Functional cyclic peptides, their preparation methods and applications

Abstract

The present invention discloses functional cyclic peptides, their preparation methods and applications, belonging to the technical field of the synthesis and application of cyclic peptides. Specifically, it involves preparing a linear peptide containing a protecting group from an amino acid reagent after activation, and then preparing a functional cyclic peptide with a cyclic structure through cyclization treatment. The functional cyclic peptides prepared by the present invention have good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good effect of inhibiting neurotransmitter release, and good whitening effect. After the linear peptide is made into a functional cyclic peptide in the present invention, the permeability is increased, the stability is improved, the activity is enhanced, and the irritation is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of the synthesis and application of cyclic peptides, and specifically relates to functional cyclic peptides, their preparation methods and applications. Background Art

[0002] Polypeptides are composed of amino acids through dehydration condensation. These amino acids themselves carry carboxyl groups and amino groups, and are generally hydrophilic compounds. The polypeptides composed of them generally have obvious hydrophilicity and ionic properties. Especially some small molecule polypeptides, due to their non-toxicity, easy absorption by the skin, and special physiological active effects after absorption, can improve certain skin problems from the surface to the essence, and have now been widely used in the formulations of beauty cosmetics. Currently, there are various cosmetics or skin care products on the market, but most skin care products only add a certain type of polypeptide with a certain efficacy as the active ingredient for the efficacy of the skin care product. Therefore, the effect obtained by the skin is relatively single and the effect is slow; furthermore, most of the current skin care products with polypeptides as the active ingredients directly add polypeptides or polypeptide stock solutions to the conventional skin care product matrix. Considering the production and stability of the entire formulation, the addition amount of polypeptides will be limited.

[0003] In conventional polypeptide modification, methods such as palmitoyl modification or myristoyl modification are usually used to complete it. The polypeptides modified with palmitoyl or myristoyl generally have hydrophilicity and lipophilicity, and often greatly increase the consumption of solvents during the separation and purification process, increasing the cost. Summary of the Invention

[0004] The purpose of the present invention is to provide a functional cyclic peptide with good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good effect of inhibiting neurotransmitter release, good whitening effect, and its preparation method and application.

[0005] By using the method of polypeptide cyclization, linear polypeptides are cyclized to obtain cyclized polypeptides. This cyclization of polypeptides dehydrates and condenses the carboxyl groups and amino groups that originally belonged to the polypeptides to form new amide bonds, increasing the lipophilicity of the polypeptides without significantly increasing the solvent consumption. The cyclized polypeptides have better stability compared to linear peptides. At the same time, by condensing carboxyl groups with amino groups or hydroxyl groups, the obtained polypeptides have better lipophilicity and better skin permeability.

[0006] The technical solution adopted by the present invention to achieve the above purpose is:

[0007] Use of the functional cyclic peptide in a moisturizing product and / or an anti-wrinkle product and / or a firming product and / or a soothing product and / or a product for inhibiting neurotransmitter release and / or a whitening product and / or an anti-glycation product and / or an antioxidant product, and the functional cyclic peptide has the following structure:

[0008] , wherein 1≤n, R m There are n in number, and R1, R2, R3 or R m is selected from H, aliphatic hydrocarbon, substituted aliphatic hydrocarbon, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidyl, substituted guanidyl, metal alkyl or an alkyl group that forms a cyclic structure with the adjacent nitrogen atom.

[0009] Preferably, n≤6; or, 1≤n≤6; or, n = 2; or, n = 3; or, n = 4; or, n = 5.

[0010] Preferably, any two or more of the groups among R1, R2, R3 and R m are the same; or, any two adjacent groups among R1, R2, R3 and R m are the same; when R1, R2, R3 or R m is selected from the -CH2-CH2-CH2- group, R1, R2, R3 or R m is connected to the adjacent N to form a ring; or, R1, R2, R3 or R m is selected from any one of the hydrogen atom group, methyl group, -CH2-CH2-CH2- group, propyl group, butyl group, hydroxymethyl group, hydroxyethyl group, acetamido group, propionamido group, CH3-S-CH2-CH2- group, HS-CH2- group, Ph-CH2- group, p-hydroxybenzyl group, acetate group, propionate group, Se-CH2- group, butylamino group, indolyl group, imidazolylmethyl group and substituted guanidyl group.

[0011] Preferably, the functional cyclic peptide is any one of the following:

[0012] .

[0013] The preparation method of the functional cyclic peptide includes: mixing a linear peptide and a cyclizing reagent in a solvent for a cyclization reaction, and performing post-treatment to prepare the functional cyclic peptide; the functional cyclic peptide has the following structure:

[0014] , wherein 1≤n, R m There are n in number, and R1, R2, R3 or R m is selected from H, aliphatic hydrocarbon, substituted aliphatic hydrocarbon, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidyl, substituted guanidyl, metal alkyl or an alkyl group that forms a cyclic structure with the adjacent nitrogen atom.

[0015] Preferably, the functional cyclic peptide is any one of the following:

[0016] .

[0017] Preferably, the preparation method of the linear peptide is solid-phase synthesis; or, the cyclization reagent is HBTU; or, the solvent is at least one of DMF, DCM and DIEA; or, the post-treatment includes post-cyclization treatment, cleavage treatment and post-cleavage treatment.

[0018] More preferably, in the post-cyclization treatment, ice water or DCM is added to the test solution after the cyclization reaction is completed to separate the cyclic peptide with protecting groups; or, the cleavage solution in the cleavage treatment includes Solution E or Solution F. Solution E is composed of TFA, anisole, EDT, PhOH and water, and TFA, anisole, EDT, PhOH and water are mixed in a volume ratio of 75-95:2-10:1-5:1-5:1-5. Solution F is composed of TFA, TIS and water, and TFA, TIS and water are mixed in a volume ratio of 80-96:2-10:2-10; or, in the post-cleavage treatment, the test solution after the cleavage treatment is added to ice ether, and the functional cyclic peptide is separated and purified.

[0019] More preferably, the post-treatment includes purifying the functional cyclic peptide by liquid chromatography; or, in the cyclization reaction, the linear peptide is added to a solvent and mixed to obtain a linear peptide solution, and then the linear peptide solution is mixed with the cyclization reagent, and the cyclization reaction is carried out at 30-50 °C; or, in the post-treatment, after the cyclization reaction is completed, the post-cyclization treatment is carried out to obtain a cyclic peptide with protecting groups, the cleavage solution is added for treatment, and finally the post-cleavage treatment is carried out to obtain the cyclic peptide.

[0020] More preferably, in the post-cyclization treatment, ice water is added to the test solution after the cyclization reaction is completed, a solid is precipitated, stirred and filtered, the solid is dissolved in EA, and then washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with protecting groups; or, in the post-cyclization treatment, DCM is added to the test solution after the cyclization reaction is completed, extracted by layering, and then washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with protecting groups; or, in the post-cleavage treatment, the test solution after the cleavage treatment is added to ice ether, a solid is precipitated, centrifuged and washed, and the solid is evaporated to dryness and purified to obtain the cyclic peptide; or, LC-MS monitoring is used in the cyclization reaction; or, the solvent is at least one of DMF, DCM and DIEA, and the solvent is based on DMF, and the relationship between the usage amount of the linear peptide and DMF is 0.1-30 mg / mL.

[0021] Preferably, in the preparation of the amino acid activation solution, the amino acid reagent and HOBt are mixed, and DMF and DIC are added at a temperature of 2-8 °C, and allowed to stand and react for 10-30 min to obtain the amino acid activation solution.

[0022] More preferably, in the preparation of the amino acid activation solution, the molar amount of HOBt used is 50-200% of the molar amount of the amino acid reagent used.

