Cyclic peptide with functions of promoting collagen synthesis, hair follicle activation and scalp aging resistance and application of cyclic peptide in cosmetics

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a cyclic peptide with functions of promoting collagen synthesis, hair follicle activation and scalp aging resistance and application of the cyclic peptide in cosmetics. According to the invention, a novel cyclic peptide capable of regulating and controlling hair follicle activation and scalp anti-aging related signal channels is found through peptide library screening, and a structurally-similar peptide of the cyclic peptide is synthesized, and the structure of the cyclic peptide is shown as a formula (I) or (II). The cyclic peptide can effectively up-regulate the expression of hair follicle activation marker genes, promote the proliferation of hair follicle dermal papilla cells and enhance the antioxidant capacity, and can also promote the synthesis of collagen, so that the cyclic peptide can be used for preparing cosmetic compositions for preventing and treating alopecia, promoting hair growth and resisting scalp aging. (I) (II)

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a cyclic peptide with the functions of promoting collagen synthesis, activating hair follicles and anti-aging of the scalp and application thereof in cosmetics. BACKGROUND

[0002] The scalp is one of the thinnest skins in the human body, yet it supports tens of thousands of hairs and is rich in hair follicles, sebaceous glands and sweat glands, and its health status directly determines the growth quality and appearance age of the hair. However, compared with the vigorous development of facial skin care, the health management of the scalp, especially systematic anti-aging care, has long been insufficiently valued.

[0003] With the influence of age growth, environmental pressure, hormone changes and bad living habits, the scalp will undergo a series of complex aging processes, mainly manifested as: scalp tissue aging and collagen loss: like facial skin, the dermal layer collagen of the scalp (especially types I and III) will be rapidly lost with age growth, resulting in thinning of the scalp, weakening of elasticity and poor microcirculation. This structural degradation not only leads to relaxation of the scalp, appearance of wrinkles, but also directly compresses and weakens the function of hair follicles, which is recognized as one of the key factors leading to and exacerbating telogen effluvium and hair thinning. Imbalance of hair follicle cycle and insufficient activation: hair follicles have the characteristic of periodic growth. In the aging process, the anagen phase of the hair follicle is shortened and the telogen phase is prolonged, resulting in gradual hair thinning. At the same time, the functional activity of hair papilla cells, hair follicle stem cells and the like decreases, and they cannot effectively start and maintain vigorous hair growth. Lack of efficient and safe active ingredients: most of the active ingredients on the market for improving scalp health and preventing hair loss have limitations. For example, minoxidil and finasteride are drugs, which have certain side effects and population restrictions; and many cosmetic raw materials such as caffeine and plant extracts, although they are relatively safe, often have single efficacy and low transdermal absorption efficiency, and it is difficult to simultaneously and efficiently solve the series of interrelated complex problems of collagen loss, hair follicle atrophy and overall microenvironment aging of the scalp.

[0004] Therefore, it has become an urgent market demand and technical bottleneck in the field of cosmetics to develop an innovative active ingredient that can target the functional unit of the dermis-hair follicle of the scalp, both reversing the aging of the dermis and directly activating the hair follicle.

[0005] In recent years, bioactive peptides, especially cyclic peptides, have shown great potential in high-end cosmetics due to their high stability, high activity and good skin permeability. Compared with linear peptides, the cyclic structure of cyclic peptides can resist the degradation of proteases in the skin, maintain a longer action time, and its three-dimensional spatial structure can more accurately bind to specific targets (such as growth factor receptors, key proteins in signal pathways). SUMMARY

[0006] Based on the above status quo, the present application targets the key pathway of hair follicle activation, i.e. Wnt / β-catenin signaling pathway, designs a LEF / TCF reporter cell system, attempts to find a brand new cyclic peptide molecule from the polypeptide library that has been internally established, which can simultaneously target and regulate multiple key signaling pathways related to collagen synthesis and hair follicle growth, thereby promoting collagen synthesis and up-regulating the expression of the marker genes of hair follicle activation and anti-aging, and evaluates its application prospect in cosmetics.

[0007] Specifically, the present application provides the use of a cyclic peptide as shown in formula (I) or (II) or a pharmaceutically acceptable salt thereof in the preparation of a reagent or composition for promoting collagen synthesis, hair follicle activation and scalp anti-aging:

[0008] (I)

[0009] (II).

[0010] Further, the composition comprises a cosmetic.

[0011] Further, the dosage form of the cosmetic comprises an ointment, a cream, a lotion, an aqueous agent, an oil agent, a gel, a powder, a tablet, a mud agent, a patch, a film agent, an aerosol, a spray, a lyophilized preparation or a nano-preparation.

[0012] Further, the cosmetic is used for anti-aging, prevention and treatment of hair loss, promotion of hair growth or scalp anti-aging.

