Cyclopeptide compound as well as preparation method and application thereof

Abstract

The invention discloses a cyclic peptide compound as well as a preparation method and application thereof, belongs to the field of polypeptide synthesis and application, and particularly relates to full-protection peptide resin prepared by solid-phase synthesis. Carrying out cutting and monocyclization on the full-protection peptide resin to obtain monocyclic peptide; the monocyclic peptide is subjected to binary cyclization treatment to obtain a cyclic peptide compound Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-, and a disulfide bond is in bridge connection with Cysamp; and the binary cyclization is cyclized under the action of iodine methanol and ascorbic acid. The cyclopeptide compound prepared by the invention has the advantages of low toxicity, good stability, good antioxidant effect and good anti-inflammatory effect, and the expression of type I collagen can be improved. Therefore, the invention has the advantages of low toxicity, good stability, good antioxidant effect and good anti-inflammatory effect, and can improve the expression of the type I collagen.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide synthesis and applications, and particularly relates to a class of cyclic peptide compounds, their preparation methods and applications. 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 by 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] Based on the above understanding, the present invention expects to construct a class of compounds with anti-wrinkle and repair effects and a long half-life through the reasonable design of the polypeptide structure and the modification of chemical bonds.

[0006] The purpose of the present invention is to provide a class of cyclic peptide compounds with low toxicity, good stability, capable of enhancing the expression of type I collagen, good antioxidant effects and good anti-inflammatory effects, their preparation methods and applications.

[0007] Peptide cyclization and side-chain binding are important means in the development of peptide drugs. The cyclic structure reduces the exposure of terminal groups, decreases the possibility of unnecessary chemical reactions, thereby improving 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 hydrolysis ability. Due to the higher stability and anti-enzyme hydrolysis ability, the cyclized and side-chain-bound peptides can maintain a longer effective concentration in vivo and reduce the dosing frequency.

[0008] The technical solution adopted by the present invention to achieve the above object is as follows: A method for preparing a fully protected peptide resin, comprising: After mixing Fmoc-Gly-OH, DIEA and DCM, add CTC resin and couple 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; Perform deprotection treatment on the first amino acid-coupled resin, and then sequentially perform the coupling of activated amino acid reagents according to the peptide sequence to obtain a fully protected peptide resin. The structure of the fully protected peptide resin is: H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0009] The present invention discloses a method for preparing a fully protected peptide resin, comprising the following steps: S1, Pretreatment of CTC resin: Mix CTC resin with DCM, and conduct nitrogen treatment at 20-40 °C for 5-30 min, remove the liquid, and obtain the pretreated CTC resin.

[0010] S2, Preparation of H-Gly-CTC resin: Mix Fmoc-Gly-OH and DCM with the pretreated CTC resin, add DIEA under a nitrogen atmosphere at 10-20 °C, then react at 25-30 °C for 1-5 h. After the reaction is completed, add methanol for capping for 10-60 min. After capping is completed, remove the liquid, wash with DMF, and obtain Fmoc-Gly-CTC resin; then add a deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Gly-CTC resin.

[0011] S3. Preparation of H-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-Cys(Trt)-OH, HOBT in DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain the activation solution. Mix the activation solution with the H-Gly-CTC resin in S2, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry to obtain H-Cys(Trt)-Gly-CTC resin.

[0012] S4. Preparation of H-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-Lys(Boc)-OH, HOBT in DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain the activation solution. Mix the activation solution with the H-Cys(Trt)-Gly-CTC resin in S3, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry to obtain H-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0013] S5. Preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-His(Trt)-OH, HOBT in DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain the activation solution. Mix the activation solution with the H-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S4, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0014] S6. Preparation of H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-Gly-OH, HOBT with DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain the activation solution. Mix the activation solution with H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S5, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0015] S7. Preparation of H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-Cys(Trt)-OH, HOBT with DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain the activation solution. Mix the activation solution with H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S6, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0016] Preparation of S8, H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-Lys(Boc)-OH, HOBT with DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain an activation solution. Mix the activation solution with H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S7, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter off the liquid by suction, wash with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add a deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0017] Preparation of S9, crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-His(Trt)-OH, HOBT with DMF, add DIC at 0 - 5 °C, activate for 1 - 5 min to obtain an activation solution. Mix the activation solution with H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S8, react at 25 - 30 °C for 20 - 60 min under a nitrogen atmosphere. After the reaction is completed, filter off the liquid by suction, wash with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add a deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain the crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0018] S10. Preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: The crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin was washed successively with methyl tert-butyl ether, tetrahydrofuran and methyl tert-butyl ether, and dried under vacuum to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin, namely the fully protected peptide resin. Appropriate amounts of methyl tert-butyl ether and tetrahydrofuran were used in the washing.

