A repair, anti-wrinkle firming peptide composition and a method for preparing the same

Abstract

The application discloses a kind of repair, anti-wrinkle firming peptide composition and preparation method thereof, belong to skin care field.The composition includes specific weight fraction oligopeptide-1, tripeptide-1 copper, palmitoyl tripeptide-1, acetyl hexapeptide-8, acetyl tetrapeptide-2 and pentapeptide-3.The application first discovers that the above six kinds of peptides can produce significant synergistic effect under accurate proportion, and through multi-level signal network synchronous realization efficient skin barrier repair, dermal matrix regeneration and nerve level wrinkle inhibition, its effect is far superior to any single peptide or the composition of missing a component.The application also provides the preparation method of the composition and the cosmetic or dermatological composition comprising it.The composition has excellent stability, high safety, and can be widely applied.

Description

Technical Field

[0001] This invention relates to the field of skin care composition technology, and more particularly to a repairing, anti-wrinkle, and firming peptide composition and its preparation method, especially a synergistic composition comprising oligopeptide-1, tripeptide-1 copper, palmitoyl tripeptide-1, acetyl hexapeptide-8, acetyl tetrapeptide-2, and pentapeptide-3. This composition exhibits unexpected and remarkable effects in repairing the skin barrier, reducing wrinkles, and improving skin firmness. The invention further relates to cosmetic or dermatological compositions comprising the said peptide composition, and the use of this composition in the preparation of related products. Background Technology

[0002] As the largest organ in the human body, the skin serves as a barrier protecting the body from external physical, chemical, and biological factors. It also acts as a crucial window into the body's aging state. With age, and the influence of various internal and external factors such as ultraviolet radiation, environmental pollution, and life stress, the skin exhibits a series of signs of aging and damage, primarily including weakened skin barrier function, dryness, roughness, decreased elasticity, sagging, and the formation of various types of wrinkles. The cellular and molecular mechanisms underlying these changes involve reduced collagen and elastin synthesis, increased activity of matrix metalloproteinases (MMPs), a decreased epidermal cell renewal rate, accumulation of oxidative stress, and a chronic micro-inflammatory state.

[0003] From a histological perspective, skin aging is a multi-level, multi-scale pathophysiological process. At the epidermal level, the proliferative capacity of keratinocytes decreases, and their differentiation process becomes disordered, leading to thinning of the stratum corneum, insufficient synthesis of barrier lipids, reduced content of natural moisturizing factors, and a significant increase in transepidermal water loss (TEWL). At the dermal-epidermal junction (DEJ) level, the expression of anchoring fibers and type IV and VII collagen decreases, the structure of the lamina densa loosens, and the interlocking structure between epidermal ridges and dermal papillae tends to flatten, resulting in weakened mechanical coupling between the epidermis and dermis, making the skin more susceptible to shear stress damage. At the dermal level, the metabolic activity of fibroblasts decreases, the synthesis rate of type I and III collagen decreases, while the expression of matrix metalloproteinases (especially MMP-1, MMP-3, and MMP-9) is upregulated, causing the degradation of collagen and elastin to exceed synthesis, and the net content of extracellular matrix (ECM) to continuously decrease. At the same time, the content of glycosaminoglycans and proteoglycans in the dermis also decreases synchronously, leading to a significant reduction in the skin's water retention capacity and viscoelasticity. At the subcutaneous tissue level, the volume and number of fat cells decrease, causing the skin to lose deep support, irreversibly exacerbating sagging and drooping. Furthermore, repeated contractions of facial muscles form dynamic wrinkles on the skin surface; as the dermal matrix degenerates and its repair capacity becomes insufficient, these dynamic wrinkles gradually solidify into static wrinkles. These changes at various levels do not occur in isolation, but rather form a positive feedback cascade through cytokine networks, integrin signaling, and mechanotransmission pathways. Therefore, interventions targeting a single point often fail to address all aspects and are unlikely to fundamentally reverse the aging process.

[0004] Driven by consumer demand for healthy and youthful skin, cosmetics and dermatological-grade products with anti-wrinkle, firming, and repairing functions have been a research hotspot. Among numerous active ingredients, bioactive peptides, due to their high activity, high specificity, and good safety profile, have become a core raw material in the fields of anti-aging and skin repair. According to different mechanisms of action, active peptides for cosmetics can be roughly divided into the following four categories: (1) Signal peptides: mimic the function of endogenous growth factors or cytokines, activate specific cell surface receptors (such as TGF-β receptors and IGF receptors) to initiate downstream signal cascades, and promote the synthesis and secretion of extracellular matrix macromolecules such as collagen, elastin, and fibronectin; (2) Neurotransmitter inhibitory peptides: by interfering with the assembly of SNARE complexes, blocking calcium ion channels or competing for neurotransmitter receptors, inhibit the release of acetylcholine at the neuromuscular junction, thereby relaxing subcutaneous muscles and reducing the production of expression lines; (3) Carrier peptides: by chelating and stabilizing metal ions (especially transition metals such as copper, manganese, and zinc), they are precisely delivered to the enzyme active center in the cell, promoting the catalytic function of metalloenzymes such as lysyl oxidase (LOX) and superoxide dismutase (SOD); (4) Enzyme inhibitory peptides: by competitively inhibiting the activity of matrix metalloproteinases, elastases, or hyaluronidase, they reduce the degradation of the extracellular matrix and maintain the net increase in dermal macromolecules. Different sequences of peptides can mimic the functions of growth factors, signaling molecules or neurotransmitters, and regulate cell behavior through specific cell signaling pathways.

[0005] In the prior art, there have been various explorations into the application of peptides in skin care. For example, Chinese patent application CN112043619A discloses an anti-wrinkle composition containing oligopeptide-1 and copper tripeptide-1, which achieves an anti-wrinkle effect by promoting collagen synthesis. Chinese patent application CN117357424A discloses a firming serum containing acetyl hexapeptide-8 and palmitoyl tripeptide-1, which utilizes a botulinum toxin-like mechanism to relax expression lines and stimulate dermal matrix regeneration. Chinese patent CN110538093A discloses a peptide repair solution that utilizes the synergistic effect of acetyl hexapeptide-8, palmitoyl tripeptide-1, copper tripeptide-1, acetyl tetrapeptide-5, palmitoyl tetrapeptide-7, and carnosine. In addition, acetyl tetrapeptide-2 has been reported to upregulate the expression of elastin and collagen, while pentapeptide-3 has been found to smooth wrinkles by blocking muscle contraction signaling pathways. Chinese patent application CN119174719A discloses an active peptide composition and its application in cosmetics, describing a scheme to achieve multi-target wrinkle reduction and firming effects by combining acetyl hexapeptide-8, tripeptide-1, tripeptide-1 copper, palmitoyl tetrapeptide-7, and palmitoyl pentapeptide-4. Internationally, skincare brands such as LOOPS have also launched peptide essence products containing acetyl hexapeptide-8, sh-oligopeptide-1, tripeptide-1 copper, and palmitoyl tripeptide-1, but their peptide combinations also include palmitoyl tetrapeptide-7 and palmitoyl pentapeptide-4. An academic paper published in 2025 reported a synergistic anti-aging combination containing seven bioactive peptides and silymarin, which achieved significant effects of 12.5% ​​increase in elasticity, 20.7% increase in firmness, and 78% increase in dermal density during a 56-day clinical observation, further demonstrating the great development potential of peptide synergistic combinations.

[0006] However, after in-depth research and large-scale screening experiments, the inventors discovered that simply combining several known anti-wrinkle or repair peptides in any way often fails to achieve satisfactory synergistic effects. Peptides may exhibit antagonistic effects due to cell membrane receptor competition, negative feedback regulation of signaling pathways, charge interactions in the formulation, or differences in stability, leading to a decrease in overall effectiveness. More importantly, existing technologies generally tend to layer peptides at the same level of action: for example, using multiple signaling peptides simultaneously at the collagen signaling level, or using multiple botulinum toxin-like peptides simultaneously at the neuroinhibition level. This "same-layer layering" strategy ignores the multidimensional nature of skin aging and fails to design combination solutions that synergistically address the three levels of epidermal barrier repair, dermal matrix regeneration, and neuromuscular inhibition. Furthermore, the pathways and durations of action of each peptide differ; without precise formulation design and functional complementarity, it is impossible to simultaneously achieve the multidimensional anti-aging goals of barrier repair, expression line inhibition, dermal matrix remodeling, and improved skin firmness within the same treatment cycle.

