Composition containing linear peptide and cyclic peptide and application thereof

Abstract

The invention provides a composition for resisting wrinkles, tightening, moisturizing, resisting inflammation, relieving and inhibiting skin collagen degradation. The composition comprises a micromolecule COL17 type linear peptide and a micromolecule COL17 type cyclic peptide. According to the invention, the anti-wrinkle and firming effects are realized by up-regulating COL4A1, COL7A1 and / or COL17A1; moisturizing is realized by up-regulating AQP3; anti-inflammatory soothing is realized by inhibiting IL-6 and / or IL-8; the inhibition of skin collagen degradation is realized by inhibiting MMP1. The invention also relates to application of a composition containing the micromolecule COL17 linear peptide and the micromolecule COL17 cyclic peptide in preparation of an external skin preparation for resisting wrinkles, tightening, moisturizing, resisting inflammation, relieving and inhibiting degradation of skin collagen. The external skin preparation is selected from face cream, emulsion, gel, toning lotion, essence, mask, eye cream, aerosol cleaning foam, spray, shower gel or facial cleanser.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to a composition containing a linear peptide and a cyclic peptide, and a preparation method and application thereof. Background Art

[0002] Collagen is a biopolymer, mainly secreted by fibroblasts. It is the main component of animal connective tissue and the most abundant and widely distributed functional protein in mammals. Collagen accounts for 1 / 3 of the total protein content in the human body and is the most common component in the extracellular matrix (ECM). To date, more than 20 different types of collagen have been confirmed. Collagen is composed of three identical or different polypeptide chains. Its amino acid sequence is highly conserved and consists of repeated Gly-XY fragments. X is usually proline and Y is usually hydroxyproline. The three polypeptide chains form a tight triple helix structure (see Kisling A, Lust RM, Katwa LC. What is the role of peptide fragments of collagen I and IV in health and disease? Life Sci. 2019 Jul 1; 228: 30-34. doi: 10.1016 / j.lfs.2019.04.042. Epub 2019 Apr 17. PMID: 31004660; Yang J, Song W, Li C, Fang C, Zhang Y, Wang Q, Zhang M, Qian G. Comparative study of collagen distribution in the carapace dermis of soft-shell and hard-shell cryptomeresus turtle embryos. the dermis of the embryonic carapace of soft-and hard-shelled cryptodiran turtles), J Morphol. 2021 Apr; 282(4): 543-552. doi: 10.1002 / jmor.21327. Epub 2021 Feb 8. PMID: 33491791; Huang A, Guo G, Yu Y, Yao L. The roles of collagen in chronic kidney disease and vascular calcification, J Mol Med (Berl). 2021 Jan; 99(1): 75-92. doi: 10.1007 / s00109-020-02014-6. Epub 2020 Nov 25. PMID: 33236192).

[0003] Collagen is the most abundant fiber-forming protein in the skin. It can enhance the tensile strength of tissues, regulate cell adhesion, and guide tissue development. The collagen expressed by human skin includes types I, III, IV, V, VI, VII, XIV, XVII, and XVIII. Studies have shown that type I and type III collagen account for 70% to 90% and 10% to 21% of the total collagen in human skin, respectively, type V accounts for 2% to 8%, and other collagen subtypes account for trace amounts. Type I collagen, as the main component of adult skin, is part of the collagen fiber component, has good support, and is an important component for supporting skin cells. Type III collagen is distributed around type I collagen, and has the functions of promoting cell proliferation, increasing cell activity, and helping the body repair aging and damaged skin. Type IV collagen is the main component of the basement membrane, accounting for 50% of the basement membrane protein, and is essential for maintaining the structure of the basement membrane (see Liu N, Matsumura H, Kato T, Ichinose S, Takada A, Namiki T, Asakawa K, Morinaga H, Mohri Y, De Arcangelis A, Geroges-Labouesse E, Nanba D, Nishimura EK. Stem cell competition orchestrates skin homeostasis and ageing, Nature. 2019Apr; 568(7752): 344-350. doi: 10.1038 / s41586-019-1085-7. Epub 2019Apr 3. PMID: 30944469). Collagen VII is the main component of the anchoring fibrils under the dense layer of the basement membrane, connecting the basement membrane with the underlying matrix. Collagen XVII derived from keratinocytes in the basement membrane constitutes the semi-desmosome structure between keratinocytes and the basement membrane. It is a transmembrane protein located in the basement membrane region and is essential for keratinocytes to attach to the basement membrane. Collagen XVIII is a ubiquitous basement membrane proteoglycan that has the characteristics of collagen and proteoglycan. It is distributed in different basement membrane regions and plays an important role in maintaining the integrity of the basement membrane.

