Complete cortex action compound peptide

Abstract

The invention provides a whole cortical action compound peptide, which comprises acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1, and is characterized in that the whole cortical action compound peptide comprises acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1; the compound peptide acts on epidermal basal layer keratinocytes, true epidermal junction (DEJ) structural proteins and / or dermal layer fibroblasts, and synergistically exerts a whole-cortex skin anti-aging effect on epidermal layer, basement membrane, dermal layer and / or nerve fibers. The compound peptide has the advantages of full-cortex three-dimensional anti-aging, layered precise targeting, multi-peptide scientific compounding, matrix synergy, clear action mechanism, efficient growth promotion of seven key proteins, remodeling of a true epidermis structure, low-concentration effect, mildness, no stimulation and the like, and is an advanced anti-aging scheme under a precise skin care trend.

Description

Technical Field

[0001] The present invention relates to a compound peptide, in particular to a compound peptide with full-cortical effect. Background Art

[0002] Aging occurs in every layer of the skin, and the aging mechanisms and characteristics of each layer of skin are different. Skin aging is mainly caused by changes in the structure and function of the epidermis, dermal-epidermal junction (DEJ) and dermis, and different layers of skin have significantly different aging characteristics. The reduction in epidermal cell vitality leads to thinning, roughness, dullness and weakened barrier function of the epidermis; the reduction of structural proteins in the DEJ layer leads to loose connection between the dermis and insufficient nutrition for the epidermis; the reduction in the vitality of fibroblasts in the dermis leads to blocked collagen synthesis, reduced support of the extracellular matrix (ECM), and signs of aging such as wrinkles, sagging and sagging of the skin.

[0003] At present, peptides have a wide range of application prospects for skin anti-aging. While recognizing that suitable peptides can play an anti-aging role on the skin, more application problems will be faced due to different choices of peptides. For example, the problem of penetration: although peptides have biological activity, their application in cosmetics needs to consider their ability to pass through the skin barrier. Due to the different sizes and hydrophobicity of peptides, they may not be able to effectively penetrate the epidermis and dermis of the skin, limiting their deep anti-aging effects. The problem of bioavailability: Even if the peptide can penetrate the surface of the skin, the bioavailability of different peptides in the skin is also an important consideration. Low bioavailability means that the peptide cannot effectively enter the deep tissue of the skin, thereby limiting its improvement of the deep skin structure and function. Safety and sensitivity issues: Different peptides may have different reactions to different skin types. The problem of the durability and delivery of the effect, the durability and delivery performance of different peptides, as well as the impact of cost and market acceptance, etc.

[0004] More importantly, there are many types of peptides, but their respective mechanisms of action are not clear. For example, although the potential of peptides in anti-aging skin has been widely recognized, how they work through different layers of skin tissue is not yet fully understood. If the synergistic effect of peptides cannot be brought into play, it will be difficult to fundamentally improve or reverse the aging process of the skin. Therefore, more in-depth research is needed to develop truly effective products that act on all levels of the skin. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention provides a full-layer skin-acting compound peptide, which synergistically exerts full-layer skin anti-aging effects on the epidermis, basement membrane, dermis and / or nerve fibers. It has the advantages of full-layer three-dimensional anti-aging, layered precise targeting, scientific compounding of multiple peptides, matrix synergy, clear mechanism of action, efficient promotion of 7 key proteins, remodeling of true epidermal structure, low concentration onset, mild and non-irritating, etc. It is an advanced anti-aging solution under the trend of precision skin care.

[0006] In order to achieve the purpose of the present invention, the present invention provides a full-thickness skin complex peptide, wherein the complex peptide includes acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1; the complex peptide acts on the keratinocytes in the basal layer of the epidermis, the structural proteins of the dermal epidermis junction (DEJ) and / or the fibroblasts in the dermis, and synergistically exerts a full-thickness skin anti-aging effect on the epidermis, basement membrane, dermis and / or nerve fibers.

[0007] After in-depth research and continuous exploration by the inventors, and through in-depth research on the mechanism of action, it was found that the complex peptides formed by acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 can act on the epidermis, the true dermal junction (DEJ) and the dermis at the same time, and accurately activate the three key targets of epidermal basal keratinocytes, DEJ structural proteins, and dermal fibroblasts; and multiple peptides work synergistically to efficiently promote the production of 7 key proteins, reshape the true epidermis structure, and synergistically exert the anti-aging effect of the entire dermis.

[0008] Preferably, the present invention provides a full-thickness complex peptide, wherein the complex peptide acts on the keratinocytes in the basal layer of the epidermis, improves the vitality of epidermal cells by promoting the expression of glycoprotein syndecan-1 on the surface of basal layer cells; the complex peptide acts on the structural protein of the dermal epidermis junction (DEJ), tightens the dermal epidermis junction (DEJ) by promoting the expression of type IV collagen, type XVII collagen, laminin and / or integrin; the complex peptide acts on the fibroblasts in the dermis, reshapes the collagen matrix by filling the extracellular matrix (ECM), and / or precisely blocking the transmission of nerve signals, thereby synergistically exerting the full-thickness skin anti-aging effect.

[0009] Syndecan-1 is the main cell surface proteoglycan in the epidermis, mainly located on the surface of basal cells, and has important functions such as promoting epidermal cell proliferation, differentiation, migration, improving intercellular adhesion and maintaining skin homeostasis. The reduction of Syndecan-1 will lead to reduced proliferation and differentiation ability of epidermal cells, weakened intercellular communication and adhesion, resulting in loose, thinning and weakened barrier function of the epidermis. Aging leads to a significant decrease of 62% in Syndecan-1. The inventors found that the compound peptide can promote the expression of basal cell surface glycoprotein Syndecan-1, thereby promoting keratinocyte proliferation, enhancing intercellular signal communication, improving intercellular tightness and adhesion, etc., thereby improving epidermal cell vitality.

[0010] Key structural proteins such as type IV and type XVII collagen, laminin, and integrin significantly decrease with age, resulting in impaired DEJ structure and function, reduced wrinkle area, loose epidermal connection, and loose and fragile skin. The inventors have found that compound peptides can promote the expression of type IV collagen, thereby increasing the three-dimensional grid density of the dense layer of the skin, improving the stability and support of the basement membrane; compound peptides can promote the expression of type XVII collagen, thereby enhancing epidermal elasticity and cohesion, and improving the adhesion between the dermis and the DEJ layer; compound peptides can promote the expression of laminin, thereby improving the stability of the epidermal-DEJ-dermal connection; compound peptides can also promote the expression of integrins, thereby improving the stability of epidermal attachment and enhancing the adhesion between the epidermis and the DEJ layer, thereby tightening the epidermal connection (DEJ).

