Preparation method and application of cyclic peptide compound

Abstract

The invention discloses a preparation method and application of a cyclic peptide compound, CTC resin is taken as a carrier, Fmoc-Gly-OH, Fmoc-Lys (Boc)-OH, Fmoc-His (Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc)-OH and Fmoc-His (Trt)-OH are sequentially coupled through solid phase synthesis, and peptide resin is obtained; the preparation method comprises the following steps: cutting to obtain a straight-chain crude peptide, carrying out cyclization reaction to generate a full-protection cyclopeptide, and finally, carrying out deprotection and purification to obtain the cyclopeptide compound. The cyclopeptide compound is low in cytotoxicity, the serum stability is remarkably improved, pancreatin can be slowly degraded into GHK to play a long-acting role, fibroblast I-type collagen expression is promoted, the SOD and GSH levels under the oxidation pressure are improved, the anti-oxidation and repair effects are better than those of GHK, and the cyclopeptide compound can be applied to the fields of cosmetics, skin repair and anti-inflammatory related medicines.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of polypeptide design, synthesis and application, and particularly relates to a preparation method of a cyclic peptide compound and application thereof. BACKGROUND

[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 extracellular matrix degradation process, the acid protein secreted by the matrix cells rich in cysteine is cleaved and released GHK, which helps tissue remodeling by regulating the level of angiogenesis. 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.

[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 the wound. GHK has antioxidant effects: it can scavenge free radicals, reduce oxidative stress damage to cells, protect cell integrity and function, 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, and reduce the damage of inflammation to tissues, and has certain therapeutic effects on skin inflammatory diseases such as acne and eczema.

[0004] Based on the above functions, GHK as a cosmetic has certain anti-wrinkle and repair effects: it can activate the synthesis of extracellular matrix, increase the content of collagen and elastic fibers, reduce the generation of wrinkles, and make the skin more compact and smooth; for damaged skin, such as sunburn and sensitive skin, GHK has good repair effect, 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 less than 1 h, and its characteristics of easy degradation affect the continuous exertion of its functions.

[0005] Polypeptide cyclization is an important means of 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-enzymatic ability. Due to higher stability and anti-enzymatic ability, the cyclized polypeptide can maintain a longer effective concentration in the body, reducing the frequency of administration.

[0006] Based on the above understanding, we expect to construct a class of compounds that inherit the functions of GHK and have a long half-life through rational design of polypeptide structure. SUMMARY

[0007] The application aims to provide a preparation method of a cyclic peptide compound with high stability and long half-life and application thereof, and aims to solve the problem that GHK has a short half-life under physiological conditions and is easily degraded, so that its function is difficult to continuously exert, while retaining the effects of stimulating fibroblast proliferation, promoting collagen synthesis, antioxidation and anti-inflammation of GHK, so that the cyclic peptide compound has a better application prospect in the fields of cosmetics, skin repair and anti-inflammation.

[0008] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows: A cyclic peptide compound, characterized in that the structure of the cyclic peptide compound is Cyclo(Gly-His-Lys-Gly-His-Lys-), and the cyclic peptide compound is cyclized through a covalent bond between N-terminal and C-terminal amino acid residues.

[0009] A preparation method of a cyclic peptide compound, comprising: According to the coupling sequence of amino acids: Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, a straight-chain crude peptide is synthesized. The straight-chain crude peptide is cyclized in a catalytic system to obtain a fully-protected cyclic peptide. The fully-protected cyclic peptide is cleaved in a cleavage solution, and after precipitation, the cyclic peptide compound is obtained by column chromatography purification and freeze-drying.

[0010] The cyclic peptide compound Cyclo(Gly-His-Lys-Gly-His-Lys-) designed by the application is cyclized to form a cyclic structure through a covalent bond between N-terminal and C-terminal amino acid residues, compared with linear oligopeptide GHK, the cyclic structure significantly reduces the exposure of terminal groups, reduces the recognition and degradation probability of terminal amino acids by proteases, and significantly improves the serum stability, solving the core problem that GHK has a short half-life and is difficult to continuously exert its function; at the same time, the cyclic structure can be slowly degraded in pancreatic enzymes and release GHK, realizing the long-acting and slow-release of GHK and prolonging the duration of physiological function.

[0011] Preferably, the use amount ratio of Fmoc-Gly-OH to CTC resin is 1.5-4 eq.

[0012] Preferably, the use amount ratio of Fmoc-Lys(Boc)-OH to CTC resin is 1.5-4 eq.

[0013] Preferably, the use amount ratio of Fmoc-His(Trt)-OH to CTC resin is 1.5-4 eq.

[0014] Preferably, the purity of the cyclic peptide compound is 95-98%, and the yield of the cyclic peptide compound is 75-85%.

[0015] Preferably, the catalytic system comprises one or more of N,N'-diisopropyl carbodiimide, 1-hydroxybenzotriazole, 2-(7-azabenzotriazolyl)-N,N,N',N'- tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and N- methylmorpholine.

[0016] The catalytic system can more efficiently activate the carboxyl group, promote the formation of amide bond between the N- and C-termini of the linear crude peptide, increase the cyclization rate and cyclization rate, reduce the polymerization by-products in the cyclization process, and improve the purity of the target product.

[0017] Preferably, the cleavage solution comprises trifluoroacetic acid, triisopropylsilane, 1,2-ethanedithiol and phenol.

[0018] Preferably, the volume ratio of triisopropylsilane to trifluoroacetic acid is 3-4:56.

[0019] Preferably, the volume ratio of 1,2-ethanedithiol to trifluoroacetic acid is 1-2:56.

[0020] Preferably, the volume ratio of phenol to trifluoroacetic acid is 1-2:56.

[0021] Triisopropylsilane and 1,2-ethanedithiol in the cleavage solution can effectively capture the carbocation generated in the cleavage process, avoiding oxidation or damage to the side chain of the peptide chain; phenol enhances the solubility of hydrophobic impurities, reduces the adsorption loss of the target peptide, and improves the purity and stability of the cyclic peptide compound.

[0022] Preferably, the synthesis comprises solid phase synthesis or liquid phase synthesis.

[0023] Preferably, the carrier in the solid phase synthesis comprises CTC resin.

[0024] Preferably, the degree of substitution of the CTC resin is 1.2-1.6 mmol / g.

[0025] Preferably, the deprotection solution in the solid phase synthesis comprises piperidine and N,N-dimethylformamide.

[0026] Preferably, the volume ratio of piperidine to N,N-dimethylformamide is 10-30:80.

[0027] Preferably, the cleavage solution in the solid phase synthesis comprises trifluoroacetic acid and dichloromethane.