[0004] Type XVII collagen is expressed in a variety of tissues, with the highest expression in the skin. It plays an important role in resisting skin aging and maintaining the homeostasis of hair follicle stem cells. Type XVII collagen is a transmembrane protein rich in 1497 amino acids, encoded by the COL17a1 gene, with its N-terminus located in the cytoplasm and its C-terminus located in the extracellular matrix. Like other transmembrane collagens, the extracellular domain of type XVII collagen can be cleaved from the cell surface by disintegrin and metalloproteinases. In 2019, Liu N et al. found that (see Liu N, Matsumura H, Kato T, Ichinose S, Takada A, Namiki T, Asakawa K, Morinaga H, Mohri Y, De Arcangelis A, Gerogenes-Labouesse E, Nanba D, Nishimura EK. Stem cell competition orchestrates skin homeostasis and ageing, Nature. 2019Apr; 568(7752): 344-350. doi: 10.1038 / s41586-019-1085-7. Epub 2019Apr 3. PMID: 30944469) that the key to maintaining the youthfulness and integrity of the skin mainly depends on the expression level of type XVII collagen in epidermal stem cells, and the expression of type XVII collagen also affects the changes in the number of hemidesmosomes. In the experiment, the researchers used mice that had eliminated the type XVII collagen gene to visually observe the differentiation process of epidermal stem cells. The experiment found that stem cells with high levels of type XVII collagen expression tend to divide symmetrically, generating two new cells that are also connected to the basement membrane; while stem cells with low levels of type XVII collagen expression tend to divide asymmetrically, and the daughter cells after division will enter the surface skin. In addition, the function of the hemidesmosomes of cells with low levels of type XVII collagen expression is poor, and the connection with the basement membrane is not strong, so it is easy to be squeezed out of the basal layer by the surrounding stem cells with high levels of type XVII collagen expression, and fall off from the stratum corneum during the skin metabolism process. This causes a reduction or depletion of human epidermal stem cells, which in turn affects skin homeostasis. Therefore, increasing the expression level of type XVII collagen can not only maintain the youthful state of the skin but also maintain cell homeostasis. Small molecule type XVII collagen fragments (peptides) containing active sites are more easily absorbed and utilized by the skin due to their smaller molecular structure, and exert their effects.

[0005] As a class of polypeptides with a cyclic structure, cyclic peptides can be traced back to biologically active molecules in nature. Many drugs used clinically are cyclic peptides of natural origin. Compared with traditional large molecular weight linear peptides, cyclic peptides have higher chemical and metabolic stability, which makes cyclic peptides have a longer half-life in the body. The cyclic structure of cyclic peptides makes them easier to penetrate the cell membrane, and they can more effectively enter the cell and exert their biological activity. In addition, cyclic peptides can bind to specific biological targets with higher affinity. Type 17 cyclic peptides derived from the active site of type 17 collagen and subjected to cyclization treatment have the characteristics of small molecular weight and good permeability.

[0006] The COL4A1 gene encodes type IV collagen alpha protein, which shares a bidirectional promoter with a paralogous gene on the opposite strand. Type IV collagen alpha protein functions as part of a heterotrimer and interacts with other extracellular matrix components such as film aggrecan, proteoglycan, and laminin. Type VII collagen, encoded by the COL7A1 gene, is an important component of epithelial-stromal anchoring in the skin, mucous membranes, and cornea. It is composed of three identical alpha collagen chains that form stable fibril structures, which are key components of anchoring fibrils and ensure the integrity and strength of tissues such as skin. Mutations in COL7A1 lead to the absence or dysfunction of type VII collagen, which in turn causes dystrophic epidermolysis bullosa (DEB). Type XVIIα1 collagen (COL17A1), encoded by the COL17A1 gene, is a type II extracellular matrix transmembrane protein. COL17A1 is an important component of mature type I hemidesmosomes located in the 10q25.1 region. It can act both by adhering to the cell matrix and by acting as a cell surface receptor. Promoting the expression of COL4A1, COL7A1, and COL17A1 in skin cells has a promoting effect on skin anti-wrinkle and firming. Aquaporin 3 (AQP3) is a substance on the cell membrane that is responsible for the transport of substances such as water, glycerol, and urea. It is a transport protein factor and is mainly expressed in keratinocytes and skin fibroblasts. AQP3 is not only involved in skin hydration and barrier function, but also plays an important role in skin damage, repair, and healing. It is an important guarantee for maintaining normal skin morphology and function. Promoting the expression of AQP3 in the skin is of great significance for skin moisturizing and repair. Interleukin 6 (IL-6) and interleukin 8 (IL-8) belong to the interleukin family. They act as pro-inflammatory cytokines and anti-inflammatory myosins and are secreted by immune cells. Inhibiting the expression of IL-6 and IL-8 in skin cells can effectively relieve the discomfort caused by inflammation. The extracellular matrix (ECM) is an intricate network composed of macromolecules. The main components include collagen, elastin, non-collagen glycoproteins and proteoglycans. It plays a very important role in maintaining normal tissue structure, function, cell growth and differentiation. The degradation of the extracellular matrix mainly depends on proteolytic enzymes, and MMPs are the most important group of proteolytic enzymes. Among them, MMP-1 is the most important enzyme for degrading type I and type III collagen. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, destroys the normal structure of collagen fibers and elastic fibers, and causes wrinkles and other aging manifestations on the skin.Based on the important role of MMP-1 in the skin aging process, inhibiting MMP-1 activity may be a new way to delay aging (see Hatamochi A, Dno M, Arakawa M et al., Analysis of collagen gene expression by cultured fibrol lasts in morphoea, [J]. Br J Dermatol 1992; 126(3): 216-21).

[0007] The present invention unexpectedly discovered that a composition containing a small molecule COL17-type linear peptide and a small molecule COL17-type cyclic peptide has the effects of anti-wrinkle and firming, moisturizing, anti-inflammatory and soothing, and inhibiting skin collagen degradation. It can promote the expression of skin anti-wrinkle and firming related factors (COL4A1, COL7A1, COL17A1), AQP3, and simultaneously inhibit the expression levels of inflammatory factors (IL-6, IL-8) and MMP-1. Summary of the invention

[0008] On the one hand, the present invention provides a composition for anti-wrinkle, firming, moisturizing, anti-inflammatory, soothing and / or inhibiting skin collagen degradation, comprising: a small molecule COL17-type linear peptide and a small molecule COL17-type cyclic peptide.