[0019] Preferably, in S1, the usage amounts of CTC resin and DCM are 1 g: 5 - 10 mL. The active site of CTC resin is 1 - 1.6 mmol / g.

[0020] Preferably, in S2, the molar usage amount of Fmoc-Gly-OH is 100 - 300% of the active site on the CTC resin; the usage amounts of Fmoc-Gly-OH and DCM are 1 mmol: 1 - 5 mL, the molar usage amount of DIEA is 100 - 500% of the molar usage amount of Fmoc-Gly-OH, and the usage amounts of Fmoc-Gly-OH and methanol are 1 mmol: 0.1 - 0.8 mL. The deprotection solution is a DMF solution containing 10 - 30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Appropriate amount is used in the DMF washing.

[0021] Preferably, in S3, the molar usage amount of Fmoc-Cys(Trt)-OH is 100 - 150% of the molar usage amount of Fmoc-Gly-OH in S2, the molar usage amount of HOBT is 80 - 120% of the molar usage amount of Fmoc-Cys(Trt)-OH, the usage amounts of Fmoc-Cys(Trt)-OH and DMF are 1 mmol: 0.5 - 1.5 mL, and the molar usage amount of DIC is 80 - 120% of the molar usage amount of Fmoc-Cys(Trt)-OH. The deprotection solution is a DMF solution containing 10 - 30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Appropriate amount is used in the DMF washing.

[0022] Preferably, in S4, the molar amount of Fmoc-Lys(Boc)-OH used is 100-150% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH used, the usage amount of Fmoc-Lys(Boc)-OH and DMF is 1 mmol: 0.5-1.5 mL, and the molar amount of DIC used is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH used. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount in the DMF washing.

[0023] Preferably, in S5, the molar amount of Fmoc-His(Trt)-OH used is 100-150% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 80-120% of the molar amount of Fmoc-His(Trt)-OH used, the usage amount of Fmoc-His(Trt)-OH and DMF is 1 mmol: 0.5-1.5 mL, and the molar amount of DIC used is 80-120% of the molar amount of Fmoc-His(Trt)-OH used. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount in the DMF washing.

[0024] Preferably, in S6, the molar amount of Fmoc-Gly-OH used is 100-150% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 80-120% of the molar amount of Fmoc-Gly-OH used, the usage amount of Fmoc-Gly-OH and DMF is 1 mmol: 0.5-1.5 mL, and the molar amount of DIC used is 80-120% of the molar amount of Fmoc-Gly-OH used. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount in the DMF washing.

[0025] Preferably, in S7, the molar amount of Fmoc-Cys(Trt)-OH used is 100-150% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 80-120% of the molar amount of Fmoc-Cys(Trt)-OH used, the usage amount of Fmoc-Cys(Trt)-OH and DMF is 1 mmol: 0.5-1.5 mL, and the molar amount of DIC used is 80-120% of the molar amount of Fmoc-Cys(Trt)-OH used. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount in DMF washing.

[0026] Preferably, in S8, the molar amount of Fmoc-Lys(Boc)-OH used is 100-150% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH used, the usage amount of Fmoc-Lys(Boc)-OH and DMF is 1 mmol: 0.5-1.5 mL, and the molar amount of DIC used is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH used. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount in DMF washing.

[0027] Preferably, in S9, the molar amount of Fmoc-His(Trt)-OH used is 100-150% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 80-120% of the molar amount of Fmoc-His(Trt)-OH used, the usage amount of Fmoc-His(Trt)-OH and DMF is 1 mmol: 0.5-1.5 mL, and the molar amount of DIC used is 80-120% of the molar amount of Fmoc-His(Trt)-OH used. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount in DMF washing.

[0028] The present invention discloses the preparation of a fully protected polypeptide. The fully protected peptide resin is mixed with a cleavage solution, reacted at 20-40 °C for 20-60 min, filtered, the filtrate is precipitated with petroleum ether to obtain a solid, and vacuum dried to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, that is, the fully protected polypeptide.

[0029] Preferably, the cutting solution is a DCM solution containing TFA. The cutting solution contains 1-3 vol% of TFA. The whole protected peptide resin is submerged in the cutting solution during use. Petroleum ether is used in an appropriate amount.

[0030] The present invention discloses a preparation method of a whole protected polypeptide. The whole protected peptide resin is mixed with the cutting solution and reacted at 20-40 °C for 20-60 min, then filtered. The filtrate is concentrated by rotary evaporation until dry to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, that is, the whole protected polypeptide.

[0031] Preferably, the cutting solution is a DCM solution containing HFIP. The cutting solution contains 20-40 vol% of HFIP. The whole protected peptide resin is submerged in the cutting solution during use.

