rapidly reduced to GSH under the action of glutathione reductase, maintaining the intracellular level of reduced glutathione and reducing oxidative stress.

[0132] This invention studies whether PR2495 and PR2495B can enhance the antioxidant capacity of cells, whether they affect the expression levels of antioxidant-related enzymes, and the final antioxidant capacity.

[0133] Fibroblasts HFF-1 in the logarithmic growth phase were seeded into 6-well plates at a density of 50,000 cells / well. 2 mL of medium was added to each well and incubated overnight in an incubator to allow the cells to adhere and grow for 24 hours. The cells were grouped and DMEM medium containing the test samples was added. In addition, a blank control group was set up, in which no polypeptide was added and no oxidative stress was applied. A negative control group was set up, in which no polypeptide was added, and 7 μg / mL vitamin E was used as the antioxidant positive control group. Oxidative stress was applied by ultraviolet irradiation with UVA. The blank control group was not irradiated. The ultraviolet irradiation with UVA was 30 J / cm 2。The cell supernatants were collected after 24 hours, and the levels of SOD and GSH were detected according to the instructions of the SOD detection kit and the GSH quantification kit. The test samples were GHK or PR2495 or PR2495B, and the concentration of the test samples was 10 μM.

[0134] T-test statistical analysis was used for comparison between groups. A significant difference was considered when P < 0.05, marked with * in the figure; a highly significant difference was considered when P < 0.01, marked with ** in the figure.

[0135] According to the formula: promotion rate = (test group - positive control group) / positive control group × 100%, the promotion levels of SOD and GSH in each group were calculated.

[0136] The results of the antioxidant experiment were as Figures 8 - 9 shown. Compared with the blank control group, the levels of SOD and GSH in the negative control group were significantly down-regulated, and the levels of both in the positive control group were significantly recovered. The SOD level was increased by about 53.7%, and the GSH level was increased by about 119.7%, proving that the oxidative stress stimulation was effective and the antioxidant positive control worked normally. The treatment with GHK showed a certain antioxidant capacity. Compared with the negative control, the SOD level was increased by about 36.5% and the GSH level was increased by about 61.0% at 10 μM. Compared with the negative control, the SOD level of PR2495B was increased by about 88.5%, and the GSH level was increased by about 124%. Compared with the negative control, the SOD level of PR2495 was increased by about 74.7%, and the GSH level was increased by about 120%. Both PR2495 and PR2495B could increase the expression level of the antioxidant key enzyme SOD, and finally increase the expression amount of the antioxidant key substance GSH.

[0137] 5. Detection of anti-inflammatory effect In the inflammatory response, cytokines such as TNF-α, IL-6, and IL-8 are important regulatory molecules. They interact with each other through a complex network and jointly participate in the regulation of immune response, tissue repair, and pathological damage. TNF-α is mainly responsible for activating immune cells, IL-6 promotes the persistence of the immune response, and IL-8 enhances the infiltration and activation of immune cells. There is a complex synergistic relationship among these three factors, which jointly constitute the core network of the inflammatory response. Under pathological conditions, such as autoimmune diseases or infections, their overexpression may lead to tissue damage and organ dysfunction. This part of the study will compare the ability of GHK and PR2495 or PR2495B to inhibit the generation of inflammatory factors in macrophages under the condition of inflammatory stress induced by lipopolysaccharide LPS.

[0138] The macrophage cell line RAW264.7 was seeded into a 6-well plate at 50,000 cells / well and cultured for 24 hours. The cells were grouped and administered drugs. The treatment group was added with DMEM medium containing the test sample. A blank control group was set up, and a negative control group was set up. The negative control group did not add the polypeptide, and 0.01% dexamethasone was used as the anti-inflammatory positive control group. After 2 hours of drug administration, 200 μL of the LPS working solution was added to each well of the groups other than the blank control group, and the cells were placed in an incubator and cultured for another 22 hours. The cell culture supernatant was collected, and TNF-α, IL-6, and IL-8 were detected and analyzed according to the operation instructions of the ELISA kit. The test samples were 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 have a significant difference, marked * in the figure; P<0.01 was considered to have a highly significant difference, marked ** in the figure.

[0139] According to the formula: Inhibition rate % = (Negative control group - Test group) / Negative control group × 100%, the inhibition rate was calculated.

[0140] The results of the anti-inflammatory experiment are as Figures 10 - 12 shown, where TNF is TNF-α, IL6 is IL-6, and IL8 is IL-8. Compared with the blank control group, the contents of TNF-α, IL-6, and IL-8 in the negative control group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the negative control group, the contents of TNF-α, IL-6, and IL-8 in the positive control group decreased significantly. The inhibition rates of TNF-α, IL-6, and IL-8 after treatment in the positive control group were approximately 34.7%, 31.8%, and 48.0% respectively, indicating that the positive control of this test 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 approximately 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 approximately 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 approximately 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.

[0141] The above embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0142] In this text, specific examples are used to elaborate on the principles and implementation modes of the present application. The description of the above embodiments is only for helping to understand the method and its core idea of the present application. The above is only the preferred implementation mode of the present application. It should be noted that due to the limitation of literal expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, retouches or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, retouches, changes or combinations, or directly applying the inventive concept and technical solution to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A method for preparing a monocyclic peptide, comprising: Mix the solid-phase synthesis resin with an amino acid reagent, and prepare a fully protected peptide resin by solid-phase synthesis. Cut and perform monocyclization on the fully protected peptide resin to obtain a monocyclic peptide. The structural formula of the monocyclic 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 derivatives.

2. The method for preparing the monocyclic peptide according to claim 1, wherein: 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.

3. The monocyclic peptide prepared by the preparation method according to 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, 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 derivatives; L1 is formed by bonding R1 and R2.

6. The bicyclic peptide compound according to claim 5, wherein: The bicyclic peptide compound is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bridge connecting Cys&Cys).

7. A method for preparing a bicyclic peptide compound, comprising: The method for preparing the monocyclic peptide according to claim 1.

8. The preparation method of the bicyclic peptide compound according to claim 7, wherein: The monocyclic peptide is subjected to bicyclization to obtain a bicyclic peptide compound; The bicyclization is carried out by cyclization under the action of iodomethanol and ascorbic acid; or, the bicyclization is carried out in DMSO.

9. The use of the monocyclic peptide according to any one of claims 3-4 in the preparation of a bicyclic peptide compound and / or a product for increasing the expression of type I collagen and / or an antioxidant product and / or an anti-inflammatory product.

10. The use of the bicyclic peptide compound according to any one of claims 5-6 in the preparation of a product for increasing the expression of type I collagen and / or an antioxidant product and / or an anti-inflammatory product.

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