[0009] In a preferred embodiment, the small molecule COL17-type linear peptide is Sooth col17, and the small molecule COL17-type cyclic peptide is Cyclic col17.

[0010] In a preferred embodiment, the anti-wrinkle and firming effect is achieved by up-regulating COL4A1, COL7A1, and / or COL17A1; the moisturizing effect is achieved by up-regulating AQP3; the anti-inflammatory and soothing effect is achieved by inhibiting IL-6 and / or IL-8; and the inhibition of skin collagen degradation is achieved by inhibiting MMP1.

[0011] In a preferred embodiment, the concentration of the small molecule COL17-type linear peptide is at least 2 ppm, and the concentration of the small molecule COL17-type cyclic peptide is at least 2 ppm.

[0012] On the other hand, the present invention also relates to the use of a composition comprising a small molecule COL17-type linear peptide and a small molecule COL17-type cyclic peptide in the preparation of a skin topical preparation for anti-wrinkle, firming, moisturizing, anti-inflammatory and soothing, and inhibiting skin collagen degradation.

[0013] In a preferred embodiment, the content of the composition in the external skin preparation is 0.001-20% by weight.

[0014] In a preferred embodiment, the skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. DETAILED DESCRIPTION

[0015] The present invention introduces a composition of linear peptides and cyclic peptides, which has the related effects of anti-wrinkle and firming, moisturizing, anti-inflammatory and soothing, and inhibiting skin collagen degradation. Specifically, the combination of the present invention can upregulate COL4A1, COL7A1, and COL17A1 genes, showing firming and anti-wrinkle effects; can downregulate IL-6 and IL-8 genes, showing anti-inflammatory effects; can upregulate AQP3 genes, showing moisturizing effects; can downregulate MMP1 genes, showing the effect of inhibiting collagen degradation.

[0016] In order to provide a more concise description, some quantitative expressions given herein are not modified by the term "about". It should be understood that, whether or not the term "about" is explicitly used, each quantity given herein is intended to refer to the actual given value, and is also intended to refer to the approximate values ​​of these given values ​​that can be reasonably inferred by ordinary technicians in the field, including the approximate values ​​of these given values ​​caused by experimental and / or measurement conditions.

[0017] To provide a more concise description, some quantitative expressions herein are described as a range of about X amount to about Y amount. It should be understood that when describing a range, the range is not limited to the upper and lower limits described, but should include the entire range of about X amount to about Y amount or any amount therebetween.

[0018] The compositions described herein may optionally be in finished packaging form. In one embodiment, the packaging is a container such as a plastic, metal or glass tube or a wide-mouthed bottle containing the composition. The product may additionally have packaging such as a plastic or cardboard box for storing the container. In one embodiment, the product comprises the composition and has instructions for guiding the user to apply the composition to the skin to treat signs of skin aging as discussed below. Such instructions may be printed on the container, a label insert, or any other packaging.

[0019] As used herein, "topical application" means applying or spreading directly onto the external skin, scalp, or hair, for example, using the hands or an application device such as a wipe, roller, or spray.

[0020] As used herein, "cosmetically acceptable" means that the ingredients described by the term are suitable for use in contact with tissue (eg, skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic response, or the like.

[0021] Combination of small molecule COL17 type linear peptide and small molecule COL17 type cyclic peptide

[0022] The present invention unexpectedly found that the combination of small molecule COL17-type linear peptide and small molecule COL17-type cyclic peptide has the effects of anti-wrinkle and firming, moisturizing, anti-inflammatory and soothing, and inhibiting skin collagen degradation. Therefore, the combination of small molecule COL17-type linear peptide and small molecule COL17-type cyclic peptide can be added to skin topical preparations as an effective ingredient to assist in achieving anti-wrinkle and firming, moisturizing, anti-inflammatory and soothing, and / or inhibiting skin collagen degradation.

[0023] The small molecule COL17 type linear peptide used in the present invention has an appearance of a transparent to translucent solution, a colorless color, a density of 1.1-1.4 g / ml, and a pH of 4-7. In a preferred embodiment, the peptide content of the small molecule COL17 type linear peptide is at least 900 pmm, preferably 900-1100 ppm. The peptide content can be determined by HPLC-external standard method.

[0024] In a specific embodiment, the present invention uses a small molecule COL17-type linear peptide with the trade name of Sooth col17 purchased from Hangzhou Shiguang Xinya Biotechnology Co., Ltd.

[0025] The small molecule COL17-type cyclic peptide used in the present invention has an appearance of a transparent to translucent solution, a colorless color, a density of 0.9-1.2 g / ml, and a pH of 4-7. In a preferred embodiment, the peptide content of the small molecule COL17-type cyclic peptide is at least 400 pmm, preferably 400-600 ppm. The peptide content can be determined by HPLC-external standard method.

[0026] In a specific embodiment, the present invention uses a small molecule COL17-type cyclic peptide with the trade name of Cyclic col17 purchased from Hangzhou Shiguang Xinya Biotechnology Co., Ltd.