[0032] The present invention discloses a preparation method of a whole protected cyclic peptide. The whole protected polypeptide is mixed with DMF to obtain a whole protected polypeptide solution; HATU, DIEA and DMF are mixed to obtain a cyclization solution; the whole protected polypeptide solution is added to the cyclization solution at 20-40 °C and reacted for 0.5-3 h. The reaction is monitored by HPLC until completion. Water and ethyl acetate are added to the reaction solution for extraction, and washed successively with saturated sodium bicarbonate aqueous solution, water and saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), that is, the whole protected cyclic peptide.

[0033] Preferably, the usage amount of the whole protected polypeptide and DMF in the whole protected polypeptide solution is 1 g: 5-15 mL; the usage amount of HATU and DMF in the cyclization solution is 1 g: 3-12 mL, and the volume ratio of DIEA to DMF in the cyclization solution is 1: 5-10; in the mixing of the whole protected polypeptide solution and the cyclization solution, the usage amount of HATU is 40-60 wt% of the whole protected polypeptide. In the liquid-liquid extraction of water and ethyl acetate, water and ethyl acetate are used in an appropriate amount at a volume ratio of 1: 0.5-2; in the washing process, saturated sodium bicarbonate aqueous solution, water and saturated sodium chloride aqueous solution are used in an appropriate amount, and anhydrous sodium sulfate is used in an appropriate amount.

[0034] The present invention discloses a method for preparing a fully protected cyclic peptide. A fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide solution; HOBT is added and dissolved, then NMM and DIC are added at 0 - 5 °C, and the reaction is carried out at 20 - 40 °C for 8 - 24 h. The reaction is monitored by HPLC until completion. Water is added to the reaction solution to precipitate solids. The solids are dissolved in ethyl acetate, and then washed successively with saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), that is, the fully protected cyclic peptide.

[0035] Preferably, the usage amounts of the fully protected polypeptide and DMF in the fully protected polypeptide solution are 1 g: 0.5 - 1.5 L, the usage amount of HOBT is 3 - 12 wt% of the fully protected polypeptide, the usage amount of NMM is 2 - 10 wt% of the fully protected polypeptide, and the usage amount of DIC is 3 - 9 wt% of the fully protected polypeptide. An appropriate amount of water is used for precipitating solids. An appropriate amount of ethyl acetate is used for dissolving the solids. Appropriate amounts of saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution are used for washing. An appropriate amount of anhydrous sodium sulfate is used.

[0036] The present invention discloses the H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin prepared by the above method.

[0037] The present invention discloses a monocyclic peptide with the structure: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

[0038] The present invention discloses a method for preparing a monocyclic peptide, including: the method for preparing a fully protected peptide resin as described above.

[0039] Preferably, the fully protected peptide resin is subjected to cleavage treatment and monocyclization to obtain a monocyclic peptide with the structure: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

[0040] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with a deprotection cleavage solution and cleaved for 1 - 4 h. After the cleavage is completed, it is precipitated with ice-cold diethyl ether, washed, centrifuged, and rotary evaporated to dryness to obtain the crude monocyclic peptide. The crude monocyclic peptide is purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-), that is, the monocyclic peptide.

[0041] More preferably, in the preparation of the monocyclic peptide, the deprotection cleavage solution is composed of TFA, TIS, EDT, PhOH and water. In the deprotection cleavage solution, TFA, TIS, EDT, PhOH and water are mixed at a volume ratio of 1:0.01 - 0.1:0.01 - 0.05:0.01 - 0.05:0.01 - 0.05. The usage amount of the fully protected cyclic peptide and the deprotection cleavage solution is 1 g:5 - 15 mL. In the sedimentation, an appropriate amount of ice ether is used. For washing, ice ether is used in an appropriate amount.

[0042] In the present invention, the monocyclic peptide is numbered PR2924B, and its structural formula is: . The monocyclic peptide exhibits the functions of inducing extracellular matrix generation, anti - inflammation and antioxidant. In the cell function test, this compound has excellent efficacy. The present invention discloses a cyclic peptide compound with the structure: Cyclo(His - Lys - Cys - Gly - His - Lys - Cys - Gly -, disulfide - bridged Cys&Cys).

[0043] The present invention discloses a preparation method of a cyclic peptide compound, including: the preparation method of a monocyclic peptide as described above.

[0044] Preferably, the monocyclic peptide is subjected to binary cyclization to obtain a cyclic peptide compound, and the binary cyclization is carried out under the action of iodomethanol and ascorbic acid.

[0045] Preferably, in the preparation of the cyclic peptide compound, the monocyclic peptide is mixed and dissolved with an acetic acid solution, and an iodomethanol solution is added at 20 - 40 °C. Stir until the reaction solution turns yellow and does not fade, indicating that the reaction is complete. Then, an ascorbic acid solution is added for reduction under stirring. Add the ascorbic acid solution until the reaction solution changes from yellow back to milky white. After stirring for 5 - 30 min without color change, sample and detect by HPLC. Then, filter the reaction solution through a filter membrane to obtain a crude product of the cyclic peptide compound. The crude product of the cyclic peptide compound is purified by reverse - phase C18 liquid chromatography and freeze - dried to obtain Cyclo(His - Lys - Cys - Gly - His - Lys - Cys - Gly -, disulfide - bridged Cys&Cys), that is, the cyclic peptide compound.

