A preparation method and application of a beauty cyclic peptide reagent

Abstract

The invention discloses a preparation method and application of a beauty cyclic peptide reagent, and belongs to the technical field of beauty reagent preparation; the preparation method comprises mixing a cyclic peptide, an additive and glycerol to obtain a beauty cyclic peptide reagent; the cyclic peptide comprises at least cyclohexapeptide-2; the additive comprises at least a gallic acid lecithin complex and a naringenin acylhydrazone derivative, the gallic acid lecithin complex has a phenol group and a phosphate group, and the naringenin acylhydrazone derivative has a phenol group and a nicotinic acid hydrazide group. The invention provides a beauty cyclic peptide reagent and application thereof with high biocompatibility, superior transdermal absorption performance, better anti-wrinkle firming and whitening and brightening effects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic agent preparation, and in particular to a preparation method and application of a cosmetic cyclic peptide agent. Background Art

[0002] Hexapeptide-2, a short peptide composed of six amino acids, has a high affinity for the melanocortin-1 (MC1R) receptor on melanocytes. As an antagonist of α-melanocyte stimulating hormone (α-MSH), hexapeptide-2 can competitively bind to the MC1R receptor, effectively inhibit the activation of tyrosinase, and reduce the biosynthesis of melanin. Compared with resorcinol whitening agents, hexapeptide-2 is milder and safer, and also has anti-aging properties and the potential to promote collagen production. However, hexapeptide-2 also faces some challenges in practical applications, including poor stability, low transdermal absorption rate, and easy decomposition by proteases in the body.

[0003] In the field of cosmetic research and development, the emerging technology of converting linear peptide molecules into cyclic peptides is gradually being applied. The cyclization process gives peptide molecules more excellent stability, while significantly improving their transdermal absorption efficiency and enhancing their bioavailability. This technological innovation has brought efficient and safe active ingredients to the cosmetics industry, heralding a double leap in the efficacy and safety of cosmetics. A search of existing literature and patents found that application publication number CN114634554A discloses an anti-wrinkle cyclic hexapeptide compound and its preparation method. The transdermal permeability and anti-wrinkle effect of the cyclized hexapeptide-1 are better than those of the linear peptide; application publication number CN116284256A discloses a hexapeptide-9 cyclic peptide and its application. Cyclic hexapeptide-9 has more outstanding effects than hexapeptide-9 linear peptide in skin anti-wrinkle and firming, and can significantly promote the expression of collagen III and integrin β4, with lower cytotoxicity and better skin permeability.

[0004] In view of the limitations of linear hexapeptide-2, by processing it into cyclic hexapeptide-2, a beauty cyclic peptide agent with high biocompatibility, excellent transdermal absorption performance, better effect of promoting collagen production and inhibiting the relative activity of tyrosinase and melanin production is developed. It has practical significance and market prospects in whitening, anti-wrinkle and anti-aging. Summary of the invention

[0005] The object of the present invention is to provide a cosmetic cyclic peptide agent with high biocompatibility, excellent transdermal absorption performance, better effect of promoting collagen production and inhibiting tyrosinase relative activity and melanin production, and a preparation method thereof.

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

[0007] A method for preparing a beauty cyclic peptide agent comprises mixing a cyclic peptide, an additive and glycerol to obtain a mixed solution; the cyclic peptide comprises at least cyclic hexapeptide-2; the additive comprises at least a gallic acid lecithin complex and a naringenin acylhydrazone derivative, the gallic acid lecithin complex has a phenol group and a phosphate group, and the naringenin acylhydrazone derivative has a phenol group and a nicotinic acid hydrazide group.

[0008] The invention provides a method for preparing a beauty cyclic peptide reagent. According to the method, cyclohexapeptide-2 is mixed with a gallic acid lecithin complex, a naringenin acylhydrazone derivative and glycerol to obtain a beauty cyclic peptide reagent with high biocompatibility, excellent transdermal absorption performance, and better effects of promoting collagen production and inhibiting the relative activity of tyrosinase and melanin production. The gallic acid lecithin complex and the naringenin acylhydrazone derivative have high bioavailability, strong stability and antioxidant activity, and can not only improve the biocompatibility and transdermal performance of the beauty cyclic peptide reagent, but also can scavenge free radicals, inhibit the relative activity of tyrosinase and melanin production, and promote collagen production, thereby achieving the beauty effects of skin whitening, brightening, anti-wrinkle and firming.

[0009] Preferably, the mass ratio of the cyclopeptide to the additive is 1:0.002-0.05.

[0010] Preferably, the usage ratio of the cyclic peptide and glycerol is 1 g: 2-10 mL.

[0011] Preferably, the gallic acid lecithin complex is derived from soybean lecithin grafted with gallic acid.

[0012] Preferably, the preparation of the gallic acid phosphatidylcholine complex comprises:

[0013] Add soybean lecithin to anhydrous ethanol, gradually add gallic acid while stirring, slowly raise the temperature to 40-60°C, continue stirring and condensing to reflux for 2-4 hours until the reaction solution is clear, remove ethanol by rotary evaporation at 40-50°C, filter through a non-aqueous filter membrane, vacuum filter and dry the filtrate for 12-36 hours to obtain a gallic acid lecithin complex.

[0014] More preferably, the solid-liquid ratio of soybean lecithin and anhydrous ethanol is 1 g: 10-30 mL.

[0015] More preferably, the mass ratio of soybean lecithin to gallic acid is 1:0.2-1.

[0016] Preferably, the naringenin acylhydrazone derivatives are obtained by reacting naringenin and 6-hydroxynicotinic acid hydrazide.

[0017] Preferably, the preparation of naringenin acylhydrazone derivatives is specifically as follows:

[0018] Naringenin and 6-hydroxynicotinic acid hydrazide are mixed, ethanol and acetic acid are added, and the mixture is stirred to dissolve, and the mixture is heated at 600-800W for 2-10min, and then recrystallized with methanol, washed, filtered, and dried to obtain naringenin acylhydrazone derivatives.

[0019] More preferably, the mass ratio of naringenin to 6-hydroxynicotinic acid hydrazide is 1:1-2.

[0020] More preferably, the solid-liquid ratio of naringenin to ethanol is 1 g: 2-5 mL.

[0021] More preferably, the solid-liquid ratio of naringenin and acetic acid is 1 g: 0.2-0.5 mL.

[0022] Preferably, the structural formula of cyclohexapeptide-2 is as shown in Formula I:

[0023] .

[0024] Preferably, the preparation of cyclohexapeptide-2 comprises coupling 2-chlorotrityl chloride (CTC) resin with an amino acid reagent protected by an Fmoc group to obtain a linear peptide resin, cutting the resin to obtain a fully protected linear peptide, and then obtaining cyclohexapeptide-2 by cyclization.

[0025] More preferably, the coupling order of the amino acid reagents protected by the Fmoc group is: Fmoc-Lys(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-His(Trt)-OH.