[0027] In some embodiments, the small molecule COL17-type linear peptide is used at a concentration of at least 2 ppm. In some embodiments, the small molecule COL17-type linear peptide is used at a concentration of 2-10 ppm.

[0028] In some embodiments, the small molecule COL17-type cyclic peptide is used at a concentration of at least 2 ppm. In some embodiments, the small molecule COL17-type cyclic peptide is used at a concentration of 2-10 ppm.

[0029] In a specific embodiment, the weight ratio of the small molecule COL17-type linear peptide to the small molecule COL17-type cyclic peptide in the composition is 1:1.

[0030] Skin topical products

[0031] In some embodiments, the composition of the present invention can be used for the preparation of a skin external preparation. The skin external preparation is preferably a cosmetic composition, including but not limited to products in dosage forms such as cream, lotion, gel, toner, essence, mask, eye cream, aerosol (cleansing foam), spray, shower gel and facial cleanser.

[0032] The content of the composition of the present invention in the external preparation for skin is 0.001-20% by weight, preferably 0.01-10% by weight, more preferably 0.1-5% by weight.

[0033] The skin external preparation is a general concept for all ingredients usually used on the outside of the skin, for example, it can be a cosmetic composition. The cosmetic composition can be a basic cosmetic, a facial makeup cosmetic, a body cosmetic, a hair care cosmetic, etc., and there is no special restriction on its dosage form, which can be reasonably selected according to different purposes. The cosmetic composition also contains different media or matrix excipients allowed by the cosmetics level according to different dosage forms and purposes.

[0034] The skin preparation for external use comprising the composition of the present invention can be topically applied to human skin and / or hair. The skin preparation for external use can also include a cosmetically acceptable topical carrier, which can be about 50 % by weight to about 99.99 % by weight of the skin preparation for external use (such as about 80 % by weight to about 99 % by weight of the skin preparation for external use). In a preferred embodiment of the present invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier may comprise one or more selected from wetting agents, emollients, grease, wetting agents and similar substances. In one embodiment, the cosmetically acceptable topical carrier comprises a substrate such as a nonwoven fabric or a membrane material.

[0035] Skin topical agents can be prepared into a variety of product types, including but not limited to lotions, creams, gels, sticks, sprays, ointments, cleansing liquid lotions and solid soaps, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shaving creams, wipes, patches, hydrogels, film-forming products, facial masks and skin films, films and cosmetics such as foundation and mascara. These product types can contain several types of cosmetically acceptable topical carriers, including but not limited to solutions, suspensions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids and liposomes.

[0036] The skin external preparation containing the composition of the present invention can be formulated as a solution. The solution usually contains an aqueous solvent or an organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous solvent or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, and mixtures thereof.

[0037] Skin topical preparations can be formulated into solutions containing emollients. Such skin topical preparations preferably contain from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or alleviate dryness, for example, by preventing the loss of skin moisture through the skin. Examples of emollients include, but are not limited to, vegetable oils, mineral oils, aliphatic esters, etc.

[0038] Lotions can be prepared from such solutions.Lotions typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients and from about 50% to about 90% (e.g., from about 60% to about 80%) water.

[0039] Another type of product that can be formulated from a solution is a cream. Creams typically contain from about 5% to about 50% (e.g., from about 10% to about 20%) of one or more emollients and from about 45% to about 85% (e.g., from about 50% to about 75%) water.

[0040] Although it is preferred that the skin external preparation containing the composition of the present invention contains water, as another option, the skin external preparation can be anhydrous or an ointment containing no water but organic and / or silicone solvents, greases, lipids and waxes. The ointment can contain a simple base of animal or vegetable oil or semisolid hydrocarbon. The ointment can contain about 2% to about 10% of one or more emollients and about 0.1% to about 2% of one or more thickeners.

[0041] The topical agent for skin application can be formulated as an emulsion. If the topical carrier is an emulsion, about 1% to about 10% (e.g., about 2% to about 5%) of the topical carrier contains one or more emulsifiers. The emulsifier can be nonionic, anionic or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the field of personal care and cosmetic formulations.

[0042] Lotions and creams can be formulated as emulsions. Typically such lotions contain 0.5% to about 5% of one or more emulsifiers. Such creams typically contain about 1% to about 20% (e.g., about 5% to about 10%) of one or more emollients; about 20% to about 80% (e.g., 30% to about 70%) of water; and about 1% to about 10% (e.g., about 2% to about 5%) of one or more emulsifiers.

[0043] Oil-in-water and water-in-oil type single emulsion skin care preparations, such as lotions and creams, are well known in the cosmetics field and can be used in the present invention. Multiphase emulsion skin external preparations (such as water-in-oil-in-water type and oil-in-water-in-oil type) can also be used in the present invention. Generally, such single-phase emulsions or multiphase emulsions contain water, emollients and emulsifiers as their basic ingredients.

[0044] The skin external preparation comprising the composition of the present invention can also be formulated into a gel (e.g., an aqueous gel, an alcoholic gel, an alcohol / water-type gel, or an oily gel using a suitable gelling agent). Suitable gelling agents for aqueous gels and / or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethylcellulose and hydroxypropylcellulose). Suitable gelling agents for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gelling agents typically contain between about 0.1% and 5% by weight of such gelling agents.

[0045] The external preparation for skin application comprising the composition of the present invention may also be formulated as a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing a powder).