[0013] As used herein, "amino acid" is referred to herein by its commonly known three-letter abbreviation or by the one-letter abbreviation recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0014] The three-letter or one-letter abbreviations of amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0015] When using three-letter abbreviations, unless preceded by an explicit "L" or "D" or clear from the context in which the abbreviation is used, the amino acid can be in the L- or D- configuration with respect to the alpha-carbon (Ca). For example, "Ala" denotes alanine without specifying the configuration with respect to the alpha-carbon, while "D-Ala" and "L-Ala" denote D-alanine and L-alanine, respectively. When using single-letter abbreviations, unless explicitly stated otherwise, the single-letter abbreviation does not specify the configuration with respect to the alpha-carbon. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxy (C) direction, according to conventional practice.

[0016] As used herein, a pharmaceutically acceptable salt can be, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic, neutral or acidic amino acid, and the like.

[0017] The method for preparing the cyclic peptide described in the present application can adopt the methods commonly used in the prior art, such as the well-known chemical synthesis method, the genetic engineering synthesis method, etc., and specifically for example, but not limited to, synthesizing a linear peptide by a solid phase method and then performing cyclization, and finally removing the solid phase to obtain the cyclic peptide described in the present application, or synthesizing a linear peptide by a solid phase method, removing the solid phase, and then performing cyclization to obtain the cyclic peptide described in the present application.

[0018] Advantages of the present application

[0019] The present application finds a new cyclic peptide capable of targeting the Wnt / β-catenin signaling pathway, a key pathway of hair follicle activation, through peptide library screening, and prepares a structural analog peptide of the cyclic peptide. Function research experiments find that the two peptides have the following technical effects:

[0020] Synergistic effect: simultaneously targeting multiple key signaling pathways related to collagen synthesis and hair follicle growth.

[0021] Multi-effect in one: achieving the triple effects of promoting collagen regeneration to reshape the structure of young scalp, activating the expression of hair follicle activation marker genes, and promoting the proliferation of hair follicle dermal papilla cells to prolong the anagen phase, and enhancing the antioxidant capacity to improve the scalp microenvironment at the molecular level.

[0022] Safe and efficient: as a cosmetic raw material, it has high safety, high permeability and excellent stability. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A synthesis process flowchart of cyclic peptide 1 is shown.

[0024] Figure 2 An HPLC chromatogram of cyclic peptide 1 is shown.

[0025] Figure 3A mass spectrum of cyclic peptide 1 is shown.

[0026] Figure 4 A synthetic process flow chart of cyclic peptide 2 is shown.

[0027] Figure 5 An HPLC chromatogram of cyclic peptide 2 is shown.

[0028] Figure 6 A mass spectrum of cyclic peptide 2 is shown.

[0029] Figure 7 A shows that cyclic peptide 1 and cyclic peptide 2 up-regulate mRNA levels of classic downstream target genes of Wnt / β-catenin pathway.

[0030] Figure 8 A shows that cyclic peptide 1 and cyclic peptide 2 up-regulate the expression of β-catenin and Cyclin D1 proteins; Figure 8 B shows that cyclic peptide 1 and cyclic peptide 2 promote the accumulation of β-catenin in the nucleus.

[0031] Figure 9 A shows that cyclic peptide 1 and cyclic peptide 2 promote the proliferation of hair follicle dermal papilla cells.

[0032] Figure 10 A shows that cyclic peptide 1 and cyclic peptide 2 inhibit the expression of senescence genes under UV irradiation.

[0033] Figure 11 A shows that cyclic peptide 1 and cyclic peptide 2 promote the expression of collagen genes under UV irradiation.

[0034] Figure 12 A shows that cyclic peptide 1 and cyclic peptide 2 promote the expression of AXIN2, LEF1, Ki-67 in hair follicle tissue cultured ex vivo.

[0035] Figure 13 A shows that cyclic peptide 1 and cyclic peptide 2 promote the synthesis of collagen. DETAILED DESCRIPTION

[0036] The application will be further described below in connection with specific embodiments, but the embodiments do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the art.