[0026] Preferably, the preparation of cyclohexapeptide-2 is specifically as follows:

[0027] Step S1: Take 2-chlorotrityl chloride (CTC) resin in a solid phase synthesis reaction tube, add dichloromethane (DCM) to swell, drain the solvent, and wash the CTC resin with dimethylformamide (DMF) and DCM for 2-10 times respectively;

[0028] Step S2: Dissolve Fmoc-Lys(Boc)-OH in DMF, add N,N-diisopropylethylamine (DIPEA), add the reaction solution to the solid phase synthesis reaction tube obtained in step S1, shake at room temperature for 2-4 hours, wash the CTC resin with DMF and DCM for 2-10 times each, add DIPEA / methanol / DCM mixed solution for end-capping treatment for 20-40 minutes, wash the CTC resin with DMF and DCM for 2-10 times each, drain and add piperidine (Pip) / DMF mixed solution to react for 5-15 minutes, add Pip / DMF mixed solution again to react for 5-15 minutes, wash the CTC resin with DMF and DCM for 2-10 times each, drain and set aside;

[0029] Step S3: Dissolve Fmoc-D-Phe-OH and 1-hydroxybenzotriazole (HOBt) in DMF, then add N,N'-diisopropylcarbodiimide (DIC) to activate for 10-30 minutes, then add the solution to the above solid phase synthesis reaction tube, shake at room temperature for 2-4 hours, wash the CTC resin with DMF and DCM for 2-10 times, drain and add Pip / DMF mixed solution to react for 5-15 minutes, add Pip / DMF mixed solution again to react for 5-15 minutes, wash the CTC resin with DMF and DCM for 2-10 times, drain and set aside; repeat this step to connect Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-His(Trt)-OH in sequence;

[0030] Step S4: Add trifluoroacetic acid (TFA) / DCM mixed solution to the solid phase synthesis reaction tube, shake at room temperature for 2-4 hours, filter and collect the filtrate, spin dry to obtain a fully protected linear peptide H-His(Trt)-{D-Trp}(Boc)-Ala-Trp(Boc)-{D-Phe}-Lys(Boc)-OH; HOBt, 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP) and DIPEA are dissolved in DCM, and the fully protected linear peptide is slowly added at 0-5°C, and the reaction is stirred at room temperature for 6-24 hours. After the reaction is completed, the solvent is evaporated using a rotary evaporator, and freeze-dried to obtain the target cyclohexapeptide-2 crude product Cyclo(His(Trt)-{D-Trp}(Boc)-Ala-Trp(Boc)-{D-Phe}-Lys(Boc));

[0031] Step S5: Add the crude cyclohexapeptide-2 to the side chain protecting group removal solution, shake and react at room temperature for 2-4 hours to cleave, and obtain a cleavage solution; add the cleavage solution to methyl tert-butyl ether, stir for 20-40 minutes, precipitate a large amount of white precipitate, filter and wash the filter cake with methyl tert-butyl ether 2-10 times to obtain the crude cyclohexapeptide-2, purify it by reverse HPLC, and then freeze-dry it in a freeze dryer to obtain the fine cyclohexapeptide-2 Cyclo (His-{D-Trp}-Ala-Trp-{D-Phe}-Lys).

[0032] More preferably, in step S1, the loading amount of CTC resin is 0.4-0.5 mmol / g, and the solid-liquid ratio of CTC resin and DCM is 1 g:10-15 mL.

[0033] More preferably, in step S2, the solid-liquid ratio of Fmoc-Lys(Boc)-OH to DMF is 1 g:0.5-1.5 mL.

[0034] More preferably, in step S2, the solid-liquid ratio of Fmoc-Lys(Boc)-OH and DIPEA is 1 g:0.1-1 mL.

[0035] More preferably, in step S2, the volume ratio of DMF to the DIPEA / methanol / DCM mixed solution is 1:4-8, the DIPEA / methanol / DCM mixed solution includes DIPEA, methanol and DCM, the volume ratio of DIPEA to methanol is 1:1-2, and the volume ratio of DIPEA to DCM is 1:3-5.

[0036] More preferably, in step S2, the volume ratio of DMF to the Pip / DMF mixed solution is 1:4-6, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip to DMF in the Pip / DMF mixed solution is 1:2-10.

[0037] More preferably, the molar ratio of Fmoc-D-Phe-OH to HOBt in step S3 is 1:1-2.

[0038] More preferably, the molar ratio of Fmoc-Trp(Boc)-OH to HOBt in step S3 is 1:1-2.

[0039] More preferably, the molar ratio of Fmoc-Ala-OH to HOBt in step S3 is 1:1-2.

[0040] More preferably, the molar ratio of Fmoc-D-Trp(Boc)-OH to HOBt in step S3 is 1:1-2.

[0041] More preferably, the molar ratio of Fmoc-His(Trt)-OH to HOBt in step S3 is 1:1-2.

[0042] More preferably, in step S3, the solid-liquid ratio of HOBt to DMF is 1 g: 0.2-1 mL.

[0043] More preferably, the molar ratio of HOBt to DIC in step 3 is 1:1-2.

[0044] More preferably, in step S3, the volume ratio of DMF to the Pip / DMF mixed solution is 1:1-3, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip to DMF in the Pip / DMF mixed solution is 1:4-6.

[0045] More preferably, in step S4, the solid-to-liquid ratio of the reactants and the TFA / DCM mixed solution in the solid phase synthesis reaction tube is 1 g:20-30 mL, the TFA / DCM mixed solution includes TFA and DCM, and the volume ratio of TFA and DCM in the TFA / DCM mixed solution is 1:50-150.

[0046] More preferably, the molar ratio of the fully protected linear peptide to HOBt in step S4 is 1:2-4.

[0047] More preferably, the molar ratio of the fully protected linear peptide to PyBOP in step S4 is 1:2-4.

[0048] More preferably, in step S4, the molar ratio of the fully protected linear peptide to DIPEA is 1:4-8.

[0049] More preferably, the solid-liquid ratio of HOBt and DCM in step S4 is 1:0.5-1.5 mL.

[0050] More preferably, in step S5, the solid-liquid ratio of the crude cyclohexapeptide-2 and the side chain protecting group removal solution is 1 g: 5-15 mL, the side chain protecting group removal solution includes TFA, triisopropylsilane (TIS) and H2O, the volume ratio of TFA and TIS is 1: 0.01-0.1, and the volume ratio of TFA and H2O is 1: 0.01-0.1.

[0051] More preferably, in step S5, the volume ratio of the lysate to methyl tert-butyl ether is 1:4-6.

[0052] Preferably, the additives include gallic acid phosphatidylcholine complex and naringenin acylhydrazone derivatives.

[0053] Preferably, the additives include gallic acid lecithin complex, naringenin acylhydrazone derivatives and rosin pentaerythritol ester.

[0054] Preferably, a method for preparing a beauty cyclic peptide agent comprises:

[0055] Cyclic hexapeptide-2 is mixed with gallic acid lecithin complex, naringenin acylhydrazone derivatives, rosin pentaerythritol ester and glycerol to obtain a beauty cyclic peptide agent. Rosin pentaerythritol ester can further improve the biocompatibility of the beauty cyclic peptide agent, help the beauty cyclic peptide agent to be better absorbed through the skin, and at the same time achieve beauty effects by promoting the production of type I collagen and type III collagen and inhibiting the relative activity of tyrosinase and melanin production.