[0046] More preferably, in the preparation of the cyclic peptide compound, acetic acid and water in the acetic acid solution are mixed at a volume ratio of 1:20 - 30. The usage amount of the monocyclic peptide and the acetic acid solution is 1 g:100 - 300 mL. The concentration of the iodomethanol solution is 0.05 - 0.5 mol / L. The usage amount of the monocyclic peptide and the iodomethanol solution is 1 g:0.5 - 5 mL. The content of ascorbic acid in the ascorbic acid solution is 0.05 - 5 wt%. The pore size of the filter membrane is 0.45 μm.

[0047] The cyclic peptide compound disclosed in this application is numbered PR2924, and its structural formula is: 。The cyclic peptide compound exhibits the functions of inducing extracellular matrix generation, anti-inflammatory and antioxidant. In cell function tests, this compound has excellent efficacy.

[0048] The present invention discloses the use of the above-mentioned monocyclic peptide in the preparation of a cyclic 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.

[0049] The present invention discloses the use of the above-mentioned cyclic peptide compound 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.

[0050] In the present invention, the H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin, namely peptide resin, is prepared by solid-phase synthesis; the peptide resin is cleaved and monocyclized to obtain a monocyclic peptide, and the structure of the monocyclic peptide is: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-); the monocyclic peptide is subjected to binary cyclization treatment to obtain a cyclic peptide compound Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bridge connecting Cys&Cys), and the binary cyclization is carried out under the action of iodomethanol and ascorbic acid. Therefore, it 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. Therefore, the present invention is a cyclic peptide compound with 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

[0051] Figure 1 It is the liquid chromatogram of PR2924B.

[0052] Figure 2 It is the mass spectrum of PR2924B.

[0053] Figure 3 It is the liquid chromatogram of PR2924.

[0054] Figure 4 It is the mass spectrum of PR2924.

[0055] Figure 5 It is the result graph of cell cytotoxicity test.

[0056] Figure 6 It is the result graph of serum stability test.

[0057] Figure 7 It is the result graph of COL1A expression test.

[0058] Figure 8It is a graph showing the test results of SOD expression level.

[0059] Figure 9 It is a graph showing the test results of GSH level.

[0060] Figure 10 It is a graph showing the expression level of TNF-α.

[0061] Figure 11 It is a graph showing the expression level of IL-6.

[0062] Figure 12 It is a graph showing the expression level of IL-8. Specific implementation manner

[0063] 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.

[0064] 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 in the present application and the features in the embodiments 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.

[0065] Example 1: A preparation method of a fully protected peptide resin S1, Pretreatment of CTC resin: Mix CTC resin with DCM, and treat it with nitrogen at 25 °C for 10 min, then remove the liquid to obtain the pretreated CTC resin. The usage amounts of CTC resin and DCM are 1 g: 8.77 mL. The active sites of CTC resin are 40 mmol, and the usage amount of CTC resin is 28.5 g.

