New use of peptide derivatives in the preparation of a composition for skin repair and firming

Abstract

New use of a peptide derivative of general formula (I) or a salt thereof in the preparation of a composition for skin repair and firming, said skin repair and firming comprising one or more of enhancing the activity of fibroblasts, promoting keratinocyte proliferation and migration, repairing photo-damage, repairing the skin barrier, increasing skin elasticity or enhancing skin firmness.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive polypeptides, and particularly relates to a new use of a peptide derivative in the preparation of a composition for skin repair and firming. Background Art

[0002] The skin is the largest and most important organ of the human body, covering the entire surface of the human body, and is composed of the epidermis, dermis and subcutaneous tissue.

[0003] Keratinocytes are the main constituent cells of the epidermis. According to different differentiation stages and characteristics, keratinocytes are, from the inside to the outside, the basal layer, spinous layer, granular layer, lucid layer and stratum corneum. The outermost stratum corneum is generally composed of 5 to 20 layers of dead flattened cells. These cells have no nucleus or other cell structures, and the cells are filled with a complex composed of keratin and amorphous matrix, becoming relatively tough, thus forming a solid skin barrier on the skin surface.

[0004] Fibroblasts in the dermis can promote the synthesis of collagen. Collagen molecules cross each other to form fibers, constituting a fine collagen fiber network in the skin, increasing the connection strength and stability of the skin. Collagen is an important component of human skin, and it has a huge water-locking function, directly determining the moisture content, smoothness, firmness and skin age of the skin.

[0005] With the increase of age, long-term exposure to the sun or ultraviolet radiation, the proliferation rate and differentiation of keratinocytes change, the expression of molecules involved in the adhesion of basal layer cells, such as integrin or keratinocyte adhesion molecule, decreases, and in addition, the skin barrier function also weakens. The composition of the extracellular matrix in the dermis also undergoes degenerative changes with the aging process, including a large loss of collagen, and at the same time, the collagen synthesized by fibroblasts also decreases, which leads to the hardening and breaking of various collagen peptide bonds that support the skin, resulting in the loosening of the reticular structure in the dermis layer of the skin, and then being damaged, collapsing, reducing the moisture retention and toughness of the skin tissue, and the skin immediately appears dry, wrinkled, loose and other problems.

[0006] With the development of the economy and the improvement of living standards, people pay more and more attention to skin care. The anti-aging and repair skin care market is huge, and more active substances are needed to be applied to products for skin repair and firming to meet the needs of consumers. Summary of the Invention

[0007] Through a large number of experimental studies, the inventor of the present invention found that a peptide derivative has the effect of repairing and firming the skin, and thus completed the present invention.

[0008] Therefore, the present invention aims to provide a new use of a peptide derivative or a salt thereof in the preparation of a composition for skin repair and firming. The skin repair and firming include one or more of enhancing the activity of fibroblasts, promoting the proliferation and migration of keratinocytes, repairing photo-damage, repairing the skin barrier, increasing skin elasticity or improving skin firmness.

[0009] The peptide derivative or a salt thereof represented by the general formula (I),

[0010]

[0011] In the general formula (I),

[0012] X represents a carbon bond or NH-CH(C=O)-(CH2) 3+n -NH-R5,

[0013] n represents 0, 1 or 2,

[0014] R1, R4 and R5 independently of one another represent hydrogen, substituted or unsubstituted C1-C 24 alkyl, substituted or unsubstituted C2-C 24 alkenyl, amidino or tetra-C1-C6-alkyl amidinium,

[0015] R2 represents hydrogen, substituted or unsubstituted C1-C 24 alkyl,

[0016] or

[0017] R1 and R4 together with the residues to which they are attached represent a 5- to 7-membered saturated ring,

[0018] R3 represents C1-C 12 -alkoxy, C1-C 12 -alkylamino, substituted or unsubstituted aryl-C1-C6-alkylamino, substituted or unsubstituted heteroaryl-C1-C6-alkylamino, substituted or unsubstituted aryl-C1-C6-alkoxy, substituted or unsubstituted heteroaryl-C1-C6-alkoxy, and

[0019] R6 represents hydrogen, or when n is 1, also represents amino, or together with R1 and the residues to which R6 and R1 are attached represents a 5- to 7-membered saturated ring,

[0020] R7 represents H, F, Cl, Br, I, -OH, -OR8 or R9-CO-O-,

[0021] R8 and R9 independently of one another represent substituted or unsubstituted C1-C 24 alkyl, substituted or unsubstituted C2-C 24 alkenyl.

[0022] The term "alkyl" refers to a saturated aliphatic straight-chain or branched-chain alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1, 2, 3, 4, 5, or 6 carbon atoms); optionally selected from: methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl.