[0028] Preferably, the volume ratio of trifluoroacetic acid to dichloromethane is 2.5-4.5:346.5.

[0029] Use of the cyclic peptide compound in cosmetics or drugs, and a dosage form of the cyclic peptide compound including oral preparations or topical preparations.

[0030] Preferably, the drug includes a skin wound repair drug for promoting healing of surgical incisions, burn and scald wound surfaces and tissue regeneration.

[0031] Preferably, the drug includes an inflammatory skin disease treatment drug for relieving inflammatory reactions of acne and eczema and performing skin barrier repair.

[0032] Preferably, the drug includes a bone and joint disease treatment drug for promoting collagen synthesis and improving joint function and bone density of patients with osteoarthritis and osteoporosis.

[0033] The present application also provides a preparation method of a peptide resin, comprising: a. dissolving Pip in DMF to obtain a deprotection solution.

[0034] Preferably, the volume ratio of Pip to DMF is 10-30:80.

[0035] b. using DMF as a washing liquid.

[0036] c. adding CTC resin into a reactor, adding DMF, passing nitrogen gas at 24-27°C for 5-15 min, and removing liquid by suction filtration.

[0037] Preferably, the degree of substitution of the CTC resin is 1.2-1.6 mmol / g, the molar amount of the CTC resin is 1 eq, and the mass of the CTC resin is W g.

[0038] Preferably, the amount of DMF is 5-10 W mL / g.

[0039] d. adding Fmoc-Gly-OH into the reactor, adding DCM, passing nitrogen gas at 10-20°C, adding DIEA, reacting at 25-28°C for 2-3 h, adding MeOH for capping for 20-40 min, removing liquid by suction filtration, washing 2-4 times with the washing liquid, adding the deprotection solution for deprotection at 25-28°C for 20-40 min, removing liquid by suction filtration, washing 5-7 times with the washing liquid, and suction drying the liquid for the next reaction.

[0040] Preferably, the amount of Fmoc-Gly-OH is 1.5-4 eq.

[0041] Preferably, the amount of DCM is 5-10 W mL / g.

[0042] Preferably, the amount of DIEA is 4-6 eq.

[0043] Preferably, the amount of MeOH used is 0.5-1.5 W mL / g.

[0044] Preferably, the amount of deprotection solution used is 7-12 W mL / g.

[0045] Preferably, the amount of washing solution used is 7-12 W mL / g.

[0046] e. Fmoc-Lys(Boc)-OH is dissolved in DMF, DIC is added and activated at 0-10°C for 3-10 min, then added to the reactor, nitrogen is introduced at 25-28°C for 30-50 min, the liquid is removed by suction filtration, washed with washing solution for 2-4 times, deprotection solution is added at 25-28°C for 20-40 min, the liquid is removed by suction filtration, washed with washing solution for 5-7 times, the liquid is sucked dry, and the next step is carried out.

[0047] Preferably, the amount of Fmoc-Lys(Boc)-OH used is 1.5-4 eq.

[0048] Preferably, the amount of HOBt used is 1.5-4 eq.

[0049] Preferably, the amount of DMF used is 3-5 W mL / g.

[0050] Preferably, the amount of DIC used is 1.5-4 eq.

[0051] Preferably, the amount of deprotection solution used is 7-12 W mL / g.

[0052] Preferably, the amount of washing solution used is 7-12 W mL / g.

[0053] f. Fmoc-His(Trt)-OH is dissolved in DMF, HOBt is added and activated at 0-10°C for 2-5 min, then added to the reactor, nitrogen is introduced at 25-28°C for 30-50 min, the liquid is removed by suction filtration, washed with washing solution for 2-4 times, deprotection solution is added at 25-28°C for 20-40 min, the liquid is removed by suction filtration, washed with washing solution for 5-7 times, the liquid is sucked dry, and the next step is carried out.

[0054] Preferably, the amount of Fmoc-His(Trt)-OH used is 1.5-4 eq.

[0055] Preferably, the amount of HOBt used is 1.5-4 eq.

[0056] Preferably, the amount of DMF used is 3-5 W mL / g.

[0057] Preferably, the amount of DIC used is 1.5-4 eq.

[0058] Preferably, the amount of deprotection solution is 7-12 W mL / g.

[0059] Preferably, the amount of washing solution is 7-12 W mL / g.

[0060] g. Dissolve Fmoc-Gly-OH and HOBt in DMF, add DIC and activate for 2-5 min at 0-10°C, then add to the reactor, pass nitrogen at 25-28°C for 30-50 min, remove the liquid by suction filtration, wash 2-4 times with washing solution, add deprotection solution at 25-28°C for 20-40 min, remove the liquid by suction filtration, wash 5-7 times with washing solution, and dry the liquid by suction, then proceed to the next step.

[0061] Preferably, the amount of Fmoc-Gly-OH is 1.5-4 eq.

[0062] Preferably, the amount of HOBt is 1.5-4 eq.

[0063] Preferably, the amount of DMF is 3-5 W mL / g.

[0064] Preferably, the amount of DIC is 1.5-4 eq.

[0065] Preferably, the amount of deprotection solution is 7-12 W mL / g.

[0066] Preferably, the amount of washing solution is 7-12 W mL / g.

[0067] h. Dissolve Fmoc-Lys(Boc)-OH and HOBt in DMF, add DIC and activate for 3-10 min at 0-10°C, then add to the reactor, pass nitrogen at 25-28°C for 30-50 min, remove the liquid by suction filtration, wash 2-4 times with washing solution, add deprotection solution at 25-28°C for 20-40 min, remove the liquid by suction filtration, wash 5-7 times with washing solution, and dry the liquid by suction, then proceed to the next step.

[0068] Preferably, the amount of Fmoc-Lys(Boc)-OH is 1.5-4 eq.

[0069] Preferably, the amount of HOBt is 1.5-4 eq.

[0070] Preferably, the amount of DMF is 3-5 W mL / g.

[0071] Preferably, the amount of DIC is 1.5-4 eq.

[0072] Preferably, the amount of deprotection solution is 7-12 W mL / g.

[0073] Preferably, the amount of washing solution is 7-12 W mL / g.

[0074] i. Dissolve Fmoc-His(Trt)-OH and HOBt in DMF, activate for 2-5 min at 0-10 DEG C, add to the reactor, pass nitrogen at 25-28 DEG C for 30-50 min, remove the liquid by suction filtration, wash 2-4 times with washing solution, add deprotection solution at 25-28 DEG C for 20-40 min, remove the liquid by suction filtration, wash 5-7 times with washing solution, and dry the liquid by suction, and proceed to the next step.

[0075] Preferably, the amount of Fmoc-His(Trt)-OH is 1.5-4 eq.