[0066] S2. Preparation of H-Gly-CTC resin: Mix Fmoc-Gly-OH and DCM with the pretreated CTC resin. Under a nitrogen atmosphere, add DIEA at 10 °C, then react at 25 °C for 2.5 h. After the reaction is completed, add methanol for capping for 30 min. After capping is completed, remove the liquid and wash with DMF to obtain Fmoc-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and drain to obtain H-Gly-CTC resin. The molar amount of Fmoc-Gly-OH used is 200% of the active sites on the CTC resin; the usage amounts of Fmoc-Gly-OH and DCM are 1 mmol: 2.5 mL, the molar amount of DIEA used is 250% of the molar amount of Fmoc-Gly-OH used, and the usage amounts of Fmoc-Gly-OH and methanol are 1 mmol: 0.38 mL. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0067] S3. Preparation of H-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-Cys(Trt)-OH and HOBT in DMF. Add DIC at 0 °C and activate for 3 min to obtain the activation solution. Mix the activation solution with the H-Gly-CTC resin in S2 and react at 25 °C for 40 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid and wash with DMF to obtain Fmoc-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and drain to obtain H-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Cys(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Cys(Trt)-OH used, the usage amounts of Fmoc-Cys(Trt)-OH and DMF are 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Cys(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0068] S4. Preparation of H-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-Lys(Boc)-OH, HOBT in DMF, add DIC at 0 °C, activate for 3 min to obtain an activation solution. Mix the activation solution with H-Cys(Trt)-Gly-CTC resin in S3, react at 25 °C for 40 min under a nitrogen atmosphere. After the reaction is completed, filter off the liquid by suction, wash with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add a deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Lys(Boc)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used, the usage amount of Fmoc-Lys(Boc)-OH and DMF is 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0069] S5. Preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-His(Trt)-OH, HOBT in DMF, add DIC at 0 °C, activate for 3 min to obtain an activation solution. Mix the activation solution with H-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S4, react at 25 °C for 40 min under a nitrogen atmosphere. After the reaction is completed, filter off the liquid by suction, wash with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add a deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-His(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-His(Trt)-OH used, the usage amount of Fmoc-His(Trt)-OH and DMF is 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-His(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0070] Preparation of S6, H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-Gly-OH, HOBT with DMF, add DIC at 0 °C, activate for 3 min to obtain the activation solution, mix the activation solution with the H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S5, react at 25 °C for 40 min under a nitrogen atmosphere, after the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Gly-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Gly-OH used, the usage amounts of Fmoc-Gly-OH and DMF are 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Gly-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0071] Preparation of S7, H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-Cys(Trt)-OH, HOBT and DMF, add DIC at 0 °C, activate for 3 min to obtain an activation solution. Mix the activation solution with H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S6, react at 25 °C for 40 min under a nitrogen atmosphere. After the reaction is completed, filter off the liquid by suction, wash with DMF to obtain Fmoc-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add a deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Cys(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Cys(Trt)-OH used, the usage amount of Fmoc-Cys(Trt)-OH and DMF is 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Cys(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0072] Preparation of S8, H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Mix and dissolve Fmoc-Lys(Boc)-OH, HOBT with DMF, add DIC at 0 °C, activate for 3 min to obtain the activation solution, mix the activation solution with H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S7, react at 25 °C for 40 min under a nitrogen atmosphere. After the reaction is completed, filter to remove the liquid, wash with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Lys(Boc)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used, the usage amounts of Fmoc-Lys(Boc)-OH and DMF are 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to immerse the resin. Use an appropriate amount during DMF washing.

[0073] Preparation of the crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Dissolve Fmoc-His(Trt)-OH, HOBT and DMF, add DIC at 0 °C, activate for 3 min to obtain the activation solution. Mix the activation solution with H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S8, react at 25 °C for 40 min under a nitrogen atmosphere. After the reaction, filter to remove the liquid, wash with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then add the deprotection solution for deprotection treatment, wash with DMF, and dry by suction to obtain the crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-His(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-His(Trt)-OH used, the usage amount of Fmoc-His(Trt)-OH and DMF is 1 mmol: 1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-His(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip, and the usage amount of the deprotection solution is to submerge the resin. Use an appropriate amount during DMF washing.

[0074] Preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Wash the crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin successively with methyl tert-butyl ether, tetrahydrofuran and methyl tert-butyl ether, and dry in vacuum to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin, that is, the fully protected peptide resin. Use an appropriate amount of methyl tert-butyl ether and tetrahydrofuran during washing.

[0075] Example 2: A preparation method of a fully protected polypeptide Preparation of fully protected polypeptide: Mix the fully protected peptide resin with the cleavage solution, react at 30 °C for 30 min, filter, concentrate the filtrate by rotary evaporation until dry to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, which is the fully protected polypeptide. The cleavage solution is a DCM solution containing TFA, and the cleavage solution contains 1 vol% of TFA. The fully protected peptide resin is submerged in the cleavage solution during use. Petroleum ether is used in an appropriate amount. The yield of the fully protected polypeptide is 107.7% and the purity is 90.9%. The fully protected peptide resin is from Example 1.

[0076] Example 3: A method for preparing a fully protected polypeptide Preparation of fully protected polypeptide: Mix the fully protected peptide resin with the cleavage solution, react at 30 °C for 30 min, filter, concentrate the filtrate by rotary evaporation until dry to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, which is the fully protected polypeptide. The cleavage solution is a DCM solution containing HFIP, and the cleavage solution contains 33 vol% of HFIP. The fully protected peptide resin is submerged in the cleavage solution during use. The yield of the fully protected polypeptide is 120.5% and the purity is 84.7%. The fully protected peptide resin is from Example 1.

[0077] Example 4: A method for preparing a fully protected cyclic peptide Preparation of fully protected cyclic peptide: Mix the fully protected polypeptide with DMF to obtain a fully protected polypeptide solution; mix HATU, DIEA and DMF to obtain a cyclization solution; add the fully protected polypeptide solution to the cyclization solution at 25 °C and react for 1 h. Monitor the reaction by HPLC until completion. Add water and ethyl acetate to the reaction solution for extraction, wash successively with saturated sodium bicarbonate aqueous solution, water and saturated sodium chloride aqueous solution, dry with anhydrous sodium sulfate, filter, and rotary evaporate to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), which is the fully protected cyclic peptide. The usage amount of the fully protected polypeptide and DMF in the fully protected polypeptide solution is 1 g:10 mL; the usage amount of HATU and DMF in the cyclization solution is 1 g:6.08 mL, and the volume ratio of DIEA to DMF in the cyclization solution is 1:7.03; in the mixing of the fully protected polypeptide solution and the cyclization solution, the usage amount of HATU is 49.33 wt% of the fully protected polypeptide. In the liquid-liquid extraction of water and ethyl acetate, water and ethyl acetate are used in an appropriate amount at a volume ratio of 1:1; in the washing, saturated sodium bicarbonate aqueous solution, water and saturated sodium chloride aqueous solution are used in an appropriate amount, and anhydrous sodium sulfate is used in an appropriate amount. The yield of the fully protected cyclic peptide is 87.5% and the purity is 91.4%. The fully protected polypeptide is from Example 2.