[0011] Collagen accounts for 72% of the entire skin, and as much as 80% of the dermis. Collagen is the main component supporting the dermis. The loss of collagen will loosen the structure of the dermis and weaken the support, thus forming a collapse and causing true wrinkles. Furthermore, the inventors have found that compound peptides can fill the extracellular matrix (ECM) and reshape the collagen matrix. For example, compound peptides can increase type I and type III collagen, thereby improving the structural support of the DEJ layer and dermis, improving skin relaxation and fading true wrinkles; compound peptides can increase elastin, thereby increasing skin elasticity, flexibility and resistance to gravity sagging. At the same time, compound peptides can accurately block the transmission of nerve signals, such as reducing the secretion of acetylcholine, thereby relaxing muscles and fading dynamic wrinkles.

[0012] Therefore, acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and acetyl hexapeptide-1 in the complex peptide can synergistically exert anti-aging effects on the entire dermis.

[0013] Preferably, the present invention provides a full-thickness skin anti-aging effect of a complex peptide, wherein the full-thickness skin anti-aging effect is achieved by affecting the complex peptide on the expression of extracellular matrix-related genes and / or proteins, on the expression of basement membrane-related genes, on the expression of cell aging marker genes, on the secretion of cellular acetylcholine and / or on the expression of cell barrier-related genes.

[0014] Preferably, the present invention provides a full-thickness effect complex peptide, wherein the effect on the expression of extracellular matrix-related genes and / or proteins includes increasing the expression of type I collagen and / or type III collagen, inhibiting the expression of matrix metalloproteinase-1 (MMP-1), increasing the expression of elastin, inhibiting the expression of matrix metalloproteinase-3 (MMP-3), and / or inhibiting the expression of matrix metalloproteinase-9 (MMP-9); the effect on the expression of basement membrane-related genes includes increasing the expression of type IV collagen, increasing the expression of laminin, and / or increasing the expression of integrin β1; the effect on the expression of cell aging marker genes includes significantly decreasing the expression of p21 (Cdkn1a); the effect on the secretion of cell acetylcholine includes significantly decreasing the secretion of cell acetylcholine; the effect on the expression of cell barrier-related genes includes increasing the expression of syndecan-1, and / or increasing the expression of type XVII collagen.

[0015] The inventors designed a precise effect comparison experiment to study the effects of the compound peptides on the expression of extracellular matrix-related genes and proteins, and found that after treatment with the compound peptides, the increase rate of type I collagen and type III collagen expression in human skin fibroblasts (HSF cells) was higher than that when palmitoyl hexapeptide-14 of the same content was used alone, the inhibition rate of MMP-1 expression in HSF cells was higher than that when palmitoyl hexapeptide-14 of the same content was used alone, the increase rate of elastin expression in HSF cells was higher than that when palmitoyl hexapeptide-12 of the same content was used alone, and the inhibition rates of MMP-3 and MMP-9 were higher than those when palmitoyl hexapeptide-14 of the same content was used alone.

[0016] Regarding the effect of compound peptides on the expression of basement membrane-related genes, it was found that after treatment with compound peptides, the increase rate of type IV collagen and laminin expression of human skin fibroblasts (HSF cells) was higher than that of using the same amount of hexapeptide-9 alone, and the increase rate of HSF cell integrin β1 expression was higher than that of using the same amount of hexapeptide-9 alone.

[0017] Regarding the effect of compound peptides on the expression of cell aging marker genes, it was found that after treatment with compound peptides, the expression of p21 (Cdkn1a) in human skin fibroblasts (HSF cells) decreased significantly in a concentration-dependent manner, indicating that the compound peptides can effectively inhibit cell aging and restore normal cell cycle.

[0018] Regarding the effect of the compound peptide on the acetylcholine secretion of rat adrenal medullary pheochromoma cells (PC12 cells), it was found that after treatment with the compound peptide, the acetylcholine secretion of PC12 cells decreased significantly, and the acetylcholine secretion was lower than that of using the same amount of acetyl hexapeptide-1 alone.

[0019] Regarding the effect of the complex peptide on the expression of cell barrier-related genes, it was found that after treatment with the complex peptide, the increase rate of syndecan-1 expression in human immortalized keratinocytes (HaCaT cells) was higher than that when the same amount of acetyl tetrapeptide-11 was used alone, and the increase rate of type XVII collagen expression in HaCaT cells was higher than that when the same amount of acetyl tetrapeptide-11 was used alone.

[0020] Preferably, the present invention provides a full-thickness complex peptide, wherein the complex peptide inhibits the degradation of extracellular matrix by activating the transforming growth factor-β1 / Smads (TGF-β1 / Smads) signaling pathway, promoting fibroblast proliferation, promoting extracellular matrix (ECM) synthesis, and / or reducing the activity of matrix metalloproteinases; the extracellular matrix (ECM) includes type I collagen, type III collagen, elastin, hyaluronic acid and / or glycosaminoglycans.

[0021] The TGF-β1 / Smads signal transduction pathway has multiple biological effects and plays an important role in tissue repair and extracellular matrix deposition. TGF-β and Wnt1 bind to their stem cell surface receptors and initiate downstream signal transduction, ultimately leading to the nuclear translocation of Smad2 / 3 and CBP / β-Catenin transcriptional regulators, respectively. This leads to upregulation of target gene expression, which further enhances fibroblast differentiation and the production and secretion of ECM proteins. Through precisely designed experiments, the inventors have shown that the compound peptide can significantly promote the phosphorylation of smad2 / 3 proteins in human skin fibroblasts (HSF cells) damaged by UVB, thereby promoting the production and secretion of ECM proteins such as collagen and elastin, achieving anti-aging effects.

[0022] Preferably, the present invention provides a full-thickness composite peptide, wherein the proportion of acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 in the composite peptide is between 0.05 and 0.80, respectively. The inventors have continuously explored and found that when the proportion of each peptide in the composite peptide is within this range, it can better synergistically exert the full-thickness skin anti-aging effect.

[0023] Preferably, the present invention provides a full-cortical effect composite peptide, wherein the proportion of acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 in the composite peptide is between 0.10 and 0.60 respectively. When the proportion of each peptide in the composite peptide is within this preferred range, the purpose of the present invention can be better achieved, and the effect is outstanding.

[0024] Preferably, the present invention provides a full-thickness composite peptide, wherein the composite peptide is used for the preparation of post-medical beauty project synergistic products, prolonging the post-operative maintenance period of medical beauty projects and / or daily anti-aging; medical beauty projects include non-invasive medical beauty and / or invasive medical beauty; non-invasive medical beauty includes photorejuvenation, acid peeling, picosecond, ultra-picosecond, Thermage, thermolift, hyaluronic acid filling, non-ablative fractional laser, face-slimming injection, and / or ultrasonic knife; invasive medical beauty includes microneedle, mesotherapy, picosecond or ultra-picosecond blasting, and / or ablative fractional laser; synergistic products include facial masks, water, lotion, cream, ointment and / or powder; daily anti-aging includes improving expression lines, preventing true lines, lifting and firming, promoting collagen regeneration and / or preventing skin sagging, which is achieved by external application and / or dermal introduction, and dermal introduction includes instrument introduction.