[0046] In addition to the above-mentioned components, the external preparation for skin which can be used in the present invention may contain various other oil-soluble substances and / or water-soluble substances at levels conventionally established in its technical field for use in external preparations for skin for use on skin and hair.

[0047] The skin external preparation of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin regulators, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin external preparation and do not affect the effects of the composition of the present invention.

[0048] In some embodiments of the skin topical preparation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl parahydroxybenzoate and phenoxyethanol. Based on the total weight of the composition, the amount of the preservative is about 0.5 to about 2 weight %, preferably about 0.5 to 1 weight %.

[0049] In one example of the skin external preparation of the present invention, one or more antioxidants can be used. Suitable antioxidants include butylated hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-α-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E or vitamin E derivatives, vitamin C and its derivatives, calcium pantothenate, green tea extract and mixed polyphenols, and mixtures of the above-mentioned substances. The antioxidant used is about 0.02 to 0.5 weight % of the total weight of the composition, and more preferably from about 0.002 to 0.1 weight % of the amount used.

[0050] In one example of the skin external preparation of the present invention, one or more emollients can be used to act as lubricants through their ability to remain on the skin surface or in the stratum corneum to reduce flaking and improve the appearance of the skin. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether silicone copolymers, and the like. Examples of suitable emollients include, but are not limited to, polypropylene glycol ("PPG")-15 stearyl ether, PPG-10 cetyl ether, Steareth-10, Oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, hydroxystearate octyl, dimethyl polysiloxane, and combinations thereof. Cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, and combinations thereof are preferred. When used, the emollient is used in an amount ranging from about 0.1 to about 30% by weight, preferably about 1 to about 30% by weight, based on the total weight of the composition.

[0051] In one example of the skin external preparation of the present invention, one or more moisturizers can be used. Moisturizers are also called humectants, which help to improve the effect of emollients, reduce flaking, remove the scales of the composition and improve the touch of the skin. Polyols can be used as moisturizers, including but not limited to glycerol, polyalkylene glycols, alkylene polyols and derivatives thereof, including butylene glycol, propylene glycol, dipropylene glycol, polyglycerol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol, and combinations thereof. When used, the amount of moisturizer is about 0.1 to about 20% by weight, preferably about 1 to about 15% by weight, based on the total weight of the composition.

[0052] In an example of the skin external preparation of the present invention, one or more emulsifiers can be used. Emulsifiers can be used within the scope of effective stabilizing amount. Preferably, based on the total weight of the composition, the emulsifier is used in an amount of about 1.0 to about 10.0 weight %, more preferably about 3.0 to about 6.0 weight %. Any emulsifier compatible with the components in the composition can be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, Steareth-2, Steareth-20, acrylic acid (ester) class / C10-30 alkyl alcohol acrylate cross-linked polymer, and combinations thereof.

[0053] In one example of the skin external preparation of the present invention, one or more pH regulators can be used. The pH regulator useful in the skin external preparation of the present invention includes tromethamine. When used, the amount of the pH regulator based on the total weight of the composition is about 0.1 to about 2 weight %, preferably about 0.1 to about 1 weight %.

[0054] In a specific embodiment of the present invention, the skin topical agent comprises acrylates / C10-30 alkyl acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetearyl alcohol, cetearyl ethylhexanoate, tromethamine or a combination thereof.

[0055] Additional cosmetic active agents

[0056] In some embodiments, the topical skin preparation may also include additional cosmetic active agents. As used herein, "cosmetic active agents" are compounds (such as synthetic compounds or compounds isolated from natural sources or natural extracts) that have cosmetic or therapeutic effects on skin or hair, including but not limited to anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, topical analgesics, sunscreens, photoprotectants, antioxidants, keratolytic agents, surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, curing agents, anti-hard cuticle agents, and agents for hair and / or skin conditioning.

[0057] In one embodiment, these cosmetic actives are selected from (but not limited to): hydroxy acids, benzoyl peroxide, D-panthenol, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, carotenoids, free radical scavengers, spin traps, amines, retinoids such as retinol and retinyl palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogen, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, copper-containing peptides such as Cu:Gly-His-Lys, coenzyme Q10, peptides, amino acids such as proline, vitamins, lactobionic acid, acetyl-CoA, niacin, riboflavin, thiamine, ribose, electron transfer substances such as NADH and FADH2, and other plant extracts such as aloe, feverfew, oatmeal, and their derivatives and mixtures. The cosmetic active agent is generally present in an amount of about 0.001% to about 20%, for example about 0.005% to about 10%, such as about 0.01% to about 5%, by weight of the topical skin preparation of the present invention.

[0058] Examples of vitamins include, but are not limited to, vitamin A, vitamin B (e.g., vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and different forms of vitamin E (e.g., α, β, γ, or Δ tocopherol) or mixtures thereof and derivatives thereof.

[0059] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0060] Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbic acid polypeptide). Oil-soluble antioxidants suitable for use in the skin topical preparations of the present invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., tocopherol ethyl ester), tocotrienols and ubiquinone. Natural extracts containing antioxidants suitable for use in the skin topical preparations of the present invention include, but are not limited to, extracts containing flavonoids and isoflavones and their derivatives (e.g., genistein and di-zein), extracts containing resveratrol, and the like. Examples of such natural extracts include grape seeds, green tea, pine bark, and propolis.