[0078] Example 5: Preparation method of a fully protected cyclic peptide Preparation of the fully protected cyclic peptide: Mix the fully protected polypeptide with DMF to obtain a fully protected polypeptide solution; add HOBT and dissolve it, then add NMM and DIC at 0 °C, react at 25 °C for 16 h, monitor the reaction completion by HPLC, add water to the reaction solution to precipitate a solid, dissolve the solid with ethyl acetate, and then wash it successively with saturated sodium bicarbonate aqueous solution, water and saturated sodium chloride aqueous solution, dry it with anhydrous sodium sulfate, filter, and rotary evaporate to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), that is, the fully protected cyclic peptide. The usage amounts of the fully protected polypeptide and DMF in the fully protected polypeptide solution are 1 g: 1 L, the usage amount of HOBT is 7.33 wt% of the fully protected polypeptide, the usage amount of NMM is 4.67 wt% of the fully protected polypeptide, and the usage amount of DIC is 6 wt% of the fully protected polypeptide. An appropriate amount of water is used for precipitating the solid. An appropriate amount of ethyl acetate is used for dissolving the solid. Appropriate amounts of saturated sodium bicarbonate aqueous solution, water and saturated sodium chloride aqueous solution are used for washing. Appropriate amount of anhydrous sodium sulfate is used. The yield of the fully protected cyclic peptide is 69.5%, and the purity is 80.3%. The fully protected polypeptide is from Example 3.

[0079] Example 6: Preparation method of a monocyclic peptide Preparation of the monocyclic peptide: Mix the fully protected cyclic peptide with the deprotection cleavage solution and cut for 2 h. After the cutting is completed, precipitate with ice-cold diethyl ether, wash, centrifuge, and rotary evaporate to dryness to obtain the crude monocyclic peptide. The crude monocyclic peptide is purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-), that is, the monocyclic peptide. The deprotection cleavage solution is composed of TFA, TIS, EDT, PhOH and water, and TFA, TIS, EDT, PhOH and water in the deprotection cleavage solution are mixed in a volume ratio of 1: 0.06: 0.03: 0.03: 0.03. The usage amounts of the fully protected cyclic peptide and the deprotection cleavage solution are 1 g: 8 mL. An appropriate amount of ice-cold diethyl ether is used for precipitation. Ice-cold diethyl ether is used for washing, and an appropriate amount is used. The purity of the monocyclic peptide is 83.2%, and the yield is 99.3%. The fully protected cyclic peptide is from Example 4. The monocyclic peptide prepared in Example 6 is numbered PR2924B.

[0080] The chromatography of the PR2924B reverse-phase C18 chromatography purification in Example 6 of the present invention is as Figure 1 shown.

[0081] The mass spectrum of the PR2924B obtained in Example 6 of the present invention is as Figure 2 shown.

[0082] Example 7: Preparation method of a cyclic peptide compound Preparation of the cyclic peptide compound: Mix and dissolve the monocyclic peptide with an acetic acid solution. Add an iodine methanol solution at 25°C and stir until the reaction solution turns yellow and does not fade, indicating that the reaction is complete. Then, add an ascorbic acid solution for reduction under stirring. Add the ascorbic acid solution until the reaction solution changes back from yellow to milky white, and stir for 10 min without color change. Take a sample for HPLC detection, and then filter the reaction solution through a filter membrane to obtain the crude cyclic peptide compound. The crude cyclic peptide compound is purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bridge Cys&Cys), that is, the cyclic peptide compound. In the acetic acid solution, acetic acid and water are mixed at a volume ratio of 1:24. The usage amount of the monocyclic peptide and the acetic acid solution is 1 g:200 mL. The concentration of the iodine methanol solution is 0.1 mol / L, and the usage amount of the monocyclic peptide and the iodine methanol solution is 1 g:1 mL. The content of ascorbic acid in the ascorbic acid solution is 1 wt%. The pore size of the filter membrane is 0.45 μm. The purity of the cyclic peptide compound is 98.9%. The monocyclic peptide is from Example 6. The cyclic peptide compound prepared in Example 7 is numbered PR2924.