[0007] Another long-standing challenge for those skilled in the art lies in the storage stability of peptide compositions. Peptide molecules are prone to degradation reactions in solution, such as aggregation and precipitation, oxidative deamidation, and β-elimination. Especially when multiple peptides coexist, hydrogen bonds, hydrophobic interactions, and electrostatic interactions between different peptide molecules can lead to non-specific intermolecular binding and the formation of irreversible aggregates, causing the active peptides to become inactive within a short period. For example, copper ions in copper peptides (such as tripeptide-1 copper) may catalyze the oxidative degradation of certain sensitive peptides (such as acetyl hexapeptide-8 containing methionine residues) in solution; while palmitoylated peptides (such as palmitoyl tripeptide-1), due to their amphiphilic nature, easily form micelles or adsorb onto container walls in aqueous solutions, resulting in uncontrollable local concentrations. Existing commercially available peptide stock solutions typically show a significant decrease in activity within 1 to 3 months under room temperature storage conditions, severely impacting product shelf life and consumer experience.

[0008] Therefore, developing a specific peptide composition that enables its individual peptide components to exert synergistic repair and anti-wrinkle firming effects across multiple targets and pathways at specific mass ratios, overcoming the limitations of single or simple peptide combinations in terms of limited effectiveness and poor durability, while simultaneously addressing long-term storage stability through scientifically sound formulation processes, and ensuring good product safety and skin permeability, has become a pressing technical challenge in this field. Furthermore, an efficient and convenient preparation method is needed to ensure the stable application of this composition in various formulations. Summary of the Invention

[0009] In view of the shortcomings of the existing technology, the primary objective of this invention is to overcome the shortcomings of the existing technology and provide a peptide composition with significant synergistic effects in repair, anti-wrinkle and firming.

[0010] The second objective of this invention is to provide a method for preparing the above-mentioned peptide composition, which is simple in process, mild in conditions, has good batch-to-batch repeatability, and is easy to scale up industrially.

[0011] A third object of the present invention is to provide cosmetic or dermatological compositions comprising the above-described peptide compositions, and their use in the preparation of related products.

[0012] The above-mentioned objective of this invention is achieved through the following technical solutions: In a first aspect, the present invention provides a repairing, anti-wrinkle, and firming peptide composition comprising the following components in parts by weight: Oligopeptide-1: 15-25 parts; Tripeptide-1 copper: 13-23 parts; Palmitoyl tripeptide-1: 15-25 parts; Acetyl hexapeptide-8: 12-20 parts; Acetyl tetrapeptide-2: 10–18 parts; Pentapeptide-3: 8 to 16 parts.

[0013] During the research process of this invention, the inventors discovered through a large number of cell models and human clinical tests that when the above six specific peptides coexist in the above weight range, they can produce a non-linear superposition cascade amplification effect on multiple signaling pathways related to skin aging and damage, thereby achieving a multiplication of skin barrier repair rate, wrinkle depth reduction and skin firmness improvement at extremely low doses.

[0014] The synergistic mechanism of this invention can be summarized as a "three-layer, six-element synergistic network": The first layer is the epidermal barrier repair layer, with oligopeptide-1 as the core. By activating the integrin α6 / β1 signaling pathway of epidermal stem cells and keratinocytes, it promotes the directed migration and proliferation of cells, and rapidly rebuilds the damaged physical barrier structure. The second layer is the dermal matrix regeneration layer, driven by tripeptide-1 copper and palmitoyl tripeptide-1 as dual cores. The former activates lysine oxidase (LOX) in a copper ion-dependent manner to achieve covalent cross-linking of newly synthesized collagen fibers and elastic fibers, while the latter enhances transdermal penetration and the ability to target fibroblast membrane receptors through palmitoylation modification, and continuously activates the TGF-β / Smad signaling pathway to promote type I and type III collagen and fibronectin formation. The de novo synthesis of proteins, with acetyl tetrapeptide-2 acting as an elastic fiber-specific assembly factor in this layer, ensures the correct deposition of newly synthesized elastic fibers in the papillary dermis by upregulating the expression of fibrillin-1 and elastin-binding protein. These three components form a ternary synergistic loop of "synthesis-crosslinking-assembly" for dermal reconstruction. The third layer is the neuromuscular inhibitory layer, where acetyl hexapeptide-8 acts as a SNARE complex assembly inhibitor, rapidly blocking neurotransmitter release at the muscle level to smooth dynamic wrinkles. Pentapeptide-3 acts on the acetylcholine receptor desensitization pathway at the postsynaptic membrane, providing a long-lasting background for muscle relaxation and creating a stable window of opportunity for the reconstruction of the first two layers, free from repeated traction by facial muscles. Positive feedback coupling is formed between the three layers through fibroblast mechanotransduction feedback: dermal matrix densification reduces the wrinkling effect of facial muscle contraction on the skin, decreasing the tendency for dynamic wrinkles to transform into static wrinkles; and the restoration of barrier integrity reduces the persistent damage to the dermal matrix caused by chronic subclinical inflammation. This multi-level, positive feedback synergistic amplification mechanism cannot be achieved by any single peptide or simple homolayer stacking combination.

[0015] In particular, oligopeptide-1, as a repair signaling peptide, can activate the migration of epidermal stem cells and fibroblasts, rapidly initiate the reconstruction of the damage barrier, and form the first synergistic loop with the copper ion transport, collagen and elastin deposition functions of tripeptide-1 copper; palmitoyl tripeptide-1 enhances transdermal penetration and affinity for fibroblasts through palmitoylation modification, and its effect of stimulating matrix macromolecule synthesis is significantly amplified by tripeptide-1 copper; acetyl hexapeptide-8, as a neurotransmitter inhibitory peptide, can rapidly smooth dynamic wrinkles at the muscle level, providing a relatively stable "wrinkle-free" window for the above-mentioned dermal remodeling process; while acetyl tetrapeptide-2 and pentapeptide-3 act on downstream targets of elastic fiber assembly and muscle contraction signals, respectively, further consolidating and prolonging the firming and anti-wrinkle effects. It is particularly noteworthy that this invention is the first to discover a unique "elasticity-relaxation" synergistic effect between acetyl tetrapeptide-2 and pentapeptide-3: acetyl tetrapeptide-2 upregulates elastin expression, enabling the skin to rebound more effectively when passively stretched, while the muscle-relaxing effect of pentapeptide-3 reduces the active stretching of the skin during daily facial expressions. The two work together—one increasing elasticity and the other reducing stretching—to exponentially amplify the firming effect, a phenomenon not disclosed or implied in any existing technology. The aforementioned six peptides form a self-consistent functional network within a defined ratio range; the absence of any one peptide prevents the achievement of a complete efficacy loop, while an imbalance in the ratio disrupts the network balance, leading to a sharp drop in efficacy.

[0016] Another unexpected advantage of the composition of this invention lies in its superior storage stability. The inventors discovered that when oligopeptide-1, tripeptide-1 copper, palmitoyl tripeptide-1, acetyl hexapeptide-8, acetyl tetrapeptide-2, and pentapeptide-3 are present in the same formulation within the proportions defined in claim 1, a protective microcluster structure spontaneously forms between the peptide components. The palmitoyl chain of palmitoyl tripeptide-1 forms a hydrophobic core with the aromatic residue side chain of acetyl hexapeptide-8 through hydrophobic interactions. The copper ions in tripeptide-1 copper form a reversible coordination with the histidine residues at the end of oligopeptide-1, effectively shielding the catalytic activity of copper ions and thus significantly reducing oxidative damage to the methionine residues of acetyl hexapeptide-8. This "endogenous stabilization" effect based on intermolecular self-assembly is something that cannot be replaced or anticipated by simply adding antioxidants or chelating agents in the prior art. Experimental data confirm that after 3 months of storage under accelerated conditions of 45°C and 75% relative humidity, the freeze-dried powder composition prepared according to the specified proportions of this invention retains more than 95% of the activity of all six peptides, which is significantly better than any single peptide or complex with an imbalanced ratio.

[0017] As a preferred technical solution, the repairing, anti-wrinkle, and firming peptide composition comprises the following components in parts by weight: Oligopeptide-1: 18-22 parts; Tripeptide-1 copper: 16-20 parts; Palmitoyl tripeptide-1: 18–22 parts; Acetyl hexapeptide-8: 14–18 parts; Acetyl tetrapeptide-2: 12-16 parts; Pentapeptide-3: 10-14 parts.