[0023] More preferably, in the preparation of the amino acid activation solution, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used, and the relationship between the amount of DMF used and the amount of the amino acid reagent used is 0.1-4.5 mL / mmol.

[0024] More preferably, in the preparation of the amino acid activation solution, the amino acid reagent includes any one of the following reagents: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Glu(otBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Phe-OH.

[0025] Preferably, in the preparation of the linear peptide, the first amino acid reagent is mixed with the CTC resin, then dichloromethane (DCM) and DIEA are added, and the reaction is carried out at 20-40 °C for 2-5 h, then methanol is added, and the reaction is carried out for 3-30 min. After the reaction is completed, filtration and washing are carried out, and deprotection solution treatment is added; in sequence according to the order of the linear peptide, the amino acid activation solution is mixed with the CTC resin bonded with the first amino acid, and the reaction is carried out for 0.5-3 h. After each reaction of the amino acid activation solution, deprotection solution treatment is carried out. After the reaction of the last amino acid reagent is completed, cleavage solution treatment is added to obtain the linear peptide.

[0026] More preferably, in the preparation of the linear peptide, the total substitution degree in the CTC resin is the product of the substitution degree and the mass of the CTC resin, that is, the total substitution degree is the molar amount of the total active reaction sites of the CTC resin, and the molar amount of the first amino acid reagent used is 50-250% of the total substitution degree in the CTC resin.

[0027] More preferably, in the preparation of the linear peptide, the relationship between the amount of dichloromethane used and the amount of the first amino acid reagent used is 1-65 mL / mmol.

[0028] More preferably, in the preparation of the linear peptide, the relationship between the amount of DIEA used and the amount of the first amino acid reagent used is 0.1-5 mL / mmol.

[0029] More preferably, in the preparation of the linear peptide, the relationship between the amount of methanol used and the amount of the first amino acid reagent used is 0.1-6.5 mL / mmol.

[0030] More preferably, in the preparation of the linear peptide, when the consumption amount of the amino acid activation solution is based on the molar amount of the amino acid reagent therein, the molar amount of the amino acid reagent used is 100-350% of the total substitution degree in the CTC resin.

[0031] More preferably, in the preparation of the linear peptide, the first amino acid reagent includes any one of the following: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH. The deprotection solution is a 10-30% Pip / DMF solution. The cleavage solution is a 30% TFE / DCM solution.

[0032] The linear peptide includes any one of H-Phe-Phe-Tyr(tBu)-Pro-OH, H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-OH, H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-OH, H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-OH, H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH, H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-OH, H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH, H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(otBu)-Glu(otBu)-Glu(otBu)-Met-OH, and H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH.

[0033] More preferably, in the preparation of the linear peptide, in the washing after the reaction of the first amino acid with the CTC resin, dichloromethane (DCM), methanol, and DMF are used for washing in sequence.

[0034] More preferably, in the preparation of the linear peptide, when adding the amino acid activation solution for reaction in the order of the linear peptide of the cyclic peptide, DMF is used for washing in the washing after each amino acid activation solution reaction.

[0035] More preferably, in the preparation of the linear peptide, during the deprotection solution treatment, after washing the CTC resin bonded with the amino acid reagent with DMF, the deprotection solution is added, and the mixture is stirred for 10 - 60 min. After the treatment is completed, the deprotection solution is removed by suction filtration, washed with DMF, and dried by suction. The deprotection solution is used in an appropriate amount.

[0036] More preferably, in the preparation of the linear peptide, during the cleavage solution treatment, the cleavage solution is added to the CTC resin bonded with the amino acid reagent, and the mixture is treated at 20 - 40 °C for 0.5 - 5 h. After the treatment is completed, the resin is removed by filtration to obtain a filtrate, and the filtrate is dried to obtain the linear peptide. The cleavage solution is used in an appropriate amount.

[0037] Preferably, in the preparation of the cyclic peptide, the linear peptide is added to a solvent to obtain a linear peptide solution, and then the linear peptide solution is mixed with a cyclization reagent and reacted at 30 - 50 °C. The reaction is monitored by LC-MS. After the reaction is completed, post-cyclization treatment is carried out to obtain a cyclic peptide with a protecting group. The cleavage solution is added for treatment, and finally post-cleavage treatment is carried out to obtain the cyclic peptide.

[0038] More preferably, in the preparation of the cyclic peptide, the solvent contains DMF and may also contain at least one of DCM and DIEA. The solvent is based on DMF for measurement. The relationship between the usage amounts of the linear peptide and DMF is 0.1 - 30 mg / mL, the relationship between the usage amounts of DCM and DMF is 0.1 - 10 mL / mL, and the relationship between the usage amounts of DIEA and DMF is 0.1 - 5 mg / mL. The cleavage solution is Solution E or Solution F, and the cleavage solution is used in an appropriate amount. The cyclization reagent is HBTU, and the relationship between the usage amounts of HBTU and DMF is 0.1 - 5 mg / mL.

[0039] More preferably, in the preparation of the cyclic peptide, Solution E is composed of TFA, anisole, EDT, PhOH, and water, and TFA, anisole, EDT, PhOH, and water are mixed in a volume ratio of 87.5:5:2.5:2.5:2.5.

[0040] More preferably, in the preparation of the cyclic peptide, Solution F is composed of TFA, TIS, and water, and TFA, TIS, and water are mixed in a volume ratio of 90:5:5; PR100

[0041] Preferably, in the post-cyclization treatment, the DMF is dried by suction, ice water is added to the test solution after the cyclization reaction is completed to precipitate a solid, the mixture is stirred and filtered, the solid is dissolved in EA, and then washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with a protecting group.

[0042] Preferably, in the post-treatment of cyclization, DMF is dried by evaporation, DCM is added to the test solution after the cyclization reaction is completed, and liquid-liquid extraction is carried out. Then, the solution is washed successively with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with a protecting group.

[0043] Preferably, in the post-treatment of cleavage, the test solution after the cleavage treatment is added to ice ether, a solid is precipitated, centrifuged and washed, and the solid is evaporated to dryness and purified to obtain a cyclic peptide.

[0044] Purification is carried out by liquid chromatography.

[0045] Since the present invention prepares a linear peptide containing a protecting group by activating an amino acid reagent, and then prepares and cleaves a functional cyclic peptide with a cyclic structure through cyclization treatment, the following beneficial effects are achieved: the functional cyclic peptide prepared by the present invention has good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good effect of inhibiting neurotransmitter release, good whitening effect, good anti-glycation effect, and good antioxidant effect. Therefore, the present invention relates to a functional cyclic peptide with good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good effect of inhibiting neurotransmitter release, and good whitening effect, and a preparation method and application thereof. Description of the Drawings

[0046] Figure 1 It is the chromatogram of PR126;

[0047] Figure 2 It is the mass spectrum of PR126;

[0048] Figure 3 It is the chromatogram of PR140;

[0049] Figure 4 It is the mass spectrum of PR140;

[0050] Figure 5 It is the chromatogram of PR100;

[0051] Figure 6 It is the mass spectrum of PR100;

[0052] Figure 7 It is the chromatogram of PR132;

[0053] Figure 8 It is the mass spectrum of PR132;

[0054] Figure 9 It is the chromatogram of PR120;

[0055] Figure 10 It is the mass spectrum of PR120;

[0056] Figure 11 It is the chromatogram of PR102;

[0057] Figure 12 is the mass spectrum of PR102;

[0058] Figure 13 is the chromatogram of PR139;

[0059] Figure 14 is the mass spectrum of PR139;

[0060] Figure 15 is the chromatogram of PR146;

[0061] Figure 16 is the mass spectrum of PR146;

[0062] Figure 17 is the chromatogram of PR116;

[0063] Figure 18 is the mass spectrum of PR116. Detailed implementation manners

[0064] The technical solutions of the present invention will be further described in detail below in combination with the specific implementation manners and the drawings:

[0065] Example 1: Preparation method of Cyclo(Tyr - Pro - Phe - Phe)

[0066] S1. The synthesis steps of the linear peptide are as follows:

[0067] S11. Place CTC resin (2.23 g, 2.5 mmol) in a 100 mL solid - phase synthesis reactor, add amino acid Fmoc - Pro - OH (1.68 g, 5 mmol), add 20 mL of dichloromethane (DCM), then add 2.0 mL of DIEA, react at 25 °C for 3 hours, add 3 mL of methanol, and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash 6 times with 20 mL of DMF solution, and drain to dry for use.