[0025] Preferably, the present invention provides a full-layer skin action complex peptide, wherein the complex peptide is used to prepare cosmetic and / or beauty and care products, including essence water / toner, lotion, face cream, beauty cream, foundation, concealer, isolation, setting makeup, eye shadow, contouring, blush, lipstick, single-use stock solution, freeze-dried powder / freeze-dried liquid, facial mask liquid / mask powder / frozen mask, and / or functional medical beauty products.

[0026] Aiming at various problems in the use of polypeptide products in the field of anti-aging, the inventors, through in-depth exploration and unremitting experiments, obtained a complex peptide containing acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1, and verified through experiments that the complex peptide can synergistically exert full-thickness skin anti-aging effects on the epidermis, basement membrane, dermis and / or nerve fibers, and has multiple advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Schematic diagram of the structure of the epidermis, dermal-epidermal junction (DEJ), and dermis; Figure 2A comparison chart showing the effect of the composite peptides provided by the present invention on the expression of syndecan-1 in HaCaT cells; Figure 3 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of type IV collagen in HSF cells; Figure 4 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of type XVII collagen in HaCaT cells; Figure 5 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of laminin in HSF cells; Figure 6 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of integrin β1 in HSF cells; Figure 7 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of type I collagen in HSF cells; Figure 8 A comparison chart of the effect of the composite peptide provided by the present invention on the expression of elastin in HSF cells; Fig. 9 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of type III collagen in HSF cells; Fig.10 A comparison chart of the effect of the composite peptide provided by the present invention on the expression of MMP-1 in HSF cells; Fig.11 A comparison chart of the effects of the composite peptides provided by the present invention on the expression of MMP-3 in HSF cells; Fig.12 A comparison chart of the effect of the composite peptide provided by the present invention on the expression of MMP-9 in HSF cells; Fig.13 A comparison chart of the effects of the composite peptides provided by the present invention on the secretion of acetylcholine in PC12 cells; Fig.14 A comparison chart showing the effect of the composite peptide provided by the present invention on the expression of p21 (Cdkn1a) in HSF cells; Fig.15 A comparison chart showing the effects of the composite peptides provided by the present invention on the expression level of p-smad2 / 3 protein. DETAILED DESCRIPTION

[0029] In order to further illustrate the present invention, examples are given below. It should be noted that these examples are purely illustrative. The purpose of providing these examples is to fully illustrate the meaning and content of the present invention, but the present invention is not limited to the scope of the embodiments. Example 1 A composite peptide with full-thickness effect

[0030] A full-thickness complex peptide, wherein the complex peptide includes acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1; the complex peptide acts on epidermal basal layer keratinocytes, dermal epidermal junction (DEJ) structural proteins and / or dermal fibroblasts, and synergistically exerts full-thickness skin anti-aging effects on the epidermis, basement membrane, dermis and / or nerve fibers. Among them, the complex peptide acts on epidermal basal layer keratinocytes, and improves epidermal cell vitality by promoting the expression of glycoprotein syndecan-1 on the surface of basal layer cells. The complex peptide acts on dermal epidermal junction (DEJ) structural proteins, and tightens the dermal epidermal junction (DEJ) by promoting the expression of type IV collagen, type XVII collagen, laminin and / or integrin. The complex peptides act on dermal fibroblasts, synergistically exerting anti-aging effects on the entire dermis by filling the extracellular matrix (ECM), reshaping the collagen matrix, and / or precisely blocking the transmission of nerve signals.

[0031] In one embodiment, the full-thickness skin anti-aging effect is achieved by affecting the complex peptide on the expression of extracellular matrix-related genes and / or proteins, the expression of basement membrane-related genes, the expression of cell aging marker genes, the secretion of cellular acetylcholine, and / or the expression of cell barrier-related genes.

[0032] Among them, the effects on the expression of extracellular matrix-related genes and / or proteins include increasing the expression of type I collagen and / or type III collagen, inhibiting the expression of matrix metalloproteinase-1 (MMP-1), increasing the expression of elastin, inhibiting the expression of matrix metalloproteinase-3 (MMP-3), and / or inhibiting the expression of matrix metalloproteinase-9 (MMP-9); the effects on the expression of basement membrane-related genes include increasing the expression of type IV collagen, increasing the expression of laminin, and / or increasing the expression of integrin β1; the effects on the expression of cell senescence marker genes include significantly decreasing the expression of p21 (Cdkn1a); the effects on the secretion of cell acetylcholine include significantly decreasing the secretion of cell acetylcholine; the effects on the expression of cell barrier-related genes include increasing the expression of syndecan-1, and / or increasing the expression of type XVII collagen.

[0033] Furthermore, the complex peptide inhibits the degradation of extracellular matrix by activating the TGF-β1 / Smads signaling pathway, promoting fibroblast proliferation, promoting extracellular matrix (ECM) synthesis, and / or reducing the activity of matrix metalloproteinases; the extracellular matrix (ECM) includes type I collagen, type III collagen, elastin, hyaluronic acid, glycosaminoglycans, etc.

[0034] In one embodiment, the proportion of acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 in the composite peptide is between 0.05 and 0.80, respectively.

[0035] In another embodiment, the proportion of acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 in the composite peptide is between 0.10 and 0.60, respectively.

[0036] The compound peptide can be used for the preparation of enhancement products after medical beauty projects, to extend the maintenance period after medical beauty projects and / or daily anti-aging. Among them, medical beauty projects include non-invasive medical beauty and / or invasive medical beauty; non-invasive medical beauty includes photorejuvenation, acid peeling, picosecond, ultra-picosecond, Thermage, thermolift, hyaluronic acid filling, non-ablative fractional laser, face-lifting needle, ultrasonic knife, etc.; invasive medical beauty includes microneedle, water light needle, picosecond or ultra-picosecond blasting, ablative fractional laser, etc. Enhanced products include facial masks, water, lotion, cream, ointment, powder, etc. Daily anti-aging includes improving expression lines, preventing true lines, lifting and firming, promoting collagen regeneration and / or preventing skin sagging, which is achieved through external application and / or skin introduction, and skin introduction includes instrument introduction.