[0018] More preferably, the repairing, anti-wrinkle, and firming peptide composition comprises the following components in parts by weight: Oligopeptide-1: 20 parts; Tripeptide-1 copper: 18 parts; Palmitoyl tripeptide-1: 20 parts; Acetyl hexapeptide-8: 16 parts; Acetyl tetrapeptide-2: 14 parts; Pentapeptide-3: 12 parts.

[0019] At this precise ratio, the synergistic coefficient of the six peptides reaches its peak, achieving an optimal balance in the improvement rates of the three dimensions of repair, anti-wrinkle and firming.

[0020] From the perspective of dosage form design, the composition of the present invention is preferably provided in the form of lyophilized powder. The lyophilized powder dosage form has the following irreplaceable advantages: (1) In a solid anhydrous environment, the molecular motion of peptide molecules is greatly restricted, and the reaction rate constant of degradation reactions (especially hydrolysis, deamidation and oxidation) is reduced by several orders of magnitude, which can achieve long-term stability at room temperature; (2) The lyophilized powder can be immediately reconstituted with the matching solvent before use, avoiding the potential irritation risk to sensitive skin from continuous contact with preservatives in liquid formulations, and is particularly suitable for use by people with sensitive skin and during the post-medical aesthetic repair period; (3) The lyophilized dosage form can be combined with the dispensing of high-concentration active ingredients, avoiding the problem of activity decay of peptides in low-concentration dilution states. When the composition is in liquid form, it is preferred to store it in a refrigerated environment of 4°C to 8°C, and to supplement the formulation with an appropriate amount of polyol (glycerol, butanediol, etc.) as a protective co-solvent and water activity regulator.

[0021] Secondly, the present invention provides a method for preparing the above-mentioned repairing, anti-wrinkle, and firming peptide composition, comprising the following steps: Oligopeptide-1, copper tripeptide-1, palmitoyl tripeptide-1, acetyl hexapeptide-8, acetyl tetrapeptide-2, and pentapeptide-3 are weighed out according to the specified ratio. They are dissolved together in a solvent at a temperature of 15°C to 35°C and a relative humidity of 30% to 60%, and the mixture is stirred continuously until a clear and homogeneous mixture is formed. Optionally, the mixture is sterilized and filtered. Optionally, the mixture or filtrate is dried to obtain a powdered composition.

[0022] Preferably, the solvent is selected from water, glycerol, propylene glycol, butanediol, pentanediol, hexanediol, or any combination thereof. A mixed solvent of ultrapure water and butanediol is particularly preferred, as this helps to pre-form a micelle-like molecular arrangement during dissolution, improving the stability of the peptide during subsequent compounding and storage. More importantly, the inventors have found that when using a mixed solvent system of ultrapure water and butanediol in a mass ratio of 8:2 to 7:3, the palmitoyl chain of palmitoyl tripeptide-1, with the assistance of butanediol, tends to form a loose pre-micelle structure rather than a tight intramolecular fold, allowing the GHK active sequence of the peptide chain to be fully exposed in the solvent, enabling faster dissociation and activation upon contact with fibroblast membrane receptors. Simultaneously, the hydrogen bond network formed by the hydroxyl groups of butanediol and water molecules effectively reduces the proportion of free water in the solution, thereby inhibiting the copper ion-catalyzed hydrolysis side reaction in the copper tripeptide-1.

[0023] The sterilization filtration step can use a 0.22 μm polyethersulfone or polyvinylidene fluoride microfiltration membrane. Drying can be achieved through freeze-drying or low-temperature spray drying. When freeze-drying is used, it is preferable to pre-freeze the mixture at -45°C to -40°C for 6 to 12 hours, then perform programmed temperature rise sublimation drying under a vacuum of 10 to 25 Pa, and finally desorption dry at 25°C to 30°C until the water activity is below 0.3. The selection of freeze-drying process parameters is crucial to the composition of this invention. The cooling rate during the pre-freezing stage directly affects the morphology of ice crystals: too fast a cooling rate (>2°C / min) will generate small and dense ice crystals, resulting in insufficient water vapor escape channels during sublimation, reduced drying efficiency, and potentially causing localized overheating and peptide denaturation; too slow a cooling rate (<0.5°C / min) will generate large ice crystals, which, while beneficial for sublimation, may cause mechanical shear stress on peptide molecules due to ice crystal expansion. The preferred cooling rate of this invention is 0.8–1.5 °C / min. Within this range, the dendritic ice crystal morphology ensures both sublimation efficiency and maximizes the protection of the peptide's structural integrity. The temperature during the desorption drying stage should not exceed 30 °C to avoid peptide amide bond hydrolysis and Maillard reactions caused by high temperatures (if the formulation contains reducing sugar protectants). The water activity (aw) of the final product should be controlled below 0.3, preferably below 0.15, to achieve optimal long-term storage stability.

[0024] Thirdly, the present invention provides a cosmetic or dermatological composition comprising the repairing, anti-wrinkle, and firming peptide composition as described above, and cosmetically or dermatologically acceptable excipients. The excipients may include, but are not limited to, solvents, thickeners, moisturizers, emulsifiers, preservatives, antioxidants, pH adjusters, fragrances, etc.

[0025] Preferably, the dosage form of the cosmetic or dermatological composition is a serum, lotion, cream, freeze-dried powder, mask, eye cream, neck cream, or ampoule-packaged stock solution.

[0026] The compositions of this invention can be combined with a variety of other active ingredients to further enhance their efficacy. For example, they can be combined with hyaluronic acid or its salts to enhance immediate hydration; with niacinamide to synergistically strengthen barrier repair and improve skin tone; with ceramides to further improve the integrity of the stratum corneum barrier; with asiaticoside or hydroxyasiaticoside to enhance anti-inflammatory and soothing effects; and with vitamin C or its derivatives to simultaneously achieve antioxidant protection. The peptide compositions of this invention exhibit good compatibility with all of the above-mentioned ingredients, without any adverse interactions such as precipitation, layering, or discoloration.

[0027] Preferably, the repairing, anti-wrinkle, and firming peptide composition is present in the cosmetic or dermatological composition at a weight percentage of 0.001% to 10%, more preferably 0.01% to 5%, and most preferably 0.1% to 2%. Even at this extremely low concentration, the peptide composition still exhibits significant repairing and anti-aging efficacy. This extremely low effective concentration is another unexpected discovery of the present invention. In conventional understanding, the effective concentration of peptide skincare ingredients is usually between 1% and 5%, while the composition of the present invention exhibits significant repairing and anti-wrinkle effects at an effective concentration of 0.1%—this is due to the positive feedback amplification mechanism of the aforementioned "three-layer hexa-element synergistic network": each peptide component plays a role at different nodes of the signaling pathway, and the cascade amplification of downstream signals significantly multiplies each upstream signal molecule, thereby triggering a complete anti-aging effect cascade at an extremely low total peptide concentration. The low effective concentration not only reduces raw material costs but also minimizes the risk of local irritation that may be caused by high concentrations of peptides, making this composition particularly suitable for long-term daily use by people with sensitive skin.

[0028] Fourthly, the present invention also provides the use of the above-mentioned peptide composition or cosmetic / dermatological composition in the preparation of products for repairing the skin barrier, reducing wrinkles, and / or improving skin firmness. The products may be pharmaceuticals, medical devices, cosmetics, or disinfectant products, etc.

[0029] The composition of the present invention is particularly suitable for the following specific groups and scenarios: (1) mature skin people aged 30 years and above whose skin has begun to show signs of early aging such as fine lines, sagging and decreased elasticity; (2) people whose skin barrier is damaged and shows signs of photoaging due to factors such as long-term sun exposure, staying up late, and high-stress life; (3) patients in the skin barrier repair period after cosmetic medical treatment (such as laser treatment, microneedling treatment, chemical peeling, etc.), in which the high efficiency of the composition of the present invention and low irritation have significant advantages; (4) people with rich facial expressions who have formed or are forming dynamic wrinkles in the corners of their eyes, forehead, and between their eyebrows; (5) people whose skin barrier function is impaired due to seasonal changes or dry environment.

[0030] In summary, compared with the prior art, the present invention has at least one of the following beneficial technical effects: For the first time, a precise six-component combination of oligopeptide-1, tripeptide-1 copper, palmitoyl tripeptide-1, acetyl hexapeptide-8, acetyl tetrapeptide-2 and pentapeptide-3 was discovered. Its repair, anti-wrinkle and firming effects are significantly higher than any combination of single peptides or missing peptides. The synergistic index Q value calculated by the Jin Zhengjun method is always greater than 1.15, showing a strong synergistic effect.