[0068] S12: Take Fmoc-Tyr(tBu)-OH (2.76 g, 6 mmol) and HOBt (0.81 g, 6 mmol) in a 50 mL beaker. Cool down to 5 °C, add 5 mL of DMF solution, and DIC (0.93 mL, 6 mmol). Let it stand for reaction for 15 minutes, and then add the solution in the 100 mL beaker to a 100 mL solid-phase synthesis reactor. Stir and react for 1.5 hours until the reaction is completed. Wash the resin three times with 20 mL of DMF solution each time. After the washing is completed, proceed to the next reaction. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash six times with 20 mL of DMF solution and drain to dry for use.

[0069] Repeat the above S12 steps. Replace Fmoc-Tyr(tBu)-OH with the following amino acid reagents in sequence: Fmoc-Phe-OH and Fmoc-Phe-OH; after the deprotection treatment is completed, then wash twice with 20 mL of methanol, twice with 20 mL of DCM solution, twice with 20 mL of methanol, and dry under vacuum to obtain H-Phe-Phe-Tyr(tBu)-Pro-CTC-resin. Treat the above peptide resin with 30 mL of 30% TFE / DCM solution, stir and react at 30 °C for 30 minutes, filter to remove the resin, and obtain the filtrate. The filtrate is dried to obtain 1.10 g of fully protected polypeptide H-Phe-Phe-Tyr(tBu)-Pro-OH, with a yield of 90% and a purity of 97.5%.

[0070] S2: The synthesis steps of the cyclic peptide with protecting groups are as follows:

[0071] S21: Weigh H-Phe-Phe-Tyr(tBu)-Pro-OH (1.02 g), dissolve it in DMF (1000 mL) until clear, add DIEA (1.674 g) and set it aside, named A; weigh HBTU (1.84 g) and add it to A. After the addition is complete, stir and react for 2 hours, and directly conduct in-process control; In-process control: LC-MS detects that the raw materials have completely reacted.

[0072] S22: Post-treatment: Drain to dry to remove most of the DMF in the reaction solution, and then add ice water (30 mL) while stirring to precipitate solids. After stirring for 10 min, filter it. Dissolve the solid in EA (20 mL) until clear, wash twice with saturated NaHCO3 aqueous solution and once with saturated brine, dry over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain 0.9 g of Cyclo(Tyr(tBu)-Pro-Phe-Phe).

[0073] S3: The synthesis steps of the cyclic peptide are as follows:

[0074] S31, Cutting: Weigh 0.9 g of Cyclo(Tyr(tBu)-Pro-Phe-Phe) and react it with cutting solution E under stirring at a controlled temperature of 30 °C. In-process control: Take a sample for MS detection. The reaction is basically complete.

[0075] S32, Post-treatment: Drop the reaction solution into ice-cold diethyl ether for precipitation. Dropwise add while shaking. Centrifuge and wash the solid 3 times. Evaporate the solid to dryness to obtain 0.68 g of Cyclo(Tyr-Pro-Phe-Phe). Detect by LC-MS and send it for purification.

[0076] The purification conditions are as follows:

[0077] Dissolution: Take 0.68 g of the crude product and dissolve it by ultrasonic treatment with 30 mL of acetic acid, 30 mL of acetonitrile, and 100 mL of water.

[0078] Filler: 50 DAC10-100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0079] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0080] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 60 mL / min;

[0081] Sample loading: Flow rate: 60 mL / min;

[0082] Elution: 30 - 50 - 80% B, 0 - 60 - 90 min;

[0083] Column cleaning: Clean with 80% acetonitrile until the baseline is balanced;

[0084] Collect the qualified product and lyophilize to obtain 55 mg. The LC diagram of the product purification is as Figure 1 shown, and the MS diagram of the purified product is as Figure 2 shown.

[0085] Example 2: Preparation method of Cyclo(Asp-Val-Lys-Tyr)

[0086] S1, The synthesis steps of the linear peptide are as follows:

[0087] S11. Place CTC resin (5.58 g, 6.25 mmol) in a 250 mL solid-phase synthesis reactor, add amino acid Fmoc-Tyr(tBu)-OH (0.459 g, 12.5 mmol), add 120 mL of dichloromethane (DCM), then add DIEA (8.7 mL), and react at 25 °C for 3 hours. Add 12.5 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 75 mL of dichloromethane (DCM), twice with 75 mL of methanol, and twice with 75 mL of DMF. Add 65 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, then wash 6 times with 120 mL of DMF solution, and drain to dryness for later use.

[0088] S12. Take Fmoc-Lys(Boc)-OH (7.03 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool down to 5 °C, add 14 mL of DMF solution, DIC (1.89 mL, 15 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 250 mL solid-phase synthesis reactor, stir and react for 1.5 hours until the reaction is complete. Wash the resin three times with 200 mL of DMF solution each time. After washing, proceed to the next reaction. Add 65 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, then wash 6 times with 120 mL of DMF solution, and drain to dryness for later use.

[0089] Repeat the above S12 step, sequentially replace Fmoc-Lys(Boc)-OH with the following amino acid reagents in order: Fmoc-Val-OH and Fmoc-Asp(OtBu)-OH; after the deprotection treatment is completed, then wash twice with 75 mL of methanol, twice with 75 mL of DCM solution, twice with 75 mL of methanol, and dry under vacuum to obtain H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-CTC-resin. Take 3.95 g of the above peptide resin, add 40 mL of 30% TFE / DCM cleavage solution, stir and react at 30 °C for 2.5 hours, filter to remove the resin, and obtain the filtrate. Dry the filtrate to obtain the crude peptide H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-OH, 1.67 g, yield 113%, purity 70.7%.

[0090] S2. The synthesis steps of the cyclic peptide with protecting groups are as follows:

[0091] S21. Weigh H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-OH(2 g), dissolve it in DMF(2000 mL) until clear, add DIEA(2.36 g), and name it as A for standby. Weigh HBTU(2.6 g), add it to A, stir for 2 hours after dropping, and directly conduct in-process control. In-process control: Detect by LC-MS that the raw materials have completely reacted.

[0092] S22. Post-treatment: Evaporate to dryness to remove most of the DMF in the reaction solution, then add ice water(30 mL)while stirring to precipitate solids. After stirring for 10 min, filter it. Dissolve the solid in EA(20 mL)until clear, wash it twice with saturated NaHCO3 aqueous solution and once with saturated brine, dry it with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain 1.22 g of Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)), with a yield of 62.5%.

[0093] S3. The synthesis steps of the cyclic peptide are as follows:

[0094] S31. Weigh 1.22 g of Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)), use the cutting solution E, control the temperature at 30 °C and stir for reaction. In-process control: Take samples to detect MS, and the reaction is basically complete.