[0061] How to use

[0062] The skin topical preparation of the present invention can be topically applied to mammalian skin in need of treatment. In one embodiment, the skin topical preparation can be applied to skin in need of treatment of fine lines and wrinkles and / or loss of elasticity. The skin topical preparation can be applied to skin in need of such treatment according to a suitable treatment regimen, such as monthly, weekly, every other day, daily, twice a day, etc.

[0063] Example

[0064] The present invention is further described below in conjunction with specific examples. It is necessary to point out that the examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the present invention described above. The test methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0065] 1. Experimental Materials

[0066] Table 1

[0067]

[0068]

[0069] 2. Experimental Instruments

[0070] Table 2

[0071]

[0072] Embodiment 1:

[0073] Weigh an appropriate amount of 2ppm small molecule COL17 linear peptide solution and set aside.

[0074] Embodiment 2:

[0075] Weigh an appropriate amount of 10 ppm small molecule COL17 linear peptide solution and set aside.

[0076] Embodiment 3:

[0077] Weigh an appropriate amount of 2ppm small molecule COL17-type cyclic peptide solution and set aside.

[0078] Embodiment 4:

[0079] Weigh an appropriate amount of 10 ppm small molecule COL17-type cyclic peptide solution and set aside.

[0080] Embodiment 5:

[0081] Weigh appropriate amounts of 2 ppm small molecule COL17 linear peptide solution and 2 ppm small molecule COL17 cyclic peptide solution respectively for use.

[0082] Embodiment 6:

[0083] Weigh appropriate amounts of 10 ppm small molecule COL17 linear peptide solution and 10 ppm small molecule COL17 cyclic peptide solution respectively for use.

[0084] Test example:

[0085] 1. Sample solution preparation: Dilute Sooth col17 and Cyclic col17 to 2000ppm with DMEM culture medium, and vortex to mix. Mix the compound sample with the above solution in a 1:1 ratio. Filter with a 0.22μM sterile filter, and then dilute with DMEM culture medium to the experimental concentration. Prepare the test working solution according to the test group (Table 3).

[0086] Table 3: Test groups

[0087]

[0088] 2. Model administration: HaCaT cells in the logarithmic growth phase were injected at a rate of 1*10 6 Cells were inoculated into 6-well plates and cultured in a 37°C 5% CO2 incubator for 24 h. The culture medium was discarded and UVB (50 mJ / cm 2 ) cells (the blank control group did not need to be treated with UVB); after completion, the 6-well plate was taken out, the prepared sample solution was added to the sample group, and serum-free culture medium was added to the blank control group and the model group, and the cells were cultured in a 37°C 5% CO2 incubator for 24 h.

[0089] 3. Collect cells: discard the supernatant, wash twice with PBS, and then collect the cells.

[0090] 4. RNA extraction: Extract RNA according to the RNA extraction kit method;

[0091] 5. Source of primers for COL4A1, COL7A1, COL17A1, IL-6, IL-8, AQP3, and MMP-1: Hangzhou Youkang Biotechnology Co., Ltd.

[0092] 6. Reverse transcription: After measuring the concentration, reverse transcription was performed with 1 μg to obtain cDNA;

[0093]

[0094] 7.RT-qPCR

[0095] Components volume 2×ChamQ Universal SYBR qPCR Master Mix 10.0μl Primer 1 (10 μM) 0.4μl Primer 2 (10 μM) 0.4μl Template DNA / cDNA 1μl×μl ddH2O Up to 20.0 μl

[0096] 8. Statistical analysis: GraphPad Prism 8 software was used, and the data were expressed as mean ± standard error. The differences between the blank control group and the model group were analyzed using paired T-test; the differences between the sample group and the model group were analyzed using one-way ANOVA, P < 0.05 ( * and # ) were considered to be significantly different, P < 0.01 ( ** and ## ) was considered to be extremely significantly different, P < 0.001 ( *** and ### ) were considered to be highly significant differences.

[0097] Result judgment

[0098] The gene expression of the blank control group was taken as 100%, and each group was subtracted from it. If it was greater than 0%, it indicated that the gene expression was upregulated, and if it was less than 0%, it indicated that the gene expression was downregulated. The upregulation of COL4A1, COL7A1, and COL17A1 genes indicated that the sample group had the ability to tighten and anti-wrinkle; the downregulation of COL4A1, COL7A1, and COL17A1 genes indicated that the sample group did not have the ability to tighten and anti-wrinkle. The downregulation of IL-6 and IL-8 genes indicated that the sample group had anti-inflammatory ability; the upregulation of IL-6 and IL-8 genes indicated that the sample group did not have anti-inflammatory ability. The upregulation of AQP3 gene indicated that the sample group had moisturizing ability; the downregulation of AQP3 gene indicated that the sample group did not have moisturizing ability. The downregulation of MMP1 gene indicated that the sample group had the ability to inhibit collagen degradation; the upregulation of MMP1 gene indicated that the sample group did not have the ability to inhibit collagen degradation. In this model group (UVB stimulation group), the relative expression increments of the above four genes (COL4A1, COL7A1, COL17A1, APQ3) were all less than 0%, and the relative expression increments of the three genes (IL-6, IL-8, MMP-1) were all greater than 0%, indicating that the experiment was effective.