[0031] The composition of this invention achieves full-chain coverage from physical repair of the skin barrier and regeneration of the dermal extracellular matrix to inhibition of expression lines at the neural level, with complete efficacy dimensions. This "three-layer six-element synergistic" architecture has not been disclosed in any existing technology, reflecting the inventors' profound understanding and systematic innovation of the multi-dimensional mechanisms of skin aging.

[0032] The preparation method is simple, and the obtained lyophilized powder and other forms retain more than 95% of their activity after 3 months of storage under accelerated conditions of 45°C and 75% relative humidity, far exceeding existing complex peptide solution products. This superior storage stability is attributed to the unique intermolecular protective microcluster structure of the peptide composition of this invention, rather than relying on the addition of additional stabilizers.

[0033] It exhibits extremely high safety, with an irritation index of zero in human patch testing, making it suitable for all skin types, including sensitive skin. Multiple repeated patch tests (48-hour closed patch, n=60) consistently showed an irritation index of zero, indicating that this composition has extremely high skin tolerance. Furthermore, the six peptides used in this invention are all mature raw materials listed by the International Committee on Nomenclature of Cosmetic Ingredients (INCI) and have a long history of safe use in the cosmetic field, exhibiting no sensitization, phototoxicity, or genotoxicity.

[0034] Extremely low effective concentration: Significant effects can be achieved with an addition of only 0.1%, which greatly reduces formulation costs and usage risks, significantly lowering the price threshold for high-end peptide skincare products and giving them broad market potential. Detailed Implementation

[0035] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as are well known to those skilled in the art. The raw materials used in this invention are commercially available or prepared using known solid-phase synthesis methods.

[0037] Main raw material information and sequence description: Oligopeptide-1 (INCI: Oligopeptide-1): The corresponding sequence is gly-His-Lysine (Gly-His-Lys), with a purity of ≥98.5%, purchased from a well-known international biochemical company.

[0038] Copper Tripeptide-1 (INCI): a copper complex of glycyl-L-histyl-L-lysine (GHK-Cu), with a purity ≥98.0%, purchased from a well-known international biochemical company.

[0039] Palmitoyl Tripeptide-1 (INCI): also known as N-palmitoyl-glycyl-L-histyl-L-lysine (Pal-GHK), with a purity ≥97.5%.

[0040] Acetyl Hexapeptide-8 (INCI): The sequence is acetyl-glutamyl-glutamyl-methionyl-glutamine-arginyl-arginine (Ac-EEMQRR-NH2), with a purity ≥98.0%.

[0041] Acetyl Tetrapeptide-2 (INCI: Acetyl Tetrapeptide-2): The sequence is acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP-OH), with a purity ≥98.0%.

[0042] Pentapeptide-3 (INCI: Pentapeptide-3): sequence glycyl-prolyl-arginyl-prolyl-alanine (GPRPA), purity ≥98.0%.

[0043] The molecular weight, isoelectric point, and hydrophilicity / hydrophobicity of the above six peptides are as follows: Oligopeptide-1 has a molecular weight of 340.4 Da and an isoelectric point of approximately 8.9; Tripeptide-1 (copper) has a molecular weight of 403.9 Da (containing a copper complex) and is blue; Palmitoyl Tripeptide-1 has a molecular weight of 578.8 Da and exhibits significant amphiphilicity due to palmitoylation modification; Acetyl Hexapeptide-8 has a molecular weight of 889.0 Da and an isoelectric point of approximately 4.5; Acetyl Tetrapeptide-2 has a molecular weight of 487.5 Da and an isoelectric point of approximately 3.8; and Pentapeptide-3 has a molecular weight of 497.5 Da. These differences in physicochemical parameters provide a molecular basis for the spatial pre-assembly of different peptides in solution.

[0044] Example 1: Preparation of Repairing, Anti-wrinkle and Firming Peptide Composition A In a cleanroom environment (temperature 22℃, relative humidity 45%), accurately weigh 20g of oligopeptide-1, 18g of tripeptide-1 copper, 20g of palmitoyl tripeptide-1, 16g of acetyl hexapeptide-8, 14g of acetyl tetrapeptide-2, and 12g of pentapeptide-3, and add them to a pre-prepared mixed solvent cooled to 20℃ (the mixed solvent is composed of ultrapure water and 1,3-butanediol in a mass ratio of 8:2, with a total mass of 2000g). Gently stir at 300rpm for 45 minutes using a magnetic stirrer to obtain a pale blue, clear, and transparent mixture. Sterilize the mixture by passing it through a 0.22μm polyethersulfone membrane syringe filter. Dispense the filtrate into vials, partially stopper them, and place them in a freeze dryer. The pre-freezing temperature was set to -45℃ for 8 hours. Then, the vacuum pump was turned on, and the vacuum degree was controlled at 15Pa. The temperature was maintained at -30℃ for 12 hours, -15℃ for 8 hours, and 0℃ for 4 hours. Finally, the sample was desorbed and dried at 25℃ for 6 hours until the moisture content was 1.2%. After capping, the lyophilized powder-type repair, anti-wrinkle and firming peptide composition A was obtained.

[0045] The mixed solvent used in this embodiment (ultrapure water: 1,3-butanediol = 8:2) was the optimal choice determined through extensive solvent screening experiments. The inventors compared various solvent systems in parallel, including pure water, water-glycerol (8:2), water-propylene glycol (8:2), water-butanediol (8:2), water-pentanediol (8:2), and water-butanediol in different ratios (9:1, 8:2, 7:3, 6:4). The results showed that the water-butanediol (8:2) system was optimal in terms of peptide dissolution rate, solution clarity, and reconstitution rate after lyophilization. Specifically, complete dissolution of palmitoyl tripeptide-1 in pure water required stirring for more than 90 minutes, and the final solution exhibited weak opalescence (indicating the presence of incompletely dispersed micelle aggregates), while the water-butanediol (8:2) system achieved optical clarity within 45 minutes. The lyophilized powder reconstitution experiment showed that the lyophilized powder prepared in the water-butanediol (8:2) system could be completely dissolved into a colorless and transparent solution within 15 seconds after the solvent was added, while the lyophilized powder in the pure water system showed a brief turbidity after reconstitution (which turned clear after about 2 minutes). This suggests that butanediol played a role in spatial isolation and protection of peptide molecules during the lyophilization process, preventing irreversible aggregation of peptide molecules during the freeze-concentration stage.

[0046] Example 2: Preparation of Repairing, Anti-wrinkle and Firming Peptide Composition B Weigh out 18g of oligopeptide-1, 20g of tripeptide-1 copper, 22g of palmitoyl tripeptide-1, 15g of acetyl hexapeptide-8, 13g of acetyl tetrapeptide-2 and 11g of pentapeptide-3, and dissolve them in 1800g of a mixed solvent of propylene glycol and water in a mass ratio of 1:9. Stir until completely dissolved, and perform sterile filtration and freeze-drying as in Example 1 to obtain composition B.

[0047] Example 3: Preparation of Repairing, Anti-wrinkle and Firming Peptide Composition C Weigh out 22g of oligopeptide-1, 16g of tripeptide-1 copper, 18g of palmitoyl tripeptide-1, 18g of acetyl hexapeptide-8, 15g of acetyl tetrapeptide-2 and 13g of pentapeptide-3, and dissolve them in 2000g of an aqueous solution containing 5% pentanediol. Prepare the solution under the same conditions as in Example 1 to obtain composition C.

[0048] Example 4: Preparation of Repairing, Anti-wrinkle, and Firming Peptide Composition D (Liquid Composition) Weigh out 20g of oligopeptide-1, 18g of tripeptide-1 copper, 20g of palmitoyl tripeptide-1, 16g of acetyl hexapeptide-8, 14g of acetyl tetrapeptide-2, and 12g of pentapeptide-3. Dissolve them in a mixed solvent of glycerol and ultrapure water in a 2:8 ratio. Add 0.45% phenoxyethanol and 0.05% octyl glycol as preservatives and solubilizers, bringing the total mass to 5000g. Stir at room temperature in the dark for 60 minutes until a homogeneous solution is obtained. Filter through a 0.22μm filter to obtain liquid peptide composition D, which can be used directly as a stock solution. Store at 4°C.

[0049] It should be noted that although the liquid formulation of peptide composition D can maintain its activity stability for up to 3 months under refrigeration conditions (4-8°C), its long-term storage stability is still not as good as that of the lyophilized powder formulation. Therefore, for commercial products requiring a longer shelf life, the lyophilized powder formulation of Examples 1-3 is preferred, along with individually packaged solvents. Liquid formulation D is more suitable as a ready-to-use product or a short-term use ampoule product.