[0095] S32. Post-treatment: Drop the reaction solution into ice ether for precipitation, shake while dropping, centrifuge and wash the solid 3 times, evaporate the solid to dryness to obtain 0.95 g of Cyclo(Asp-Val-Lys-Tyr), with a yield of 90%. Detect by LC-MS and send it for purification.

[0096] The purification conditions are as follows:

[0097] Dissolution: Take 0.95 g of the crude product and dilute it with 100 mL of water;

[0098] Filler: 50 DAC10 - 100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0099] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0100] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0101] Sample loading: Flow rate: 60 mL / min;

[0102] Elution: 0 - 20%B for 60 min;

[0103] Column cleaning: Wash with 80% acetonitrile until the baseline is balanced;

[0104] Lyophilization of the qualified product gave 202 mg. The LC chromatogram of the product purification is as shown in Figure 3 and the MS chromatogram of the purified product is as shown in Figure 4 .

[0105] Example 3: Preparation method of Cyclo(Lys-Thr-Thr-Lys-Ser)

[0106] S1. The synthesis steps of the linear peptide are as follows:

[0107] S11. Place CTC resin (6.25 g, 7 mmol) in a 250 mL solid-phase synthesis reactor, add amino acid Fmoc-Ser(tBu)-OH (8.42 g, 7 mmol), add 75 mL of dichloromethane (DCM), add DIEA (8.7 mL), and react at 25 °C for 3 hours. Then add 12.5 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 75 mL of dichloromethane (DCM), twice with 75 mL of methanol, and twice with 75 mL of DMF. Add 40 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash with 75 mL of DMF solution six times and drain for later use.

[0108] S12. Take Fmoc-Lys(Boc)-OH (9.84 g, 21 mmol) and HOBt (2.84 g, 21 mmol) in a 100 mL beaker, cool down to 5 °C, add 50 mL of DMF solution, DIC (3.2 mL, 21 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 250 mL solid-phase synthesis reactor, stir and react for 1.5 hours until the reaction is completed. Wash the resin three times with 75 mL of DMF solution each time. After washing, proceed to the next reaction. Add 40 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash with 65 mL of DMF solution six times and drain for later use.

[0109] Repeat the above S12 step, and sequentially replace Fmoc-Lys(Boc)-OH with the following amino acid reagents in order: Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH; after the deprotection treatment is completed, then wash with 130 mL of methanol twice, wash with 130 mL of DCM solution twice, wash with 130 mL of methanol twice, and dry under vacuum to obtain H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-CTC-resin. Use 3.5 g of cleavage solution 30% TFE / DCM, 40 mL for the above peptide resin, stir and react at 30 °C for 2.5 hours, filter to remove the resin, and obtain the filtrate. The filtrate is dried to obtain 1.42 g of crude peptide H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-OH. The yield is 50.9% and the purity is 88.5%.

[0110] S2. The synthesis steps of the cyclic peptide with a protecting group are as follows:

[0111] S21. Feeding: Weigh H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-OH (1.42 g), dissolve it clearly with DMF (750 mL) + DCM (750 mL), add DIEA (2.94 g), and set it aside for naming A; weigh HBTU (1.73 g), add it to A, stir and react for 2 hours after dropping, and directly perform in-process control; In-process control: LC-MS detects that the raw materials have completely reacted.

[0112] S22. Post-treatment: Dry to remove most of the DMF in the reaction solution, then add ice water (30 mL) while stirring, and no solid can be precipitated. Add DCM (20 mL) to the reaction solution, extract by layering, wash twice with saturated NaHCO3 aqueous solution, wash once with saturated brine, dry with anhydrous sodium sulfate, filter and evaporate to dryness to obtain 1.3 g of Cyclo(Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)), and the yield is 93%.

[0113] S3. The synthesis steps of the cyclic peptide are as follows:

[0114] S31. Feeding: Weigh 1.22 g of Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)) and use cleavage solution E, control the temperature at 30 °C and stir and react. In-process control: Take a sample to detect MS, and the reaction is basically complete.

[0115] S32, Post-treatment: The reaction solution was added dropwise to ice-cold diethyl ether for precipitation, shaking while adding dropwise, and the solid was centrifugally washed 3 times. The solid was dried by evaporation to obtain 0.83 g of Cyclo(Lys-Thr-Thr-Lys-Ser), with a yield of 75.5%. LC-MS was detected and sent for purification.

[0116] Purification conditions:

[0117] Dissolution: 0.83 g of the crude product was diluted with 100 mL of water;

[0118] Packing: 50 DAC10 - 100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0119] Mobile phase: A: Water; B: Acetonitrile;

[0120] Equilibration: A:B = 100:0, equilibration for 10 min, flow rate: 60 mL / min;

[0121] Sample loading: Flow rate: 60 mL / min;

[0122] Elution: 0 - 10% B for 60 min;

[0123] Column cleaning: Cleaning with 80% acetonitrile until baseline equilibrium;

[0124] The qualified product was collected and lyophilized to obtain 49 mg. The LC diagram of the product purification is as Figure 5 shown, and the MS diagram of the purified product is as Figure 6 shown.

[0125] Example 4: Preparation method of Cyclo(Arg-Lys-Asp-Val-Tyr)

[0126] S1, The synthesis steps of the linear peptide are as follows:

[0127] S11, Place CTC resin (5.58 g, 6.25 mmol) in a 100 mL solid-phase synthesis reactor, add amino acid Fmoc-Tyr(tBu)-OH (4.21 g, 12.5 mmol), add 20 mL of dichloromethane (DCM), then add DIEA (5.0 mL), and react at 25 °C for 3 hours. Add 6 mL of methanol and react for 5 minutes. Filter, wash the resin 2 times with 50 mL of dichloromethane (DCM), 2 times with 50 mL of methanol, and 2 times with 50 mL of DMF. Add 50 mL of 20% Pip / DMF solution, stir and react for 30 min, filter by suction to remove the deprotection solution, and then wash 6 times with 50 mL of DMF solution and drain for use.

[0128] S12. Take Fmoc-Val-OH (5.09 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF solution, add DIC (2.3 mL, 7.5 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 100 mL solid-phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin three times with DMF solution, 20 mL each time. After washing, proceed to the next reaction. Add 50 mL of 20% Pip / DMF solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with 50 mL of DMF solution 6 times, and drain and set aside.

[0129] Repeat the above S12 steps, and sequentially replace Fmoc-Val-OH with the following amino acid reagents in order: Fmoc-Asp(otBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH; after the deprotection treatment is completed, then wash with 50 mL of methanol 2 times, wash with 20 mL of DCM solution 2 times, wash with 50 mL of methanol 2 times, and dry in vacuum to obtain H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-CTC-resin. React the above peptide resin with 120 mL of 30% TFE / DCM solution at 30 °C for 30 minutes with stirring, filter to remove the resin, and obtain the filtrate. The filtrate is dried to obtain 4.2 g of fully protected polypeptide H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-OH, with a yield of 74% and a purity of 92.5%.

[0130] S2. The synthesis steps of the cyclic peptide with protecting groups are as follows:

[0131] S21. Charging: Weigh H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-OH (1.5 g), dissolve it clearly with DMF (1500 mL), add DIEA (1.35 g), and reserve it for naming A; weigh HBTU (1.49 g), add it to A, after dropping, stir and react for 2 hours, and directly carry out in-process control; In-process control: LC-MS detects that the raw materials have completely reacted

[0132] S22, Post-treatment: Remove most of the DMF in the reaction solution by evaporation to dryness, then add ice water (80 mL) with stirring, precipitate the solid, after stirring for 10 min, filter it, dissolve the solid in EA (40 mL), wash it twice with saturated aqueous NaHCO3 solution and once with saturated brine, dry it over anhydrous sodium sulfate, filter and evaporate to dryness to obtain 1 g of Cyclo(Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)), with a yield of 67.7%.