[0023] The term "alkenyl" refers to a straight-chain or branched-chain alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms, optionally having 2, 3, 4, 5, or 6 carbon atoms), said "alkenyl" having one or more carbon-carbon double bonds, optionally having 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; said "alkenyl" is bonded to the rest of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl.

[0024] As understood in the art, there can be a certain degree of substitution in the above groups. Thus, there can be substitution in any group in the present invention. The substituted groups mentioned in this document in the groups of the present invention indicate that the specified group can be substituted by one or more substituents at one or more available positions, preferably at 1, 2, or 3 positions, more preferably at 1 or 2 positions, still more preferably at 1 position. These substituents include, for example and without limitation: C1-C4 alkyl; hydroxy; C1-C4 alkoxy; amino; C1-C4 aminoalkyl: C1-C4 carbonyloxy; C1-C4 oxycarbonyl; halogen (such as fluorine, chlorine, bromine, and iodine); cyano; nitro; azide; C1-C4 alkylsulfonyl; mercapto; C1-C4 alkylthio; C6-C 30 aryloxy such as phenoxy; -NR b (C=NR b )NR b R c , where R b and R c are independently selected from: H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C 10 cycloalkyl, C6-C 18 aryl, C7-C 17 aralkyl, a heterocyclic group having three to ten members, or a protecting group for an amino group.

[0025] Optionally, R1 represents H.

[0026] Optionally, R2 represents H or methyl.

[0027] Optionally, R3 represents aryl-C1-C6-alkylamino.

[0028] Optionally, R7 represents H or -OH.

[0029] The peptide derivative represented by the general formula (I) or a salt thereof is selected from:

[0030] (1) H-Ala-Hyp-Arg-Arg-NH-benzyl;

[0031] (2) H-(β-Ala)-Hyp-Dab-NH-benzyl;

[0032] (3) H-Dap-Hyp-Dab-NH-benzyl;

[0033] (4) H-Ala-Hyp-Arg-NH-(CH2)2-phenyl;

[0034] (5) H-Ala-Pro-Arg-Arg-NH-benzyl;

[0035] (6) H-(β-Ala)-Pro-Dab-NH-benzyl;

[0036] (7) H-Dap-Pro-Dab-NH-benzyl;

[0037] (8) H-Ala-Pro-Arg-NH-(CH2)2-phenyl.

[0038] The amino acid abbreviations used in the present invention follow the rules specified by the Commission on Biochemical Nomenclature of IUPAC-IUB in Eur J Biochem. (1984) 138: 9-37 and J Chem (1989) 264: 633-673.

[0039] Ala refers to alanine, β-Ala refers to β-alanine, Pro refers to proline, Hyp refers to hydroxyproline, Arg refers to arginine, Dap refers to 2,3-diaminopropionic acid, and Dab refers to 2,4-diaminobutyric acid.

[0040] These peptides of the present invention can exist as stereoisomers or mixtures of stereoisomers; for example, these amino acids containing them can have L-, D-configurations, or can be racemic independently of each other. Thus, it is possible to obtain isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereoisomers or enantiomers, depending on the number of asymmetric carbons and what isomeric or isomeric mixtures are present. The preferred structures of these peptides of the present invention are pure isomers, i.e., enantiomers or diastereoisomers.

[0041] Optionally, the composition contains a peptide derivative represented by the general formula (I) or a salt thereof at a mass percentage concentration of 0.0001% - 5%;

[0042] Optionally, the composition contains a peptide derivative represented by the general formula (I) or a salt thereof at a mass percentage concentration of 0.0005% - 1%;

[0043] Optionally, the composition contains a peptide derivative represented by the general formula (I) or a salt thereof at a mass percentage concentration of 0.001% - 0.1%;

[0044] Optionally, the composition contains a peptide derivative represented by the general formula (I) or a salt thereof at a mass percentage concentration of 0.005% - 0.01%.

[0045] The peptide derivative represented by the above general formula (I) or a salt thereof can improve the activity of fibroblasts, promote the proliferation and migration of keratinocytes, repair photo-damage, repair the skin barrier, increase skin elasticity or improve skin firmness.

[0046] The salts of the peptide derivative represented by the above general formula (I) include metal salts of the peptide derivative represented by the general formula (I), and the metals include: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;

[0047] Optionally, the salts of the peptide derivative represented by the above general formula (I) include salts formed by the peptide derivative represented by the general formula (I) and an organic base, and the organic bases include: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;

[0048] Optionally, the salts of the peptide derivative represented by the above general formula (I) include salts formed by the peptide derivative represented by the general formula (I) and an inorganic acid or an organic acid, and the organic acids include: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid;

[0049] Optionally, the inorganic acids include: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0050] The peptide derivative represented by the above general formula (I) or a salt thereof can be completely formed into the compound of the general formula (I) by methods known per se in the field of peptide chemistry, such as solid-phase synthesis method, liquid-phase synthesis method or a method combining solid phase and liquid phase, and can also be prepared by a biotechnological method aimed at generating the desired sequence, or by controlled hydrolysis of proteins of animal, fungal or plant origin.