[0050] Example 5: Preparation of an essence containing peptide composition Prepare the repairing, anti-wrinkle, and firming serum according to the following formula (by weight percentage): The peptide composition A obtained in Example 1 consisted of 1.0% glycerol, 5.0% glycerol, 4.0% butylene glycol, 0.1% sodium hyaluronate, 0.4% polyacrylamide dimethyl taurate, 0.2% PEG-40 hydrogenated castor oil, 0.5% phenoxyethanol / caprylyl glycol, 0.05% xanthan gum, 0.03% disodium EDTA, 0.01% fragrance, and the balance being deionized water.

[0051] Preparation process: Deionized water, glycerin, butylene glycol, sodium hyaluronate, xanthan gum and disodium EDTA are added to an aqueous phase pot, heated to 80°C and stirred for 30 minutes. After cooling to 45°C, pre-dispersed thickener polyacrylamide dimethyl taurate is added and stirred evenly. When the temperature drops to 30°C, phenoxyethanol / octyl glycol, PEG-40 hydrogenated castor oil, fragrance and peptide composition A of Example 1 are added in sequence, and the mixture is stirred continuously for 20 minutes to obtain a transparent essence.

[0052] Temperature control during peptide composition addition is crucial. The inventors discovered that when the material temperature exceeds 40°C, the methionine residues of acetyl hexapeptide-8 are easily oxidized, leading to decreased activity and a slight yellow discoloration of the solution. Therefore, the addition of the peptide composition is strictly controlled when the material temperature drops below 30°C to ensure the thermal stability of the active peptide. Simultaneously, the disodium EDTA in this formulation not only acts as a conventional metal ion chelating agent but also preferentially complexes with trace amounts of transition metal ions (such as iron and manganese) in the presence of the formulation, thereby indirectly protecting the coordination structure of copper ions in tripeptide-1 from being replaced by external metal ions.

[0053] Example 6: Preparation of a repair cream containing peptide composition Formulation (by weight percentage): Example 2 Peptide Composition B 0.5%, Cetearyl Oleate / Sorbitan Oleate 4.5%, Squalane 8.0%, Caprylic / Capric Triglyceride 5.0%, Shea Butter 3.0%, Glycerin 7.0%, Propylene Glycol 3.0%, Hydroxyethyl Acrylate / Sodium Acryloyl Dimethyl Taurate Copolymer 0.8%, Phenoxyethanol 0.7%, Dipotassium Glycyrrhizate 0.1%, Deionized Water to 100%.

[0054] Process: The oil phase (emulsifier, squalane, caprylic / capric triglyceride, shea butter) is heated to 80°C, and the aqueous phase (water, glycerin, propylene glycol, dipotassium glycyrrhizate) is heated to 80°C. The aqueous phase is slowly drawn into the oil phase and homogenized for 4 minutes. The temperature is lowered to 45°C and the thickener copolymer is added. The mixture is stirred until it is uniform and forms a paste. The peptide composition B from Example 2 and the preservative are added below 40°C. The cream is obtained by vacuum degassing.

[0055] Cream formulations present additional challenges to the stability and transdermal delivery of peptide compositions. Because creams are oil-in-water or water-in-oil emulsions, peptide molecules are primarily distributed in the continuous aqueous phase, and their interaction with the oil phase interface can affect activity. The inventors, through experimental screening, determined the optimal timing for adding the peptide composition to the cream—after the emulsion has formed and cooled to below 40°C. At this point, the interfacial film of the emulsion is sufficiently stable, allowing the peptide molecules to be uniformly dispersed in the outer aqueous phase without being encapsulated or inactivated by the emulsifier's interfacial film. Furthermore, squalane and shea butter, as emollient oils, form a semi-occlusive film on the skin surface, helping to reduce transdermal water loss and indirectly enhancing the repairing effect of the peptide composition.

[0056] Example 7: Lyophilized Powder Ampoules of Peptide Compositions and Solvent Compatibility The lyophilized powder is 50mg / bottle directly dispensed from peptide composition A of Example 1; the solvent is an aqueous solution of 0.05% sodium hyaluronate, 2.0% glycerin, and 1.0% trehalose. When using, the solvent is injected into the lyophilized powder, and after complete dissolution, it is applied to the face.

[0057] The selection of components in the solvent formulation is not arbitrary. Sodium hyaluronate (molecular weight 1000-1500kDa) provides adequate viscosity at a low concentration of 0.05%, facilitating even application and forming a breathable moisturizing film. Glycerin's strong water absorption ensures immediate hydration upon application to the skin after reconstitution. Trehalose, a natural disaccharide, has been proven by numerous studies to stabilize the natural conformation of proteins and peptides in dry and low-temperature environments. Its hydroxyl groups form hydrogen bonds with the amide groups of peptide chains by replacing water molecules, effectively preventing conformational relaxation and subsequent aggregation and precipitation of peptide molecules in solution after reconstitution of the lyophilized powder. The separate packaging design of the solvent and lyophilized powder not only maximizes the storage stability of the active peptides but also gives consumers a sense of "freshly prepared" ritual, enhancing the user experience.

[0058] Comparative Example To verify the synergistic effect and the criticality of the proportions of the compositions of the present invention, the following comparative examples were set up (all were prepared as lyophilized powders according to the process of Example 1, with the total peptide mass controlled at 100 parts, and the volume was adjusted to equal amounts using a mixed solvent of water and butanediol): Comparative Example 1 (Oligopeptide-1 missing): Tripeptide-1 copper 18g, palmitoyl tripeptide-1 20g, acetyl hexapeptide-8 16g, acetyl tetrapeptide-2 14g, pentapeptide-3 12g.

[0059] Comparative Example 2 (without tripeptide-1 copper): Oligopeptide-1 20g, Palmitoyl tripeptide-1 20g, Acetyl hexapeptide-8 16g, Acetyl tetrapeptide-2 14g, Pentapeptide-3 12g.

[0060] Comparative Example 3 (without palmitoyl tripeptide-1): Oligopeptide-1 20g, Tripeptide-1 copper 18g, Acetyl hexapeptide-8 16g, Acetyl tetrapeptide-2 14g, Pentapeptide-3 12g.

[0061] Comparative Example 4 (without Acetyl Hexapeptide-8): Oligopeptide-1 20g, Tripeptide-1 Copper 18g, Palmitoyl Tripeptide-1 20g, Acetyl Tetrapeptide-2 14g, Pentapeptide-3 12g.

[0062] Comparative Example 5 (without Acetyl Tetrapeptide-2): Oligopeptide-1 20g, Tripeptide-1 Copper 18g, Palmitoyl Tripeptide-1 20g, Acetyl Hexapeptide-8 16g, Pentapeptide-3 12g.

[0063] Comparative Example 6 (without pentapeptide-3): Oligopeptide-1 20g, Tripeptide-1 copper 18g, Palmitoyl Tripeptide-1 20g, Acetyl Hexapeptide-8 16g, Acetyl Tetrapeptide-2 14g.

[0064] Comparative Example 7 (ratios outside the preferred range): Oligopeptide-1 30g, Tripeptide-1 Copper 5g, Palmitoyl Tripeptide-1 25g, Acetyl Hexapeptide-8 5g, Acetyl Tetrapeptide-2 20g, Pentapeptide-3 15g (the ratios of each component deviate significantly from the limits defined in this invention).

[0065] Comparative Example 8 (Commercial Reference Product): A hexapeptide anti-wrinkle serum from a well-known international brand. According to its product label, the main peptides include acetyl hexapeptide-8, palmitoyl tripeptide-1, and pentapeptide-3, but it does not contain oligopeptide-1, tripeptide-1 copper, or acetyl tetrapeptide-2. Use it according to its recommended dosage.

[0066] The comparative examples of this invention follow the principle of "systematic subtraction," that is, subtracting each component one by one from the optimal six-component combination to verify the indispensability of each component to the overall efficacy. This method can clearly reveal the specific role played by each peptide in the synergistic network: Comparative Example 1 (deficient oligopeptide-1) is used to verify the damage to the overall synergistic network caused by the absence of the epidermal repair layer; Comparative Example 2 (deficient tripeptide-1 copper) is used to assess the impact of the loss of copper ion-dependent cross-linking function; Comparative Example 3 (deficient palmitoyl tripeptide-1) is used to examine the consequences of the loss of transdermal and signal peptide functions; Comparative Example 4 (deficient acetyl hexapeptide-8) is used to measure the irreplaceability of the neuromuscular rapid inhibitory layer; Comparative Example 5 (deficient acetyl tetrapeptide-2) is used to assess the impact of the loss of elastic fiber assembly function; Comparative Example 6 (deficient pentapeptide-3) is used to confirm the necessity of the long-acting muscle relaxation background. Comparative Example 7 further verifies that even if all six peptides are present, an imbalance in their proportions will lead to "synergistic network imbalance" and a significant decrease in efficacy. This systematic comparative example setup ensures a rigorous demonstration of the synergistic mechanism of this invention.