[0133] S3, The synthesis steps of the cyclic peptide are as follows:

[0134] S31, Charging: Weigh 1 g of Cyclo(Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)), react it with cutting solution E at a controlled temperature of 30 °C with stirring; In-process control: Take a sample for MS detection, and the reaction is basically complete.

[0135] S32, Post-treatment: Drop the reaction solution into ice-cold diethyl ether for precipitation, shake it while dropping, centrifuge and wash the solid 3 times, evaporate the solid to dryness to obtain 0.7 g of Cyclo(Arg-Lys-Asp-Val-Tyr), with a yield of 87.5%, detect LC-MS, and send it for purification.

[0136] Purification conditions:

[0137] Dissolution: Take 0.7 g of the crude product and dilute it with 100 mL of water;

[0138] Packing: 50 DAC10-100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0139] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0140] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0141] Sample loading: Flow rate: 60 mL / min;

[0142] Elution: 0-20% B for 60 min;

[0143] Column cleaning: Clean with 80% acetonitrile until the baseline is balanced;

[0144] Collect the qualified product and freeze-dry to obtain 156 mg. The LC diagram of the product purification is as shown in Figure 7 shown, and the MS diagram of the purified product is as shown in Figure 8 shown.

[0145] Example 5: Preparation method of Cyclo(Ser-Val-Val-Val-Arg-Thr)

[0146] S1. The synthesis steps of the linear peptide are as follows:

[0147] S11. Place CTC resin (5.58 g, 6.25 mmol) in a 100 mL solid-phase synthesis reactor, add amino acid Fmoc-Arg(Pbf)-OH (8.11 g, 12.5 mmol), add 20 mL of dichloromethane (DCM), then add DIEA (5.0 mL), and react at 25 °C for 3 hours. Add 6 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 50 mL of dichloromethane (DCM), twice with 50 mL of methanol, and twice with 50 mL of DMF. Add 50 mL of 20% Pip / DMF solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with 50 mL of DMF solution six times, and drain to dryness for standby.

[0148] S12. Take Fmoc-Val-OH (5.09 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF solution, DIC (2.3 mL, 7.5 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 100 mL solid-phase synthesis reactor, stir and react for 1.5 hours to complete the reaction. Wash the resin three times with 20 mL of DMF solution each time. After washing, proceed to the next step of the reaction. Add 50 mL of 20% Pip / DMF solution, stir and react for 30 min, filter by suction to remove the deprotection solution, then wash with 50 mL of DMF solution six times, and drain to dryness for standby.

[0149] Repeat the above S12 steps, and sequentially replace Fmoc-Val-OH with the following amino acid reagents in order: Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH; after the deprotection treatment is completed, then wash twice with 50 mL of methanol, twice with 20 mL of DCM solution, twice with 50 mL of methanol, and dry under vacuum to obtain H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-CTC-resin. Stir and react the above peptide resin with 100 mL of 30% TFE / DCM solution at 30 °C for 30 minutes, filter to remove the resin, and obtain the filtrate. The filtrate is dried to obtain 3.96 g of fully protected polypeptide H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH, with a yield of 79% and a purity of 92.1%.

[0150] S2. The synthesis steps of the cyclic peptide with protecting groups are as follows:

[0151] S21, Feeding: Weigh H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH (1 g), dissolve it in DMF (1000 mL) until clear, add DIEA (1.48 g) and DCM (10 mL), and set it aside named A; Weigh HBTU (1.12 g), add it to A, after dropping, stir and react for 2 hours, and directly conduct in-process control; In-process control: Detect by LC-MS that the raw materials have completely reacted.

[0152] S22, Post-treatment: Evaporate to dryness to remove most of the DMF in the reaction solution, then add ice water (30 mL) while stirring. No solid can be precipitated. Add DCM (20 mL) to the reaction solution, perform liquid-liquid extraction, wash twice with saturated NaHCO3 aqueous solution, wash once with saturated brine, dry with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain 0.66 g of Cyclo(Ser(tBu)-Val-Val-Val-Arg(Pbf)-Thr(tBu)).

[0153] S3, The synthesis steps of the cyclic peptide are as follows:

[0154] S31, Feeding: Weigh 0.66 g of Cyclo(Ser(tBu)-Val-Val-Val-Arg(Pbf)-Thr(tBu)), use the cutting solution E, control the temperature at 30 °C and stir to react; In-process control: Take a sample to detect MS, and the reaction is basically complete.

[0155] S32, Post-treatment: Drop the reaction solution into ice ether for precipitation, shake while dropping, centrifuge and wash the solid 3 times, evaporate the solid to dryness to obtain 0.28 g of Cyclo(Ser-Val-Val-Val-Arg-Thr), detect by LC-MS, and send it for purification.

[0156] Purification conditions:

[0157] Dissolution: Take 0.28 g of the crude product and dilute it with 100 mL of water;

[0158] Column packing: 21.2 * 250 mm, 10 - 120, C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0159] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0160] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 10 mL / min;

[0161] Sample loading: Flow rate: 10 mL / min;

[0162] Elution: 4 - 24%B for 60 min;

[0163] Column cleaning: Wash with 80% acetonitrile until the baseline is balanced;

[0164] Collect the qualified product and freeze-dry it to obtain 28 mg. The LC chromatogram of the product purification is as shown in Figure 9 shown, and the MS chromatogram of the purified product is as shown in Figure 10 shown.

[0165] Example 6: Preparation method of Cyclo(Glu-Glu-Met-Gln-Arg-Arg)

[0166] S1. The synthesis steps of the linear peptide are as follows:

[0167] S11. Place CTC resin (2.23 g, 2.5 mmol) in a 100 mL solid-phase synthesis reactor, add amino acid Fmoc-Met-OH (1.85 g, 5 mmol), add 20 mL of dichloromethane (DCM), add DIEA (2.0 mL), and react at 25 °C for 3 hours. Then add 3 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash with 20 mL of DMF solution six times and dry by suction for later use.

[0168] S12. Take Fmoc-Glu(otBu)-OH (2.55 g, 6 mmol) and HOBt (0.81 g, 6 mmol) in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF solution, DIC (0.93 mL, 6 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 100 mL solid-phase synthesis reactor, stir and react for 1.5 hours until the reaction is completed. Wash the resin three times with 20 mL of DMF solution each time. After washing, proceed to the next reaction. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash with 20 mL of DMF solution six times and dry by suction for later use.

[0169] Repeat the above S12 step, and sequentially replace Fmoc-Glu(otBu)-OH with the following amino acid reagents in order: Fmoc-Glu(otBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH; after the deprotection treatment is completed, then wash with 20 mL of methanol twice, 20 mL of DCM solution twice, 20 mL of methanol twice, and dry under vacuum to obtain H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-CTC-resin. React the above peptide resin with 40 mL of 30% TFE / DCM solution at 30 °C with stirring for 30 minutes, filter to remove the resin, and obtain a filtrate. The filtrate is dried to obtain 2.0 g of fully protected polypeptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-OH, with a yield of 58.8% and a purity of 95.6%.

[0170] S2. The synthesis steps of the cyclic peptide with a protecting group are as follows:

[0171] S21. Charging: Weigh H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-OH (1.62 g), dissolve it in DMF (1620 mL) until clear, add DIEA (0.98 g), and set it aside as named A; weigh HBTU (1.08 g), add it to A, and stir and react for 2 hours after dropping. Conduct in-process control directly; In-process control: Detect by LC-MS that the raw materials have completely reacted.

