Application of blue copper peptide composition in prevention of hyperpigmentation and acceleration of wound healing after phototherapy microneedle operation

Abstract

The invention belongs to the technical field of skin repair and medical skin care products, and discloses application of a blue copper peptide composition in prevention of hyperpigmentation and acceleration of wound healing after a phototherapy microneedle operation. The composition comprises the following active components: blue copper peptide, line particle energy peptide, SnMS protein, nicotinamide and hyaluronic acid, and the components are compounded according to a specific ratio. Through gene reset and system repair, matrix'removal-reconstruction 'biphasic regulation and control, an SIRT1 / STAT3 repair axis is activated, and a multi-target linkage mechanism of multi-channel inhibition of hyperpigmentation is realized, so that high-quality wound healing and hyperpigmentation prevention are realized. The composition has no cytotoxicity, can significantly reduce the ROS content, inhibit the expression of inflammatory factors, and improve the melanin inhibition rate and collagen synthesis amount, solves the problems of single target spot and function fragmentation of the existing postoperative repair product, and upgrades the postoperative care from passive injury repair to active regeneration guidance.

Description

Technical Field

[0001] This invention belongs to the field of skin repair and medical skin care technology, specifically relating to a blue copper peptide composition, and more particularly to the application of a blue copper peptide composition in preventing pigmentation and accelerating wound healing after phototherapy microneedling. Background Technology

[0002] With the popularization and refinement of cosmetic dermatology techniques, fractional laser, intense pulsed light (IPL), radiofrequency microneedling, and various minimally invasive phototherapy methods have been widely used for skin improvement, pigmentation removal, scar repair, and skin rejuvenation, becoming mainstream choices in both clinical practice and among consumers. These techniques create controllable, minimally invasive damage to skin tissue, activating the skin's own repair and collagen remodeling mechanisms to improve skin structure and appearance. However, in clinical applications, the integrity of the skin barrier is disrupted after phototherapy and microneedling, accompanied by significant acute inflammatory reactions, oxidative stress damage, and abnormal activation of melanocytes. This leads to two prominent clinical problems: First, the incidence of post-inflammatory hyperpigmentation (PIH) is high. Asian populations, due to higher sensitivity of skin melanocytes, are more prone to localized hyperpigmentation and recurrence of pigmentation under inflammatory stimulation, significantly reducing treatment effectiveness and patient compliance. Second, wound healing is slow, repair quality is insufficient, epidermal barrier reconstruction is delayed, and dermal collagen fiber arrangement is disordered, easily leading to dryness, desquamation, redness, sensitivity, and even scar hyperplasia. Traditional care methods struggle to achieve efficient and orderly tissue regeneration. (Mei, XL, & Wang, L. (2018). Ablative fractional carbon dioxide lasercombined with intense pulsed light for the treatment of photoaging skin inChinese population: A split-face study. Medicine, 97(3), e9494. https: / / doi.org / 10.1097 / MD.000000000009494).

[0003] Current clinical postoperative repair products and solutions still have significant shortcomings: most repair products are based on single functional ingredients, such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which mainly act on epidermal cell proliferation and have limited effects on dermal collagen remodeling, inflammatory signal regulation, and pigmentation prevention (Hu F, Wang X, Liang G, Lv L, Zhu Y, Sun B, Xiao Z. Effects of epidermal growth factor and basic fibroblast growth factor on the proliferation and osteogenic and neural differentiation of adipose-derived stem cells. Cell Reprogram. 2013 Jun;15(3):224-32. doi:10.1089 / cell.2012.0077. PMID: 23713433; PMCID: PMC3666248.); Dipotassium glycyrrhizate, bisabolol, and other anti-inflammatory and soothing ingredients can only relieve acute symptoms such as redness, swelling, heat, and pain, and cannot actively guide the precise repair of skin tissue (CN116509752A Publication Date: 2023.08.01); Niacinamide, arbutin, and other whitening ingredients mostly target existing melanin and are difficult to inhibit melanocyte activation from the source of inflammation, resulting in weak preventive effects (Burger, P.; Landreau, A.; Azoulay, S.; Michel, T.; Fernandez, X. Skin Whitening Cosmetics: Feedback and Challenges in the Development of Natural Skin Lighteners. Cosmetics 2016, 3,36. https: / / doi.org / 10.3390 / cosmetics3040036). Overall, existing technologies mostly remain at the level of passive moisturizing, anti-inflammation, and anti-infection, with single targets and independent pathways. They lack an integrated synergistic mechanism that goes from inhibiting excessive inflammation, regulating cell repair, promoting orderly collagen production to blocking abnormal pigmentation. They have failed to form a synergistic repair network from gene regulation → inflammation suppression → precise matrix remodeling → pigmentation prevention, and cannot simultaneously and efficiently address the core needs of accelerated wound healing and pigmentation prevention after phototherapy microneedling.