[0083] In the present invention, the chromatography of PR2924 purified by reverse-phase C18 chromatography in Example 7 is as Figure 3 shown.

[0084] In the present invention, the mass spectrum of PR2924 obtained in Example 7 is as Figure 4 shown.

[0085] Test example: To verify the physicochemical properties and physiological functions of the cyclic peptide compound and the monocyclic peptide 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 is DMEM medium. The cyclic peptide compound was prepared by the method of Example 7, and the cyclic peptide compound is numbered PR2924. The monocyclic peptide was prepared by the method of Example 6, and the monocyclic peptide is numbered PR2924B.

[0086] 1. Cytotoxicity test The present invention evaluated the potential toxicity of PR2924 and PR2924B to cells and determined their possible safe concentration ranges.

[0087] 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 approximately 10,000 cells / well was added to each well to ensure uniform 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 then returned to the incubator for further 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 range. The test samples were PR2924 or PR2924B, 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 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%.

[0088] The results of the cytotoxicity experiment are as Figure 5 shown. For epithelial cells treated with different concentrations of PR2924 and PR2924B, the cell viability did not show a significant decrease. At a concentration of 500 μM, the cell viability remained at 95% relative to the control group, confirming that PR2924 and PR2924B have extremely low cytotoxicity.

[0089] 2. Serum stability detection The present invention evaluated the degradation of PR2924 and PR2924B in a serum environment to predict their metabolism in vivo.

[0090] The lyophilized test samples were directly dissolved in 10 vol% fetal bovine serum at a final concentration of 1 mg / mL. The lyophilized oligopeptide GHK was dissolved under the same conditions as the control group. The above-prepared solutions were filtered through a 0.22 μM sterile filter, filled into sterile containers, and incubated in a 37°C incubator. Sampling was performed at the set time points. The residual amount of the polypeptide was determined using analytical high-performance liquid chromatography. Taking the content at 0 time as 100%, the degradation 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 samples were PR2924 or PR2924B. The set time points were 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h.

[0091] The results of the serum stability experiment are as Figure 6As shown, among them, GHK in the control group was almost completely degraded in 10 vol% serum conditions for 4 hours, and its degradation half-life was calculated to be about 0.7 hours according to the degradation curve. Under the same conditions, the half-life of PR2924 was about 19 hours, and the half-life of PR2924B was about 15 hours. After cyclization, the stability was greatly improved. On the basis of PR2924B, the half-life of PR2924 was further improved after intramolecular disulfide bond secondary cyclization.

[0092] 3. Test on the promotion rate of type I collagen expression This invention studied the effects of PR2924 and PR2924B on the expression level of COL1A in human fibroblasts HFF-1.

[0093] The fibroblasts HFF-1 in the logarithmic growth phase were seeded into a 6-well plate at 50,000 cells / well. 2 mL of culture medium was added to each well and placed in an incubator overnight to allow the cells to adhere and grow for 24 hours. Grouped, DMEM medium containing the test sample was added, and 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 PR2924 or PR2924B, and the concentrations of the test samples were 0, 10, 20, 40 μM.

[0094] After 24 hours of treatment, the culture medium was removed, and the RNA of the cells in each well was extracted. 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. Calculate the expression level according to the formula: Relative RNA expression level = 2ΔΔC(t).

[0095] The t-test statistical analysis was used for comparison between groups. P < 0.05 was considered to have a significant difference, marked with * in the figure; P < 0.01 was considered to have a highly significant difference, marked with ** in the figure.

[0096] According to the calculation formula: Upregulation rate = (test group - blank control group) / blank control group × 100%, calculate the expression upregulation rate.

[0097] The experimental results of type I collagen expression are as Figure 7As shown, compared with the blank control group, after treatment with TGF-β1 in the positive control group, the expression level of COL1A increased significantly, demonstrating that the cell model and qPCR system were working properly. Compared with the blank control group, the treatment with PR2924B significantly positively regulated the expression of COL1A, with an up-regulation rate of 29% at 10 μM, 40% at 20 μM, and 44% at 40 μM; the treatment with PR2924 significantly positively regulated the expression of COL1A, with an up-regulation rate of 36% at 10 μM, 50% at 20 μM, and 63% at 40 μM. Both PR2924 and PR2924B could promote the expression of type I collagen.

[0098] 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 to maintain the level of reduced glutathione in cells and reduce oxidative stress.

[0099] This invention studied whether PR2924 and PR2924B could enhance the antioxidant capacity of cells, whether they affected the expression levels of antioxidant-related enzymes and the final antioxidant capacity.