[0172] S22. Post-treatment: Dry to remove most of the DMF in the reaction solution, then add ice water (30 mL) while stirring to precipitate a solid. After stirring for 10 min, filter it. Dissolve the solid in EA (15 mL) until clear, wash it twice with saturated aqueous NaHCO3 solution and once with saturated brine, dry it over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain 1.57 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)), with a yield of 98%.

[0173] S3. The synthesis steps of the cyclic peptide are as follows:

[0174] S31. Charging: Weigh 1.57 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)) and use cutting solution E, control the temperature at 30 °C and stir and react for 2 hours; In-process control: Take a sample to detect MS, and the reaction is basically complete.

[0175] S32, Post-treatment: The reaction solution was added dropwise to ice-cold diethyl ether for precipitation, while shaking during the addition. The solid was centrifuged and washed 3 times, and then dried in a vacuum drying oven for 16 hours to obtain 0.98 g of Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala) with a yield of 99.9%. It was detected by LC-MS and sent for purification.

[0176] Purification conditions:

[0177] Dissolution: 1.5 g of the crude product was diluted with 200 mL of water.

[0178] Packing: 50 DAC10-100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0179] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0180] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0181] Sample loading: Flow rate: 60 mL / min;

[0182] Elution: 0 - 20% B for 60 min;

[0183] Column cleaning: Wash with 80% acetonitrile until baseline equilibrium.

[0184] The qualified product was collected and freeze-dried to obtain 200 mg. The LC diagram of the product purification is as Figure 11 shown, and the MS diagram of the purified product is as Figure 12 shown.

[0185] Example 7: Preparation method of Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala)

[0186] S1, The synthesis steps of the linear peptide are as follows:

[0187] S11, Place the CTC resin (2.23 g, 2.5 mmol) in

[0188] a 100 mL solid-phase synthesis reactor, add the amino acid Fmoc-Met-OH (1.85 g, 5 mmol), add 20 mL of dichloromethane (DCM), then add DIEA (2.0 mL), and react at 25 °C for 3 hours. Add 3 mL of methanol and react for 5 minutes. Filter, wash the resin with 20 mL of dichloromethane (DCM) 2 times, 20 mL of methanol 2 times, and 20 mL of DMF 2 times. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash with 20 mL of DMF solution 6 times and drain for use.

[0189] S12: Take Fmoc-Glu(otBu)-OH (2.55 g, 6 mmol) and HOBt (0.81 g, 6 mmol) in a 50 mL beaker. Cool down to 5 °C, add 5 mL of DMF solution, and DIC (0.93 mL, 6 mmol). Let it stand for reaction for 15 minutes, and then add the solution in the 100 mL beaker to a 100 mL solid-phase synthesis reactor. Stir and react for 1.5 hours until the reaction is completed. Wash the resin three times with 20 mL of DMF solution each time. After the washing is completed, proceed to the next reaction. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter to remove the deprotection solution, and then wash six times with 20 mL of DMF solution and drain to dry for later use.

[0190] Repeat the above S12 steps. Replace Fmoc-Glu(otBu)-OH with the following amino acid reagents in sequence: Fmoc-Glu(otBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH; after the deprotection treatment is completed, then wash twice with 20 mL of methanol, twice with 20 mL of DCM solution, twice with 20 mL of methanol, and dry under vacuum to obtain H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-CTC-resin. React the above peptide resin with 40 mL of 30% TFE / DCM solution at 30 °C for 30 minutes with stirring, filter to remove the resin, and obtain the filtrate. Dry the filtrate to obtain 1.79 g of fully protected polypeptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH, with a yield of 50.8% and a purity of 89.8%.

[0191] S2: The synthesis steps of the cyclic peptide with protecting groups are as follows:

[0192] S21: Charging: Weigh H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH (1.56 g), dissolve it clearly with DMF (1500 mL), add DIEA (0.9 g), and reserve it for naming A; weigh HBTU (0.99 g), add it to A, and stir and react for 2 hours after dropping. Conduct in-process control directly; In-process control: LC-MS detects that the raw materials have completely reacted.

[0193] S22, Post-treatment: Remove most of the DMF in the reaction solution by evaporation to dryness, then add ice water (36 mL) with stirring, and a solid will precipitate. After stirring for 10 min, filter it. Dissolve the solid with EA (18 mL), wash it twice with saturated aqueous NaHCO3 solution and once with saturated brine, dry it over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain 1.36 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala), with a yield of 88%.

[0194] S3, The synthesis steps of the cyclic peptide are as follows:

[0195] S31, Charging: Weigh 1.36 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala), react it with cutting solution E, control the temperature at 30 °C and stir for 2 hours; In-process control: Take a sample for MS detection, and the reaction is basically complete.

[0196] S32, Post-treatment: Drop the reaction solution into ice ether for precipitation, shake it while dropping, centrifuge and wash the solid 3 times, and dry it in a vacuum drying oven for 16 hours to obtain 1.15 g of Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala), with a yield of 132%, and send it for purification.

[0197] Purification conditions:

[0198] Dissolution: Take 1.5 g of the crude product and dilute it with 200 mL of water;

[0199] Packing: 50 DAC10-100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0200] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0201] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0202] Sample loading: Flow rate: 60 mL / min;

[0203] Elution: 0-20% B for 60 min;

[0204] Column cleaning: Clean with 80% acetonitrile until the baseline is balanced;

[0205] Collect the qualified product and lyophilize to obtain 154 mg. The LC diagram of the product purification is as shown in Figure 13 shown, and the MS diagram of the purified product is as shown in Figure 14 shown.

[0206] Example 8: Preparation Method of Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp)

[0207] S1. The synthesis steps of the linear peptide are as follows:

[0208] S11. Place CTC resin (2.23 g, 2.5 mmol) in a 100 mL solid-phase synthesis reactor, add amino acid Fmoc-Met-OH (1.85 g, 5 mmol), add 20 mL of dichloromethane (DCM), then add DIEA (2.0 mL), and react at 25 °C for 3 hours. Add 3 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, then wash with 20 mL of DMF solution 6 times, and filter with suction until dry for use.

[0209] S12. Take Fmoc-Glu(otBu)-OH (2.55 g, 6 mmol) and HOBt (0.81 g, 6 mmol) in a 50 mL beaker, cool down to 5 °C, add 5 mL of DMF solution, DIC (0.93 mL, 6 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 100 mL solid-phase synthesis reactor, stir and react for 1.5 hours until the reaction is complete. Wash the resin three times with 20 mL of DMF solution each time. After the washing is completed, proceed to the next reaction. Add 20 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, then wash with 20 mL of DMF solution 6 times, and filter with suction until dry for use.

[0210] Repeat the above S12 step, and sequentially replace Fmoc-Lys(Boc)-OH with the following amino acid reagents in order: Fmoc-Glu(otBu)-OH, Fmoc-Asp(otBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH; after the deprotection treatment is completed, then wash with 20 mL of methanol twice, wash with 20 mL of DCM solution twice, wash with 20 mL of methanol twice, and dry under vacuum to obtain H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-CTC-resin. React the above peptide resin with 40 mL of 30% TFE / DCM solution at 30 °C with stirring for 30 minutes, filter to remove the resin, and obtain a filtrate. The filtrate is dried to obtain 2.83 g of fully protected polypeptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(otBu)-Glu(otBu)-Glu(otBu)-Met-OH, with a yield of 73.1% and a purity of 93.3%.