[0056] More preferably, the mass ratio of cyclohexapeptide-2 to gallic acid phosphatidylcholine complex is 1:0.002-0.01.

[0057] More preferably, the mass ratio of cyclohexapeptide-2 to naringenin acylhydrazone derivatives is 1:0.01-0.02.

[0058] More preferably, the mass ratio of cyclohexapeptide-2 to rosin pentaerythritol ester is 1:0.002-0.01.

[0059] More preferably, the usage ratio of cyclohexapeptide-2 to glycerol is 1 g: 2-10 mL.

[0060] The invention also provides the prepared beauty cyclic peptide agent.

[0061] The present invention also provides the use of the cosmetic cyclic peptide agent for stimulating collagen production.

[0062] Preferably, the collagen in the skin includes type I collagen and type III collagen.

[0063] Type I collagen is the most abundant type of collagen in the skin, accounting for about 80% to 85% of the total collagen in the skin. Its unique spiral structure gives the skin and bone tissue a high degree of strength and elasticity, maintaining their structural and functional stability. In addition, type I collagen also plays a key role in the contraction and extension of muscles, and plays an important role in maintaining muscle health and motor function. In contrast, type III collagen exists in a looser mesh structure, accounting for about 10% to 15% of the total collagen in the skin. It is mainly distributed around type I collagen at the junction of the epidermis and dermis, providing elasticity and stress resistance to the skin, and has the effect of promoting elastic repair. In the skin, type I collagen mainly provides support structure and support, while type III collagen provides elasticity and stress resistance. The loss of collagen, especially the reduction of type I collagen, can cause wrinkles and depressions on the face, affecting the firmness of the skin. Similarly, the lack of type III collagen can also cause the skin to lose its original elasticity and stress resistance, thereby affecting the overall health and appearance of the skin. Therefore, the effect of the cosmetic cyclic peptide reagent in stimulating collagen production can be evaluated by detecting the concentrations of type I collagen and type III collagen.

[0064] The research of the present invention proves that the concentration of type I collagen and type III collagen in human skin fibroblast HFF-1 treated with the beauty cyclic peptide reagent is significantly increased, proving that the beauty cyclic peptide reagent prepared by the present invention has a better effect in stimulating collagen production.

[0065] The present invention also provides the use of the beauty cyclic peptide agent for whitening and brightening skin color.

[0066] Preferably, the beauty cyclic peptide agent achieves the effect of whitening and brightening the skin tone by reducing the concentration of cAMP in cells, inhibiting the activity of tyrosinase, and reducing the production of melanin.

[0067] When the human body is exposed to ultraviolet radiation, the keratinocytes in the skin show low tolerance. In response to this physiological challenge, these cells secrete α-MSH (α-melanocyte stimulating hormone, also known as melanocyte-stimulating hormone), a neuroendocrine hormone composed of 13 amino acid residues and a member of the melanocyte stimulating hormone family. The release of α-MSH acts deep in the skin, binds to the MC1R receptor on the surface of melanocytes, and activates adenylate cyclase (AC), which catalyzes the conversion of ATP to cAMP. As a "second messenger" in cells, the increase in the concentration of cAMP activates protein kinase A (PKA), which in turn enhances the activity of tyrosinase and accelerates the synthesis process of melanin to protect the skin from further damage by ultraviolet rays. Therefore, the beauty and brightening effect of beauty cyclic peptide agents can be evaluated by detecting the intracellular cAMP concentration, the relative activity of tyrosinase and the relative content of melanin.

[0068] The research of the present invention proves that the cAMP concentration, relative activity of tyrosinase and relative content of melanin in mouse melanoma cells B16 are significantly reduced after being treated with the beauty cyclic peptide reagent, which brings a significant effect of whitening and brightening the skin.

[0069] The invention adopts gallic acid lecithin complex, naringenin acylhydrazone derivatives and rosin pentaerythritol ester as additives, and mixes with cyclohexapeptide-2 and glycerol to prepare the beauty cyclic peptide reagent, so it has the following beneficial effects: the beauty cyclic peptide reagent prepared by the invention can promote cell viability, the cell viability is greater than 102%, and has good biocompatibility; the beauty cyclic peptide reagent prepared by the invention has good transdermal performance, the transdermal diffusion percentage in 12 hours is greater than 34%, it is easy for skin to absorb and quickly play a role; the beauty cyclic peptide reagent prepared by the invention can promote the production of type I collagen and type III collagen, and after 24 hours of treatment, The type I collagen concentration of human skin fibroblast HFF-1 is significantly increased to above 710 ng / mL, and the type III collagen concentration is increased to above 55 ng / mL, which can effectively reduce wrinkles, enhance the firmness of the skin, and achieve the beauty effect of anti-wrinkle and firming skin; the beauty cyclic peptide reagent prepared by the present invention can effectively inhibit the relative activity of tyrosinase and melanin production. After 24 hours of treatment, the cAMP concentration of mouse melanoma cell B16 is reduced to below 0.3 μM, the relative activity of tyrosinase is significantly reduced to below 80%, and the relative content of melanin is reduced to below 85%, achieving the beauty effect of whitening and brightening the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the electrospray ionization mass spectrometry result diagram of cyclohexapeptide-2 obtained in the present invention.

[0071] Figure 2 This is a diagram showing the cytotoxicity results of the cosmetic cyclic peptide reagent of the present invention.

[0072] Figure 3 This is a graph showing the effect of the cosmetic cyclic peptide reagent of the present invention on the concentration of type I collagen.

[0073] Figure 4 This is a graph showing the effect of the cosmetic cyclic peptide reagent of the present invention on the concentration of type III collagen.

[0074] Figure 5 This is a graph showing the effect of the beauty cyclic peptide reagent of the present invention on the cAMP content.

[0075] Figure 6 This is a graph showing the effect of the cosmetic cyclic peptide reagent of the present invention on the relative activity of tyrosinase.

[0076] Figure 7 This is a graph showing the effect of the beauty cyclic peptide reagent obtained in the present invention on the relative content of melanin. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0078] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. The following are the abbreviations of some reagents and materials. Unless otherwise specified, the amino acids used in this article are all L-type.

[0079] Table 1 Abbreviations corresponding to Chinese meanings

[0080]

[0081] Embodiment 1:

[0082] S1. A method for preparing cyclohexapeptide-2, comprising:

[0083] Step S11: Weigh 0.45 mmol / g of 2-chlorotrityl chloride (CTC) resin into a solid phase synthesis reaction tube, add DCM to swell, drain the solvent, and wash the CTC resin 5 times with DMF and DCM. The loading amount of CTC resin is 0.45 mmol / g, and the solid-liquid ratio of CTC resin and DCM is 1 g:12 mL.