[0037] Example 1: Screening of polypeptides related to hair follicle activation

[0038] (1) A linear polypeptide library comprising at least 5 x 10 20 polypeptide members was constructed according to the prior art, wherein each member in the peptide library is present in substantially equal amounts and each has an amino acid sequence Kmix - (X) m- Emix or Emix -(X) n - Kmix, Kmix is lysine (50% ratio) or other naturally occurring amino acid, Emix is glutamic acid (50% ratio) or other naturally occurring amino acid, X is naturally occurring amino acid, m and n are each independently an integer from 0 to 20, linear polypeptides in the polypeptide library are subjected to common chemical modifications including acetylation, palmitoylation, myristoylation, biotinylation, etc. by chemical methods, and are subjected to chemical cyclization to obtain a polypeptide library with diverse structures including uncyclized peptides, head-to-tail cyclized peptides, head-to-side cyclized peptides, side-to-tail cyclized peptides, and side chain cyclized peptides. Then, based on a high-throughput molecular interaction screening platform, polypeptides with target affinity are screened from the obtained polypeptide library for cosmetic targets such as whitening, anti-wrinkle, anti-aging, ion channel, anti-inflammatory, antibacterial, hair growth, etc., as candidate polypeptide molecules for functional screening.

[0039] (2) The 8x LEF / TCF response element was cloned into the pGL4.1[luc2P] plasmid, and the reporter plasmid was co-transfected with a plasmid containing a puromycin resistance gene into human hair follicle dermal papilla cells. After 48 hours of transfection, puromycin was added for pressure screening, which lasted for 2-3 weeks until all untransfected control cells died. Single cell clones were selected by limiting dilution and expanded. The LEF / TCF reporter cell line was treated with Wnt3a protein, and luciferase activity was detected. The clone with the highest induction fold and the lowest background was selected as the final reporter cell line for subsequent screening.

[0040] (3) The freeze-dried powder of the candidate polypeptide molecule was resuspended with sterile DMSO to prepare a uniform stock solution concentration (10 mM). The final concentration of DMSO was kept consistent and below 0.1% (v / v) in all experiments to avoid cytotoxicity. The stock solution was diluted with cell culture medium to prepare a working solution concentration (10 µM) for screening.

[0041] (4) The reporter cells were seeded in a 96-well white transparent bottom cell culture plate at a density of 1×10 4 After the cells adhered, different polypeptide working solutions were added to each well using an automated liquid handling workstation, and the following controls were set: positive control, 10 µM Wnt3a protein was added; negative control, the same volume of solvent (culture medium containing 0.1% DMSO) was added. After co-culturing the cells with the polypeptides for 48 hours, the old culture medium was discarded, luciferase detection reagent was added, and after incubation on a shaker in the dark, the luminescence value of each well was read using a chemiluminescence detector.

[0042] (5) The luminescence value data was normalized to the fold change relative to the negative control, and the sample wells with an activation fold greater than 2 times were determined as positive wells.

[0043] Thus, the individual peptides related to hair follicle activation are obtained, and then each hit polypeptide is dose-dependently verified. The final polypeptide capable of promoting the expression of hair follicle activation-related genes is compared with a known cosmetic peptide library, and a novel functional cyclic peptide molecule is found, the structure of which is shown as formula (I) in the specification (named as cyclic peptide 1 in the present application).

[0044] Example 2: Synthesis of a cyclic peptide related to hair follicle activation

[0045] The synthesis process flow chart of cyclic peptide 1 is shown in Figure 1 .

[0046] The detailed experimental process is as follows:

[0047] (1) Preparation of Fmoc-Glu(OAll)-CTC resin

[0048] ① 2-CTC resin 1.030 g (1% DVB, 100-200 mesh, 1.0 mmol / g) was weighed into a 20 mL solid-phase reactor, 10 mL DCM solution was added to the reactor, the shaking speed was set to 550 r / min, and after shaking for 45 min, the solution was drained.

[0049] Fmoc-Glu(OAll)-OH (2 eq, 2.0 mmol) 0.818 g was weighed into a 50 mL beaker, dissolved in DCM solution (5 mL), and then added to the resin, DIEA (3 eq, 1.8 mmol) was added to the resin reaction solution during shaking, and the reaction was carried out at 500 r / min and 25°C for 1 h. Then anhydrous methanol 0.8 mL was directly added to the reaction solution to close the incompletely reacted active sites, and the reaction was carried out at room temperature for 45 min. After the reaction was completed, the resin was washed with DMF solution (10 mL / 3 min / time) for 3 times. Then the resin was washed alternately according to the following procedure: DCM x 5 times (10 mL / 3 min / time), MeOH x 5 times (10 mL / 3 min / time), and finally the resin was in a shrunk state, which was placed in a vacuum drying box and dried at 25°C until the weight was constant. The substitution value was measured by weight gain method to be 0.62 mmol / g.

[0050] ② Deprotection

[0051] The Fmoc-Glu(OAll)-CTC resin obtained in the process ① was added to DCM (10 mL), and the shaking table was set at a speed of 550 r / min. After oscillation for 45 min, the solution was drained. 10 mL of 20% piperidine / DMF solution was added to the resin in the reactor to remove the Fmoc protecting group of the resin, and the reaction was carried out at 500 r / min and 25°C for 5 min. The solution was drained. Then 10 mL of 20% piperidine / DMF solution was added to the resin again, and the reaction was carried out at 500 r / min and 25°C for 15 min. The solution was drained. The resin was washed with DMF solution (10 mL / 3 min / time) for 5 times.