[0076] Preferably, the amount of HOBt is 1.5-4 eq.

[0077] Preferably, the amount of DMF is 3-5 W mL / g.

[0078] Preferably, the amount of DIC is 1.5-4 eq.

[0079] Preferably, the amount of deprotection solution is 7-12 W mL / g.

[0080] Preferably, the amount of washing solution is 7-12 W mL / g.

[0081] j. Wash 1-3 times with methyl tert-butyl ether, remove the liquid by suction filtration, wash 1-3 times with tetrahydrofuran, remove the liquid by suction filtration, wash 1-3 times with methyl tert-butyl ether, remove the liquid by suction filtration, and vacuum dry for 10-15 h to obtain the peptide resin; Preferably, the amount of methyl tert-butyl ether is 7-12 W mL / g.

[0082] Preferably, the amount of tetrahydrofuran is 7-12 W mL / g.

[0083] The application also provides a preparation method of a linear crude peptide, comprising: Mix TFA and DCM to obtain a cleavage solution, add the peptide resin, and shake bed react at 30 DEG C for 30 min; after filtration, the mother liquor is subjected to sedimentation to precipitate solid, centrifugal separation of the supernatant, and centrifugal washing with washing solution for 3 times; and vacuum dry for 10-15 h to obtain the crude peptide.

[0084] Preferably, the volume ratio of TFA to DCM is 2.5-4.5:346.5.

[0085] Preferably, the volume-mass ratio of TFA to the peptide resin is 2.5-4.5 mL:35 g.

[0086] Preferably, the volume-mass ratio of petroleum ether to the peptide resin is 2600-3000 mL:35 g.

[0087] Preferably, the washing liquid is petroleum ether, and the volume-mass ratio of the washing liquid to the peptide resin is 250-350 mL:35 g.

[0088] The application further provides a preparation method of a fully-protected cyclic peptide, comprising: The linear crude peptide is dissolved in DMF to obtain solution A; HATU and DIEA are dissolved in DMF to obtain solution B; solution A is added to solution B at 24-27°C, and the reaction is carried out for 50-70 min; the reaction solution is extracted with a primary extractant, and then extracted with a secondary extractant in an aqueous phase; the organic phases are combined, washed with saturated sodium bicarbonate aqueous solution for 1-2 times, washed with deionized water for 1-3 times, washed with saturated sodium chloride aqueous solution for 1-3 times, dried with anhydrous sodium sulfate, filtered, and spin-dried to obtain the fully-protected cyclic peptide.

[0089] Preferably, in solution A, the mass-volume ratio of the linear crude peptide to DMF is 10 g:100 mL.

[0090] Preferably, in solution B, the mass-volume ratio of HATU to DMF is 7-8 g:30 mL, and the volume-mass ratio of HATU to DIEA is 7-8 g:6.8 mL.

[0091] Preferably, the mass of solution A is measured by the mass of the linear crude peptide therein, the mass of solution B is measured by the mass of HATU therein, and the mass ratio of HATU to the linear crude peptide is 7-8:10.

[0092] Preferably, the primary extractant is 40-60 v / v% ethyl acetate aqueous solution.

[0093] Preferably, the secondary extractant is ethyl acetate.

[0094] The application further provides a preparation method of a cyclic peptide compound, comprising: TFA, TIS, EDT and PhOH are dissolved in deionized water to obtain a cleavage solution, and the fully-protected cyclic peptide is added; the cleavage is carried out for 1-3 h; ice ethyl ether is added to precipitate the solid; the supernatant is separated by centrifugation; the washing liquid is used for centrifugal washing for 2-4 times; after spin-drying, RP-HPLC purification is carried out; the eluate is collected and freeze-dried to obtain the cyclic peptide compound.

[0095] Preferably, the volume ratio of deionized water to TFA is 1-2:56. Preferably, the volume ratio of TIS to TFA is 3-4:56.

[0096] Preferably, the volume ratio of EDT to TFA is 1-2:56.

[0097] Preferably, the volume ratio of PhOH to TFA is 1-2:56.

[0098] Preferably, the mass volume ratio of the fully protected cyclic peptide to TFA is 5-10 g:56 mL.

[0099] Preferably, the volume mass ratio of ethyl ether to the fully protected cyclic peptide is 600-700 mL:8 g.

[0100] Preferably, the washing liquid is ethyl ether, and the volume mass ratio of the washing liquid to the fully protected cyclic peptide is 80-120 mL:8 g.

[0101] Preferably, the chromatographic column is a C18 reverse phase chromatographic column with a size of 2-6 mm x 150-300 mm.

[0102] Preferably, the mobile phase A is a 0.05-0.15 wt% TFA aqueous solution, and the mobile phase B is a 0.05-0.15 wt% TFA acetonitrile solution.

[0103] Preferably, the volume ratio of the mobile phase A to the mobile phase B is 70-90:20, and the flow rate is 0.8-1.2 mL / min.

[0104] Preferably, the injection amount is 5-15 μL.

[0105] The present application has the following beneficial effects: high purity and yield of the product, low cytotoxicity of the cyclic peptide compound, significantly improved serum stability, slow degradation into GHK in pancreatic enzymes for long-acting effect, and better antioxidant capacity than GHK in promoting type I collagen synthesis. Therefore, the present application is a cyclic peptide compound preparation method and its application, which is efficient, stable, biocompatible, has long functional duration, and is suitable for the fields of cosmetics, skin repair, and anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS

[0106] Figure 1 is a schematic diagram of the molecular structure of the cyclic peptide compound.

[0107] Figure 2 is a schematic diagram of the liquid chromatogram of the cyclic peptide compound.

[0108] Figure 3 is a schematic diagram of the mass spectrum of the cyclic peptide compound.

[0109] Figure 4 is a schematic diagram of the test results of the cytotoxicity of the cyclic peptide compound.

[0110] Figure 5 The stability test results of the cyclic peptide compound in 10% fetal bovine serum are shown in the following schematic diagram.

[0111] Figure 6 The stability test results of the cyclic peptide compound in 100% fetal bovine serum are shown in the following schematic diagram.

[0112] Figure 7 The stability test results of the cyclic peptide compound in pancreatin are shown in the following schematic diagram.

[0113] Figure 8 The mass spectrum of unknown peaks generated in the degradation of the cyclic peptide compound in pancreatin test is shown in the following schematic diagram.

[0114] Figure 9 The liquid phase comparison diagram of the unknown peaks generated in the degradation of the cyclic peptide compound in pancreatin test and the retention time of GHK is shown in the following schematic diagram.

[0115] Figure 10 The stability test results of the cyclic peptide compound in pepsin are shown in the following schematic diagram.