[0037] In addition, the composite peptide can be used to prepare cosmetic and / or beauty and care products, including essence water / toner, lotion, face cream, beauty cream, foundation, concealer, isolation, setting makeup, eye shadow, contouring, blush, lipstick, single-use stock solution, freeze-dried powder / freeze-dried liquid, facial mask liquid / mask powder / frozen mask, and / or functional medical beauty products. Example 2 The compound peptide acts on the keratinocytes in the basal layer of the epidermis to promote the expression of glycoprotein Syndecan-1 on the surface of the basal layer cells

[0038] Studies have shown that acetyl tetrapeptide-11 can promote the expression of glycoprotein syndecan-1 on the surface of basal cells, improve the vitality of epidermal cells, and revitalize and renew the epidermis. The inventors designed relevant experiments and used PCR to detect the effect of the complex peptide on the expression level of syndecan-1 gene in human immortalized keratinocytes (HaCaT cells), and compared it with the effect of acetyl tetrapeptide-11 single peptide.

[0039] Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a ratio of 5:5:5:5:80 in the form of a solution at a process temperature below 40°C to prepare compositions containing different concentrations of compound peptides (containing acetyl tetrapeptide-11 at 0.5 μg / mL, 1 μg / mL, 2 μg / mL and 5 μg / mL, respectively), without direct contact with aldehyde-containing compounds, metal ions, strong acids, strong bases and / or active enzymes during the process.

[0040] Experimental process: Control group: untreated HaCaT cells, PCR method was used to detect the expression of syndecan-1 gene.

[0041] Single peptide group: HaCaT cells were treated with 5 μg / mL acetyl tetrapeptide-11 single peptide solution for 24 h, and the syndecan-1 gene expression level was detected by PCR.

[0042] Experimental group: HaCaT cells were treated with four groups of compound peptide solutions containing 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 5 μg / mL of acetyl tetrapeptide-11, respectively, for 24 h, and the expression of syndecan-1 gene was detected by PCR.

[0043] Experimental results: The syndecan-1 gene expression level of cells in the control group was set to 1.0; The relative expression of syndecan-1 gene in the single peptide group treated with 5 μg / mL acetyl tetrapeptide-11 solution was 1.6; In the experimental groups treated with composite peptide solutions containing 0.5μg / mL, 1μg / mL, 2μg / mL, and 5μg / mL of acetyl tetrapeptide-11, the relative expression levels of syndecan-1 gene were 1.7, 1.8, 2.2, and 3.9, respectively, which were increased by 70%, 80%, 120%, and 290% compared with the control group.

[0044] It can be seen that the composite peptide provided by the present invention has a beneficial effect on the expression of syndecan-1 gene at low concentrations and at the same concentration, and the impact value is greater than that of acetyl tetrapeptide-11 single peptide, and has a stronger effect on epidermal basal layer keratinocytes.

[0045] Therefore, the composite peptide provided by the present invention acts on the keratinocytes in the basal layer of the epidermis, and improves the activity of the epidermal cells by promoting the expression of glycoprotein Syndecan-1 on the surface of the basal layer cells. Example 3 The complex peptide acts on the structural protein of the dermal epidermal junction (DEJ) to promote the expression of type IV collagen, type XVII collagen, laminin and / or integrin

[0046] Studies have shown that acetyl tetrapeptide-11 and hexapeptide-9 can promote the expression of type IV and type XVII collagen, laminin, integrin, etc., and tighten DEJ. The inventors designed relevant experiments and used PCR to detect the effects of the compound peptides on the gene expression levels of type IV collagen, laminin, and integrin β1 in HSF cells and the gene expression levels of type XVII collagen in HaCaT cells, and compared them with the effects of acetyl tetrapeptide-11 and hexapeptide-9 single peptides.

[0047] 1. Effect of compound peptides on the expression level of type IV collagen gene in HSF cells Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a ratio of 1:1:1:1:6 in the form of a solution at a process temperature below 40ºC to prepare compositions containing different concentrations of compound peptides (containing hexapeptide-9 at 2μg / mL, 8μg / mL and 20μg / mL, respectively). During the process, there is no direct contact with aldehyde-containing compounds, ketones, metal ions, strong acids, strong bases and / or active enzymes.

[0048] Experimental process: Skin damage modeling: Prepare human skin fibroblasts (HSF cells), cause cell damage through ultraviolet irradiation, and establish a cell damage model.

[0049] The control group, model group, single peptide group and experimental group were set up: Control group: The expression of type IV collagen gene was detected by PCR in human skin fibroblasts (HSF cells) that were not irradiated with ultraviolet light.

[0050] Model group: The damaged cells were cultured for 24 hours, and the expression of type IV collagen gene was detected by PCR.

[0051] Single peptide group: HSF damaged cells were treated with 20 μg / mL hexapeptide-9 single peptide solution for 24 h, and the expression of type IV collagen gene was detected by PCR.

[0052] Experimental group: Three groups of compound peptide solutions containing 2μg / mL, 8μg / mL, and 20μg / mL of hexapeptide-9 were used to treat HSF damaged cells for 24 h, and the expression of type IV collagen gene was detected by PCR.

[0053] Experimental results: The gene expression of type IV collagen in the control group cells was set to 1.0; Under UVB conditions, the relative gene expression of type IV collagen in the model group cells was significantly reduced to 0.28; In the single peptide group treated with 20 μg / mL hexapeptide-9, the relative gene expression of type IV collagen was 0.64; In the experimental groups treated with three groups of composite peptide solutions containing 2μg / mL, 8μg / mL, and 20μg / mL of hexapeptide-9, the relative gene expression levels of type IV collagen were 0.92, 1.14, and 1.52, respectively, which increased by 229%, 307%, and 443% compared with the model group.

[0054] It can be seen that the complex peptide provided by the present invention has a beneficial effect on the gene expression of type IV collagen at low concentrations and at the same concentration, and the impact value is greater than that of hexapeptide-9 single peptide, and can better act on the dermal epidermal junction (DEJ) structural protein.

[0055] 2. Effect of the complex peptide on the expression level of type XVII collagen gene in HaCaT cells Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a ratio of 2:1:1:1:5 in the form of a solution at a process temperature below 40°C to prepare compositions containing different concentrations of compound peptides (containing acetyl tetrapeptide-11 at 0.5 μg / mL, 1 μg / mL, 2 μg / mL and 5 μg / mL, respectively), without direct contact with aldehyde-containing compounds, metal ions, strong acids, strong bases and / or active enzymes during the process.

[0056] Experimental process: Control group: The gene expression of type XVII collagen was detected by PCR in untreated HaCaT cells.

[0057] Single peptide group: HaCaT cells were treated with 5 μg / mL acetyl tetrapeptide-11 single peptide solution for 24 h, and the gene expression of type XVII collagen was detected by PCR.

[0058] Experimental group: HaCaT cells were treated with four groups of composite peptide solutions containing 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 5 μg / mL of acetyl tetrapeptide-11, respectively, for 24 h, and the gene expression of type XVII collagen was detected by PCR.