[0051] For example, a method for obtaining a peptide derivative having the general formula (I) includes the following steps:

[0052] -Couple an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a protected or solid support-bound C-terminus;

[0053] -Remove the group protecting the N-terminus;

[0054] -Repeat the coupling sequence and the removal of the group protecting the N-terminus until the desired peptide sequence is obtained;

[0055] -Remove the group protecting the C-terminus or cleave from the solid support.

[0056] Preferably, the C-terminus is bound to a solid support and the method is carried out on solid phase, including coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus bound to a polymeric support; removing the group protecting the N-terminus; and repeating this sequence the required number of times so as to thereby obtain a peptide of the desired length, followed by cleaving the synthesized peptide from the initial polymeric support.

[0057] During the entire synthesis, the functional groups of the side chains of these amino acids are kept sufficiently protected with temporary or permanent protecting groups and can be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric support.

[0058] The method may include the following additional steps: deprotecting the N-terminus and C-terminus and / or cleaving the peptide from the polymeric support in a non-determined order using standard conditions and methods known in the art, and subsequently the functional groups of said termini may be modified. Optional modifications of the N-terminus and C-terminus may be carried out on the peptide of general formula (I) bound to the polymeric support, or optional modifications of the N-terminus and C-terminus may be carried out after the peptide has been cleaved from the polymeric support.

[0059] The peptide derivative or its salt of the above general formula (I) may be incorporated into a cosmetically or pharmaceutically acceptable delivery system or sustained release system.

[0060] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier administered together with the peptide derivative of the present invention, which are selected from: water, oil or surfactant, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyethylene oxides, polyethylene glycols, dextrans, glycerol, digitonin and the like. Those of ordinary skill in the art know the diluents that can be used in different delivery systems in which the peptide derivative of the present invention can be administered.

[0061] The term "sustained release" is used in its conventional meaning to refer to a delivery system of a compound that provides for the gradual release of the compound over a period of time, and preferably, but not necessarily, has a relatively constant level of compound release over the entire period.

[0062] Examples of delivery systems or sustained release systems are liposomes, oleosomes, nonionic surfactant vesicles, ethosomes, millimeter capsules, micron capsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, lipovesicles, micelles, millimeter spheres, micron spheres, nanospheres, lipid spheres, micron emulsions, nanoemulsions, millimeter particles, micron particles or nanoparticles. Preferred delivery systems or sustained release systems are liposomes and micron emulsions, and more preferably water-in-oil micron emulsions having an internal structure of reverse micelles.

[0063] Sustained release systems can be prepared by methods known in the prior art and can be administered, for example, by topical or transdermal administration, including adhesive patches, non-adhesive patches, occlusive patches, and microelectronic patches; or by systemic administration such as and not limited to, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into a specific body site, and preferably should release a relatively constant amount of these peptide derivatives of the present invention. The amount of peptide derivative contained in the sustained release system will depend, for example, on the site to which the composition will be administered, the release kinetics and duration of the peptide derivative of the present invention, and the nature of the condition, disorder and / or disease to be treated and / or cared for.

[0064] The peptide derivative represented by the above general formula (I) or a salt thereof can also be adsorbed on a solid organic polymer or a solid inorganic support, such as and not limited to, talc, bentonite, silica, starch, or maltodextrin, etc.

[0065] The composition according to the present invention is a cosmetic composition or a pharmaceutical composition.

[0066] The composition is present in a formulation selected from: creams, oils, milks, balms, foams, lotions, gels, liniments, slurries, soaps, shampoos, conditioners, serums, ointments, mousses, hair oils, powders, sticks, pens, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, jellies or gelatin.

[0067] The composition according to the present invention further comprises at least one other active agent for enhancing the effect of skin repair and firming according to the present invention, and the other active agent is selected from peptides, natural plant ingredients, vitamin C and its derivatives or retinoids.

[0068] The beneficial effects achieved by the present invention compared with the prior art include:

[0069] The peptide derivative represented by the general formula (I) of the present invention or a salt thereof can improve the activity of fibroblasts, promote the proliferation and migration of keratinocytes, repair photo-damage, repair the skin barrier, increase skin elasticity, and improve skin firmness, thus having a good skin repair and firming effect and being applicable to skin repair and firming products. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is a diagram showing the effect of peptide (2) and peptide (6) on the proliferation of NIH3T3 cells. * indicates that there is a statistical difference between the administration group and the blank control group, p < 0.05 (n = 4). ** indicates that there is a significant difference between the administration group and the blank control group, p < 0.01 (n = 4). *** indicates that there is an extremely significant difference between the administration group and the blank control group, p < 0.001 (n = 4).