[0116] Figure 11 The test results of the cyclic peptide compound on the expression promotion rate of collagen type I gene are shown in the following schematic diagram.

[0117] Figure 12 The test results of the cyclic peptide compound on the expression level of SOD under oxidative stress of fibroblasts are shown in the following schematic diagram.

[0118] Figure 13 The test results of the cyclic peptide compound on the expression level of GSH under oxidative stress of fibroblasts are shown in the following schematic diagram. DETAILED DESCRIPTION

[0119] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0120] The concepts involved in the present application will be described below in combination with the drawings. It should be pointed out here that the descriptions of the various concepts below are only to make the content of the present application easier to understand, and do not represent a limitation on the scope of protection 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.

[0121] The abbreviations used in the present application correspond to the Chinese meanings shown in the following Table 1.

[0122] Table 1: Chinese meanings of abbreviations

[0123] Example 1: Provide a cyclic peptide compound, specifically including the following preparation steps: Preparation of peptide resin: a. Dissolve 20 mL of Pip in 80 mL of DMF to obtain a deprotection solution; b. Use DMF as a washing liquid; c. Add 28.6 g of CTC resin to the reactor, add 250 mL of DMF, stir under the condition of 25°C with nitrogen for 10 min, and remove the liquid by suction filtration; d. Add 23.8 g of Fmoc-Gly-OH to the reactor, add 200 mL of DCM, stir under the condition of 15°C with nitrogen, add 33 mL of DIEA, react under the condition of 26°C for 2.5 h, add 30 mL of MeOH to cap for 30 min, remove the liquid by suction filtration, wash with 300 mL of washing liquid for 3 times, add 300 mL of deprotection solution to deprotect under the condition of 26°C for 30 min, remove the liquid by suction filtration, wash with 300 mL of washing liquid for 6 times, and dry the liquid for the next reaction; e. Dissolve 45 g of Fmoc-Lys(Boc)-OH and 13 g of HOBt in 100 mL of DMF, activate at 4°C for 5 min after adding 14.8 mL of DIC, add to the reactor, stir under the condition of 26°C with nitrogen for 40 min, remove the liquid by suction filtration, wash with 300 mL of washing liquid for 3 times, add 300 mL of deprotection solution to deprotect under the condition of 26°C for 30 min, remove the liquid by suction filtration, wash with 300 mL of washing liquid for 6 times, and dry the liquid for the next reaction; f. Dissolve 59.5 g of Fmoc-His(Trt)-OH and 13 g of HOBt in 100 mL of DMF, activate at 4°C for 3 min after adding 14.8 mL of DIC, add to the reactor, stir under the condition of 26°C with nitrogen for 40 min, remove the liquid by suction filtration, wash with 300 mL of washing liquid for 3 times, add 300 mL of deprotection solution to deprotect under the condition of 26°C for 30 min, remove the liquid by suction filtration, wash with 300 mL of washing liquid for 6 times, and dry the liquid for the next reaction; g. 28.54 g of Fmoc-Gly-OH was dissolved in 100 mL of DMF, 14.8 mL of DIC was added at 4°C for 3 min, then added to the reactor, nitrogen was introduced at 26°C, reacted for 40 min, the liquid was removed by suction filtration, washed with 300 mL of washing liquid for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing liquid for 6 times, the liquid was sucked dry, and the next step was carried out; h. 45 g of Fmoc-Lys(Boc)-OH was dissolved in 100 mL of DMF, 14.8 mL of DIC was added at 4°C for 5 min, then added to the reactor, nitrogen was introduced at 26°C, reacted for 40 min, the liquid was removed by suction filtration, washed with 300 mL of washing liquid for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing liquid for 6 times, the liquid was sucked dry, and the next step was carried out; i. 59.5 g of Fmoc-His(Trt)-OH was dissolved in 100 mL of DMF, 14.8 mL of DIC was added at 4°C for 3 min, then added to the reactor, nitrogen was introduced at 26°C, reacted for 40 min, the liquid was removed by suction filtration, washed with 300 mL of washing liquid for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing liquid for 6 times, the liquid was sucked dry, and the next step was carried out; j. 300 mL of methyl tert-butyl ether was added and washed for 2 times, the liquid was removed by suction filtration, 300 mL of tetrahydrofuran was added and washed for 2 times, the liquid was removed by suction filtration; 300 mL of methyl tert-butyl ether was added and washed for 2 times, the liquid was removed by suction filtration, and vacuum drying was carried out for 12 h to obtain the peptide resin.

[0124] Preparation of linear crude peptide: TFA was mixed with DCM to obtain a cutting liquid, the peptide resin was added, and the reaction was carried out on a shaking bed at 30°C for 30 min. After filtration, the mother liquor was precipitated with petroleum ether to obtain a solid, the supernatant was separated by centrifugation, and then washed with washing liquid by centrifugation for 3 times. Vacuum drying was carried out for 12 h to obtain the crude peptide. The volume ratio of TFA to DCM was 3.5:346.5, the mass volume ratio of peptide resin to TFA was 35 g:3.5 mL, the volume mass ratio of petroleum ether to peptide resin was 2800 mL:35 g; the washing liquid was petroleum ether, and the volume mass ratio of washing liquid to peptide resin was 300 mL:35 g.

[0125] Preparation of the full-protected cyclic peptide: dissolve the linear crude peptide in DMF as solution A; dissolve HATU and DIEA in DMF as solution B; add solution A to solution B at 25°C, react for 60 min, extract the reaction solution in a primary extraction agent, extract in a secondary extraction agent in the aqueous phase, combine the organic phases, wash once with saturated aqueous sodium bicarbonate solution, wash twice with deionized water, wash twice with saturated aqueous sodium chloride solution, dry over anhydrous sodium sulfate and filter, and spin dry to obtain the full-protected cyclic peptide. In solution A, the mass / volume ratio of the linear crude peptide to DMF is 10 g: 100 mL; in solution B, the mass / volume ratio of HATU to DMF is 7.8 g: 30 mL, and the volume / mass ratio of DIEA to HATU is 6.8 mL: 7.8 g; the mass of solution A is measured based on the mass of the linear crude peptide therein, and the mass of solution B is measured based on the mass of HATU therein, and the mass ratio of the linear crude peptide to HATU is 10:7.8; the primary extraction agent is 50 v / v% ethyl acetate aqueous solution, and the secondary extraction agent is ethyl acetate.