[0052] ③Coupling

[0053] Fmoc-Dab(Boc)-OH (3 eq, 1.86 mmol) 0.845 g, Oxyma (3 eq, 1.86 mmol) 0.264 g were weighed into a 50 mL beaker, dissolved in DMF solution (5 mL), and then added to the amino acid solution. DIC (3 eq, 1.86 mmol) 0.288 mL was added to activate the reaction for 5 min, and then added to the above deprotected resin. The reaction was carried out at 500 r / min and 25°C for 1 h. After the reaction was completed, the resin was washed with DMF solution (10 mL / 3 min / time) for 5 times.

[0054] ④Peptide chain extension

[0055] According to the sequence composition, the subsequent steps ② and ③ were repeated until the coupling of the first amino acid at the N-terminal was completed. The Fmoc protecting group was removed, and the resin was washed with DMF solution (15 mL / 3 min / time) for 5 times.

[0056] ⑤Solid phase ring formation

[0057] Allyl removal solution was added to the resin: tetraphenylphosphonium palladium (0.2 eq, 0.12 mmol) 0.143 g was weighed, dissolved in 10 mL of DCM, and then added to the resin. Then benzene silane (15 eq, 9.3 mmol) 1.15 mL was added dropwise, and the reaction was carried out at room temperature for 30 min to remove the allyl (All) protecting group in the sequence.

[0058] PyBop (3 eq, 1.86 mmol) 0.967 g was weighed, dissolved in DMF 10 mL, and the completely dissolved PyBoP solution was added to the resin, followed by the addition of DIEA (3 eq, 1.86 mmol) 0.307 mL, and the reaction was allowed to proceed at room temperature for 12 h. After the reaction was completed, the resin was washed 5 times with DMF solution (10 mL / 3 min / time). Then the resin was washed alternately according to the following procedure: DCM x 5 times (10 mL / 3 min / time), MeOH x 5 times (10 mL / 3 min / time), and finally the resin was shrunk and placed in a vacuum drying box, and dried at 25°C to constant weight. Finally, the peptide resin 1.238 g was obtained.

[0059] ⑥ Cleavage of the fully protected peptide to obtain the fully protected peptide cyclo(β-Ala-Pro-Dab(Boc)-Glu)-OH

[0060] The dried resin 1.238 g obtained in ⑤ was added to the cleavage solution (1% TFA / DCM) (g 肽树脂 :V 裂解液 =1:10), and the reaction was allowed to proceed at room temperature for 3 h. After the reaction was completed, the product was concentrated to obtain the fully protected peptide cyclo(β-Ala-Pro-Dab(Boc)-Glu)-OH, which was weighed to obtain 0.211 g of yellow oily liquid.

[0061] ⑦ Condensation with benzylamine in liquid phase

[0062] The fully protected peptide fragment obtained in ⑥ was weighed as 0.100 g (0.18 mmol), dissolved in DCM 5 mL, followed by the addition of PyBop (2 eq, 0.36 mmol) 0.0.187 g, DIEA (4 eq, 0.72 mmol) 0.12 mL, and finally benzylamine (5 eq, 0.9 mmol) 98.4 μL, and the reaction was allowed to proceed at room temperature for 12 h. After the reaction was completed, the product was concentrated, dissolved in a small amount of DMF, precipitated in ice water, and finally the fully protected peptide 1 with a protecting group was obtained as a cyclic peptide 0.110 g with a yield of 95.6%.

[0063] ⑧ Cleavage

[0064] Take 0.110 g of the fully protected peptide obtained in the above-mentioned process ⑦, add 10 mL of the freshly prepared and pre-cooled K reagent cleavage solution, and the K reagent cleavage solution is prepared according to the volume ratio of TFA: phenol: water: benzyl thioether: ethanedithiol = 82.5: 5: 5: 5: 2.5 (TFA / phenol / water / benzyl thioether / ethanedithiol = 82.5 / 5 / 5 / 5 / 2.5 (v / v / v / v / v)), and the reaction is carried out at 25°C under 300 r / min oscillation and light shielding for 3 h. After the reaction is completed, the cleavage solution is slowly added to the pre-cooled anhydrous ether solution according to the ratio of cleavage solution / anhydrous ether = 1:10 (v / v), and a white precipitate is generated. Then, centrifugation is carried out at 500 rpm / min, the supernatant is discarded, and new anhydrous ether solution is added, oscillated, centrifuged, and the supernatant is discarded. The above-mentioned centrifugation process is repeated for 5 times, and the muddy white precipitate is collected and vacuum dried at 25°C to constant weight. Finally, 91.2 mg of white solid crude peptide is obtained, and the yield is 94.0 %.