[0071] Figure 2 It is a diagram showing the effect of peptide (2) and peptide (6) on the proliferation of HaCaT cells. * indicates that there is a statistical difference between the administration group and the blank control group, p < 0.05 (n = 4). ** indicates that there is a significant difference between the administration group and the blank control group, p < 0.01 (n = 4). *** indicates that there is an extremely significant difference between the administration group and the blank control group, p < 0.001 (n = 4).

[0072] Figure 3 It is a diagram showing the effect of 100 ppm of peptide (1) - peptide (6) on the proliferation of NIH3T3 cells. *** indicates that there is an extremely significant difference between the administration group and the blank control group, p < 0.001 (n = 4).

[0073] Figure 4 It is a diagram showing the effect of 100 ppm of peptide (1) - peptide (6) on the proliferation of HaCaT cells. * indicates that there is a statistical difference between the administration group and the blank control group, p < 0.05 (n = 4). *** indicates that there is an extremely significant difference between the administration group and the blank control group, p < 0.001 (n = 4).

[0074] Figure 5 It is a diagram showing the results of the scratch assay of HaCaT cells observed under a 100-fold microscope.

[0075] Figure 6 It is a diagram showing the repair effect of peptide (2) and peptide (6) on photo-damaged HaCaT cells. ## indicates a significant difference compared with the non-irradiated normal control group, p < 0.01 (n = 4). * indicates a statistical difference compared with the control group after irradiation, p < 0.05 (n = 4). ** indicates a significant difference compared with the control group after irradiation, p < 0.01 (n = 4).

[0076] Figure 7It is the diagram showing the effects of peptide (2) and peptide (6) on the adhesion of HaCaT cells. * indicates that there is a statistical difference between the administration group and the blank control group, p < 0.05 (n = 4). *** indicates that there is a highly significant difference between the administration group and the blank control group, p < 0.001 (n = 4).

[0077] Figure 8 It is the diagram showing the effects of peptide (2) and peptide (6) on the contraction of collagen lattice. A: The diagram of the contraction trend of collagen area at 24 h; B: The statistical chart of the difference in the contraction of collagen area at 24 h. * indicates that there is a statistical difference between the administration group and the blank control group, p < 0.05. ** indicates that there is a significant difference between the administration group and the blank control group, p < 0.01.

[0078] Figure 9 It is the morphological diagram showing the promotion of collagen lattice contraction by peptide (2) and peptide (6).

[0079] Figure 10 It is the diagram of the results of the transdermal water loss test.

[0080] Figure 11 It is the diagram of the results of the skin elasticity test. Specific implementation manners

[0081] To better understand the present invention, the invention will be described in detail below in combination with embodiments and drawings. However, it should be understood that these embodiments and drawings are only for illustrative purposes and are not intended to limit the scope of the present invention.

[0082] Example I Cell proliferation experiment

[0083] 1.1 Reagents and materials

[0084] Fetal bovine serum (Gibco), DMEM medium (Gibco), penicillin, streptomycin, MTT (Sigma).

[0085] 1.2 Instruments

[0086] Microplate reader (MD, USA), CO2 incubator (Yiheng, Shanghai), laminar flow hood (Jinghua, Suzhou).

[0087] 1.3 Cell lines

[0088] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences' Committee for the Preservation of Type Cultures. Mouse skin fibroblasts (NIH3T3) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences' Committee for the Preservation of Type Cultures.

[0089] 1.4 Samples to be tested

[0090] Drug administration group: 12.5 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl, 25 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl, 50 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl, 100 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl; 12.5 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl, 25 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl, 50 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl, 100 ppm of peptide (6)H-β-Ala)-Pro-Dab-NH-benzyl.

[0091] 100 ppm of peptide (1)H-Ala-Hyp-Arg-Arg-NH-benzyl; 100 ppm of peptide (3)H-Dap-Hyp-Dab-NH-benzyl; 100 ppm of peptide (4)H-Ala-Hyp-Arg-NH-(CH2)2-phenyl; 100 ppm of peptide (5)H-Ala-Pro-Arg-Arg-NH-benzyl; 100 ppm of peptide (7)H-Dap-Pro-Dab-NH-benzyl; 100 ppm of peptide (8)H-Ala-Pro-Arg-NH-(CH2)2-phenyl.

[0092] Control group: PBS blank control.

[0093] 1.5 Experimental purpose

[0094] The purpose of this experiment is to evaluate the cell viability 72 h after drug administration and determine the effect of the peptide derivative of the present invention on cell proliferation, using NIH3T3 fibroblasts and HaCaT keratinocytes as experimental subjects.

[0095] 1.6 Experimental method

[0096] Take cryopreserved NIH3T3 and HaCaT cells for culture, passage them at a ratio of 1:2 to about 5 generations, and select cells with good growth as experimental subjects.