[0100] HFF-1 fibroblasts 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, where no polypeptide was added and no oxidative stress was applied. The negative control group was without polypeptide, and 7 μg / mL vitamin E was used as the antioxidant positive control group. Oxidative stress was applied by ultraviolet irradiation with UVA, and the blank control group was not irradiated. After 24 hours, the cell supernatants were collected, and the levels of SOD and GSH were detected according to the instructions of the SOD detection kit and GSH quantification kit. The test samples were GHK, PR2924, or PR2924B, and the concentration of the test samples was 10 μM. The unit of ultraviolet irradiation with UVA was 30 J / cm 2 。

[0101] The t-test statistical analysis was used for comparison between groups. P<0.05 was considered to have a significant difference, marked with * in the figure; P<0.01 was considered to have a highly significant difference, marked with ** in the figure.

[0102] According to the formula: Uplift rate = (Test group - Positive control group) / Positive control group × 100%, calculate the SOD and GSH elevation levels of each group.

[0103] The results of the antioxidant experiment are as Figures 8 - 9 shown. Among them, 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 increased. The SOD level in the positive control group increased by 53.7%, and the GSH level increased by 119.7%, proving that the oxidative stress stimulation was effective and the antioxidant positive control worked properly. The GHK treatment showed a certain antioxidant capacity. Compared with the negative control, at 10 μM, the SOD level increased by 36.5% and the GSH level increased by 61.0%. Compared with the negative control, for PR2924B, the SOD level increased by 61% and the GSH level increased by 110%; for PR2924, compared with the negative control, the SOD level increased by 80% and the GSH level increased by 117%. The antioxidant capacities of PR2924 and PR2924B both exceeded those of the positive control group. It was proved that PR2924 and PR2924B could increase the expression level of the antioxidant key enzyme SOD, and finally increase the expression amount of the antioxidant key substance GSH.

[0104] 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 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 abilities of GHK, PR2924 and PR2924B to inhibit the production of inflammatory factors in macrophages under the condition of inflammatory stress induced by lipopolysaccharide LPS.

[0105] 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 treated with drugs. The treatment group was added with DMEM medium containing the test sample. In addition, a blank control group and a negative control group were 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 working solution of LPS was added to each well of the remaining groups except the blank control group, and the cells were placed in an incubator and cultured for 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 t-test statistical analysis was used for comparison among groups. P<0.05 was considered to have a significant difference, marked with * in the figure; P<0.01 was considered to have a highly significant difference, marked with ** in the figure. The test samples were GHK or PR2924 or PR2924B, and the concentration of the test sample was 10 μM.

[0106] According to the formula: Inhibition rate % = (Negative control group - Test group) / Negative control group × 100%, the inhibition rate was calculated.

[0107] 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, 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 the positive control group were 34.7%, 31.8%, and 48.0% respectively; the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with 10 μM GHK were 15.1%, 34.2%, and 19.4% respectively; the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with 10 μM PR2924B were 28.6%, 51.9%, and 21.8% respectively; the inhibition rates of TNF-α, IL-6, and IL-8 after treatment with 10 μM PR2924 were 29%, 54.5%, and 33.7% respectively. PR2924 and PR2924B both showed stronger effects than GHK at the same concentration and both showed significant anti-inflammatory abilities.

[0108] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for implementing 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.

[0109] In this article, specific examples are used to elaborate on the principles and implementation manners of this application. The description of the above embodiments is only used to help understand the method and its core idea of this application. The above is only the preferred implementation manner of this application. It should be noted that due to the limited nature of written expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of this application, several 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 directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of this application.

Claims

1. A method for preparing a fully protected peptide resin, comprising: Mix Fmoc-Gly-OH, DIEA and DCM, then add CTC resin and couple 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; Perform deprotection treatment on the first amino acid-coupled resin, and then sequentially couple activated amino acid reagents according to the peptide sequence to obtain a fully protected peptide resin. The structure of the fully protected peptide resin is: H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

2. The fully protected peptide resin prepared by the method according to claim 1, with the structure: H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

3. A cyclic peptide with the structure: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

4. A method for preparing a monocyclic peptide, comprising: The method for preparing a fully protected peptide resin according to claim 1.

5. The preparation method of a single-ring peptide according to claim 4, characterized in that: Perform cleavage treatment and cyclization on the fully protected peptide resin to obtain a cyclic peptide with the structure: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

6. A cyclic peptide compound with the structure: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bridge connecting Cys&Cys).

7. A method for preparing a cyclic peptide compound, comprising: The method for preparing a cyclic peptide according to claim 4.

8. The preparation method of a cyclic peptide compound according to claim 7, wherein: Perform bicyclization treatment on the cyclic peptide to obtain a cyclic peptide compound, and the bicyclization is carried out by the action of iodomethanol and ascorbic acid.

9. The use of the cyclic peptide according to claim 3 in the preparation of a cyclic peptide compound and / or a product for enhancing the expression of type I collagen and / or an antioxidant product and / or an anti-inflammatory product.

10. The use of the cyclic peptide compound according to claim 6 in the preparation of a product for enhancing the expression of type I collagen and / or an antioxidant product and / or an anti-inflammatory product.

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