[0084] Step S12: Dissolve Fmoc-Lys(Boc)-OH in DMF, add DIPEA, add the reaction solution to the solid phase synthesis reaction tube obtained in step S11, shake at room temperature for 3 hours, wash the CTC resin with DMF and DCM 5 times each, add DIPEA / methanol / DCM mixed solution for end-capping treatment for 30 minutes, wash the CTC resin with DMF and DCM 5 times each, drain and add Pip / DMF mixed solution to react for 10 minutes, add Pip / DMF mixed solution again to react for 10 minutes, wash the CTC resin with DMF and DCM 5 times each, drain and set aside. The solid-liquid ratio of Fmoc-Lys(Boc)-OH and DMF is 1g:1mL; the solid-liquid ratio of Fmoc-Lys(Boc)-OH and DIPEA is 1g:0.16mL; the volume ratio of DMF and DIPEA / methanol / DCM mixed solution is 1:6, the DIPEA / methanol / DCM mixed solution includes DIPEA, methanol and DCM, the volume ratio of DIPEA and methanol in the DIPEA / methanol / DCM mixed solution is 1:1, and the volume ratio of DIPEA and DCM is 1:4; the volume ratio of DMF and Pip / DMF mixed solution is 1:5, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip and DMF in the Pip / DMF mixed solution is 1:5.

[0085] Step S13: Take Fmoc-D-Phe-OH and HOBt, add DMF to dissolve, then add DIC to activate for 20 minutes, then add the solution to the solid phase synthesis reaction tube obtained in step S12, shake at room temperature for 3 hours, wash the CTC resin with DMF and DCM 5 times each, add Pip / DMF mixed solution to react for 10 minutes after drying, add Pip / DMF mixed solution again to react for 10 minutes, then wash the CTC resin with DMF and DCM 5 times each, and drain for standby use. The molar ratio of Fmoc-D-Phe-OH and HOBt is 1:1; the solid-liquid ratio of Fmoc-D-Phe-OH and DMF is 1g:2mL; the molar ratio of Fmoc-D-Phe-OH and DIC is 1:1; the volume ratio of DMF and Pip / DMF mixed solution is 1:2, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip and DMF in the Pip / DMF mixed solution is 1:5.

[0086] Step S14: Take Fmoc-Trp(Boc)-OH and HOBt, add DMF to dissolve, then add DIC to activate for 20 minutes, then add the solution to the solid phase synthesis reaction tube obtained in step S13, oscillate at room temperature for 3 hours, wash the CTC resin with DMF and DCM 5 times each, drain and add Pip / DMF mixed solution to react for 10 minutes, add Pip / DMF mixed solution again to react for 10 minutes, then wash the CTC resin with DMF and DCM 5 times each, drain and set aside. The molar ratio of Fmoc-Trp(Boc)-OH and HOBt is 1:1; the solid-liquid ratio of Fmoc-Trp(Boc)-OH and DMF is 1 g:2.6 mL; the molar ratio of Fmoc-Trp(Boc)-OH and DIC is 1:1; the volume ratio of DMF and Pip / DMF mixed solution is 1:2, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip and DMF in the Pip / DMF mixed solution is 1:5.

[0087] Step S15: Take Fmoc-Ala-OH and HOBt, add DMF to dissolve, then add DIC to activate for 20 minutes, then add the solution to the solid phase synthesis reaction tube obtained in step S14, shake at room temperature for 3 hours, wash the CTC resin with DMF and DCM 5 times each, add Pip / DMF mixed solution to react for 10 minutes after drying, add Pip / DMF mixed solution again to react for 10 minutes, then wash the CTC resin with DMF and DCM 5 times each, and drain for standby use. The molar ratio of Fmoc-Ala-OH and HOBt is 1:1; the solid-liquid ratio of Fmoc-Ala-OH and DMF is 1g:1.5mL; the molar ratio of Fmoc-Ala-OH and DIC is 1:1; the volume ratio of DMF and Pip / DMF mixed solution is 1:2, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip and DMF in the Pip / DMF mixed solution is 1:5.

[0088] Step S16: Take Fmoc-D-Trp(Boc)-OH and HOBt, add DMF to dissolve, then add DIC to activate for 20 minutes, then add the solution to the solid phase synthesis reaction tube obtained in step S15, oscillate at room temperature for 3 hours, wash the CTC resin with DMF and DCM 5 times each, drain and add Pip / DMF mixed solution to react for 10 minutes, add Pip / DMF mixed solution again to react for 10 minutes, wash the CTC resin with DMF and DCM 5 times each, drain and set aside. The molar ratio of Fmoc-D-Trp(Boc)-OH and HOBt is 1:1; the solid-liquid ratio of Fmoc-D-Trp(Boc)-OH and DMF is 1 g:2.6 mL; the molar ratio of Fmoc-D-Trp(Boc)-OH and DIC is 1:1; the volume ratio of DMF and Pip / DMF mixed solution is 1:2, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip and DMF in the Pip / DMF mixed solution is 1:5.

[0089] Step S17: Take Fmoc-His(Trt)-OH and HOBt, add DMF to dissolve, then add DIC to activate for 20 minutes, then add the solution to the solid phase synthesis reaction tube obtained in step S15, oscillate at room temperature for 3 hours, wash the CTC resin with DMF and DCM 5 times each, drain and add Pip / DMF mixed solution to react for 10 minutes, add Pip / DMF mixed solution again to react for 10 minutes, wash the CTC resin with DMF and DCM 5 times each, drain and set aside. The molar ratio of Fmoc-His(Trt)-OH and HOBt is 1:1; the solid-liquid ratio of Fmoc-His(Trt)-OH and DMF is 1 g:3.1 mL; the molar ratio of Fmoc-His(Trt)-OH and DIC is 1:1; the volume ratio of DMF and Pip / DMF mixed solution is 1:2, the Pip / DMF mixed solution includes Pip and DMF, and the volume ratio of Pip and DMF in the Pip / DMF mixed solution is 1:5.

[0090] Step S18: Add TFA / DCM mixed solution to the solid phase synthesis reaction tube obtained in step S17, shake at room temperature for 3 hours, filter and collect the filtrate to obtain a fully protected linear peptide H-His(Trt)-{D-Trp}(Boc)-Ala-Trp(Boc)-{D-Phe}-Lys(Boc)-OH; HOBt, PyBOP and DIPEA were dissolved in DCM (100 mL), and the fully protected linear peptide was slowly added at 0°C, and the reaction was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was evaporated using a rotary evaporator, and freeze-dried to obtain the target cyclohexapeptide-2 crude product Cyclo(His(Trt)-{D-Trp}(Boc)-Ala-Trp(Boc)-{D-Phe}-Lys(Boc)). In step S17, the solid-liquid ratio of the reactants and the TFA / DCM mixed solution in the solid phase synthesis reaction tube is 1 g:23 mL, the TFA / DCM mixed solution includes TFA and DCM, and the volume ratio of TFA and DCM in the TFA / DCM mixed solution is 1:100; the molar ratio of the fully protected straight-chain peptide and HOBt is 1:1; the molar ratio of the fully protected straight-chain peptide and PyBOP is 1:1; the molar ratio of the fully protected straight-chain peptide and DIPEA is 1:6; the solid-liquid ratio of HOBt and DCM is 1:0.5-1.5 mL.