[0067] The novelty, inventiveness, and utility of the compositions of this invention are demonstrated below through in vitro cell experiments, ex vivo skin models, and human clinical trials. In all tests, except for Comparative Example 8, which was added in the weighed amount of liquid stock solution, all other lyophilized powder samples were first dissolved in PBS or culture medium to a uniform total peptide concentration before application.

[0068] Experimental Example 1: Fibroblast Proliferation and Scratch Repair Assay Human dermal fibroblasts (HDF) were seeded in 24-well plates and, after reaching 90% confluence, uniform "wounds" were created using a sterile pipette tip. Exfoliated cells were washed away with PBS. Serum-free DMEM medium (total peptide concentration of 5 μg / mL) containing samples from each example and comparative example was added, while the blank control group received only medium. After 24 hours of culture, the scratch area was measured under a microscope, and the migration rate (repair rate) was calculated.

[0069] Repair rate (%) = (0h scratch area - 24h scratch area) / 0h scratch area × 100%.

[0070] The results are shown in Table 1.

[0071] Table 1. Scratch repair rate and proliferation activity of fibroblasts Blank control 18.3±2.5 100.0±5.2 Example 1 (Composition A) 78.6±4.2 162.4±8.9 Example 2 (Composition B) 74.8±3.8 156.1±7.5 Example 3 (Composition C) 71.9±4.0 150.7±8.1 Comparative Example 1 (lacking oligopeptide-1) 45.2±3.5 124.3±6.4 Comparative Example 2 (lacking tripeptide-1 copper) 52.1±3.9 130.8±7.1 Comparative Example 3 (Palmitoyl Tripeptide-1 Deficit) 47.8±4.3 128.9±6.8 Comparative Example 4 (lacking acetyl hexapeptide-8) 53.4±3.1 136.5±6.0 Comparative Example 5 (Acetyl Tetrapeptide-2 Deprived) 50.5±4.1 132.0±7.3 Comparative Example 6 (lacking pentapeptide-3) 56.7±3.7 141.2±5.9 Comparative Example 7 (Disproportionate) 62.4±3.3 145.8±7.6 Comparative Example 8 (Commercially Available Comparison) 38.9±3.0 115.6±5.1 As shown in Table 1, the repair rate of Example 1 was as high as 78.6%, almost 1.5 times that of any combination of missing peptides in Comparative Examples 1-6, and more than twice that of the commercially available control, demonstrating that any single deletion would lead to a precipitous drop in the repair effect. This fully confirms that the hexapeptides must coexist and be in the correct proportions to trigger a complete barrier repair process. Although Comparative Example 7 contained all six peptides, the proportions were unbalanced, and the repair rate was only 62.4%, still significantly lower than that of Example 1, indicating that the proportion range of the present invention is crucial for activating the synergistic effect.

[0072] Further analysis of the data in Table 1 reveals an interesting phenomenon: the repair rates of Comparative Example 4 (lacking acetyl hexapeptide-8) and Comparative Example 6 (lacking pentapeptide-3) were 53.4% ​​and 56.7%, respectively, both significantly lower than Example 1 but slightly higher than the other comparative examples. This indicates that although the two peptides in the neuromuscular inhibitory layer do not directly participate in the barrier repair signaling pathway, their presence indirectly promotes the migration and repair efficiency of fibroblasts by reducing the mechanical stress disturbance caused by spontaneous cell contraction during cell culture. This finding further corroborates the scientific validity of the "three-layer synergistic network" theory of this invention; inhibition at the neuromuscular level not only directly contributes to wrinkle smoothing but also has an indirect promoting effect on barrier repair.

[0073] Experiment Example 2: Determination of Collagen and Elastin Synthesis HDF cells were loaded at 2×10 4 / wells were seeded, and when confluence reached 80%, culture medium containing the test sample (total peptide concentration 3 μg / mL) was added and cultured for 72 hours. The supernatant was collected, and the content of type I procollagen C-terminal peptide (PICP) and elastin was detected using an ELISA kit. The results are shown in Table 2.

[0074] Table 2. Collagen and elastin secretion levels Blank control 18.2±3.1 2.4±0.5 Example 1 86.5±5.4 14.8±1.3 Example 2 82.1±4.8 13.9±1.1 Example 3 79.4±5.0 13.2±1.2 Comparative Example 1 42.6±4.2 6.2±0.9 Comparative Example 2 39.5±3.7 5.8±0.8 Comparative Example 3 44.3±4.0 5.1±0.7 Comparative Example 4 51.7±3.9 7.3±0.9 Comparative Example 5 48.2±4.5 6.9±1.0 Comparative Example 6 55.9±4.1 8.0±0.8 Comparative Example 7 68.1±4.9 11.0±1.1 Comparative Example 8 35.8±3.2 4.3±0.6 As can be seen, the synthesis of type I collagen precursor peptide and elastin in Example 1 was significantly increased compared to the comparative example, demonstrating the decisive advantage of the composition in dermal matrix reconstruction. Of particular note is that the elastin synthesis in Comparative Example 5 (acetyl tetrapeptide-2 deficient) was only 6.9 ng / mL, only 46.6% of that in Example 1, while its impact on collagen synthesis was relatively small (PICP was 55.7% of Example 1), which fully verifies the specific role of acetyl tetrapeptide-2 in the elastic fiber synthesis pathway. Meanwhile, Comparative Example 2 (copper tripeptide-1 deficient) showed the largest decreases in both PICP and elastin, dropping to 45.7% and 39.2% of that in Example 1, respectively, indicating that the copper ion-dependent signaling pathway is the core hub for the co-synergistic synthesis of collagen and elastin. These data not only validated the functional localization of each peptide in the synergistic network, but also further demonstrated that there is a positive feedback regulatory axis of "copper-dependent elastic assembly" between tripeptide-1 copper and acetyl tetrapeptide-2. Copper ions are not only a necessary cofactor for LOX activity, but may also upregulate the transcription level of elastin genes through some unknown mechanism. The presence of acetyl tetrapeptide-2 ensures that newly synthesized elastin can be effectively integrated into the elastic fiber network.

[0075] Experimental Example 3: Tightening and Contraction Test of 3D Skin Model A commercially available 3D full-thickness skin model (8 mm in diameter) was used. A sample solution (total peptide concentration 0.02%) was uniformly applied to the model surface once daily for 5 consecutive days. On the 6th day, samples were taken, and the diameter of the skin model was measured using calipers. The shrinkage rate (tightness index) was calculated as: shrinkage rate = (original diameter - treated diameter) / original diameter × 100%. The fibronectin content in the tissue homogenate was also measured. The results are shown in Table 3.

[0076] Table 3. Skin model contraction rate and fibronectin expression Blank control 2.1±0.6 4.2±0.5 Example 1 18.7±1.5 21.4±2.0 Example 2 17.5±1.3 19.8±1.8 Example 3 16.9±1.4 18.9±1.7 Comparative Example 1 8.3±1.2 10.6±1.4 Comparative Example 2 7.6±1.1 9.5±1.3 Comparative Example 4 10.4±1.0 12.3±1.5 Comparative Example 6 11.2±1.3 13.1±1.6 Comparative Example 7 13.8±1.2 16.0±1.7 Comparative Example 8 6.5±0.9 8.2±1.1 The contraction of the model directly reflects the skin's tightening and lifting ability. The composition of this invention achieves a contraction rate of nearly 20%, a significant leap compared to the missing or imbalanced groups. The contraction of the 3D skin model is a combined result of fibroblast-mediated collagen matrix remodeling and myofibroblast contraction. Fibronectin, as a key bridging molecule for cell-matrix adhesion, directly affects the efficiency of force transmission and the final contraction amplitude. In Example 1, the fibronectin expression level (21.4 ng / mg protein) increased by 4.1 times compared to the blank control. This increase significantly exceeded the increases in collagen and elastin (3.8 times and 5.2 times, respectively), suggesting that the upregulation of fibronectin by this composition may be independent of its effect on collagen synthesis and involves the activation of the integrin-FAK signaling pathway. High expression of fibronectin not only enhances the contractile force of fibroblasts but also provides a more complete assembly template for newly synthesized collagen and elastin fibers, ultimately resulting in a significantly enhanced tightening effect at the macroscopic level.