[0065] Process ⑨: preparative HPLC for purification of the peptide

[0066] Take 30.0 mg of the above-mentioned crude product, dissolve in 2 mL of water, and purify by preparative HPLC. The sample purification is completed according to the gradient elution program in Table 1, wherein the mobile phase A is 80% acetonitrile / water (containing 0.1% TFA), the mobile phase B is water (containing 0.1% TFA), the detection wavelength is 220 nm, the flow rate is 10 mL / min, and the column specifications are 20×250 mm, 10 µm, and 120 Å.

[0067] Table 1: Elution program for purification of the crude product

[0068]

[0069] The chromatogram of the cyclic peptide 1 is shown in Figure 2 , the target fractions are combined, and the cyclic peptide 1 is finally obtained by freeze-drying, with a yield of 57.3% and a purity of 98.753%. The mass spectrum is shown in Figure 3 . The MS result shows that [M+H] + = 487.2697, which is correct for the molecular weight.

[0070] According to the structural analysis of the cyclic peptide 1, the NHBzl group has two connection sites on Glu. In order to prove whether the obtained cyclic peptide also has the same function as the cyclic peptide 1 when the NHBzl group is connected to the side chain carboxyl group on Glu, the inventors also synthesized a cyclic peptide 2 having a structure as shown in formula (II) in the specification.

[0071] The process flow chart for synthesis of the cyclic peptide 2 is shown in Figure 4 .

[0072] The detailed experimental process is as follows:

[0073] (1) Preparation of Fmoc-Glu(CTC-Resin)-OAll

[0074] ① Take 2-CTC resin 1.020 g (1% DVB, 100-200 mesh, 1.0 mmol / g) into a 20 mL solid-phase reactor, add 10 mL DCM solution to the reactor, set the shaking speed of the shaking table to 550 r / min, shake for 45 min, and then drain the solution.

[0075] Take Fmoc-Glu-OAll (2 eq, 2.0 mmol) 0.818 g into a 50 mL beaker, dissolve in DCM solution (5 mL), then add to the resin, and add DIEA (3 eq, 1.8 mmol) to the resin reaction solution during shaking, 500 r / min, 25°C, shake for 1 h. Then add anhydrous methanol 0.8 mL directly to the reaction solution to close the incompletely reacted active sites, and shake at room temperature for 45 min. After the reaction is completed, wash the resin with DMF solution (10 mL / 3 min / time) for 3 times. Then wash the resin alternately according to the following procedure: DCM x 5 times (10 mL / 3 min / time), MeOH x 5 times (10 mL / 3 min / time), finally the resin is in a shrunk state, and is placed in a vacuum drying box and dried at 25°C under vacuum to a constant weight. The substitution value is 0.615 mmol / g measured by weight gain method.

[0076] ② Deprotection

[0077] Add the Fmoc-Glu(CTC Resin)-OAll) resin obtained in the process of ① to DCM (10 mL), set the shaking speed of the shaking table to 550 r / min, shake for 45 min, and then drain the solution. Add 10 mL of 20% piperidine / DMF solution to the resin in the reactor to remove the Fmoc protecting group of the resin, 500 r / min, 25°C, shake for 5 min, drain the solution; then add 10 mL of 20% piperidine / DMF solution to the resin again, 500 r / min, 25°C, shake for 15 min, drain the solution. Wash the resin with DMF solution (10 mL / 3 min / time) for 5 times.

[0078] ③ Coupling

[0079] Fmoc-Dab(Boc)-OH (3 eq, 1.67 mmol) 0.782 g, Oxyma (3 eq, 1.67 mmol) 0.237 g into a 50 mL beaker, dissolved with DMF solution (5 mL), activated with condensing agent DIC (3 eq, 1.67 mmol) 0.288 mL into the amino acid solution for 5 min, then added to the above deprotected resin, 500 r / min 25°C oscillation reaction for 1 h; after the reaction was completed, the resin was washed 5 times with DMF solution (10 mL / 3 min / time).

[0080] ④Peptide chain extension

[0081] According to the sequence composition, repeat steps ② and ③ subsequently until the coupling of the first amino acid at the N-terminus is completed. Remove the Fmoc protecting group, wash the resin 5 times with DMF solution (15 mL / 3 min / time).

[0082] ⑤Solid phase ring formation

[0083] Add allyl removal solution to the resin: weigh tetrakis triphenylphosphine palladium (0.2 eq, 0.11 mmol) 0.14 g, dissolve with 10 mL DCM, add to the resin, then add dropwise phenylsilane (15 eq, 8.4 mmol) 1.04 mL, react at room temperature for 30 min, remove the allyl (All) protecting group in the sequence.