[0126] Preparation of the cyclic peptide compound: dissolve TFA, TIS, EDT and PhOH in deionized water to obtain a cleavage solution, add the full-protected cyclic peptide, cut for 2 h, precipitate the solid by adding ethyl ether, centrifuge to separate the supernatant, centrifuge to wash with a washing solution for 3 times, spin dry, and purify by RP-HPLC, elute and collect, and freeze dry to obtain the cyclic peptide compound. The volume ratio of TFA to deionized water is 56:1.6, the volume ratio of TIS to TFA is 3.2:56, the volume ratio of EDT to TFA is 1.6:56, the volume ratio of PhOH to TFA is 1.6:56, the mass / volume ratio of the full-protected cyclic peptide to TFA is 8 g: 56 mL, the volume / mass ratio of ethyl ether to the full-protected cyclic peptide is 640 mL: 8 g, the washing solution is ethyl ether, and the volume / mass ratio of the washing solution to the full-protected cyclic peptide is 100 mL: 8 g; the chromatographic column is a C18 reversed-phase chromatographic column with a size of 4.6 mm x 250 mm, the mobile phase A is 0.1 wt% TFA aqueous solution, the mobile phase B is 0.1 wt% TFA acetonitrile solution, the volume ratio of the mobile phase A to the mobile phase B is 80:20, the flow rate is 1 mL / min, and the injection amount is 10 μL.

[0127] Example 2: This example is different from Example 1 only in the preparation of the full-protected cyclic peptide.

[0128] Preparation of the full-protected cyclic peptide: dissolve the linear crude peptide in DMF as A solution; dissolve HATU and NMM in DMF as B solution; add A solution to B solution at 25°C, react for 60 min, extract the reaction solution in one extraction agent, extract in the second extraction agent in the aqueous phase, combine the organic phases, wash once with saturated aqueous sodium bicarbonate solution, wash twice with deionized water, wash twice with saturated aqueous sodium chloride solution, dry with anhydrous sodium sulfate, filter, and spin dry to obtain the full-protected cyclic peptide. In the A solution, the mass / volume ratio of the linear crude peptide to DMF is 10 g: 100 mL; in the B solution, the mass / volume ratio of HATU to DMF is 7.8 g: 30 mL, and the volume / mass ratio of NMM to HATU is 6.8 mL: 7.8 g; the mass of the A solution is measured based on the mass of the linear crude peptide therein, and the mass of the B solution is measured based on the mass of HATU therein, and the mass ratio of the linear crude peptide to HATU is 10:7.8; the first extraction agent is 50 v / v% ethyl acetate aqueous solution, and the second extraction agent is ethyl acetate.

[0129] Example 3: This example is compared with Example 1, and the only difference is the preparation of the peptide resin.

[0130] Preparation of the peptide resin: a. dissolve 20 mL of Pip in 80 mL of DMF to obtain a deprotection solution; b. use DMF as a washing solution; c. add 28.6 g of CTC resin to the reactor, add 250 mL of DMF, stir under nitrogen at 25°C for 10 min, and remove the liquid by suction filtration; d. add 35.7 g of Fmoc-Gly-OH to the reactor, add 200 mL of DCM, stir under nitrogen at 15°C, add 49.5 mL of DIEA, react at 26°C for 2.5 h, add 45 mL of MeOH to cap for 30 min, remove the liquid by suction filtration, wash 3 times with 300 mL of the washing solution, add 300 mL of the deprotection solution to deprotect at 26°C for 30 min, remove the liquid by suction filtration, wash 6 times with 300 mL of the washing solution, and dry the liquid for the next reaction; e. dissolve 67.5 g of Fmoc-Lys(Boc)-OH and 19.5 g of HOBt in 150 mL of DMF, activate at 4°C for 5 min after adding 22.2 mL of DIC, add to the reactor, stir under nitrogen at 26°C for 40 min, remove the liquid by suction filtration, wash 3 times with 300 mL of the washing solution, add 300 mL of the deprotection solution to deprotect at 26°C for 30 min, remove the liquid by suction filtration, wash 6 times with 300 mL of the washing solution, and dry the liquid for the next reaction; f. 89.25 g of Fmoc-His(Trt)-OH was dissolved in 150 mL of DMF, 22.2 mL of DIC was added at 4°C for 3 min, and then added to the reactor, nitrogen was introduced at 26°C, and the reaction was carried out for 40 min. The liquid was removed by suction filtration, washed with 300 mL of washing solution for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing solution for 6 times, the liquid was sucked dry, and the next step was carried out. g. 42.81 g of Fmoc-Gly-OH was dissolved in 150 mL of DMF, 22.2 mL of DIC was added at 4°C for 3 min, and then added to the reactor, nitrogen was introduced at 26°C, and the reaction was carried out for 40 min. The liquid was removed by suction filtration, washed with 300 mL of washing solution for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing solution for 6 times, the liquid was sucked dry, and the next step was carried out. h. 67.5 g of Fmoc-Lys(Boc)-OH was dissolved in 150 mL of DMF, 22.2 mL of DIC was added at 4°C for 5 min, and then added to the reactor, nitrogen was introduced at 26°C, and the reaction was carried out for 40 min. The liquid was removed by suction filtration, washed with 300 mL of washing solution for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing solution for 6 times, the liquid was sucked dry, and the next step was carried out. i. 89.25 g of Fmoc-His(Trt)-OH was dissolved in 150 mL of DMF, 22.2 mL of DIC was added at 4°C for 3 min, and then added to the reactor, nitrogen was introduced at 26°C, and the reaction was carried out for 40 min. The liquid was removed by suction filtration, washed with 300 mL of washing solution for 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, washed with 300 mL of washing solution for 6 times, the liquid was sucked dry, and the next step was carried out. j. 300 mL of methyl tert-butyl ether was added for washing for 2 times, the liquid was removed by suction filtration, 300 mL of tetrahydrofuran was added for washing for 2 times, the liquid was removed by suction filtration, 300 mL of methyl tert-butyl ether was added for washing for 2 times, the liquid was removed by suction filtration, and vacuum drying was carried out for 12 h to obtain the peptide resin.

[0131] Example 4: Compared with Example 1, the difference is only in the preparation of the peptide resin.