[0077] Experiment Example 4: Quantitative Analysis of Synergistic Effect (Jin Zhengjun Q-value Method) The synergistic coefficient was calculated using the Jin Zhengjun method: Q = EA + B / (EA + EB - EA × EB), where EA is the percentage of the measured effect of a single peptide, EB is another component, and the molecule represents the combined experimental effect. Significant synergy is indicated when Q > 1.15. Comparisons were made between Example 1 and the individual applications and pairwise combinations of the constituent hexapeptides. The overall synergistic Q value of Example 1 was calculated to be 1.37, and the Q value for any five peptides missing was below 0.9. This confirms the synergistic innovation of the present invention from a quantitative pharmacological perspective.

[0078] To further verify the specificity of the synergistic effect, the inventors also calculated the individual effect values ​​of each peptide at the same total peptide concentration. The results showed that: oligopeptide-1 alone achieved a repair rate of 28.4% and collagen synthesis of 32.6 ng / mL; tripeptide-1 copper alone achieved a repair rate of 30.1% and collagen synthesis of 35.8 ng / mL; palmitoyl tripeptide-1 alone achieved a repair rate of 33.7% and collagen synthesis of 38.2 ng / mL; acetyl hexapeptide-8 alone achieved a repair rate of 22.5% (its anti-wrinkle effect is mainly achieved through neuromuscular inhibition rather than promoting repair); acetyl tetrapeptide-2 alone achieved a repair rate of 25.9% and elastin synthesis of 5.4 ng / mL; and pentapeptide-3 alone achieved a repair rate of 24.1%. If a simple additive model is used, the sum of the effects of the six peptides used alone would result in a repair rate of 165.7%, while the measured repair rate in Example 1 was 78.6% (based on the relative reduction of the scratch area). However, if the repair rate is converted to a repair rate and normalized with the blank control, the weighted average effect value of the six peptides used alone is approximately 52.3%, while Example 1 showed 78.6%—the Q-value calculation confirms that 1.37 > 1.15, clearly ruling out a simple additive effect. This indicates that the synergy of the present invention is not a simple accumulation of peptide types, but rather the result of nonlinear coupling between molecular pathways at a precise ratio.

[0079] Experiment Example 5: Human Clinical Efficacy Test Sixty female subjects aged 35-55 years were recruited and randomly divided into 6 groups of 10 each. Group 1 used the serum from Example 5, Group 2 used the serum containing Comparative Example 1, Group 3 used the serum containing Comparative Example 2, Group 4 used the serum containing Comparative Example 4, Group 5 used the serum containing Comparative Example 7, and Group 6 used the commercially available product from Comparative Example 8. All subjects used the product twice daily, morning and evening, for 8 consecutive weeks. The depth of crow's feet (using the PRIMOS high-resolution skin texture analyzer) and the skin elasticity R² value (Cutometer) were assessed at weeks 0, 4, and 8 after use. The data are shown in Table 4.

[0080] Table 4. Results of human clinical tests (mean ± standard deviation) Group 1 (Formula of Example 1) Week 0 68.7±12.5 0.642±0.048 Week 4 52.1±10.3 0.701±0.042 Week 8 41.2±8.7 0.735±0.035 Wrinkle reduction rate 40.0％ - elasticity increase rate - 14.5％ Group 2 (lacking oligopeptide-1) Week 0 67.9±13.1 0.638±0.052 Week 8 58.3±11.2 0.668±0.046 Wrinkle reduction rate 14.1％ Elasticity increased by 4.7%. Group 3 (lacking tripeptide-1 copper) Week 8 56.5±10.9 0.672±0.044 Reduction rate 16.8％ Increased by 5.3% Group 4 (lacking acetyl hexapeptide-8) Week 8 57.8±11.5 0.678±0.039 Reduction rate 14.9％ Increased by 6.3% Group 5 (Disproportionate) Week 8 52.9±10.8 0.695±0.041 Reduction rate 22.0％ Increased by 8.9% Group 6 (Commercially Available) Week 8 59.1±12.0 0.665±0.047 Reduction rate 12.9％ Increased by 4.2% The results were highly convincing: the first group showed a 40% reduction in wrinkle depth and a nearly 15% improvement in skin elasticity. In comparison, the improvements achieved by any group lacking peptides, the group with imbalanced proportions, and commercially available products were all less than 50% of those of this invention. In the self-assessment questionnaire, the first group showed a satisfaction rate of over 92% for "improved skin firmness," "reduction of fine lines and wrinkles," and "smooth skin texture," with no complaints of irritation.

[0081] It is worth noting the time-matter data in the first group: by week 4, wrinkle depth had already decreased by 23.9% and elasticity increased by 9.2%, indicating that the composition of this invention exhibits rapid onset of action within 4 weeks. This is closely related to the immediate muscle relaxation effects of acetyl hexapeptide-8 and pentapeptide-3. The further improvement at week 8 compared to week 4 (wrinkle depth decreased from 52.1 μm to 41.2 μm, and elasticity increased from 0.701 to 0.735) reflects the sustained cumulative effect of dermal matrix reconstruction. This dual-phase efficacy of "immediate + long-lasting" is precisely the core clinical application advantage of the composition of this invention—consumers can feel the improvement in skin smoothness in the early stages of use (1-4 weeks), building confidence in its use; after continuous use (5-8 weeks), the effect of deep tissue reconstruction begins to appear, and the skin becomes firmer and more elastic.

[0082] Experiment Example 6: Transdermal Water Loss (TEWL) Repair Effect Test A standardized skin barrier injury model was created on the inner forearm of 20 subjects using an adhesive tape peeling method. A 0.5% aqueous solution containing the peptide composition of Example 1 and a control solution (containing only solvent) were applied to both. TEWL values ​​were measured before treatment and at 1 h, 4 h, and 8 h after treatment using a Tewameter TM300. The results showed that the TEWL value in the Example 1 treatment group decreased by 42.3% after 4 h compared to immediately after injury, and by 65.7% after 8 h, while the control group only decreased by 15.1% and 28.4%, respectively. This data directly confirms the highly efficient repair ability of the composition of this invention on physical skin barrier damage and provides strong evidence for the rapid activation of the oligopeptide-1-led epidermal repair layer synergistic network.

[0083] Experiment Example 7: Fibroblast contractility test (cellular mechanical verification of the tightening mechanism) Human dermal fibroblasts were seeded in a collagen gel matrix to construct a three-dimensional cell-collagen contraction model. Culture medium containing samples from each example and comparative example (total peptide concentration 5 μg / mL) was added, and after 48 h of culture, the surface area change of the collagen gel was measured to calculate the contraction rate. Results: The contraction rate of the blank control group was 15.7%, the contraction rate of Example 1 group reached 48.3%, the contraction rate of Comparative Example 2 (deficient in tripeptide-1 copper) was 22.8%, the contraction rate of Comparative Example 5 (deficient in acetyl tetrapeptide-2) was 26.1%, and the contraction rate of Comparative Example 6 (deficient in pentapeptide-3) was 30.4%. This result indicates that tripeptide-1 copper plays a central role in the mechanical coupling of fibroblasts and collagen (its deficiency leads to a sharp drop in contraction rate of 53%), while the contributions of acetyl tetrapeptide-2 and pentapeptide-3 to cell contraction are also significant. The synergistic destructive effect of the loss of these three components is far greater than the algebraic sum of the effects of individual loss.

[0084] Stability assessment: The lyophilized powder composition A prepared in Example 1 was subjected to accelerated testing at 45°C and 75% RH. Samples were taken at 0, 1, 2, and 3 months, and the changes in the content of six peptides were determined by high-performance liquid chromatography (HPLC). The results showed that after 3 months, the residual contents of oligopeptide-1, tripeptide-1 copper, palmitoyl tripeptide-1, acetyl hexapeptide-8, acetyl tetrapeptide-2, and pentapeptide-3 were 96.2%, 95.8%, 97.1%, 96.9%, 95.4%, and 96.5% of the initial contents, respectively, with no significant degradation. In contrast, the content of acetyl hexapeptide-8 in the commercially available stock solution decreased by approximately 18% after 1 month under the same conditions. This indicates that the solid form of the composition of the present invention has excellent long-term stability.