[0059] Experimental results: The gene expression of type XVII collagen in the control group was set to 1.0; In the single peptide group treated with 5 μg / mL acetyl tetrapeptide-11 solution, the relative gene expression of type XVII collagen was 1.31; In the experimental groups treated with the composite peptide solutions containing 0.5μg / mL, 1μg / mL, 2μg / mL, and 5μg / mL of acetyl tetrapeptide-11, the relative gene expressions of type XVII collagen were 1.01, 1.01, 1.05, and 1.45, respectively, which were increased by 1%, 1%, 5%, and 45% compared with the control group.

[0060] It can be seen that the complex peptide provided by the present invention has a beneficial effect on the gene expression of type XVII collagen; at the same concentration, the impact value is greater than that of acetyl tetrapeptide-11 single peptide, and can better act on the dermal epidermal junction (DEJ) structural protein.

[0061] 3. Effects of compound peptides on the gene expression levels of laminin and integrin β1 in HSF cells Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a ratio of 2:2:1:1:4 in the form of a solution at a process temperature below 40ºC to prepare compositions containing different concentrations of compound peptides (containing hexapeptide-9 at 2μg / mL, 8μg / mL and 20μg / mL, respectively). During the process, there is no direct contact with aldehyde-containing compounds, metal ions, strong acids, strong bases and / or active enzymes.

[0062] Experimental process: Skin damage modeling: Prepare HSF cells, cause cell damage through ultraviolet irradiation, and establish a cell damage model.

[0063] The control group, model group, single peptide group and experimental group were set up: Control group: The gene expression levels of laminin and integrin β1 were detected by PCR in HSF cells that were not irradiated with UV light.

[0064] Model group: The damaged cells were cultured for 24 h, and the gene expression levels of laminin and integrin β1 were detected by PCR.

[0065] Single peptide group: HSF damaged cells were treated with 20 μg / mL hexapeptide-9 single peptide solution for 24 h, and the gene expression levels of laminin and integrin β1 were detected by PCR.

[0066] Experimental group: Three groups of mixed peptide solutions containing 2μg / mL, 8μg / mL, and 20μg / mL of hexapeptide-9 were used to treat HSF damaged cells for 24 h, and the gene expression levels of laminin and integrin β1 were detected by PCR.

[0067] Experimental results: The expression level of each gene in the control group cells was set to 1.0.

[0068] (1) Effects of complex peptides on the gene expression of laminin Under UVB conditions, the relative gene expression of laminin in the model group cells was significantly reduced to 0.35; In the single peptide group treated with 20 μg / mL hexapeptide-9, the relative expression of the laminin gene was 0.79; In the experimental groups treated with three mixed peptide solutions containing 2μg / mL, 8μg / mL, and 20μg / mL of hexapeptide-9, the relative gene expression levels of laminin were 0.84, 1.10, and 1.81, respectively, which increased by 140%, 214%, and 417% relative to the model group.

[0069] (2) Effect of compound peptides on the gene expression of integrin β1 Under UVB conditions, the relative gene expression of integrin β1 in the model group cells was significantly reduced to 0.75; In the single peptide group treated with 20 μg / mL hexapeptide-9, the relative gene expression of integrin β1 was 1.2; In the experimental groups treated with three groups of composite peptide solutions containing 2μg / mL, 8μg / mL, and 20μg / mL of hexapeptide-9, the relative gene expression levels of integrin β1 were 0.92, 1.25, and 1.83, respectively, which increased by 23%, 67%, and 144% relative to the model group.

[0070] It can be seen that the complex peptide provided by the present invention has a beneficial effect on the gene expression of laminin and integrin β1 at low concentrations and the same concentration, and the impact value is greater than that of hexapeptide-9 single peptide, and can better act on the true epidermal junction (DEJ) structural protein.

[0071] Therefore, the complex peptide acts on the structural proteins of the dermal epidermal junction (DEJ) and tightens the dermal epidermal junction (DEJ) by promoting the expression of type IV collagen, type XVII collagen, laminin and / or integrin. Example 4 The compound peptide acts on dermal fibroblasts, fills the extracellular matrix (ECM), and reshapes the collagen matrix

[0072] Studies have shown that palmitoyl hexapeptide-12 and palmitoyl hexapeptide-14 have a certain effect of filling the extracellular matrix (ECM). The inventors designed relevant experiments and used PCR to detect the effects of the compound peptides on the extracellular matrix (ECM) of human skin fibroblasts (HSF cells), including the expression levels of type I collagen and / or type III collagen, elastin, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and / or matrix metalloproteinase-9 (MMP-9), and compared them with the effects of palmitoyl hexapeptide-12 and palmitoyl hexapeptide-14 single peptides.

[0073] Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a solution form at a ratio of 2:1:1:2:4 at a process temperature below 40ºC to prepare compositions containing different concentrations of compound peptides (containing 0.5μg / mL, 1μg / mL, 5μg / mL of palmitoyl hexapeptide-14; 1μg / mL, 2μg / mL, 10μg / mL of palmitoyl hexapeptide-12), without direct contact with aldehydes and ketones, metal ions, strong acids, strong bases and / or active enzymes during the process.

[0074] Experimental process: Skin damage modeling: Prepare HSF cells, cause cell damage through ultraviolet irradiation, and establish a cell damage model.

[0075] The control group, model group, single peptide group and experimental group were set up: Control group: The gene expression levels of type I collagen, type III collagen, elastin, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9) were detected by PCR in HSF cells that were not irradiated with ultraviolet light.

[0076] Model group: The damaged cells were cultured for 24 h, and the gene expression levels of type I collagen, type III collagen, elastin, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9) were detected by PCR.

[0077] Single peptide group: (1) HSF damaged cells were treated with 5 μg / mL palmitoyl hexapeptide-14 single peptide solution for 24 h, and the gene expression levels of type I collagen, type III collagen, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9) were detected by PCR.

[0078] (2) HSF damaged cells were treated with a 10 μg / mL palmitoyl hexapeptide-12 single peptide solution for 24 h, and the gene expression of elastin was detected by PCR.

[0079] Experimental group: Three groups of compound peptide solutions containing 0.5μg / mL, 1μg / mL, and 5μg / mL of palmitoyl hexapeptide-14 and 1μg / mL, 2μg / mL, and 10μg / mL of palmitoyl hexapeptide-12 were used to treat HSF damaged cells for 24 h, and the gene expression levels of type I collagen, type III collagen, elastin, matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9) were detected by PCR.

[0080] Experimental results: The expression level of each gene in the control group cells was set to 1.0.