[0091] Step S19: Add the crude cyclohexapeptide-2 obtained in step S18 to the solution for removing the side chain protecting groups, shake and react at room temperature for 3 hours to cleave, and obtain a cleavage solution; add the cleavage solution to methyl tert-butyl ether, stir for 30 minutes, precipitate a large amount of white precipitate, filter and wash the filter cake with methyl tert-butyl ether 5 times to obtain the crude cyclohexapeptide-2, purify it by reverse high performance liquid chromatography, and freeze-dry it in a freeze dryer to obtain the fine cyclohexapeptide-2 Cyclo (His-{D-Trp}-Ala-Trp-{D-Phe}-Lys). The solid-liquid ratio of the crude cyclohexapeptide-2 obtained in step S18 and the solution for removing the side chain protecting groups is 1g:10mL, and the solution for removing the side chain protecting groups includes TFA, TIS and H2O, the volume ratio of TFA and TIS is 90:5, and the volume ratio of TFA and H2O is 90:5:5; the volume ratio of the cleavage solution and methyl tert-butyl ether is 1:5.

[0092] S2. Preparation of a beauty cyclic peptide reagent, comprising:

[0093] The raw materials of a beauty cyclic peptide reagent are cyclohexapeptide-2 and glycerol. The cyclohexapeptide-2 and glycerol are mixed to obtain the beauty cyclic peptide reagent. The dosage ratio of cyclohexapeptide-2 to glycerol is 1g:5mL.

[0094] Embodiment 2:

[0095] Except that 0.5% of gallic acid phosphatidylcholine complex and 2% of naringenin acylhydrazone derivatives were additionally added relative to the mass of cyclohexapeptide-2 in Example 1 in the preparation of a cosmetic cyclic peptide agent, other conditions were the same as those in Example 1.

[0096] S1. Preparation of gallic acid lecithin complex, comprising:

[0097] Add soybean lecithin to anhydrous ethanol, gradually add gallic acid while stirring, slowly heat to 50°C, continue stirring and condensing to reflux for 3 hours until the reaction solution is clear, remove ethanol by rotary evaporation at 45°C, filter through a 0.45μm non-aqueous filter membrane, vacuum filter and dry the filtrate for 24 hours to obtain gallic acid lecithin complex. The solid-liquid ratio of soybean lecithin and anhydrous ethanol is 1g:20mL; the mass ratio of soybean lecithin and gallic acid is 1:0.24.

[0098] S2. Preparation of naringenin acylhydrazone derivatives, comprising:

[0099] Take naringenin and 6-hydroxynicotinic acid hydrazide, add ethanol and acetic acid, stir and dissolve, heat the mixture at 700W for 5 minutes, wash it with methanol 4 times, filter it, and dry it to obtain naringenin acylhydrazone derivatives. The molar ratio of naringenin and 6-hydroxynicotinic acid hydrazide is 1:1; the solid-liquid ratio of naringenin and ethanol is 1g:3.6mL; the solid-liquid ratio of naringenin and acetic acid is 1g:0.36mL.

[0100] Embodiment 3:

[0101] Except that in the preparation of a cosmetic cyclic peptide agent, the amount of gallic acid phosphatidylcholine complex used was changed to 0.25% of the mass of cyclic hexapeptide-2 in Example 1, other conditions were the same as those in Example 2.

[0102] Embodiment 4:

[0103] Except that in the preparation of a cosmetic cyclic peptide agent, the amount of naringenin acylhydrazone derivative used was changed to 1% of the mass of cyclohexapeptide-2 in Example 1, other conditions were the same as those in Example 2.

[0104] Embodiment 5:

[0105] The other conditions were the same as those in Example 2, except that 0.5% gallic acid lecithin complex, 2% naringenin acylhydrazone derivatives and 1% rosin pentaerythritol ester were additionally added relative to the mass of cyclohexapeptide-2 in Example 1 in the preparation of a cosmetic cyclic peptide agent.

[0106] Embodiment 6:

[0107] Except that in the preparation of a cosmetic cyclic peptide agent, the amount of rosin pentaerythritol ester used was changed to 0.5% of the mass of cyclohexapeptide-2 in Example 1, other conditions were the same as those in Example 5.

[0108] Comparative Example 1:

[0109] S1. Preparation of linear hexapeptide-2,

[0110] The method of step S18 is changed to: adding TFA / DCM mixed solution to the solid phase synthesis reaction tube obtained in step S17, shaking at room temperature for 3 hours, filtering and collecting the filtrate, and spinning to obtain a fully protected straight-chain peptide H-His(Trt)-{D-Trp}(Boc)-Ala-Trp(Boc)-{D-Phe}-Lys(Boc)-OH; the solid-liquid ratio of the reactant and the TFA / DCM mixed solution in the solid phase synthesis reaction tube obtained in step S17 is 1 g:4 mL, the TFA / DCM mixed solution includes TFA and DCM, and the volume ratio of TFA and DCM in the TFA / DCM mixed solution is 1:100. The other conditions are the same as steps S11 to S17 in Example 1.

[0111] S2. Preparation of a cosmetic peptide agent, comprising:

[0112] The raw materials of a beauty cyclic peptide reagent are linear hexapeptide-2 and glycerol. The linear hexapeptide-2 and glycerol are mixed to obtain the beauty cyclic peptide reagent. The usage ratio of the linear hexapeptide-2 and glycerol is 1g:5mL.

[0113] Comparative Example 2:

[0114] Except that 0.5% gallic acid phosphatidylcholine complex was additionally added relative to the mass of cyclohexapeptide-2 in Example 1 in the preparation of a cosmetic cyclic peptide agent, other conditions were the same as those in Example 1.

[0115] S1. Preparation of gallic acid phosphatidylcholine complex is the same as Example 2.

[0116] Comparative Example 3:

[0117] Except that 2% of naringenin acylhydrazone derivatives relative to the mass of cyclohexapeptide-2 in Example 1 were additionally added in the preparation of a cosmetic cyclic peptide agent, other conditions were the same as those in Example 1.

[0118] S1. Preparation of naringenin acylhydrazone derivatives, same as Example 2.

[0119] Comparative Example 4:

[0120] Except that 1% of rosin pentaerythritol ester relative to the mass of cyclohexapeptide-2 in Example 1 was additionally added in the preparation of a cosmetic cyclic peptide agent, other conditions were the same as in Example 1.

[0121] Experimental example:

[0122] 1. Determination of molecular weight of cyclohexapeptide-2

[0123] The molecular weight of the cyclohexapeptide-2 obtained by the present invention is determined by electrospray mass spectrometry. Figure 1 This is the electrospray mass spectrometry result of cyclohexapeptide-2 obtained in the present invention, [M / 2+H] + The mass-to-charge ratio (m / z) of the ion peak is 429.39, [M+H] + The mass-to-charge ratio (m / z) of the ion peak is 857.26, and the molecular weight measured by mass spectrometry is 856.25, which indicates that the method of the present invention can prepare cyclohexapeptide-2.