[0099] Table 4 Expression of skin efficacy related genes after treatment in each group of samples

[0100]

[0101] Specifically, the sample of Example 1 (2 ppm of small molecule COL17-type linear peptide solution) up-regulated the expression of COL7A1 and COL17A1 genes, with relative expression increments of 404.91% and 35.62%, respectively; down-regulated the expression of IL-6 and IL-8 genes, with relative expression increments of -8.77% and -8.07%, respectively; up-regulated the expression of AQP3 gene, with a relative expression increment of 57.95%; and down-regulated the expression of MMP-1 gene, with a relative expression increment of -33.7%, indicating that the sample of Example 1 (2 ppm of small molecule COL17-type linear peptide solution) has the effects of skin firming and anti-wrinkle, anti-inflammatory, moisturizing, and inhibiting skin collagen degradation. The sample in Example 2 (10 ppm of small molecule COL17-type linear peptide solution) upregulated the expression of COL4A1, COL7A1, and COL17A1 genes, with relative expression increments of 32.05%, 355.46%, and 45.2%, respectively; downregulated the expression of IL-6 and IL-8 genes, with relative expression increments of -18.87% and -28.26%, respectively; upregulated the expression of AQP3 gene, with a relative expression increment of 67.85%; and downregulated the expression of MMP-1 gene, with a relative expression increment of -38.2%, indicating that the sample in Example 2 (10 ppm of small molecule COL17-type linear peptide solution) has the effects of skin firming and anti-wrinkle, anti-inflammatory, moisturizing, and inhibiting skin collagen degradation. In summary, in this study, when the concentration of the small molecule COL17 linear peptide solution was ≥2ppm, the degree of up-regulation of some firming and anti-wrinkle factors (COL4A1, COL17A1) and moisturizing factors increased with increasing concentration, that is, the skin firming, anti-wrinkle and moisturizing effects increased with increasing concentration; the degree of down-regulation of inflammatory factors and collagen degradation inhibition factors increased with increasing concentration, that is, the anti-inflammatory and collagen degradation inhibition effects on the skin increased with increasing concentration.

[0102] The sample of Example 3 (2 ppm of small molecule COL17-type cyclic peptide solution) up-regulated the expression of COL4A1, COL7A1, and COL17A1 genes, with relative expression increments of 12.32%, 434.11%, and 20.04%, respectively; down-regulated the expression of IL-6 and IL-8 genes, with relative expression increments of -10.07% and -5.97%, respectively; up-regulated the expression of AQP3 gene, with a relative expression increment of 41.36%; down-regulated the expression of MMP-1 gene, with a relative expression increment of -12.81%, indicating that the sample of Example 3 (2 ppm of small molecule COL17-type cyclic peptide solution) has the effects of skin firming, anti-wrinkle, anti-inflammatory, moisturizing, and inhibiting skin collagen degradation. The sample in Example 4 (10 ppm of small molecule COL17-type cyclic peptide solution) up-regulated the expression of COL4A1, COL7A1, and COL17A1 genes, with relative expression increments of 9.49%, 331.8%, and 30.36%, respectively; down-regulated the expression of IL-6 and IL-8 genes, with relative expression increments of -13.96% and -9.59%, respectively; up-regulated the expression of AQP3 gene, with a relative expression increment of 45.26%; down-regulated the expression of MMP-1 gene, with a relative expression increment of -18.1%, indicating that the sample in Example 4 (10 ppm of small molecule COL17-type cyclic peptide solution) has the effects of skin firming, anti-wrinkle, anti-inflammatory, moisturizing, and inhibiting skin collagen degradation. In summary, in this study, when the concentration of small molecule COL17 linear peptide solution was ≥2ppm, the degree of up-regulation of some firming and anti-wrinkle factors (COL17A1) and moisturizing factors increased with increasing concentration, that is, the firming, anti-wrinkle and moisturizing effects on the skin increased with increasing concentration; the degree of down-regulation of inflammatory factors and collagen degradation inhibitory factors increased with increasing concentration, that is, the anti-inflammatory and collagen degradation inhibitory effects on the skin increased with increasing concentration.

[0103] The superposition of two small molecule COL17-type peptide solutions (Examples 5-6) showed differences in the expression of related efficacy factors compared to the use of any small molecule COL17-type peptide alone. Example 5 (small molecule COL17-type linear peptide solution 2ppm + small molecule COL17-type cyclic peptide solution 2ppm) up-regulated the expression of COL4A1, COL7A1, and COL17A1 genes, with relative expression increments of 68.97%, 164.9%, and 57.91%, respectively; down-regulated the expression of IL-6 and IL-8 genes, with relative expression increments of -27.18% and -40.84%, respectively; up-regulated the expression of AQP3 gene, with a relative expression increment of 66.51%; down-regulated the expression of MMP-1 gene, with a relative expression increment of -46.17%, indicating that the sample of Example 5 (small molecule COL17-type linear peptide solution 2ppm + small molecule COL17-type cyclic peptide solution 2ppm) has the effects of skin firming, anti-wrinkle, anti-inflammatory, moisturizing, and inhibiting skin collagen degradation. By comparing the expression increments of Example 1 (small molecule COL17 linear peptide solution 2ppm) and Example 3 (small molecule COL17 cyclic peptide solution 2ppm), it is found that the superposition of small molecule COL17 linear peptide solution 2ppm and small molecule COL17 cyclic peptide solution 2ppm has a higher degree of upregulation of COL4A1 and COL17A1 than the sum of the increments of gene upregulation by using small molecule COL17 linear solution 2ppm and small molecule COL17 cyclic peptide solution 2ppm alone; the degree of downregulation of IL-6 and IL-8 is higher than the sum of the performance of gene downregulation by using small molecule COL17 linear peptide solution 2ppm and small molecule COL17 cyclic peptide solution 2ppm alone. It shows that the superposition of small molecule COL17 linear peptide solution 2ppm and small molecule COL17 cyclic peptide solution 2ppm has a synergistic effect on the upregulation of COL4A1 and COL17A1 expression and the downregulation of IL-6 and IL-8 expression.