[0097] Inoculate 2000 cells per well in a 96-well plate. After the cells adhere, add the samples of the drug administration group and the control group respectively according to the serial dilution method, supplement the medium to 200 μL, and incubate in an incubator at 37 °C and 5% CO2 for 72 h.

[0098] Subsequently, add 22 μL of 5 mg / ml MTT to each well and continue to incubate in an incubator at 37 °C with 5% CO2 for 4 h. Discard the original solution and add 150 μL / well of DMSO. After 5 min, use a microplate reader to read the reference OD values at wavelengths of 490 nm and 630 nm.

[0099] 1.7 Results

[0100] The MTT method is a method for detecting cell survival and growth, and the measured OD value is proportional to cell activity.

[0101] Figure 1 Shown are the detection results of the effects of peptide (2) and peptide (6) on NIH3T3 cells. The results show that, compared with the normal control group, peptides (2) and (6) at each dose can increase the activity of NIH3T3 cells, and show a dose-dependent manner.

[0102] Figure 2 Shown are the detection results of the effects of peptide (2) and peptide (6) on HaCaT cells. The results show that, compared with the normal control group, peptides (2) and (6) at each dose can increase the activity of HaCaT cells, and show a dose-dependent manner.

[0103] Figure 3 Shown are the detection results of the effects of peptides (1) - (6) at 100 ppm on NIH3T3 cells. The results show that at a concentration of 100 ppm, peptides (1) - (6) have no toxic effect on NIH3T3 cells, but can also increase their cell activity.

[0104] Figure 4 Shown are the detection results of the effects of peptides (1) - (6) at 100 ppm on HaCaT cells. The results show that at a concentration of 100 ppm, peptides (1) - (6) have no toxic effect on HaCaT cells, but can also increase their cell activity.

[0105] In summary, the peptide derivatives of the present invention can increase the activity of NIH3T3 fibroblasts and HaCaT keratinocytes, and promote their proliferation, indicating that the peptide derivatives of the present invention have a skin repair effect.

[0106] Example 2 Scratch Assay

[0107] 2.1 Reagents and Materials

[0108] Fetal bovine serum (Gibco), DMEM medium (Gibco), penicillin, streptomycin.

[0109] 2.2 Instruments

[0110] Optical microscope, CO2 incubator.

[0111] 2.3 Cell Lines

[0112] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences.

[0113] 2.4 Samples to be tested

[0114] Drug administration group: 100 ppm of peptide (2) H-(β-Ala)-Hyp-Dab-NH-benzyl, 100 ppm of peptide (6) H-(β-Ala)-Pro-Dab-NH-benzyl, 50 U / mL of EGF.

[0115] Control group: PBS blank control.

[0116] 2.5 Experimental purpose

[0117] The purpose of this experiment is to observe the cell migration under an optical microscope 24 hours after drug administration by performing a scratch experiment on confluent HaCaT cells in a culture dish, so as to determine whether the peptide of the present invention can promote cell proliferation and migration.

[0118] 2.6 Experimental method

[0119] Take the cryopreserved HaCaT human stratum corneum cells for culture, passage them at a ratio of 1:2 to about 5 generations, and select the cells with better growth as the experimental subjects. Inoculate the cells into a 12-well plate at a density of 200,000 cells / well, with 2 mL of cell suspension in each well. After the cells adhere and grow confluently, change to a medium containing 0.5% - 1% fetal bovine serum to maintain the cells for 24 hours to synchronize them. Use a sterilized pipette tip to make a scratch, wash off the detached cells with PBS, add the sample to be tested, and continue to incubate in an incubator at 37°C and 5% CO2. After 24 hours, place it under an optical microscope to analyze the wound healing area.

[0120] 2.7 Results

[0121] The scratch migration experiment of cells can reflect the cell proliferation and repair ability. The results are as Figure 5 shown. The cell scratch in the normal control group is still obvious, and the scratch spacing is normal; compared with the control group, the cell proliferation and migration in the EGF positive group are obvious, the spacing basically disappears, and they are connected into one piece; while the scratch spacing of peptide (2) and peptide (6) is shorter than that of the cell scratch in the normal control group, and cell migration trajectories appear. From this, it can be seen that the peptide derivative of the present invention can promote the proliferation and migration of HaCaT stratum corneum cells and has a skin repair effect.

[0122] Example 3 Photodamage repair experiment

[0123] 3.1 Reagents and materials

[0124] Fetal bovine serum (Gibco), DMEM medium (Gibco), penicillin, streptomycin, MTT (Sigma).

[0125] 3.2 Instruments

[0126] Microplate reader (MD, USA), CO2 incubator (Yiheng, Shanghai), laminar flow hood (Jinghua, Suzhou).

[0127] 3.3 Cell line

[0128] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences.