[0132] Preparation of the peptide resin: a. 20 mL of Pip was dissolved in 80 mL of DMF to obtain a deprotection solution; b. DMF was used as a washing liquid; c. 28.6 g of CTC resin was added to the reactor, 250 mL of DMF was added, nitrogen was introduced at 25°C for 10 min, and the liquid was removed by suction filtration; d. 47.6 g of Fmoc-Gly-OH was added to the reactor, 200 mL of DCM was added, nitrogen was introduced at 15°C, 66 mL of DIEA was added, and the reaction was carried out at 26°C for 2.5 h, 60 mL of MeOH was added for capping for 30 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 6 times, the liquid was sucked dry, and the next step was carried out; e. 90 g of Fmoc-Lys(Boc)-OH and 26 g of HOBt were dissolved in 200 mL of DMF, 29.6 mL of DIC was added at 4°C for activation for 5 min, then added to the reactor, nitrogen was introduced at 26°C for 40 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 6 times, the liquid was sucked dry, and the next step was carried out; f. 119 g of Fmoc-His(Trt)-OH and 26 g of HOBt were dissolved in 200 mL of DMF, 29.6 mL of DIC was added at 4°C for activation for 3 min, then added to the reactor, nitrogen was introduced at 26°C for 40 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 6 times, the liquid was sucked dry, and the next step was carried out; g. 57.08 g of Fmoc-Gly-OH and 26 g of HOBt were dissolved in 200 mL of DMF, 29.6 mL of DIC was added at 4°C for activation for 3 min, then added to the reactor, nitrogen was introduced at 26°C for 40 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 3 times, 300 mL of deprotection solution was added at 26°C for deprotection for 30 min, the liquid was removed by suction filtration, 300 mL of washing liquid was used for washing 6 times, the liquid was sucked dry, and the next step was carried out; h. 90 g of Fmoc-Lys(Boc)-OH was dissolved in 200 mL of DMF, 29.6 mL of DIC was added at 4°C for 5 min, and then added to the reactor, nitrogen was introduced at 26°C for 40 min, and then filtered to remove the liquid, washed with 300 mL of washing solution for 3 times, 300 mL of deprotection solution was added at 26°C for 30 min, and then filtered to remove the liquid, washed with 300 mL of washing solution for 6 times, and then the liquid was dried, and then the next step was carried out; i. 119 g of Fmoc-His(Trt)-OH was dissolved in 200 mL of DMF, 29.6 mL of DIC was added at 4°C for 3 min, and then added to the reactor, nitrogen was introduced at 26°C for 40 min, and then filtered to remove the liquid, washed with 300 mL of washing solution for 3 times, 300 mL of deprotection solution was added at 26°C for 30 min, and then filtered to remove the liquid, washed with 300 mL of washing solution for 6 times, and then the liquid was dried, and then the next step was carried out; j. 300 mL of methyl tert-butyl ether was added and washed for 2 times, and then filtered to remove the liquid, 300 mL of tetrahydrofuran was added and washed for 2 times, and then filtered to remove the liquid; 300 mL of methyl tert-butyl ether was added and washed for 2 times, and then filtered to remove the liquid, and then vacuum dried for 12 h to obtain the peptide resin.

[0133] Test Example 1: Purity and yield test of the cyclic peptide compound.

[0134] Test sample: The cyclic peptide compound prepared in Example 1.

[0135] Test method: According to the peak area of the cyclic peptide compound after purification, the purity R(%)=S1 / S0x100% was calculated, wherein S1 is the main peak area of the cyclic peptide compound, and S0 is the total peak area. According to m 纯品 =m1xR, the quality of the pure product was calculated, and according to S(%)=m 纯品 / m0x100%, the yield was calculated. Wherein m1 is the mass of the cyclic peptide compound after purification, R is the purity of the cyclic peptide compound after purification, and m0 is the mass of the theoretical cyclic peptide compound.

[0136] The molecular structure of the cyclic peptide compound prepared by the application is shown in Figure 1 The liquid chromatogram of the cyclic peptide compound prepared by the application is shown in Figure 2 The mass spectrum of the cyclic peptide compound prepared by the application is shown in Figure 3 The purity is 96.8%, and the yield is 80.5%, which indicates that the cyclic peptide compound with the structure shown in Figure 1 is successfully prepared.

[0137] Test Example 2: Cytotoxicity test of cyclic peptide compounds.

[0138] Test sample: cyclic peptide compounds prepared in Example 1.

[0139] Test method: HFF-1 cells were inoculated into a 96-well plate, 200 μL of medium was added to each well, containing 10,000 cells / well, to ensure uniform cell density; incubated in a 37°C, 5% CO2 incubator overnight to allow the cells to adhere and grow, 24 h later, the cell culture medium was aspirated, 200 μL of DMEM medium was added as a control group, and 200 μL of DMEM medium containing 1, 5, 10, 50, 100 and 500 μM of cyclic peptide compounds was added as an experimental group, the 96-well plate was returned to the incubator for continued incubation for 24 h, the absorbance value was measured at 490 nm wavelength by MTT method using a microplate reader, and a group of only medium without cells was designed as a blank group, the cell viability (%) = (A1-A0 / A2-A0) x 100% was calculated to obtain the cell viability, wherein A1 is the absorbance of the experimental group, A2 is the absorbance of the blank group, and A0 is the absorbance of the control group, to determine whether the compound has cytotoxicity and its safe concentration range.

[0140] The test results of the cytotoxicity of the cyclic peptide compounds prepared by the present application are shown in Table 1, and the cell viability of the epithelial cells treated with different concentrations of different cyclic peptides does not show a significant decrease in cell viability, and the cell viability at a concentration of 500 μM is still maintained at 95% relative to the control group, confirming that the cyclic peptide compounds have very low cytotoxicity. Figure 4

[0141] Test Example 3: Serum stability test of cyclic peptide compounds.

[0142] Test sample: cyclic peptide compounds prepared in Example 1.

[0143] Test method: The freeze-dried cyclic peptide compounds were dissolved in 10% and 100% fetal bovine serum, respectively, at a final concentration of 1 mg / mL; the freeze-dried oligopeptide GHK was also dissolved under the same conditions as a control group; the above prepared solution was filtered through a 0.22 μM sterile filter and placed in a sterile container, incubated in a 37°C incubator, sampled at the set time points of 0, 0.25, 0.5, 1, 2, 4, 8, 24 and 48 h, and the residual amount of the polypeptide was determined using analytical high performance liquid chromatography, the decomposition ratio was calculated by comparing the peak areas at different time points, the relative content (%) = (S1 / S0) x 100% was calculated to obtain the relative content, wherein S1 is the peak area of the test sample at different times, and S0 is the peak area of the test sample at 0 h, the residual percentage-time curve was drawn, the half-life was calculated, and the decomposition of the polypeptide in the serum environment was evaluated to predict its metabolism in vivo.

[0144] ​The stability test results of the cyclic peptide compound prepared by the present application in 10% fetal bovine serum are shown in Figure 5 As shown, under the condition of 10% serum, the GHK in the control group is almost completely degraded in 4h, and the degradation half-life is about 0.7h according to the degradation curve, while under the same conditions, the cyclic peptide compound still remains more than 95% after 48h; the stability test results of the cyclic peptide compound prepared by the present application in 100% fetal bovine serum are shown in Figure 6 As shown, under the condition of 100% serum, the relative content of the cyclic peptide compound decreases by 0.9% in 24h, and still remains more than 95% after 48h.