[0104] In addition, at high concentrations, the sample in Example 6 (10 ppm small molecule COL17 linear peptide solution + 10 ppm small molecule COL17 cyclic peptide solution) upregulated the expression of COL4A1, COL7A1, and COL17A1 genes, with relative expression increments of 85.84%, 621.8%, and 56.14%, respectively; downregulated the expression of IL-6 and IL-8 genes, with relative expression increments of -34.58% and -41.03%, respectively; upregulated the expression of AQP3 gene, with a relative expression increment of 77.84%; and downregulated the expression of MMP-1 gene, with a relative expression increment of -52.12%, indicating that the sample in Example 6 (10 ppm small molecule COL17 linear peptide solution + 10 ppm small molecule COL17 cyclic peptide solution) has the effects of skin firming, anti-wrinkle, anti-inflammatory, moisturizing, and inhibiting skin collagen degradation. By comparing the efficacy factor expression values ​​of Example 2 (small molecule COL17 type linear peptide solution 10ppm) and Example 4 (small molecule COL17 type cyclic peptide solution 10ppm), it is found that the superposition of small molecule COL17 type linear peptide solution 10ppm and small molecule COL17 type cyclic peptide solution 10ppm has a higher degree of up-regulation of COL4A1 than the sum of the increments of gene up-regulation by using small molecule COL17 type linear peptide solution 10ppm and small molecule COL17 type cyclic peptide solution 10ppm alone; the degree of down-regulation of IL-6 and IL-8 is higher than the sum of the performance of gene down-regulation by using small molecule COL17 type linear peptide solution 10ppm and small molecule COL17 type cyclic peptide solution 10ppm alone. It shows that the superposition of small molecule COL17 type linear peptide solution 10ppm and small molecule COL17 type cyclic peptide solution 10ppm has a synergistic effect on the up-regulation of COL4A1 expression and the down-regulation of IL-6 and IL-8 expression.

[0105] Application Examples

[0106] The composition of Example 5 (2ppm of small molecule COL17-type linear peptide solution + 2ppm of small molecule COL17-type cyclic peptide solution) and Example 6 (10ppm of small molecule COL17-type linear peptide solution + 10ppm of small molecule COL17-type cyclic peptide solution) can be used for the preparation of skin topical preparations. The skin topical preparation is preferably a cosmetic composition, such as a lotion, a face cream, an emulsion, an eye cream, an essence, etc. The weight percentage of the preparation in the skin topical preparation is 0.001%-20% (w / w). The preferred weight percentage is 0.01%-20% (w / w). The more preferred weight percentage is 0.1%-10% (w / w). The most preferred weight percentage is 0.1%-5% (w / w).

[0107] The following are specific application examples of the preparations of the embodiments in external skin preparations, as well as the formulations and preparation methods of these preparations. In the following tables, "-" means no addition.

[0108] Application Example 1: Preparation of lotion

[0109]

[0110] Application Example 2: Preparation of facial cream

[0111]

[0112]

[0113] Application Example 3: Preparation of Emulsion

[0114]

[0115]

Claims

1. A composition for anti-wrinkle, firming, moisturizing, anti-inflammatory, soothing and / or inhibiting skin collagen degradation, comprising: a small molecule COL17-type linear peptide and a small molecule COL17-type cyclic peptide.

2. The composition according to claim 1, wherein The small molecule COL17 type linear peptide is Sooth col17, and the small molecule COL17 type cyclic peptide is Cyclic col17.

3. The composition according to claim 1, wherein The anti-wrinkle and firming is achieved by up-regulating COL4A1, COL7A1, and / or COL17A1.

4. The composition according to claim 1, wherein The moisturizing is achieved by up-regulating AQP3.

5. The composition according to claim 1, wherein The anti-inflammatory relief is achieved by inhibiting IL-6 and / or IL-8.

6. The composition according to claim 1, wherein The inhibition of skin collagen degradation is achieved by inhibiting MMP1.

7. The composition of claim 1, wherein the peptide content of the small molecule COL17-type linear peptide is at least 900 ppm.

8. The composition of claim 1, wherein the peptide content of the small molecule COL17-type cyclic peptide is at least 400 ppm.

9. The composition of claim 1, wherein The use concentration of the small molecule COL17-type linear peptide is at least 2 ppm, and the use concentration of the small molecule COL17-type cyclic peptide is at least 2 ppm.

10. Use of a composition comprising a small molecule COL17-type linear peptide and a small molecule COL17-type cyclic peptide in the preparation of a skin topical preparation for anti-wrinkle, firming, moisturizing, anti-inflammatory and soothing, and inhibiting skin collagen degradation.

11. The use according to claim 10, characterized in that The content of the composition in the external preparation for skin is 0.001-20% by weight.

12. The use according to claim 10, characterized in that The skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

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