[0211] S2. The synthesis steps of the cyclic peptide with a protecting group are as follows:

[0212] S21. Feeding: Weigh H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(otBu)-Glu(otBu)-Glu(otBu)-Met-OH (1.5 g), dissolve it in DMF (1500 mL) until clear, add DIEA (1 mL), and reserve it for naming A; weigh HBTU (0.87 g), add it to A, stir and react for 2 hours after dropping, and directly perform in-process control; In-process control: LC-MS detects that the raw materials have completely reacted.

[0213] S22. Post-treatment: Dry to remove most of the DMF in the reaction solution, then add ice water (40 mL) while stirring to precipitate a solid. After stirring for 10 min, filter it. Dissolve the solid in EA (20 mL) until clear, wash it twice with saturated aqueous NaHCO3 solution and once with saturated brine, dry it over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain 0.94 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(OtBu)), with a yield of 63%.

[0214] S3. The synthesis steps of the cyclic peptide are as follows:

[0215] S31, Feeding: Weigh 0.94 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(OtBu)) and use cutting solution E. Stir and react at a controlled temperature of 30 °C; In-process control: Take a sample for MS detection. The reaction is basically complete.

[0216] S32, Post-treatment: Drop the reaction solution into ice ether for precipitation. Shake while dropping. Centrifuge and wash the solid 3 times. Evaporate the solid to dryness to obtain 0.52 g of Cyclo(Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val), with a yield of 86%. Detect by LC-MS and send it for purification.

[0217] Purification conditions:

[0218] Dissolution: Take 0.52 g of the crude product and dilute it with 100 mL of water;

[0219] Filler: 50 DAC10-100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0220] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0221] Equilibration: A:B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0222] Sample loading: Flow rate: 60 mL / min;

[0223] Elution: 0 - 20% B for 60 min;

[0224] Column cleaning: Clean with 80% acetonitrile until the baseline is balanced;

[0225] Collect the qualified product and freeze-dry to obtain 170 mg. The LC chart of the product purification is as Figure 15 shown, and the MS chart of the purified product is as Figure 16 shown.

[0226] Example 9: Preparation method of Cyclo(Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val)

[0227] S1, The synthesis steps of the linear peptide are as follows:

[0228] S11. Place CTC resin (2.77 g, 2 mmol) in a 250 mL solid-phase synthesis reactor, add amino acid Fmoc-Pro-OH (0.674 g, 2 mmol), add 120 mL of dichloromethane (DCM), add DIEA (8.7 mL), and react at 25 °C for 3 hours. Then add 12.5 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 75 mL of dichloromethane (DCM), twice with 75 mL of methanol, and twice with 75 mL of DMF. Add 25 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash 6 times with 130 mL of DMF solution and drain to dryness for later use.

[0229] S12. Take Fmoc-Lys(Boc)-OH (2.814 g, 6 mmol) and HOBt (0.81 g, 6 mmol) in a 100 mL beaker, cool down to 5 °C, add 25 mL of DMF solution, add DIC (3.2 mL, 21 mmol), let it stand and react for 15 minutes, and add the solution in the 100 mL beaker to a 250 mL solid-phase synthesis reactor, stir and react for 1.5 hours until the reaction is completed. Wash the resin three times with 25 mL of DMF solution each time. After washing, proceed to the next reaction. Add 25 mL of 20% Pip / DMF solution, stir and react for 30 min, filter with suction to remove the deprotection solution, and then wash 6 times with 25 mL of DMF solution and drain to dryness for later use.

[0230] Repeat the above S12 step. Sequentially replace Fmoc-Lys(Boc)-OH with the following amino acid reagents in order: Fmoc-Phe-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-Phe-OH, Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Val-OH; after the deprotection treatment is completed, then wash twice with 50 mL of methanol, twice with 50 mL of DCM solution, twice with 50 mL of methanol, and dry in vacuum to obtain H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-CTC-resin. Treat the above peptide resin with 40 mL of 30% TFE / DCM cleavage solution, stir and react at 30 °C for 2.5 hours, filter to remove the resin, and obtain the filtrate. Dry the filtrate to obtain 2.1 g of crude peptide H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH. The yield is 64.02% and the purity is 95.3%.

[0231] S2. The synthesis steps of the cyclic peptide with protecting groups are as follows:

[0232] S21, Feeding: Weigh H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH (1 g), dissolve it in DMF (1000 mL) until clear, add DIEA (0.64 g), and reserve it as named A; Weigh HBTU (0.65 g), add it to A, stir and react for 2 hours after dropping, and directly conduct in-process control; In-process control: Detect by LC-MS that the raw materials have completely reacted.

[0233] S22, Post-treatment: Evaporate to dryness to remove most of the DMF in the reaction solution, then add ice water (30 mL) while stirring to precipitate solids. After stirring for 10 min, filter it. Dissolve the solid in EA (20 mL) until clear, wash it twice with saturated aqueous NaHCO3 solution and once with saturated brine, dry it over anhydrous sodium sulfate, filter and evaporate to dryness to obtain 0.85 g of Cyclo(Met-Pro-D-Phe-Arg(Pbf)-D-Trp(Boc)-Phe-Lys(Boc)-Pro-Val), with a yield of 86%.

[0234] S3, The synthesis steps of the cyclic peptide are as follows:

[0235] S31, Feeding: Weigh 0.85 g of Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)), react with cutting solution E, control the temperature at 30 °C and stir; In-process control: Take samples to detect MS, and the reaction is basically complete.

[0236] S32, Post-treatment: Drop the reaction solution into ice ether for precipitation, shake while dropping, centrifuge and wash the solid 3 times, dry it in a vacuum drying oven for 16 hours to obtain 0.62 g of Cyclo(Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val), with a yield of 84.6%. Detect by LC-MS and send it for purification.

[0237] Purification conditions:

[0238] Dissolution: Take 0.62 g of the crude product, add 160 mL of water and 10 mL of acetic acid, and dissolve it by ultrasonic;

[0239] Filler: 50 DAC10-100 C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

[0240] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0241] Equilibration: A:B = 95:5, equilibrate for 10 min, flow rate: 60 mL / min;

[0242] Loading: Flow rate: 60 mL / min;

[0243] Elution: 16 - 36% B for 60 min;

[0244] Column cleaning: Clean with 80% acetonitrile until baseline equilibrium;

[0245] Collect qualified products and lyophilize to obtain 160 mg. The LC chromatogram of product purification is as shown in Figure 17 shown, and the MS chromatogram of the purified product is as shown in Figure 18 shown.

[0246] Test example:

[0247] Moisturizing test method: Test for the content of AQP3 (aquaporin 3)

[0248] (1) Cell seeding: Seed cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2).

[0249] (2) Solution preparation: Prepare the working solution of the test substance according to the experimental design.

[0250] Table 1 AQP3 experimental design table

[0251]

[0252] The diluent in the AQP3 experiment is cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, PR157.

[0253] (3) Add the test substance: After culturing in an incubator (37 °C, 5% CO2) for 24 h, add the test substance according to the table and continue culturing for 24 h.

[0254] (4) Sample collection: Discard the supernatant and rinse the cells 3 times with PBS.

[0255] (5) Immunofluorescence staining:

[0256] a. Add methanol to fix the cells, rinse 3 times with PBS, and add 1 mL of BSA to each well to block for 1 hour.

[0257] b. Discard the blocking solution, add the primary antibody to each well, and place it in a 4 °C refrigerator overnight. Discard the primary antibody and rinse 3 times with PBS.

[0258] c. Add the secondary antibody to each well and incubate for 2 hours. Discard the secondary antibody and rinse 3 times with PBS.

[0259] d. Add DAPI to each well for nuclear staining for 10 min, discard the DAPI, wash 3 times with PBS, and then take pictures using a fluorescence microscope.