[0145] Test Example 4: Trypsin stability test of cyclic peptide compound.

[0146] Test sample: cyclic peptide compound prepared in Example 1.

[0147] Test method: The freeze-dried cyclic peptide compound is dissolved in deionized water to 10mg / ml, and then diluted with 0.25% trypsin cell digestion solution to a final concentration of 1mg / mL; the freeze-dried oligopeptide GHK is also dissolved under the same conditions as the control group; the above prepared solution is filtered through a 0.22μM sterile filter and then placed in a sterile container and incubated in a 37°C incubator; sampling is performed at 0, 1, 2, 4, 8, 12, 16 and 24h according to the set time points, and the residual amount of the polypeptide is determined by using analytical high performance liquid chromatography; the decomposition ratio is calculated by comparing the peak areas at different time points, and the relative content (%)=(S1 / S0)×100% is calculated to obtain the relative content, wherein S1 is the peak area of the test sample at different times, and S0 is the peak area of the test sample at 0h; the residual percentage-time curve is drawn, and the half-life is calculated to study the stability of the cyclic peptide compound in 0.25% trypsin cell digestion solution and evaluate the degradation rate thereof in the simulated intestinal digestion environment. When the high performance liquid chromatography detection of the cyclic peptide compound is performed under the condition of 0.25% trypsin digestion, a new generated chromatographic peak appears before the retention time of the main peak with the decrease of the peak area of the main peak, the molecular weight of the unknown peak is determined by using liquid chromatography-mass spectrometry, and the high performance liquid chromatography retention time is compared by using the GHK standard.

[0148] The stability test results of the cyclic peptide compound prepared by the present application in trypsin are shown in Figure 7 As shown, the relative content of the GHK in the control group is almost unchanged under the condition of 0.25% trypsin cell digestion solution for 24h, indicating that the GHK can exist stably under the condition of 0.25% trypsin cell digestion solution for 24h, and under the same conditions, the relative content of the cyclic peptide compound decreases by 68.6% under the condition of 0.25% trypsin cell digestion solution for 24h, and the degradation half-life thereof is about 8.8h according to the degradation curve.

[0149] The mass spectrum of the unknown peak generated by the degradation of the cyclic peptide compound prepared by the present invention in the pancreatic enzyme test is as follows: Figure 8 As shown, the molecular weight of the unknown peak determined by liquid chromatography-mass spectrometry is consistent with the molecular weight of GHK; the unknown peak generated by the degradation of the cyclic peptide compound prepared by the present invention in the pancreatic enzyme test is compared with the GHK peak retention time liquid phase comparison as shown in the figure. Figure 9 As shown, in the retention time comparison experiment in the HPLC spectrum, the retention time of the unknown peak was consistent with the retention time of GHK, confirming that the cyclic peptide compound would be decomposed into GHK under the conditions of 0.25% trypsin cell digestion solution and exist stably in the form of GHK.

[0150] Test Example 5: Pepsin stability test of cyclic peptide compounds.

[0151] Test sample: the cyclic peptide compound prepared in Example 1.

[0152] Test method: The cyclic peptide compound was dissolved in deionized water to 10 mg / ml, and then diluted with 9 times the volume of artificial gastric fluid containing pepsin to a final peptide concentration of 1 mg / mL; the lyophilized oligopeptide GHK was dissolved under the same conditions as the control group; the above-prepared solution was sterile-filtered through 0.22 μM, placed in a sterile container, and incubated in a 37°C thermostat. Samples were taken at the set time points of 0, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours. The residual amount of the peptide was determined by analytical high-performance liquid chromatography. The decomposition ratio was calculated by comparing the peak areas at different time points. The relative content was calculated according to relative content (%) = (S1 / S0) × 100%, where S1 is the peak area of ​​the test sample at different times and S0 is the peak area of ​​the test sample at 0 hours. The residual percentage versus time curve was plotted and the half-life was calculated. The stability of the cyclic peptide compound in pepsin / artificial gastric fluid was studied and its degradation rate in the simulated gastric digestion environment was evaluated.

[0153] The stability test results of the cyclic peptide compound prepared by the present invention in pepsin are as follows: Figure 10 As shown, the relative levels of GHK and cyclic peptide compounds in the control group remained stable in the pepsin / artificial gastric fluid conditions within 24 hours in both experimental groups. Combining all the above stability data: GHK is stable in gastric fluid and stable under the action of trypsin, but has extremely poor stability in serum; cyclic peptide compounds, while maintaining stability in gastric fluid, greatly improve their stability in serum, showing good potential as oral drugs; cyclic peptide compounds are unstable under the action of trypsin and will slowly degrade and release GHK, thereby exerting corresponding physiological functions for a long time.

[0154] Experimental Example 6: Test on the effect of cyclic peptide compounds on the expression enhancement of type I collagen gene.

[0155] Test sample: cyclopeptide compound prepared in Example 1.

[0156] Test method: fibroblast HFF-1 in logarithmic growth phase was inoculated into a 6-well plate at 50000 cells / well, 2mL of culture medium was added to each well, and the plate was placed in an incubator overnight to allow the cells to adhere and grow for 24h; DMEM medium containing 10, 20 and 40μM of GHK or cyclopeptide compound was used as the experimental group, blank DMEM medium was used as the blank control group, and DMEM medium containing 100 ng / mL of TGF-β1 was used as the positive control group; after 24h 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, the expression level of type I collagen mRNA of HFF-1 cells at different concentrations was determined, and the expression level was calculated according to the formula: RNA relative expression level = 2 -ΔΔC(t) , ΔΔC(t) = ΔCt 实验组 -ΔCt 空白对照组 , ΔCt =Ct 目的基因 -Ct 内参基因 , the results are expressed as Mean ± SD, t-test statistical analysis was used for comparison between groups, P<0.05 was considered to have significant difference, marked with * in the figure; P<0.01 was considered to have extremely significant difference, marked with ** in the figure. According to the calculation formula: up-regulation rate (%) = (R1-R0) / R0x100%, wherein R1 is the RNA relative expression level of the experimental group, R0 is the RNA relative expression level of the blank control group, the expression up-regulation rate is calculated, and the effect of the cyclopeptide compound on the expression level of COL1A gene in human fibroblast HFF-1 is studied.