[0129] 3.4 Samples to be tested

[0130] Peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl at 12.5 ppm, 25 ppm, 50 ppm, 100 ppm; peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl at 12.5 ppm, 25 ppm, 50 ppm, 100 ppm. The above samples were dissolved in PBS.

[0131] 3.5 Experimental purpose

[0132] The purpose of this experiment was to determine the repair ability of the peptides of the present invention on photo-damaged cells by establishing a UVB photo-damaged cell model, administering the drug 24 h after ultraviolet radiation, and using the MTT method to detect cell proliferation activity.

[0133] 3.6 Experimental method

[0134] Take cryopreserved HaCaT cells for culture, passage them at a ratio of 1:2 to about 5 generations, and select cells with good growth as experimental subjects.

[0135] Inoculate the cells at 10,000 cells / well into a 96-well plate. When the cells grow to about 80%, establish a UVB photo-damage model. Add 50 μL of medium and do not irradiate with UVB as the blank control group; after adding an appropriate amount of PBS to the experimental group and washing repeatedly until colorless, add 50 μL of PBS, and irradiate with 80 J / cm 3Irradiate under a UVB lamp with a distance of 15 cm between the light source and the culture flask. After irradiation of the experimental group, discard the PBS, add the culture medium and serially diluted drugs to 200 μL, and continue to incubate in an incubator at 37 °C and 5% CO2 for 24 h.

[0136] After 24 h, add 22 μL of 5 mg / mL MTT to each well, incubate in the incubator for 4 h, then discard the original solution and add 150 μL / well of DMSO. After 5 min, use a microplate reader to read the reference OD values at wavelengths of 490 nm and 630 nm.

[0137] 3.7 Results

[0138] The results of cell photodamage repair are shown in Figure 6 . The results showed that compared with the non-irradiated normal control group, the cell viability was significantly decreased after UVB irradiation, indicating that the photodamage model was successfully established. After administration, peptides (2) and (6) at each dose could significantly increase the viability of photodamaged HaCaT cells. This indicates that the peptide derivatives of the present invention can repair photodamaged cells, improve their viability, and have good skin repair ability.

[0139] Example 4 Test for promoting cell adhesion

[0140] 4.1 Reagents and materials

[0141] Fetal bovine serum (Gibco), DMEM medium (Gibco), penicillin, streptomycin, MTT (Sigma).

[0142] 4.2 Instruments

[0143] Microplate reader (MD, USA), CO2 incubator (Yiheng, Shanghai), laminar flow hood (Suzhou Jinghua).

[0144] 4.3 Cell line

[0145] Human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences' Committee for the Preservation of Type Cultures.

[0146] 4.4 Test samples

[0147] Drug administration group: 12.5 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl, 25 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl, 50 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl, 100 ppm of peptide (2)H-(β-Ala)-Hyp-Dab-NH-benzyl; 12.5 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl, 25 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl, 50 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl, 100 ppm of peptide (6)H-(β-Ala)-Pro-Dab-NH-benzyl. The above samples were all dissolved in PBS.

[0148] Control group: PBS blank control.

[0149] 4.5 Experimental purpose

[0150] The purpose of this experiment is to evaluate the effect of the drug on cell adhesion by selecting HaCaT keratinocytes, plating them in a 96-well plate coated with the drug, incubating and applying an external force for a period of time, so as to determine whether the peptide of the present invention can improve cell elasticity.

[0151] 4.6 Experimental method

[0152] Take the cryopreserved HaCaT human keratinocytes for culture, passage them at a ratio of 1:2 to about 5 generations, and select the cells with better growth as the experimental subjects.

[0153] Add the samples to be tested into a 96-well plate at 20 μL / well and dry them overnight in a constant temperature oven at 37°C. On the next day, digest the HaCaT cells with better growth, seed them at a density of 10,000 HaCaT cells / well, and supplement the medium to 200 μL. Incubate them in a 37°C, 5% CO2 incubator for 3 h. After the incubation, take out the culture plate and continue to supplement the medium until the liquid level just overflows, seal it with a sealing film to ensure there are no bubbles. Flip it clockwise for 20 min. Discard the original medium, add 90 μL of fresh medium and 10 μL of 5 mg / mL MTT to each well, and incubate them in a 37°C, 5% CO2 incubator for 3 h. Discard the solution and add 150 μL of DMSO. Use an enzyme-linked immunosorbent assay (ELISA) reader to read the reference OD values at wavelengths of 490 nm and 630 nm.

[0154] 4.7 Results

[0155] After the action of three-dimensional force, the cells with strong adhesiveness can remain on the 96-well plate. By performing MTT quantitative analysis on the live cells on the plate, the adhesion of the cells can be reflected. The more live cells remain on the 96-well plate, the larger the measured OD value, indicating stronger cell adhesion.

[0156] The experimental results are as Figure 7 shown. Compared with the normal control group, both peptide (2) and peptide (6) can improve the adhesion ability of HaCaT cells, thus being beneficial to improving their elasticity.