[0081] (1) Effect of compound peptides on gene expression of type I collagen Under UVB conditions, the relative gene expression of type I collagen in the model group cells was significantly reduced to 0.43; In the single peptide group treated with 5 μg / mL palmitoyl hexapeptide-14, the relative gene expression of type I collagen was 0.87; In the experimental groups treated with three groups of composite peptide solutions containing 0.5μg / mL, 1μg / mL, and 5μg / mL of palmitoyl hexapeptide-14, the relative gene expression levels of type I collagen were 0.90, 0.92, and 1.20, respectively, which increased by 109%, 114%, and 180% relative to the model group.

[0082] It can be seen that the complex peptide provided by the present invention has a beneficial effect on the gene expression of type I collagen at low concentrations and at the same concentration, and the impact value is greater than that of palmitoyl hexapeptide-14 single peptide, and can better act on dermal fibroblasts and fill the extracellular matrix (ECM).

[0083] (2) Effect of compound peptides on the gene expression of elastin Under UVB conditions, the relative gene expression of elastin in the model group cells was significantly reduced to 0.51; In the single peptide group treated with 10 μg / mL palmitoyl hexapeptide-12, the relative gene expression of elastin was 0.95; In the experimental groups treated with three groups of compound peptide solutions containing 1μg / mL, 2μg / mL, and 10μg / mL of palmitoyl hexapeptide-12, the relative gene expression of elastin was 0.93, 0.94, and 1.01, respectively, which increased by 82%, 84%, and 98% relative to the model group.

[0084] It can be seen that the complex peptide provided by the present invention has a beneficial effect on the gene expression of elastin; at the same concentration, the impact value is greater than that of palmitoyl hexapeptide-12 single peptide, and can better act on dermal fibroblasts and fill the extracellular matrix (ECM).

[0085] (3) Effect of compound peptides on gene expression of type III collagen Under UVB conditions, the relative gene expression of type III collagen in the model group cells was significantly reduced to 0.46; In the single peptide group treated with 5 μg / mL palmitoyl hexapeptide-14, the relative gene expression of type III collagen was 0.64; In the experimental groups treated with three groups of composite peptide solutions containing 0.5μg / mL, 1μg / mL, and 5μg / mL of palmitoyl hexapeptide-14, the relative gene expression levels of type III collagen were 0.65, 0.77, and 0.90, respectively, which increased by 41%, 67%, and 96% relative to the model group.

[0086] It can be seen that the complex peptide provided by the present invention has a beneficial effect on the gene expression of type III collagen at low concentrations and at the same concentration, and the impact value is greater than that of palmitoyl hexapeptide-14 single peptide, and can better act on dermal fibroblasts and fill the extracellular matrix (ECM).

[0087] (4) Effect of the complex peptide on the gene expression of matrix metalloproteinase-1 (MMP-1) Under UVB conditions, the relative expression level of MMP-1 in the model group cells increased significantly to 1.96; In the single peptide group treated with 5 μg / mL palmitoyl hexapeptide-14, the relative gene expression of MMP-1 was 1.52; In the experimental groups treated with three groups of composite peptide solutions containing 0.5μg / mL, 1μg / mL, and 5μg / mL of palmitoyl hexapeptide-14, the relative gene expression levels of MMP-1 were 1.71, 1.42, and 1.26, respectively, which were reduced by 13%, 28%, and 36% relative to the model group.

[0088] It can be seen that the composite peptide provided by the present invention has an inhibitory effect on the gene expression of MMP-1 at medium and low concentrations and at the same concentration, and the impact value is greater than that of palmitoyl hexapeptide-14 single peptide, and can better act on dermal fibroblasts and fill the extracellular matrix (ECM).

[0089] (5) Effect of the complex peptide on the gene expression of matrix metalloproteinase-3 (MMP-3) Under UVB conditions, the relative expression level of MMP-3 in the model group cells increased significantly to 1.18; In the single peptide group treated with 5 μg / mL palmitoyl hexapeptide-14, the relative gene expression of MMP-3 was 0.78; In the experimental groups treated with three groups of composite peptide solutions containing 0.5μg / mL, 1μg / mL, and 5μg / mL of palmitoyl hexapeptide-14, the relative gene expression levels of MMP-3 were 0.99, 0.92, and 0.73, respectively, which were reduced by 16%, 22%, and 38% relative to the model group.

[0090] It can be seen that the composite peptide provided by the present invention has an inhibitory effect on the gene expression of MMP-3; at the same concentration, the impact value is greater than that of palmitoyl hexapeptide-14 single peptide, and can better act on dermal fibroblasts and fill the extracellular matrix (ECM).

[0091] (6) Effect of the complex peptide on the gene expression of matrix metalloproteinase-9 (MMP-9) Under UVB conditions, the relative expression level of MMP-9 in the model group cells increased significantly to 1.58; In the single peptide group treated with 5 μg / mL palmitoyl hexapeptide-14, the relative gene expression of MMP-9 was 0.94; In the experimental groups treated with three groups of composite peptide solutions containing 0.5μg / mL, 1μg / mL, and 5μg / mL of palmitoyl hexapeptide-14, the relative gene expression levels of MMP-9 were 1.04, 0.94, and 0.75, respectively, which were reduced by 34%, 41%, and 53% relative to the model group.

[0092] It can be seen that the composite peptide provided by the present invention has an inhibitory effect on the gene expression of MMP-9; at the same concentration, the impact value is greater than that of palmitoyl hexapeptide-14 single peptide, and can better act on dermal fibroblasts and fill the extracellular matrix (ECM).

[0093] Therefore, this complex peptide acts on dermal fibroblasts to fill the extracellular matrix (ECM) and reshape the collagen matrix. Example 5: Compound peptides can accurately block the transmission of neural signals

[0094] Studies have shown that acetyl hexapeptide-1 can block the transmission of nerve signals and has a certain effect on reducing wrinkles. The inventors designed a related experiment, using the ELISA method to detect the effect of the compound peptide on the acetylcholine secretion of rat adrenal medullary pheochromocytoma cells (PC12 cells), and compared the effect with that of acetyl hexapeptide-1 alone.

[0095] Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a ratio of 1:1:1:1:1 in the form of a solution at a process temperature below 40ºC to prepare compositions containing different concentrations of composite peptides (containing acetyl hexapeptide-1 at 1μg / mL, 2μg / mL and 10μg / mL, respectively), without direct contact with aldehyde-containing compounds, metal ions, strong acids, strong bases and / or active enzymes during the process.

[0096] Experimental process: Control group: The secretion of acetylcholine in untreated PC12 cells was detected by ELISA.

[0097] Single peptide group: PC12 cells were treated with 10 μg / mL acetyl hexapeptide-1 single peptide solution for 24 h, and the acetylcholine secretion was detected by ELISA.

[0098] Experimental group: PC12 cells were treated with three groups of mixed peptide solutions containing 1μg / mL, 2μg / mL, and 10μg / mL of acetyl hexapeptide-1 for 24 h, and the acetylcholine secretion was detected by ELISA.