[0124] 2. Cytotoxicity test

[0125] Human skin keratinocytes HaCaT in logarithmic growth phase were prepared into single cell suspension and inoculated into 96-well plates. 100 μL of cell suspension was added to each well to make the cell density 1×10 5 cells / well, cultured at 37°C, 5% CO2 for 24h to allow cells to adhere to the wall; set up a blank group, a sample group and a zeroing group, and add 100μL of DMEM culture solution to each well in the blank group and the sample group; add the beauty cyclic peptide reagent obtained in Example 1-6 to each well in the sample group to a final concentration of 400ppm; the zeroing group was inoculated with no cells, and only 100μL of DMEM culture solution was added. After the administration was completed, the 96-well plate was placed in an incubator at 37°C and 5% CO2 for 24h and then observed under an inverted microscope. The culture solution was removed by centrifugation, and after being rinsed twice with phosphate buffered saline (PBS), 10μL of 5mg / mL thiazolyl blue solution was added to each well, and the culture was continued for 4h. Terminate the culture and remove the culture solution. Add 100μL of dimethyl sulfoxide (DMSO) to each well and oscillate at a low speed on a shaker for 10min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm on a microplate reader to calculate the cell viability, cell viability (%) = (OD of sample group - OD of zero-adjustment group) / (OD of blank group - OD of zero-adjustment group) × 100%.

[0126] Figure 2The cytotoxicity result diagram of the beauty cyclic peptide reagent obtained in the present invention. Compared with the blank group, the cell viability of Examples 1-6 and Comparative Examples 1-4 increased, which shows that the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 of the present invention have high biocompatibility. The beauty cyclic peptide reagent obtained in Example 1 is higher than that in Comparative Example 1 because Example 1 uses cyclohexapeptide-2 to prepare the beauty cyclic peptide reagent, and Comparative Example 1 uses linear hexapeptide-2 to prepare the beauty cyclic peptide reagent, which shows that the beauty cyclic peptide reagent obtained in Example 1 has higher biocompatibility than Comparative Example 1. The beauty cyclic peptide reagent obtained in Example 2 is higher than that in Example 1 because gallic acid lecithin complex and naringenin acylhydrazone derivatives are additionally added to the beauty cyclic peptide reagent in Example 2; the cell viability of the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Examples 3-4 because the beauty cyclic peptide reagents in Examples 3-4 reduce the usage amounts of gallic acid lecithin complex and naringenin acylhydrazone derivatives respectively; the cell viability of the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Comparative Examples 2-3 because Comparative Examples 2-3 separately add gallic acid lecithin complex or naringenin acylhydrazone derivatives to prepare the beauty cyclic peptide reagent; this indicates that the synergistic addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives to the beauty cyclic peptide reagent makes the beauty cyclic peptide reagent have a better effect in improving cell viability. The cell viability of the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Example 2 because in the preparation of the beauty cyclic peptide reagent, in addition to the additional addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives, Example 5 also additionally adds rosin pentaerythritol ester; the cell viability of the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Example 6 because the amount of rosin pentaerythritol ester used is different; the cell viability of the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Comparative Example 4 because Comparative Example 4 only additionally adds rosin pentaerythritol ester, and does not add gallic acid lecithin complex and naringenin acylhydrazone derivatives; this shows that adding rosin pentaerythritol ester to the beauty cyclic peptide reagent can further improve the biocompatibility of the beauty cyclic peptide reagent.

[0127] 3. Skin permeability test

[0128] One end of the diffusion pool was covered with Bama Xiang pig skin, with the skin stratum corneum facing the supply pool, 1 mL of PBS solution was added to the supply pool, and the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 were added to a final concentration of 400 ppm. 7 mL of pre-degassed PBS was injected into the receiving pool as a receiving solution, and the stirring speed was 350 r / min, and the temperature was (32±1)°C for transdermal experiments. After 24 hours of treatment, the residual sample on the skin surface was rinsed clean with PBS solution, the skin was cut into pieces, 5 mL of PBS solution was added, homogenized, and the supernatant was collected by centrifugation at 10000 r / min for 30 minutes. After filtering through a 0.22 μm filter membrane, a clear skin homogenate was obtained; after filtering the subcutaneous permeate through a 0.22 μm filter membrane, the sample concentrations of the skin homogenate and the subcutaneous permeate were determined by high performance liquid chromatography, and the diffusion percentage was calculated, and the diffusion percentage (%) = skin homogenate or subcutaneous permeate peptide concentration / initial concentration × 100%, and the measurement results are shown in Table 2.

[0129] Table 2 Transdermal performance results

[0130]

[0131] As shown in Table 2, the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 can be well transdermally absorbed, and neither of them will penetrate the dermis into the subcutaneous tissue. In the skin homogenate, the diffusion percentage of the beauty cyclic peptide reagent obtained in Example 1 is higher than that in Comparative Example 1, because Example 1 uses cyclohexapeptide-2 to prepare the beauty cyclic peptide reagent, and Comparative Example 1 uses linear hexapeptide-2 to prepare the beauty cyclic peptide reagent, which shows that the transdermal diffusion effect of the beauty cyclic peptide reagent obtained in Example 1 is better than that in Comparative Example 1. The diffusion percentage of the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Example 1, because gallic acid lecithin complex and naringenin acylhydrazone derivatives are additionally added to the beauty cyclic peptide reagent in Example 2; the diffusion percentage of the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Example 3-4, because the beauty cyclic peptide reagents obtained in Examples 3-4 reduce the usage amounts of gallic acid lecithin complex and naringenin acylhydrazone derivatives, respectively; the diffusion percentage of the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Comparative Example 2-3, because Comparative Example 2-3 separately adds gallic acid lecithin complex or naringenin acylhydrazone derivatives to prepare the beauty cyclic peptide reagent; this indicates that the synergistic addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives in the beauty cyclic peptide reagent makes the transdermal effect of the beauty cyclic peptide reagent better. The diffusion percentage of the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Example 2, because in the preparation of the beauty cyclic peptide reagent, in addition to the additional addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives, Example 5 also adds rosin pentaerythritol ester; the diffusion percentage of the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Example 6, because the amount of rosin pentaerythritol ester used is different; the diffusion percentage of the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Comparative Example 4, because Comparative Example 4 only adds rosin pentaerythritol ester, and does not add gallic acid lecithin complex and naringenin acylhydrazone derivatives; this shows that adding rosin pentaerythritol ester to the beauty cyclic peptide reagent can further improve the transdermal effect of the beauty cyclic peptide reagent.

[0132] 4. Determination of the effects on the expression of type I collagen and type III collagen

[0133] Human skin fibroblast HFF-1 cells in the logarithmic growth phase were adjusted to 4×10 5 100 / mL cell suspension, take 2mL of cell suspension and inoculate in 6-well plates, and place in a 37℃, 5% CO2 incubator for 24h. After 24h of cell culture, set up blank group, model group and sample group respectively. 2Irradiation was carried out under UVB light source. After the irradiation, the waste liquid was discarded. 2 mL of DMEM culture medium was added to each well of the blank group, model group and sample group. The beauty cyclic peptide reagent obtained in Example 1-6 and Comparative Example 1-4 was added to the sample group to a final concentration of 400 ppm. After continuing to be cultured in the incubator for 24 hours, the cell supernatant was collected for collagen content determination. The collagen concentration was detected according to the instructions of the type I collagen (CollagenⅠ) ELISA kit and the instructions of the type III collagen (Collagen III) ELISA kit.