[0084] Weigh PyBop (3 eq, 1.67 mmol) 0.900 g, dissolve with 10 mL DMF, add the completely dissolved PyBoP solution to the resin, finally add DIEA (3 eq, 1.67 mmol) 0.276 mL, react at room temperature for 12 h, after the reaction is completed, wash the resin 5 times with DMF solution (10 mL / 3 min / time). Then wash the resin alternately according to the following procedure: DCM x 5 times (10 mL / 3 min / time), MeOH x 5 times (10 mL / 3 min / time), finally the resin is in a shrunk state, placed in a vacuum drying oven, dried at 25°C under vacuum to constant weight, finally obtain the peptide resin 1.210 g.

[0085] ⑥Full protection peptide cleavage to obtain full protection peptide cyclo(β-Ala-Pro-Dab(Boc)-Glu)

[0086] Add the dried resin obtained in ⑤ 1.210 g to the cleavage solution (1% TFA / DCM) (g 肽树脂 :V 裂解液=1:10), room temperature reaction 3 h. After the reaction is completed, concentrated, to obtain the full protection of the peptide cyclo (β-Ala-Pro-Dab(Boc)-Glu) weighing 0.176 g yellow oily liquid.

[0087] ⑦Liquid phase with benzylamine condensation

[0088] The full protection of the peptide fragment obtained in the process of ⑥ 0.100 g (0.18 mmol) was weighed, dissolved in DCM 5 mL, followed by PyBop (2 eq, 0.36 mmol) 0.0.187 g, DIEA (4 eq, 0.72 mmol) 0.12 mL, and finally benzylamine (5 eq, 0.9 mmol) 98.4 μL, room temperature reaction 12 h, after the reaction is completed, concentrated, dissolved in a small amount of DMF, ice water precipitation, finally obtained with the protection group of the cyclic peptide 2 full protection of the peptide 0.106 g, yield 94.7%.

[0089] ⑧cleavage

[0090] The full protection of the peptide obtained in the process of ⑦ above 0.106 g was weighed, 10 mL of the newly prepared pre-cooled K reagent cleavage solution was added, and the K reagent cleavage solution was prepared according to the volume ratio TFA: phenol: water: benzyl thioether: ethanedithiol = 82.5:5:5:5:2.5 (TFA / phenol / water / benzyl thioether / ethanedithiol = 82.5 / 5 / 5 / 5 / 2.5 (v / v / v / v / v)), 300 r / min 25℃ oscillation reaction for 3 h in the dark. After the reaction is completed, according to the cleavage solution / anhydrous ether = 1:10 (v / v) ratio, the cleavage solution is slowly added to the pre-cooled anhydrous ether solution, and a white precipitate is generated. Then centrifuged at 500 rpm / min, discard the supernatant, add new anhydrous ether solution, shake, centrifuge, discard the supernatant, repeat the above centrifugation process 5 times, collect the muddy white precipitate, 25℃ vacuum drying to constant weight, finally obtained white solid crude peptide 89.3 mg, yield 93.2 %.

[0091] ⑨Preparative HPLC for purification of peptide

[0092] The above crude product 30.0 mg was weighed and dissolved in 2 mL of water, and preparative HPLC purification was performed. The sample purification was completed according to the gradient elution program in Table 2, wherein the mobile phase A: 80% acetonitrile / water (containing 0.1% TFA), the mobile phase B: water (containing 0.1% TFA); detection wavelength: 220 nm; flow rate: 10 mL / min; column specifications: 20×250 mm, 10 μm, 120 Å.

[0093] Table 2: Elution program for crude product purification

[0094]

[0095] The chromatogram of cyclopeptide 2 is shown in Figure 5 The target fractions were combined and freeze-dried to obtain cyclopeptide 2 17.5 mg, with a yield of 58.3%, and a purity = 99.138 %. The mass spectrum is shown in Figure 6 The MS result shows: [M+H] + = 487.2702, which is correct for the molecular weight.

[0096] Example 3: Functional verification of two cyclopeptides

[0097] 1. Activation of Wnt / β-catenin pathway and downstream effects at the molecular and cellular levels by two cyclopeptides

[0098] Human hair follicle dermal papilla cells were seeded in 6-well plates. After the cells adhered, the medium was changed to serum-free medium, and different concentrations of cyclopeptide 1 and cyclopeptide 2 (0.1, 1, 10 µM) were added, respectively, and a solvent control group (0.1% DMSO) and a positive control group (100 ng / mL Wnt3a protein) were set. After 24 hours of treatment, total RNA was extracted and reverse transcribed into cDNA. qPCR was used to detect the mRNA levels of the classic downstream target genes of the Wnt / β-catenin pathway, including AXIN2, LEF1, CYCLIN D1 and VERSICAN, and the relative expression was calculated. The results are shown in Figure 7 As can be seen, compared with the control group, the treatment groups of cyclopeptide 1 and cyclopeptide 2 can dose-dependently up-regulate the mRNA expression levels of these target genes, among which cyclopeptide 1 has a more significant up-regulation, and cyclopeptide 2 has a slightly weaker performance but still has an up-regulation effect.