[0157] The test results of the expression promotion rate of the cyclopeptide compound prepared in the application on type I collagen gene are shown in Table 1. Figure 11 As shown in Table 1, compared with the blank control group, the expression of COL I gene was significantly up-regulated after treatment with TGF-β1 in the positive control group, proving that the cell model and qPCR system are working normally; the expression of COL I gene was significantly up-regulated after treatment with GHK, the expression up-regulation rate was 25% at 10μM, 36% at 20μM, and 41% at 40μM; the expression of COL I gene was significantly up-regulated after treatment with the cyclopeptide compound, the expression up-regulation rate was 33% at 10μM, 43% at 20μM, and 54% at 40μM; it is proved that the cyclopeptide compound prepared in the application has better promotion effect on the expression of type I collagen than GHK.

[0158] Test example 7: antioxidant effect test of cyclopeptide compound.

[0159] Test sample: cyclopeptide compound prepared in Example 1.

[0160] Test method: fibroblast HFF-1 in the logarithmic growth phase is inoculated into a 6-well plate at 50000 cells per well, 2 mL of culture medium is added to each well, and the cells are allowed to adhere and grow for 24 h in an incubator; 10 muM GHK or cyclic peptide compound is added to the DMEM culture medium as the experimental group, no polypeptide is added to the blank control group, no oxidative stress is applied, 7 muM vitamin E is added as the antioxidant positive control group; oxidative stress is applied by 30 J / cm 2 UVA ultraviolet irradiation, the blank control group is not irradiated; the cell supernatant is collected after 24 h, and the SOD and GSH levels are detected according to the kit instructions; the results are plotted and expressed as Mean+SD. The t-test statistical analysis is used for comparison between groups. P<0.05 is considered to have a significant difference, marked with * in the figure, and P<0.01 is considered to have a very significant difference, marked with ** in the figure; the expression up-regulation rate is calculated according to the formula: up-regulation rate (%)=(R1-R0) / R0x100%, wherein R1 is the SOD and GSH levels of the experimental group, and R0 is the SOD and GSH levels of the positive control group, to determine whether the cyclic peptide compound can improve the antioxidant capacity of the cells, whether it affects the expression level of antioxidant-related enzymes, and the final antioxidant capacity.

[0161] The test results of the cyclic peptide compound prepared in the application on the SOD expression level of fibroblasts under oxidative stress are shown in Figure 12 The test results of the cyclic peptide compound prepared in the application on the GSH expression level of fibroblasts under oxidative stress are shown in Figure 13 Compared with the blank control group, the SOD and GSH levels of the negative control group are significantly down-regulated, and the levels of the positive control group are significantly up-regulated, with the SOD level up-regulated by 51.2% and the GSH level up-regulated by 116.4%, proving that the oxidative stress stimulation is effective and the antioxidant positive control is working normally; GHK treatment shows a certain antioxidant capacity, with the SOD level up-regulated by 38.4% and the GSH level up-regulated by 57.4% at 10 muM compared with the negative control; the cyclic peptide compound has the SOD level up-regulated by 75.2% and the GSH level up-regulated by 108.9% compared with the negative control. The SOD and GSH levels of the experimental groups of GHK and the cyclic peptide compound are both up-regulated. It is proved that the cyclic peptide compound can up-regulate the expression level of the antioxidant key enzyme SOD and ultimately up-regulate the expression amount of the antioxidant key substance GSH.

[0162] The above-described embodiments and / or implementations are only used to illustrate the preferred embodiments and / or implementations of the present application, and do not limit the embodiments of the present application in any form, and any person skilled in the art can make some changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the present application, but should be considered as the same technology or embodiment as the present application.

[0163] The principles and implementations of the present application are described herein with specific examples. The above examples are only used to help understand the method of the present application and its core idea. The above description is only the preferred embodiments of the present application. It should be pointed out that, due to the limitation of language expression, there are infinite specific structures objectively. For ordinary skilled persons in the art, without departing from the principles of the present application, some improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner. These improvements, refinements, changes or combinations, or the 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 cyclic peptide compound, characterized in that: The structure of the cyclic peptide compound is Cyclo(Gly-His-Lys-Gly-His-Lys-), and the cyclic peptide compound is cyclized through a covalent bond connection between the N-terminal and C-terminal amino acid residues.

2. The method for preparing the cyclic peptide compound according to claim 1, characterized in that: include, The crude linear peptide was synthesized according to the coupling sequence of amino acids: Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH; The straight-chain crude peptide is placed in a catalytic system for cyclization to obtain a fully protected cyclic peptide; The fully protected cyclic peptide is placed in a lysis solution for cleavage, precipitated, purified by column chromatography, and freeze-dried to obtain a cyclic peptide compound.

3. The preparation method according to claim 2, wherein: The amount ratio of the Fmoc-Gly-OH to the CTC resin is 1.5-4eq, the amount ratio of the Fmoc-Lys(Boc)-OH to the CTC resin is 1.5-4eq, the amount ratio of the Fmoc-His(Trt)-OH to the CTC resin is 1.5-4eq, the purity of the cyclic peptide compound is 95-98%, and the yield of the cyclic peptide compound is 75-85%.

4. The preparation method according to claim 2, wherein: The catalytic system includes one or more of N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and N-methylmorpholine.

5. The preparation method according to claim 2, wherein: The lysis solution includes trifluoroacetic acid, triisopropylsilane, 1,2-ethanedithiol and phenol, the volume ratio of the triisopropylsilane to trifluoroacetic acid is 3-4:56, the volume ratio of the 1,2-ethanedithiol to trifluoroacetic acid is 1-2:56, and the volume ratio of the phenol to trifluoroacetic acid is 1-2:

56.

6. The preparation method according to claim 2, wherein: The synthesis includes solid phase synthesis or liquid phase synthesis.

7. The preparation method according to claim 6, characterized in that: The carrier in the solid phase synthesis comprises CTC resin, and the substitution degree of the CTC resin is 1.2-1.6 mmol / g.

8. The preparation method according to claim 6, characterized in that: The deprotection solution in the solid phase synthesis comprises piperidine and N,N-dimethylformamide, and the volume ratio of the piperidine to N,N-dimethylformamide is 10-30:

80.

9. The preparation method according to claim 6, characterized in that: The cutting solution in the solid phase synthesis includes trifluoroacetic acid and dichloromethane, and the volume ratio of the trifluoroacetic acid to dichloromethane is 2.5-4.5:346.

5.

10. Use of the cyclic peptide compound according to claim 1 in cosmetics or medicines, characterized in that: The dosage forms of the cyclic peptide compound include oral preparations or topical preparations; the drugs include skin wound repair drugs, which are used to promote the healing of surgical wounds and burn wounds and tissue regeneration; the drugs include inflammatory skin disease treatment drugs, which are used to relieve the inflammatory response of acne and eczema and repair the skin barrier; the drugs include bone and joint disease treatment drugs, which promote collagen synthesis and are used to improve joint function and bone density in patients with osteoarthritis and osteoporosis.

Images