[0099] Experimental results: The acetylcholine secretion of cells in the control group was set to 1.0; In the single peptide group treated with 10 μg / mL acetyl hexapeptide-1 solution, the relative secretion of acetylcholine was 0.65; The relative secretions of acetylcholine in the experimental groups treated with the composite peptide solutions containing 1 μg / mL, 2 μg / mL, and 10 μg / mL of acetyl hexapeptide-1 were 0.93, 0.72, and 0.59, respectively, which were 7%, 28%, and 41% lower than those in the control group.

[0100] It can be seen that the composite peptide provided by the present invention has an inhibitory effect on the secretion of acetylcholine; at the same concentration, the impact value is greater than that of acetyl hexapeptide-1 single peptide, and has a significant wrinkle-lightening effect. Example 6 Effect of compound peptides on the expression of cell senescence marker genes

[0101] p21 (Cdkn1a) is a cell cycle-dependent kinase inhibitor that is usually highly expressed during cell senescence and serves as a biological marker of cell senescence. In p53-mediated cell senescence, p21 (Cdkn1a) plays a key role and can be used as an important marker for identifying cell senescence. With the progression of aging, the expression level of p21 (Cdkn1a) in senescent cells increases significantly. In experimental studies, by intervening in aging models, such as administering specific drugs or treatments, if a significant decrease in the expression of p21 (Cdkn1a) is observed, it can be suggested that the intervention can alleviate cell cycle arrest and delay cell senescence.

[0102] The inventors designed relevant experiments to verify the effect of the complex peptide on the expression of cell aging marker genes.

[0103] Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a ratio of 0.5:2:2:3:2.5 in the form of a solution at a process temperature below 40ºC to prepare a composition containing different concentrations of compound peptides (5μg / mL, 10μg / mL, 20μg / mL, 50 μg / mL), and there is no direct contact with aldehyde-containing compounds, metal ions, strong acids, strong bases and / or active enzymes during the process.

[0104] Experimental process: Skin damage modeling: Prepare human skin fibroblasts (HSF cells), cause cell damage through ultraviolet irradiation, and establish a cell damage model.

[0105] Set up control group, model group and experimental group: Control group: The gene expression of p21 (Cdkn1a) was detected by PCR in HSF cells that were not irradiated with UV light.

[0106] Model group: The damaged cells were cultured for 24 hours, and the gene expression of p21 (Cdkn1a) was detected by PCR.

[0107] Experimental group: Four groups of compound peptide solutions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL were used to treat HSF damaged cells for 24 h, and the gene expression of p21 (Cdkn1a) was detected by PCR.

[0108] Experimental results: The gene expression of p21 (Cdkn1a) in the control group cells was set to 1.0; Under UVB conditions, the relative gene expression of p21 (Cdkn1a) in the model group cells increased significantly to 3.6; In the experimental groups treated with 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL of the four mixed peptide solutions, the relative expression levels of p21 (Cdkn1a) gene were 1.83, 1.60, 1.48, and 1.13, respectively, which were decreased by 49%, 56%, 59%, and 69% compared with the model group.

[0109] It can be seen that the compound peptide provided by the present invention significantly inhibits the expression of p21 (Cdkn1a) gene at low concentrations and the same concentration, produces significant beneficial effects, and shows concentration dependence, indicating that the anti-aging peptide can effectively inhibit cell aging and restore normal cell cycle. Example 7 Compound peptide activates TGF-β1 / Smads signaling pathway

[0110] Signals are transduced through the Smads protein family, of which Smad2 and Smad3 are the main transmission molecules. They form complexes after being phosphorylated, and Smad4 assists in forming stable complexes to enter the cell nucleus. After entering the cell nucleus, these complexes bind to specific sequences on DNA and act as transcription factors to regulate the transcription of target genes, such as interacting with nuclear transcription factors such as connective tissue growth factor (CTGF). By regulating the transcription and expression of multiple genes such as CTGF and NF-κB, the TGF-β1 / Smads signaling pathway affects cell growth, signal transduction networks, extracellular matrix (ECM), and negative feedback systems. The inventors designed relevant experiments to verify the effect of the complex peptide on the TGF-β1 / Smads signaling pathway.

[0111] Experimental preparation: Acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl hexapeptide-14, palmitoyl hexapeptide-12 and hexapeptide-9 are mixed in a solution form at a ratio of 2:0.5:0.5:3:4 at a process temperature below 40ºC to prepare compositions containing different concentrations of compound peptides (5μg / mL, 10μg / mL, 20μg / mL, 50 μg / mL), without direct contact with aldehyde-containing compounds, metal ions, strong acids, strong bases and / or active enzymes during the process.

[0112] Experimental process: Skin damage modeling: Prepare human skin fibroblasts (HSF cells), cause cell damage through ultraviolet irradiation, and establish a cell damage model.

[0113] Set up control group, model group and experimental group: Control group: Normal HSF cells that were not irradiated with ultraviolet light were used to detect the effect of the compound peptides on the expression of p-smad2 / 3 protein in HSF cells using Western blot.

[0114] Model group: The damaged cells were cultured for 24 h, and the expression of p-smad2 / 3 protein in HSF cells was detected by Western blot.

[0115] Experimental group: Four groups of compound peptide solutions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL were used to treat HSF damaged cells for 24 h, and the effect of compound peptides on the expression of p-smad2 / 3 protein in HSF cells was detected by Western blot.

[0116] Experimental results: Under UVB conditions, the relative expression of p-smad2 / 3 protein in the control group cells was 0.60; Under UVB conditions, the expression of p-smad2 / 3 protein in the model group cells was significantly reduced, with a relative expression of 0.49; In the experimental groups treated with 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL of the four mixed peptide solutions, the relative expression levels of p-smad2 / 3 protein were 0.68, 0.75, 1.10, and 1.41, respectively, which increased by 39%, 53%, 124%, and 188% relative to the model group.

[0117] It can be seen that the composite peptide provided by the present invention can significantly promote the phosphorylation of smad2 / 3 protein in UVB-damaged HSF cells at a lower concentration, activate the TGF-β1 / Smads signaling pathway, and then promote the production and secretion of ECM proteins such as collagen and elastin, thereby promoting fibroblast proliferation, promoting extracellular matrix (ECM) synthesis, and reducing the activity of matrix metalloproteinases to inhibit the degradation of extracellular matrix, thereby achieving the purpose of resisting skin aging. Among them, the extracellular matrix (ECM) includes type I collagen, type III collagen, elastin, hyaluronic acid, glycosaminoglycans, etc.