[0099] At the protein level, similarly, the hair follicle dermal papilla cells were treated with cyclopeptide 1 and cyclopeptide 2 (10 µM). Total protein was extracted, and the total amount of non-phosphorylated β-catenin and Cyclin D1 protein was detected by Western Blot. The results are shown in Figure 8 As can be seen, the total amount of β-catenin and Cyclin D1 protein increases. In addition, the nuclear protein of untreated and cyclopeptide 1 and cyclopeptide 2 treated hair follicle dermal papilla cells was extracted to detect the aggregation of β-catenin in the nucleus, and the results are shown in Figure 8 B, it can be seen that the level of β-catenin in the nucleus is significantly higher than that of the control.

[0100] 2. Promotion of hair follicle activation and anti-aging phenotype by two cyclopeptides

[0101] 2.1 Cell proliferation and viability detection

[0102] Hair follicle dermal papilla cells were treated with cyclic peptide 1 and cyclic peptide 2 (10 µM), and cell viability was detected by CCK-8 at 24, 48, and 72 hours after treatment. The results are shown in Figure 9 As can be seen, both cyclic peptide 1 and cyclic peptide 2 can promote the proliferation of hair follicle dermal papilla cells.

[0103] 2.2 Anti-aging phenotype detection

[0104] Human scalp fibroblasts were pretreated with cyclic peptide 1 and cyclic peptide 2 (10 µM), and then irradiated with ultraviolet light to induce aging. The expression of aging markers p16 and p21 was detected by qPCR. The results are shown in Figure 10 As can be seen, both cyclic peptide 1 and cyclic peptide 2 can down-regulate p16 / p21 expression, suggesting that cyclic peptide 1 and cyclic peptide 2 have good anti-aging effects. In addition, the expression of COL1A1, COL3A1, and ELN genes was also detected, and the results are shown in Figure 11 As can be seen, cyclic peptide 1 and cyclic peptide 2 can restore the damage to collagen production caused by ultraviolet irradiation.

[0105] 3. Hair follicle growth evaluation of the two cyclic peptides

[0106] Human hair follicles in the early growth phase were isolated and randomly divided into groups and cultured in air-liquid interface medium. Cyclic peptide 1 or cyclic peptide 2 was added to the medium in the experimental group, and ordinary medium was used in the control group. The medium was replaced every 2 days and photographed for record. The culture was continued for 14 days, and the length of the hair shaft extension was measured. As can be seen, the hair shaft extension rate of the cyclic peptide-treated group was significantly faster than that of the control group. After the culture ended, the hair follicle tissue was collected, RNA was extracted, and the expression of AXIN2, LEF1, and Ki-67 was detected by qPCR. The results are shown in Figure 12 As can be seen, higher expression of AXIN2, LEF1, and Ki-67 was detected in the cyclic peptide 1 and cyclic peptide 2-treated hair follicles.

[0107] 4. Collagen synthesis promotion evaluation of the two cyclic peptides

[0108] Human dermal fibroblasts (HSF) were cultured in 6-well plates, and when the cell density reached 70%-80%, the cells were treated with different concentrations of cyclic peptide 1 and cyclic peptide 2 (0.1, 1, 10 µM). A negative control group (0.1% DMSO) and a positive control group (10 ng / mL TGF-β) were set up. After 48 hours of treatment, the cell culture supernatant was collected. The concentration of collagen in the supernatant was detected using type I and III collagen ELISA kits. The total protein concentration of the cells was also determined by BCA method to standardize the collagen secretion amount. The ELISA results are shown in Figure 13As shown, it can be seen that the treatment groups of cyclic peptide 1 and cyclic peptide 2 can dose-dependently up-regulate the expression level of collagen type I and III compared with the control group, proving that they can promote collagen synthesis.