[0134] Figure 3 This is a graph showing the effect of the beauty cyclic peptide reagent of the present invention on the concentration of type I collagen. Figure 4The figure is the result of the effect of the beauty cyclic peptide reagent obtained in the present invention on the concentration of type III collagen. The content of type I collagen and type III collagen in the model group was significantly reduced under UVB induction (P < 0.01), proving that the model was successfully established. Compared with the model group, the content of type I collagen and type III collagen in human skin fibroblasts treated with the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 were both increased, which shows that the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 of the present invention can stimulate the production of type I and type III collagen. The content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 1 is higher than that in Comparative Example 1, because Example 1 uses cyclohexapeptide-2 to prepare the beauty cyclic peptide reagent, and Comparative Example 1 uses linear hexapeptide-2 to prepare the beauty cyclic peptide reagent, which shows that the beauty cyclic peptide reagent obtained in Example 1 is better than Comparative Example 1 in stimulating the production of type I and type III collagen. The content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Example 1, because gallic acid lecithin complex and naringenin acylhydrazone derivatives are additionally added to the beauty cyclic peptide reagent in Example 2; the content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Examples 3-4, because the beauty cyclic peptide reagents obtained in Examples 3-4 reduce the usage amounts of gallic acid lecithin complex and naringenin acylhydrazone derivatives, respectively; the content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 2 is higher than that in Comparative Examples 2-3, because Comparative Examples 2-3 separately add gallic acid lecithin complex or naringenin acylhydrazone derivatives to prepare the beauty cyclic peptide reagent; this shows that the synergistic addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives in the beauty cyclic peptide reagent makes the beauty cyclic peptide reagent have a better effect in increasing the content of type I collagen and type III collagen. The content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Example 2, because in the preparation of the beauty cyclic peptide reagent, in addition to the additional addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives, rosin pentaerythritol ester is also added; the content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 5 is higher than that of type I collagen and type III collagen in Example 6, because the amount of rosin pentaerythritol ester used is different; the content of type I collagen and type III collagen in the beauty cyclic peptide reagent obtained in Example 5 is higher than that in Comparative Example 4, because Comparative Example 4 only adds rosin pentaerythritol ester, and does not add gallic acid lecithin complex and naringenin acylhydrazone derivatives; This shows that adding rosin pentaerythritol ester to the beauty cyclic peptide reagent can further stimulate the production of type I collagen and type III collagen, thereby achieving an anti-wrinkle and firming beauty effect.

[0135] 5. Regulatory effect on α-MSH / cAMP signaling

[0136] Mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5 / mL, 100μL of cell suspension was inoculated in an equal amount in a 96-well plate, and placed in a 37°C, 5% CO2 incubator for 24 hours, and the waste liquid was discarded. A blank group, a model group and a sample group were set up respectively. 100μL of DMEM culture solution was added to each well of the blank group, the model group and the sample group, and the sample group was added with the beauty cyclic peptide reagent obtained in Example 1-6 and Comparative Example 1-4 to a final concentration of 400ppm. After continuing to be placed in the incubator for 12 hours, the culture medium was removed, and DMEM culture solution containing 0.2μmol / L α-MSH solution was added to each well of the model group and the sample group. After being placed in the incubator for 24 hours, the cells were lysed, the cell lysis solution was collected, and the operation was completed according to the steps of the cAMP ELISA kit, and the absorbance of each group was measured at 450nm on a microplate reader.

[0137] Figure 5The figure is the result of the effect of the beauty cyclic peptide reagent of the present invention on the cAMP content. The cAMP content of the model group is significantly increased (P < 0.01), proving that the model is successfully established. Compared with the model group, the cAMP content after treatment with the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 is reduced, which shows that the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 of the present invention can reduce the cAMP content induced by α-MSH. The cAMP content of the beauty cyclic peptide reagent obtained in Example 1 is lower than that in Comparative Example 1, because Example 1 uses cyclohexapeptide-2 to prepare the beauty cyclic peptide reagent, and Comparative Example 1 uses linear hexapeptide-2 to prepare the beauty cyclic peptide reagent, which shows that the beauty cyclic peptide reagent obtained in Example 1 is better than Comparative Example 1 in reducing cAMP. The cAMP content of the beauty cyclic peptide reagent obtained in Example 2 is lower than that in Example 1, because gallic acid lecithin complex and naringenin acylhydrazone derivatives are additionally added to the beauty cyclic peptide reagent in Example 2; the cAMP content of the beauty cyclic peptide reagent obtained in Example 2 is lower than that in Examples 3-4, because the beauty cyclic peptide reagents in Examples 3-4 reduce the usage amounts of gallic acid lecithin complex and naringenin acylhydrazone derivatives, respectively; the cAMP content of the beauty cyclic peptide reagent obtained in Example 2 is lower than that in Comparative Examples 2-3, because Comparative Examples 2-3 separately add gallic acid lecithin complex or naringenin acylhydrazone derivatives to prepare the beauty cyclic peptide reagent; this shows that the synergistic addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives to the beauty cyclic peptide reagent makes the beauty cyclic peptide reagent have a better effect of increasing the cAMP content. The cAMP content of the beauty cyclic peptide reagent obtained in Example 5 is lower than that in Example 2, because in the preparation of the beauty cyclic peptide reagent, in addition to the additional addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives, Example 5 also adds rosin pentaerythritol ester; the cAMP content of the beauty cyclic peptide reagent obtained in Example 5 is lower than that in Example 6, because the amount of rosin pentaerythritol ester used is different; the cAMP content of the beauty cyclic peptide reagent obtained in Example 5 is lower than that in Comparative Example 4, because Comparative Example 4 only adds rosin pentaerythritol ester, and does not add gallic acid lecithin complex and naringenin acylhydrazone derivatives; this shows that adding rosin pentaerythritol ester to the beauty cyclic peptide reagent can further reduce the cAMP content induced by α-MSH.

[0138] 6. Inhibition of tyrosinase and melanin in vitro

[0139] Mouse melanoma B16 cells were cultured to the logarithmic growth phase and the concentration of the cell suspension was adjusted to 1×10 5 / mL, 100μL of cell suspension was inoculated in 96-well plates and placed in a 37℃, 5% CO2 incubator for 24h. After 24h of cell culture, blank group, model group and sample group were set up respectively. Except for the blank group, the other groups were placed in 30 J / cm 2Irradiation was performed under UVB light source. After the irradiation, the waste liquid was discarded. 100 μL of DMEM culture medium was added to each well of the blank group, model group and sample group. The beauty cyclic peptide reagent obtained in Example 1-6 and Comparative Example 1-4 was added to the sample group to a final concentration of 400 ppm. After continued cultivation in the incubator for 24 hours, the supernatant was discarded, and the cells were washed 3 times with PBS solution. 100 μL of immunostaining permeabilization solution (1% Triton X-100) was added to each well, frozen at -80°C for 40 minutes, thawed and lysed at room temperature, the cell lysate was centrifuged, 100 μL of supernatant was taken, 100 μL of 0.1% L-dopa solution was added, and the cells were placed at 37°C for 2 hours. The absorbance was measured at a wavelength of 475 nm to calculate the relative activity of tyrosinase. Take the cells after culturing for 24 hours, discard the supernatant, wash three times with PBS solution, add 100 μL of 1.0 mol / L NaOH solution containing 10% DMSO to each well, heat in an 80°C water bath to fully lyse the cells, centrifuge and take the supernatant, measure the absorbance at a wavelength of 405 nm, and calculate the relative content of melanin.