[0085] To further investigate the molecular mechanism of the stability of the compositions of this invention, the inventors performed circular dichroism (CD) spectroscopy analysis on samples before and after the accelerated testing. The results showed that after 3 months of accelerated testing, the CD spectrum of the composition of Example 1 highly overlapped with the initial sample, with no significant changes in the peak shape and position (deviation less than 3%), indicating that the secondary structure of each peptide remained stable during storage. However, the CD spectrum of Comparative Example 7 (with an unbalanced ratio) showed a significant shift in the 195-210 nm region, suggesting the formation of disordered β-sheet aggregates between peptide molecules. This data explains the structural basis for the excellent stability of the compositions of this invention at the conformational level—only under specific proportions can the intermolecular protective microclusters formed by the six peptides through hydrophobic pairing, hydrogen bonding networks, and metal coordination remain stable. In addition, the inventors conducted a "proportional stability" experiment: the six peptides were dissolved and freeze-dried according to the proportions of Example 1 and Comparative Example 7, respectively. The activity retention rate of the freeze-dried powder after 3 months of storage under accelerated conditions of 45°C / 75%RH was measured—Example 1 showed over 95.4%, while Comparative Example 7 showed only 68.3% (oligopeptide-1) to 79.5% (palmitoyl tripeptide-1), a significant difference. This result, from another perspective, proves that the proportion range defined in this invention is not only the optimal range for efficacy but also the optimal range for stability.

[0086] To further verify the stability of the composition of the present invention under real shelf conditions, the inventors also conducted a long-term storage experiment: the lyophilized powder composition A of Example 1 was stored at 25°C / 60%RH and 4°C in the dark for 24 months, with samples taken and tested every 3 months. After 24 months, the content retention rate of the six peptides was between 91.3% and 94.7% at 25°C and between 97.1% and 98.4% at 4°C. This result fully demonstrates that the lyophilized powder composition of the present invention can achieve a shelf life of at least 24 months under actual shelf conditions, far exceeding the typical shelf life of existing commercially available liquid peptide products (usually 6 to 12 months).

[0087] Safety assessment: In addition to human patch testing, the inventors also conducted the following safety evaluation experiments: (1) Repeated skin irritation test: In accordance with the method of the "Cosmetic Safety Technical Specifications" (2015 edition), the essence of Example 5 was continuously applied to the skin of the back of guinea pigs once a day for 14 consecutive days. No irritation reactions such as erythema and edema were observed, and the irritation index was zero. (2) Acute eye irritation test: 0.1 mL of the essence of Example 5 was dripped into the conjunctival sac of rabbit eyes and observed at 1h, 24h, 48h, and 72h. No eye irritation reactions such as conjunctival hyperemia, edema, or corneal opacity were observed. According to the Draize scoring method, it was determined to be non-irritating. (3) Phototoxicity test: In accordance with the 3T3 neutral red uptake phototoxicity test method of OECD TG432, the IC50 value of the composition of Example 1 on Balb / c 3T3 fibroblasts under UVA irradiation (5J / cm²) was not significantly different from that under no irradiation conditions, and it was determined to be non-potentially phototoxic. (4) Human Repeated Injury Patch Test (HRIPT): 100 healthy volunteers underwent 9 induction patch tests and 1 provocation patch test over 6 weeks. No subjects showed any sensitization reaction, indicating that the composition of the present invention is non-sensitizing to the skin. This series of safety evaluation data provides sufficient safety assurance for the widespread application of the composition of the present invention in cosmetics and dermatological products.

[0088] Transdermal absorption evaluation: The transdermal permeability of the six peptides in the composition of Example 1 was evaluated using the Franz diffusion cell method with isolated rat abdominal skin as the transdermal barrier. A 0.1% PBS solution containing the peptide composition was added to the supply cell, and PBS (pH 7.4) was added to the receiving cell. Samples were taken at 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and the concentration of each peptide in the receiving solution was quantified by HPLC-MS / MS. The results showed that palmitoyl tripeptide-1, due to palmitoylation modification, had a cumulative transdermal permeability of 68.3±5.2% at 24 h, significantly higher than that of unmodified oligopeptide-1 (32.7±4.1% at 24 h) and tripeptide-1 copper (29.5±3.8%). The 24 h transdermal permeability of acetyl hexapeptide-8, acetyl tetrapeptide-2, and pentapeptide-3 were 41.2±4.5%, 37.8±4.0%, and 44.6±4.9%, respectively. Comprehensive transdermal data show that each peptide component in the composition of the present invention has good transdermal performance. Palmitoyl tripeptide-1, as a "transdermal pioneer", can reach the dermis first to activate fibroblasts, creating an active cellular response environment for the peptide components that arrive later, thereby achieving synergy in the time dimension.

[0089] In summary, based on a deep understanding of multi-target anti-aging signaling pathways, this invention has screened out the optimal synergistic ratio of six specific peptides, exhibiting significantly superior repair, anti-wrinkle, and firming properties compared to existing technologies at the cellular, tissue, and human levels. The preparation process is simple, stable, safe, and efficient, showing promising prospects for industrial application. The "three-layer hexa-peptide synergistic network" theoretical framework and intermolecular protective microcluster structure stabilization strategy established in this invention are not only applicable to the specific hexa-peptide combination of this invention but also provide methodological guidance for the future development of more multi-target anti-aging compound solutions based on synergistic mechanisms.

[0090] It should be noted that although the present invention has been described in detail through the above specific embodiments, those skilled in the art should understand that various modifications and changes can be made to the present invention without departing from its spirit and scope, and such modifications and changes also fall within the protection scope of the present invention. The protection scope of the present invention is defined by the appended claims.

Claims

1. A repairing, anti-wrinkle, and firming peptide composition, characterized in that, It contains the following components in parts by weight: Oligopeptide-1: 15-25 parts; Tripeptide-1 copper: 13-23 parts; Palmitoyl tripeptide-1: 15-25 parts; Acetyl hexapeptide-8: 12-20 parts; Acetyl tetrapeptide-2: 10–18 parts; Pentapeptide-3: 8 to 16 parts.

2. The repairing, anti-wrinkle, and firming peptide composition according to claim 1, characterized in that, It contains the following components in parts by weight: Oligopeptide-1: 18-22 parts; Tripeptide-1 copper: 16-20 parts; Palmitoyl tripeptide-1: 18–22 parts; Acetyl hexapeptide-8: 14–18 parts; Acetyl tetrapeptide-2: 12-16 parts; Pentapeptide-3: 10-14 parts.

3. The repairing, anti-wrinkle, and firming peptide composition according to claim 2, characterized in that, It contains the following components in parts by weight: Oligopeptide-1: 20 parts; Tripeptide-1 copper: 18 parts; Palmitoyl tripeptide-1: 20 parts; Acetyl hexapeptide-8: 16 parts; Acetyl tetrapeptide-2: 14 parts; Pentapeptide-3: 12 parts.

4. A method for preparing a repairing, anti-wrinkle, and firming peptide composition according to any one of claims 1-3, characterized in that, The process includes the following steps: weighing each peptide component according to the ratio, dissolving it in a solvent, stirring and mixing it evenly to obtain a mixture; and subjecting the mixture to sterile filtration and / or drying.

5. The method for preparing a repairing, anti-wrinkle, and firming peptide composition according to claim 4, characterized in that, The solvent is selected from water, glycerol, propylene glycol, butylene glycol, pentanediol, hexanediol, or any combination thereof.

6. The method for preparing a repairing, anti-wrinkle, and firming peptide composition according to claim 4, characterized in that, The drying process is freeze drying or low-temperature spray drying.

7. A cosmetic or dermatological composition, characterized in that, It comprises a repairing, anti-wrinkle, and firming peptide composition according to any one of claims 1-3, and cosmetically or dermatologically acceptable excipients.

8. A cosmetic or dermatological composition according to claim 7, characterized in that, The dosage form of the cosmetic or dermatological composition is a serum, lotion, cream, freeze-dried powder, mask, eye cream, or neck cream.

9. A cosmetic or dermatological composition according to claim 7 or 8, characterized in that, The repairing, anti-wrinkle, and firming peptide composition is present in the cosmetic or dermatological composition at a weight percentage of 0.001% to 10%.

10. The use of a repairing, anti-wrinkle, and firming peptide composition according to any one of claims 1-3 or a cosmetic or dermatological composition according to any one of claims 7-9 in the preparation of products for repairing the skin barrier, reducing wrinkles, and / or firming the skin.