[0109] Example 4: Study on the application potential of two cyclic peptides in cosmetics

[0110] 1. Formulation stability study

[0111] Formulation stability:

[0112] Emulsion preparation: The water phase containing water, disodium EDTA (mass concentration of 0.1%), glycerol (mass concentration of 5%), carbomer (mass concentration of 0.3%), xanthan gum (mass concentration of 0.2%), and (sucrose stearate + sorbitan stearate + butylene glycol + sorbitol) (mass concentration of 1.5%), and the oil phase containing cetyl stearyl alcohol (mass concentration of 1%), 26# white oil (mass concentration of 5%), dimethicone (mass concentration of 1%), and olive fruit (mass concentration of 2%) were heated to 80°C and stirred to dissolve. At 80°C, the water phase and the oil phase were mixed and homogenized to emulsify at a homogenization speed of 9000 rpm for 3 min. Then, the temperature was slowly reduced to 60°C at a speed of 900-1000 rpm, and potassium hydroxide (mass concentration of 0.1%) was added for neutralization. The temperature was continuously reduced to 45°C, and propylene glycol (mass concentration of 0.5%), phenoxyethanol (mass concentration of 0.5%), fragrance (mass concentration of 0.06%), and cyclic peptide 1 or cyclic peptide 2 (mass concentration of 2%) were added and stirred uniformly. The emulsion was added to a test tube and sealed. The emulsion was stored at 4°C, 25°C, and 40°C, and the physical properties were observed at 0, 1, 2, and 4 weeks. The results showed that there were no significant changes in color, odor, and layering of the three creams after 4 weeks.

[0113] 2. Skin irritation evaluation

[0114] Skin irritation test was performed using commercial reconstructed human epidermis model EpiSkin™. Briefly, cyclopeptide 1 and cyclopeptide 2 were dissolved in Dulbecco's Phosphate Buffered Saline (DPBS) respectively, with mass concentration of 0.5%, 1% and 2% set. 15 μL cyclopeptide solution was applied directly to the surface of epidermis model, while negative control (DPBS solution only) and positive control (DPBS solution containing 5% SDS) were set. Incubate for 42 minutes at 37°C, 5% CO2, immediately rinse the surface of each model repeatedly and gently with plenty of DPBS to completely remove all test substances. Transfer the model to a culture plate containing fresh maintenance medium, return to the incubator and continue to culture for 42 hours. After 42 hours of culture, cell viability was determined using MTT method. Calculate the average net absorbance value of each group (test group, negative control group, positive control group), and calculate the relative cell viability (%) = (test group average OD value / negative control group average OD value) x 100%, if the average relative viability of test substance < 50%, it can be predicted that the substance is "irritating", if the average relative viability of test substance ≥ 50%, it can be predicted that the substance is "non-irritating".

[0115] The results show that the average relative viability of cyclopeptide 1 and cyclopeptide 2 treatment groups are both higher than 50%, indicating that they are non-irritating.

[0116] 3. Transdermal evaluation:

[0117] EpiSkin™ model was cultured in a special medium at 37°C, 5% CO2 for 24 hours. Cyclopeptide 1 and cyclopeptide 2 were dissolved in DPBS respectively, with mass concentration of 2% set. 20 μL cyclopeptide preparation was evenly applied to the surface of EpiSkin™ model. At least 3 biological replicates were set. Incubate in the incubator for 6, 12, 24 hours. After incubation, wash the surface of the model gently but thoroughly with DPBS, then use transparent tape to adhere to the surface of the model and then tear off, repeat 15 times, the remaining EpiSkin™ model tissue itself after tape stripping is homogenized or dissolved, high performance liquid chromatography (HPLC) is used to quantify the cyclopeptide in the obtained sample, and the skin absorption rate = content in active epidermis / total drug amount x 100% is calculated. The results show that cyclopeptide 1 and cyclopeptide 2 both have good skin permeability, with 24-hour skin absorption rate higher than 3%.

[0118] It should be noted that the preferred embodiments of the present application are described in the specification and its drawings only for the purpose of better understanding the present application, and the present application can be implemented in many different forms, and is not limited to the embodiments described in the specification, and these embodiments are not intended to be additional limitations on the content of the present application, and the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive. Furthermore, each of the above technical features continues to be combined with each other, forming various embodiments not listed above, which are all considered to be within the scope of the present application specification; further, for those skilled in the art, the above description can be improved or changed, and all these improvements and changes shall fall within the scope of protection of the claims of the present application.

Claims

1. Use of the cyclic peptide of formula (I) or (II) or a pharmaceutically acceptable salt thereof in the preparation of reagents or compositions for promoting collagen synthesis, hair follicle activation, and scalp anti-aging: (I) (II)。 2. The use according to claim 1, characterized in that, The composition includes cosmetics.

3. The use according to claim 2, characterized in that, The dosage forms of the cosmetics include ointments, creams, emulsions, liquids, oils, gels, powders, tablets, muds, patches, films, aerosols, sprays, freeze-dried preparations, or nano-preparations.

4. The use according to claim 2, characterized in that, The cosmetics are used for anti-aging, prevention and treatment of hair loss, promotion of hair growth, or anti-aging of the scalp.

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