[0157] Example 5 Collagen Lattice Test Experiment

[0158] 5.1 Reagents and Materials

[0159] Fetal bovine serum (Gibco), DMEM medium (Gibco), penicillin, streptomycin.

[0160] 5.2 Instruments

[0161] CO2 incubator (Shanghai Yiheng).

[0162] 5.3 Cell Lines

[0163] Mouse skin fibroblasts (NIH3T3) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection.

[0164] 5.4 Samples to be Tested

[0165] Peptide (2) H-(β-Ala)-Hyp-Dab-NH-benzyl at 100 ppm, peptide (6) H-(β-Ala)-Pro-Dab-NH-benzyl at 100 ppm, EGF at 50 U / mL.

[0166] 5.5 Experimental Purpose

[0167] The purpose of this experiment is to use the collagen lattice experiment to test the effect of drugs on cell density in order to evaluate their ability to tighten the skin.

[0168] 5.6 Experimental Method

[0169] Take the cryopreserved NIH3T cells and passage them at a ratio of 1:2 to about 5 generations, and select the cells with good growth as the experimental subjects.

[0170] Coat the following drugs 12 hours in advance: peptide (2) at 100 ppm, peptide (6) at 100 ppm, and EGF at 50 U / mL. The group without coated drugs is used as the blank control group.

[0171] Prepare 1.76× lattice medium: Dissolve 0.38 g of NaHCO3 in 10 mL of ultrapure water, add 17.6 mL of 10× DMEM to 100 mL. Place a 15 mL sterile plastic tube on ice, add pre-cooled reagents, and add samples strictly in the following order: ① 0.92 mL of 1.76× lattice medium, ② 0.2 mL of 3% collagen stock solution, ③ 0.04 mL of 0.1 M NaOH, ④ 0.19 mL of fetal bovine serum, ⑤ 0.67 mL of cell suspension (about 10,000 cells). After completing the sample addition, invert and mix well to avoid generating bubbles. Transfer the mixed medium solution to a 12-well plate culture dish and incubate it in an incubator at 37°C and 5% CO2 for 24 h. Take pictures and record at different time points.

[0172] 5.7 Results

[0173] The aggregation of collagen lattice is actually the result of the interaction between the cytoplasmic matrices, and the lattice shrinkage rate of the collagen lattice experiment is proportional to the cell density. In this experiment, the area of collagen aggregation was measured by a semi-quantitative method to reflect the degree of collagen contraction.

[0174] The results are as Figure 8 、 9 shown. The results show that after the action of peptide (2), peptide (6) and EGF of the present invention, the area of the collagen lattice is significantly reduced. Thus, it is indicated that the peptide derivatives of the present invention can increase the compactness of NIH3T3 fibroblasts and have the effect of tightening the skin.

[0175] Example 6 Preparation of Liposomes Containing Peptide (2)

[0176]

[0177] Weigh dipalmitoyl phosphatidylcholine and dissolve it in chloroform. Evaporate the solvent under vacuum until a thin layer of phospholipid is obtained. Hydrate this layer with an aqueous solution of the peptide at the required concentration at 55°C to obtain multilamellar liposomes. Subject the multilamellar liposomes to high-pressure homogenization treatment to obtain unilamellar liposomes with smaller and more uniform sizes.

[0178] Example 7 Preparation of a Micron Emulsion Composition Containing Peptide (6)

[0179]

[0180] Weigh the components of phase B according to the prescription dosage and add them to a container. Then, add phase D to phase B and homogenize under continuous stirring. Subsequently, add phase A to the mixture. Finally, add phase C to obtain a micron emulsion composition containing peptide (6).

[0181] Example 8 Preparation of a Serum Containing Peptide (2)

[0182]

[0183]

[0184] Add the prescribed amount of sodium hyaluronate to water, stir to mix evenly, then heat to 80 - 85 °C, and keep stirring while maintaining the temperature to disperse evenly. When the temperature drops below 40 °C, add glycerin, aloe vera gel, peptide (2), vitamin C, caprylyl glycol, and 1,2 - hexanediol, and stir evenly. Adjust the pH value of the solution to about 5.5 with 15% triethanolamine to obtain the product.

[0185] Example 9 Functional Test for Repairing Skin Barrier and Increasing Skin Elasticity

[0186] 9.1 Subjects

[0187] 60 subjects aged 30 - 50 were randomly divided into 2 groups, with an average of 30 people in each group.

[0188] 9.2 Samples and Grouping

[0189] Select Example 8 as the administration group, and set up a placebo group at the same time.

[0190] 9.3 Test Instruments

[0191] Trans - epidermal water loss tester Tewameter TM300 and skin elasticity tester Cutometer dualMPA580.