[0118] Experiments show that the compound peptide has a synergistic beneficial effect on the expression of extracellular matrix-related genes or proteins, such as the increase rate of type I collagen and type III collagen expression is higher than that of palmitoyl hexapeptide-14 used alone at the same content, the inhibition rate of MMP-1, MMP-3, and MMP-9 expression is higher than that of palmitoyl hexapeptide-14 used alone at the same content, and the increase rate of elastin expression is higher than that of palmitoyl hexapeptide-12 used alone at the same content. The compound peptide has a synergistic beneficial effect on the expression of basement membrane-related genes, such as the increase rate of type IV collagen and laminin expression is higher than that of hexapeptide-9 used alone at the same content, and the increase rate of integrin β1 expression is higher than that of hexapeptide-9 used alone at the same content.

[0119] The compound peptide has a synergistic beneficial effect on the expression of cell aging marker genes, such as significantly reducing the expression of p21 (Cdkn1a) in a concentration-dependent manner, indicating that the compound peptide can effectively inhibit cell aging and restore normal cell cycles. The compound peptide has a synergistic beneficial effect on the secretion of acetylcholine in rat adrenal medullary pheochromocytoma cells (PC12 cells), such as significantly reducing the secretion of acetylcholine in PC12 cells, and its acetylcholine secretion is lower than that of acetyl hexapeptide-1 used alone at the same content. The compound peptide has a synergistic beneficial effect on the expression of cell barrier-related genes, such as the increase rate of syndecan-1 expression is higher than that of acetyl tetrapeptide-11 used alone at the same content, and the increase rate of type XVII collagen expression is higher than that of acetyl tetrapeptide-11 used alone at the same content. Therefore, this complex peptide can simultaneously exert anti-aging effects on the skin from multiple dimensions such as the epidermis, basement membrane, dermis and even nerve fibers, and achieve comprehensive synergistic functions in multiple aspects.

[0120] Therefore, the composite peptide provided by the present invention acts on the keratinocytes in the basal layer of the epidermis, the structural proteins of the dermal epidermis junction (DEJ) and / or the fibroblasts in the dermis, and achieves its full-thickness skin anti-aging effect on the epidermis, basement membrane, dermis and / or nerve fibers by affecting the expression levels of extracellular matrix-related genes and / or proteins, the expression levels of basement membrane-related genes, the expression levels of cell aging marker genes, the secretion of cell acetylcholine and the expression levels of cell barrier-related genes.

[0121] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A full-thickness complex peptide, characterized in that: The complex peptides include acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1; the complex peptides act on epidermal basal layer keratinocytes, dermal epidermal junction (DEJ) structural proteins and / or dermal fibroblasts, and synergistically exert full-thickness skin anti-aging effects on the epidermis, basement membrane, dermis and / or nerve fibers.

2. The complex peptide according to claim 1, characterized in that The compound peptide acts on the keratinocytes in the basal layer of the epidermis, and improves the activity of the epidermal cells by promoting the expression of glycoprotein syndecan-1 on the surface of the basal layer cells; The complex peptide acts on the structural protein of the dermal epidermal junction (DEJ), and tightens the dermal epidermal junction (DEJ) by promoting the expression of type IV collagen, type XVII collagen, laminin and / or integrin; The complex peptide acts on dermal fibroblasts, synergistically exerts an anti-aging effect on the entire dermis by filling the extracellular matrix (ECM), reshaping the collagen matrix, and / or precisely blocking the transmission of nerve signals.

3. The complex peptide according to claim 1, characterized in that: The full-thickness skin anti-aging effect is achieved by affecting the complex peptide on the expression of extracellular matrix-related genes and / or proteins, the expression of basement membrane-related genes, the expression of cell aging marker genes, the secretion of cell acetylcholine and / or the expression of cell barrier-related genes.

4. The complex peptide according to claim 3, characterized in that: The effects on the expression of extracellular matrix-related genes and / or proteins include increasing the expression of type I collagen and / or type III collagen, inhibiting the expression of matrix metalloproteinase-1 (MMP-1), increasing the expression of elastin, inhibiting the expression of matrix metalloproteinase-3 (MMP-3), and / or inhibiting the expression of matrix metalloproteinase-9 (MMP-9); The effect on the expression of basement membrane-related genes includes increasing the expression of type IV collagen, increasing the expression of laminin, and / or increasing the expression of integrin β1; The effect on the expression of cell senescence marker genes includes a significant decrease in the expression of p21 (Cdkn1a); The effect on the secretion of acetylcholine in cells includes significantly reducing the secretion of acetylcholine in cells; The effect on the expression of cell barrier-related genes includes increasing the expression of Syndecan-1 and / or increasing the expression of type XVII collagen.

5. The complex peptide according to claim 1, characterized in that: The composite peptide inhibits the degradation of extracellular matrix by activating TGF-β1 / Smads signaling pathway, promoting fibroblast proliferation, promoting extracellular matrix (ECM) synthesis, and / or reducing the activity of matrix metalloproteinases; the extracellular matrix (ECM) includes type I collagen, type III collagen, elastin, hyaluronic acid and / or glycosaminoglycans.

6. The complex peptide according to claim 1, characterized in that: The proportions of acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 in the composite peptide are respectively between 0.05 and 0.

80.

7. The complex peptide according to claim 6, characterized in that: The proportions of acetyl tetrapeptide-11, hexapeptide-9, palmitoyl hexapeptide-12, palmitoyl hexapeptide-14 and / or acetyl hexapeptide-1 in the composite peptide are respectively between 0.10 and 0.

60.

8. The complex peptide according to claim 1, characterized in that: The compound peptide is used for the preparation of products for enhancing the efficacy of medical beauty projects, extending the maintenance period of medical beauty projects after surgery and / or daily anti-aging; The medical aesthetics items include non-invasive medical aesthetics and / or invasive medical aesthetics; the non-invasive medical aesthetics include photorejuvenation, acid peeling, picosecond, ultra-picosecond, Thermage, thermolift, hyaluronic acid filling, non-ablative fractional laser, face-lifting injection, and / or ultrasonic knife; the invasive medical aesthetics include microneedle, water light needle, picosecond or ultra-picosecond blasting, and / or ablative fractional laser; The synergistic products include facial masks, lotions, milks, creams, ointments and / or powders; The daily anti-aging treatment includes improving expression lines, preventing true lines, lifting and firming, promoting collagen regeneration and / or preventing skin sagging, which is achieved through external application and / or dermal introduction, and the dermal introduction includes instrument introduction.

9. The complex peptide according to claim 1, characterized in that: The composite peptide is used to prepare cosmetic and / or beauty and care products, including essence water / toner, lotion, face cream, beauty cream, base makeup, concealer, isolation, setting makeup, eye shadow, contouring, blush, lipstick, single-use stock solution, freeze-dried powder / freeze-dried liquid, facial mask liquid / mask powder / frozen mask, and / or functional medical beauty products.

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