[0260] (6)Result analysis: Quantitatively analyze the fluorescence intensity of AQP3 using Image Pro Plus software.

[0261] Table 2 Test results of AQP3 content

[0262]

[0263] The following conclusions can be obtained through testing of the present invention: The PR095 sample has a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR100 sample has a moisturizing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. The PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, and PR157 sample all have a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0264] Moisturizing test method: HA (hyaluronic acid) content test

[0265] (1)Cell seeding: Seed cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2).

[0266] (2)Prepare the solution: Prepare the test article working solution according to the experimental design.

[0267] Table 3 HA experimental design table

[0268]

[0269] The diluent in the experiment is cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157.

[0270] (3)Add the test article: After culturing for 24 h in an incubator (37 °C, 5% CO2), add the test article according to the table and continue culturing for 24 h.

[0271] (4)Collect the samples: Collect the supernatant and measure the HA content using an ELISA kit.

[0272] Table 4 Test results of HA content

[0273]

[0274] The following conclusions can be obtained through the tests of the present invention: The PR095 sample has a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR100 sample, PR102 sample, PR116 sample, PR120 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, and PR157 sample have a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR126 sample has a moisturizing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL.

[0275] Test method for anti-wrinkle and firming effect: Measurement of type I collagen and MMP-1 content

[0276] (1) Cell seeding: Seed cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2).

[0277] (2) Solution preparation: Prepare the test substance working solution according to the experimental design.

[0278] Table 5 Experimental design table

[0279]

[0280] The diluent in the experiment is cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157. In the test, CollagenI is type I collagen; UVA is ultraviolet A; VC is vitamin C; VE is vitamin E.

[0281] (3) UVA irradiation: After culturing for 24 hours, the negative control group, positive control group, and sample groups are irradiated with UVA at a total dose of 9 J / cm2. At the same time, the blank control group is placed in the same environment (UVA irradiation dose is 0 J / cm2).

[0282] (4)Add the test substance: According to the experimental design, after irradiation, add the test substance in groups. Add 1 mL of cell culture medium to each well in the blank control group and the negative control group; add 1 mL of cell culture medium containing vitamin C and vitamin E to each well in the positive control group; add 1 mL of culture medium containing the test substance at the corresponding concentration to each well in the sample group. After adding the test substance, place the 24-well plate in an incubator (37 °C, 5% CO2) and culture for 24 h.

[0283] (5)Collect the supernatant for determination of type I collagen and MMP-1 content.

[0284] (6)Result analysis: The t-test statistical analysis was used for comparison between groups, and all statistical analyses were two-tailed.

[0285] Table 6 Test results of type I collagen content

[0286]

[0287] The following conclusions can be obtained through testing in this invention: Samples PR095, PR100, PR102, PR116, PR120, PR126, PR132, PR139, PR140, PR146, and PR157 have anti-wrinkle and firming effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL.

[0288] Soothing test method: IL-6 content test

[0289] (1)Cell seeding: Seed cells into a 24-well plate and incubate overnight in an incubator (37 °C, 5% CO2).

[0290] Table 7 Experimental design table for IL-6 synthesis

[0291]

[0292] The diluent in the experiment is cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157. In the test, IL-6 is interleukin-6 (cytokine), and LPS is lipopolysaccharide.

[0293] (2)Add the test substance: According to the experimental grouping, when the cell confluence rate in the 24-well plate reaches 40% - 60%, add the test substance in groups, with 3 replicates in each group. Place the 24-well plate in an incubator (37 °C, 5% CO2) and incubate for 24 h.

[0294] (3)Detection: After culturing for 24 hours, collect the supernatant and determine the IL-6 content using an ELISA kit.

[0295] Table 8 Test results of IL-6 content

[0296]

[0297] The following conclusions can be drawn from the tests of the present invention: The PR095 sample has a soothing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL. The PR100 sample has a soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. The PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, and PR146 sample all have a soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR157 sample has a soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL.

[0298] Test method for inhibiting neurotransmitter release: Norepinephrine test

[0299] (1)Cell seeding: Seed cells into a 24-well plate and culture in an incubator (37 °C, 5% CO2) for 6 days.

[0300] (2)Cell treatment: To study the inhibitory effect on NA release, discard the culture medium and wash the cells with HBSS. Before inducing exocytosis, pre-incubate the cells with different concentrations of the test samples dissolved in HBSS for 60 minutes. Take out the supernatant and proceed according to the norepinephrine release induction protocol described below.

[0301] The diluent in the experiment is cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group is selected from PR102.

[0302] (3)Induce human norepinephrine release: Treat with HBSS containing TPA for 8 minutes, remove TPA, and continue to incubate in HBSS containing ION and TPA for 5 minutes for the induced release of norepinephrine (NA). This group is the positive control for the test. The basal control group is treated with HBSS containing 100 nM TPA for 8 + 5 minutes, and the remaining steps are the same as the positive control group. Immediately collect the supernatant containing the released NA after incubation and store it at -80 °C for further ELISA test analysis.

[0303] (4)Extraction of norepinephrine (NA) from the supernatant: On the day of measurement, the sample was thawed at room temperature for 1 hour, and the NA sample was extracted using a 24-well plate coated with boric acid gel. First, it was incubated with extraction buffer, then washed with double-distilled water, then incubated with an acylation reagent under the extraction buffer, and finally washed with double-distilled water. Finally, after adding the release buffer, the sample was quantitatively analyzed for NA using the ELISA method.

[0304] (5)Determination of NA content by ELISA method.

[0305] (6)Data analysis: The t-test statistical analysis was used for comparison between groups, and all statistical analyses were two-tailed.

[0306] Table 9 Test results

[0307]

[0308] The above tests were conducted on PR-102 in the present invention. The PR-102 sample could inhibit the release of the neurotransmitter norepinephrine at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. It was shown that the PR-102 sample could inhibit the release of the neurotransmitter norepinephrine.

[0309] Melanin content test

[0310] Table 10 Experimental design table for melanin content

[0311]

[0312] The diluent in the experiment was cell culture medium, such as DMEM medium. The concentrations of the sample groups were 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups were selected from PR116.

[0313] Logarithmic growth phase cells were collected and inoculated into a 24-well plate. After culturing in an incubator (37 °C, 5% CO2) for 24 h, according to the cytotoxicity results, the test substances were added as shown in the table, and the untreated cells were used as the blank control. Three parallels were set for each group.

[0314] After adding the drugs, the cells were further cultured in an incubator (37 °C, 5% CO2) for 24 h. The supernatant was discarded, 0.5 mL of 1 M NaOH containing 10% DMSO was added, and the mixture was incubated at 80 °C for 1 h. Using 1 M NaOH containing 10% DMSO as the solvent control, the absorbance value was read on an enzyme-linked immunosorbent assay (ELISA) reader and the relative inhibition rate of cellular melanin was calculated.

[0315]

[0316] Table 11 Melanin content test results

[0317]

[0318] The present invention has conducted the above tests on PR116. The PR-116 samples have whitening effects at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL, indicating that the PR-116 samples have whitening effects.

[0319] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those of ordinary skill in the art can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the patent protection scope of the present invention should be defined by the claims.

[0320] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, rather than imposing any formal restrictions on the implementation manners of the technology of the present invention. 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 content of the present invention, but should still be regarded as the technology or embodiment substantially the same as the present invention.

[0321] Specific examples are used in this article to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of written expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present 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 within the protection scope of the present application.

Claims

1. Use of a functional cyclic peptide in the preparation of a moisturizing product and / or an anti-wrinkle product and / or a firming product and / or a soothing product, the structure of the functional cyclic peptide being as follows: 。

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