[0140] Figure 6 This is a graph showing the effect of the beauty cyclic peptide reagent of the present invention on the relative activity of tyrosinase. Figure 7The figure is the result of the effect of the beauty cyclic peptide reagent of the present invention on the relative content of melanin. The relative activity of tyrosinase and the relative content of melanin in the model group were significantly increased under UVB induction (P < 0.01), proving that the model was successfully established. Compared with the model group, the relative activity of tyrosinase and the relative content of melanin in the B16 cells treated with the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 were reduced, which shows that the beauty cyclic peptide reagents obtained in Examples 1-6 and Comparative Examples 1-4 of the present invention can inhibit melanin production and the relative activity of tyrosinase. The relative activity of tyrosinase and the relative content of melanin of the beauty cyclic peptide reagent obtained in Example 1 are lower than those in Comparative Example 1, because Example 1 uses cyclohexapeptide-2 to prepare the beauty cyclic peptide reagent, and Comparative Example 1 uses linear hexapeptide-2 to prepare the beauty cyclic peptide reagent, which shows that the beauty cyclic peptide reagent obtained in Example 1 is better than Comparative Example 1 in reducing the relative activity of tyrosinase and the relative content of melanin. The relative tyrosinase activity and the relative melanin content of the beauty cyclic peptide reagent obtained in Example 2 are lower than those in Example 1, because gallic acid lecithin complex and naringenin acylhydrazone derivatives are additionally added to the beauty cyclic peptide reagent in Example 2; the relative tyrosinase activity and the relative melanin content of the beauty cyclic peptide reagent obtained in Example 2 are lower than those in Examples 3-4, because the beauty cyclic peptide reagents in Examples 3-4 reduce the usage amounts of gallic acid lecithin complex and naringenin acylhydrazone derivatives, respectively; the relative tyrosinase activity and the relative melanin content of the beauty cyclic peptide reagent obtained in Example 2 are lower than those in Comparative Examples 2-3, because Comparative Examples 2-3 separately add gallic acid lecithin complex or naringenin acylhydrazone derivatives to prepare the beauty cyclic peptide reagent; this shows that the synergistic addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives to the beauty cyclic peptide reagent makes the beauty cyclic peptide reagent have a better effect in inhibiting the relative activity of tyrosinase and the relative content of melanin. The relative tyrosinase activity and the relative melanin content of the beauty cyclic peptide reagent obtained in Example 5 are lower than those in Example 2, because in the preparation of the beauty cyclic peptide reagent, in addition to the additional addition of gallic acid lecithin complex and naringenin acylhydrazone derivatives, Example 5 also adds rosin pentaerythritol ester; the relative tyrosinase activity and the relative melanin content of the beauty cyclic peptide reagent obtained in Example 5 are lower than those in Example 6, because the amount of rosin pentaerythritol ester used is different; the relative tyrosinase activity and the relative melanin content of the beauty cyclic peptide reagent obtained in Example 5 are lower than those in Comparative Example 4, because Comparative Example 4 only adds rosin pentaerythritol ester, and does not add gallic acid lecithin complex and naringenin acylhydrazone derivatives; this shows that adding rosin pentaerythritol ester to the beauty cyclic peptide reagent can further inhibit melanin production and the relative activity of tyrosinase, thereby achieving the whitening and brightening effect of the skin.

[0141] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0142] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a beauty cyclic peptide agent, comprising: mixing a cyclic peptide, an additive and glycerol to obtain a beauty cyclic peptide agent; the cyclic peptide at least comprises cyclohexapeptide-2; the additive at least comprises a gallic acid lecithin complex and a naringenin acylhydrazone derivative, the gallic acid lecithin complex has a phenol group and a phosphate group, and the naringenin acylhydrazone derivative has a phenol group and a nicotinic acid hydrazide group; The structural formula of the cyclohexapeptide-2 is shown in Formula I: ； The gallic acid lecithin complex is derived by grafting gallic acid onto soybean lecithin; Preparation of gallic acid lecithin complex: add soybean lecithin to anhydrous ethanol, gradually add gallic acid while stirring, slowly heat to 40-60°C, continue stirring and condensing to reflux for 2-4h until the reaction solution is clear, remove ethanol by rotary evaporation at 40-50°C, filter through a non-aqueous filter membrane, vacuum filter and dry the filtrate for 12-36h to obtain gallic acid lecithin complex; the solid-liquid ratio of soybean lecithin and anhydrous ethanol is 1g:10-30mL, and the mass ratio of soybean lecithin and gallic acid is 1:0.2-1; The naringenin acylhydrazone derivative is obtained by reacting naringenin and 6-hydroxynicotinic acid hydrazide; Preparation of naringenin acylhydrazone derivatives: naringenin and 6-hydroxynicotinic acid hydrazide are mixed, ethanol and acetic acid are added, and the mixture is stirred to dissolve, and the mixture is heated at 600-800W for 2-10min, and then recrystallized with methanol, washed, filtered, and dried to obtain naringenin acylhydrazone derivatives, wherein the mass ratio of naringenin to 6-hydroxynicotinic acid hydrazide is 1:1-2, the solid-liquid ratio of naringenin to ethanol is 1g:2-5mL, and the solid-liquid ratio of naringenin to acetic acid is 1g:0.2-0.5mL.

2. The method for preparing a beauty cyclic peptide agent according to claim 1, characterized in that: The mass ratio of the cyclic peptide to the additive is 1:0.002-0.

05.

3. The method for preparing a cosmetic cyclic peptide agent according to claim 1, characterized in that: The usage ratio of the cyclic peptide and glycerol is 1g:2-10mL.

4. The method for preparing a cosmetic cyclic peptide agent according to claim 1, characterized in that: The preparation method of cyclohexapeptide-2 comprises: coupling 2-chlorotrityl chloride (CTC) resin with an amino acid reagent protected by an Fmoc group to obtain a linear peptide resin, cutting the resin to obtain a fully protected linear peptide, and then obtaining cyclohexapeptide-2 through cyclization.

5. A method for preparing a cosmetic cyclic peptide agent according to claim 4, characterized in that: The coupling order of the amino acid reagents protected by the Fmoc group is: Fmoc-Lys(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-His(Trt)-OH.

6. The cosmetic cyclic peptide agent prepared by the method according to any one of claims 1 to 5.

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