[0192] 9.4 Objectives

[0193] If the skin barrier function is intact, less trans - epidermal water loss of the skin occurs, and the measured trans - epidermal water loss value is small. By testing the trans - epidermal water loss values and skin elasticity values of the subjects before and after using the samples, the skin repair and firming effects of the peptide derivative of the present invention are evaluated.

[0194] 9.5 Methods

[0195] After the subjects' faces were cleaned, they sat still for 30 min. First, measure the initial trans - epidermal water loss values and skin elasticity values of specific areas of their faces. Apply the sample evenly to the test area, twice a day, and keep using it morning and evening. Do not use other cosmetics. Continuously use it for 8 weeks, and respectively test and record the trans - epidermal water loss values and skin elasticity values of the test area. The tests of the same subject were completed by the same measurement personnel. Finally, statistically analyze the values measured each time in the test area of the subjects, and analyze the variation rules of their trans - epidermal water loss values and skin elasticity values.

[0196] 9.6 Results

[0197] The test results of trans - epidermal water loss and skin elasticity are shown in Figure 10 、 11。

[0198] Figure 10 The results showed that, compared with the placebo group, after continuous use of the peptide (2) of the present invention for 8 weeks, the value of transepidermal water loss decreased significantly, indicating that the peptide (2) has a significant effect on repairing the skin barrier.

[0199] Figure 11 The results showed that after continuous use of the placebo for 8 weeks, there was no significant change in skin elasticity. Compared with the placebo group, after continuous use of the peptide (2) of the present invention for 8 weeks, the skin elasticity was significantly restored.

[0200] This shows that continuous use of the peptide derivative of the present invention can promote the proliferation of keratinocytes, repair the skin barrier, improve the activity of fibroblasts, promote the secretion of extracellular matrix, increase the content of collagen, thereby improving the skin quality from the source, repairing the skin, increasing skin elasticity, and improving firmness. Therefore, it has a good skin repair and firming effect and can be applied to skin repair and firming products.

[0201] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, but it does not mean that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the art to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the protection scope of the present invention.

Claims

1. Use of a peptide derivative or a salt thereof in the preparation of a composition for skin repair and firming, wherein the skin repair and firming is to promote the proliferation and migration of keratinocytes; The peptide derivative is H-(β-Ala)-Hyp-Dab-NH-benzyl.

2. The use according to claim 1, wherein The composition contains 0.0001%-5% by mass concentration of the peptide derivative or a salt thereof.

3. The use according to claim 1, wherein The composition contains 0.0005%-1% by mass concentration of the peptide derivative or a salt thereof.

4. The use according to claim 1, characterized in that, The composition contains 0.001%-0.1% by mass concentration of the peptide derivative or a salt thereof.

5. The use according to claim 1, characterized in that, The composition contains 0.005%-0.01% by mass concentration of the peptide derivative or a salt thereof.

6. The use according to claim 1, characterized in that, The salt of the peptide derivative includes a metal salt of the peptide derivative, and the metal includes: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

7. The use according to claim 1, wherein, The salt of the peptide derivative includes a salt formed by the peptide derivative and an organic base, and the organic base includes: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine.

8. The use according to claim 1, characterized in that, The salt of the peptide derivative includes a salt formed by the peptide derivative and an inorganic acid or an organic acid, and the organic acid includes: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; The inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

9. The use according to claim 1, characterized in that, The peptide derivative or a salt thereof is incorporated into a cosmetically or pharmaceutically acceptable delivery system or sustained-release system, or adsorbed onto a cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support; The cosmetically or pharmaceutically acceptable delivery system or sustained-release system is selected from: liposomes, oil bodies, nonionic surfactant liposome vesicles, ethosomes, millimeter capsules, micron capsules, nanocapsules, nanostructured lipid carriers, sponges, liposomes, micelles, millimeter spheres, micron spheres, nanospheres, lipid spheres, micron emulsions or nanoemulsions; The cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support is selected from: talc, bentonite, silica, starch or maltodextrin.

10. The use according to claim 9, wherein, The cosmetically or pharmaceutically acceptable delivery system or sustained-release system is a liposome or a micron emulsion.

11. The use according to claim 9, characterized in that, The cosmetically or pharmaceutically acceptable delivery system or sustained-release system is a water-in-oil type micron emulsion having an internal structure of reverse micelles.

12. The use according to claim 1, characterized in that, The composition is a cosmetic composition or a pharmaceutical composition, and the preparation of the composition is selected from: creams, oils, foams, lotions, gels, liniments, ointments, mousses, powders, sticks, pens, sprays, aerosols, capsules, tablets, granules, suspensions, emulsions, elixirs, polysaccharide films or jellies.

13. According to the use described in claim 1, it is characterized in that, The composition further contains at least one other active agent for enhancing the effect of skin repair and firming, and the other active agent is selected from peptides, natural plant ingredients, vitamin C or retinoids.

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