[0004] Blue copper peptide, as a tripeptide-copper complex with multiple biological activities, can participate in the regulation of skin repair-related signaling pathways, promote fibroblast proliferation and collagen synthesis, and also has the potential for anti-inflammatory, antioxidant and wound repair promotion (Pickart, L., Vasquez-Soltero, JM, & Margolina, A. (2015). GHK Peptide as a Natural Modulator of Multiple Cellular Pathways in Skin Regeneration. BioMed research international, 2015, 648108. https: / / doi.org / 10.1155 / 2015 / 648108). However, when used alone, its mode of action is singular and its targeting is insufficient, making it difficult to systematically cover the entire process of postoperative inflammation control, barrier reconstruction, collagen remodeling and pigmentation prevention. Existing single-component products containing copper peptides or nicotinamide have not been optimized for the characteristics of post-phototherapy microneedling damage, and cannot achieve synergistic effects of repair and color inhibition. They have limited effects on improving the quality of post-operative wound healing and preventing post-inflammatory hyperplasia (PIH), and cannot meet the clinical demand for efficient, safe, and integrated post-operative repair solutions.

[0005] Therefore, developing a blue copper peptide composition that can synergistically inhibit inflammatory responses, accelerate wound healing, improve repair quality, and prevent post-inflammatory hyperpigmentation from the source, and applying it to post-phototherapy microneedling care to overcome the shortcomings of existing technologies such as single post-operative repair targets, fragmented functions, and insufficient repair efficiency, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an application of a blue copper peptide composition in preventing pigmentation and accelerating wound healing after phototherapy microneedling. Its active ingredients include blue copper peptide (GHK-Cu), mitochondrial energy peptide, SnMS protein, nicotinamide, and hyaluronic acid. The components, when combined in a specific ratio, produce a synergistic effect, achieving high-quality wound healing and preventing pigmentation through multi-target and multi-pathway linkage.

[0007] The mechanism of action of this composition is as follows: (1) Gene resetting and system repair: As a core signaling molecule, copper peptide can induce a 31.2% shift in the gene expression profile of skin cells toward rejuvenation. Mitochondrial energy peptide can specifically enhance this effect, synergistically upregulate DNA repair-related genes (such as XRCC1) and antioxidant genes (such as SOD2), and downregulate key inflammatory factors (such as IL-6, TNF-α) and fibrosis-related genes (such as TGF-β1), thereby optimizing the cell's repair potential from the root. (2) Matrix "clearance-reconstruction" biphasic regulation: Copper peptide can stimulate the activity of matrix metalloproteinase-2 (MMP-2), promoting the "clearance" of denatured collagen fragments caused by photothermal damage. At the same time, copper peptide, SnMS protein and nicotinamide work synergistically to strongly stimulate the synthesis of type I and type III collagen and core proteoglycans (Decorin). The synchronous increase of Decorin is crucial. It can guide the orderly arrangement of newly formed collagen fibers like a "spacer" and prevent their cross-linking from becoming disordered, thereby structurally reducing scar formation and promoting the normalization of skin texture. (3) Activation of the synergistic repair signal axis: Blue copper peptide is an effective activator of SIRT1 (deacetylase 1). Activated SIRT1 can further regulate the STAT3 signaling pathway to form the "SIRT1 / STAT3 repair axis". This axis can significantly reduce the inflammatory response, promote the rapid reconstruction of the mucosa and skin barrier, and enhance the cell's self-renewal ability. (4) Multi-pathway inhibition of pigmentation: a) Reduce the stimulation of melanocytes from the source through the above-mentioned potent anti-inflammatory mechanism (SIRT1 / STAT3 axis activation and downregulation of inflammatory genes); b) Nicotinamide can inhibit the transport of melanosomes from melanocytes to keratinocytes; c) The antioxidant effects of blue copper peptide and mitochondrial energy peptide can reduce the induction of pigment synthesis by oxidative stress. The combination of the three can achieve preventive melanin inhibition.

[0008] On one hand, the present invention provides the application of a blue copper peptide composition in the preparation of a product for preventing pigmentation after phototherapy microneedling.

[0009] On the other hand, the present invention provides the use of a blue copper peptide composition in the preparation of a product for accelerating wound healing after phototherapy microneedling.

[0010] Specifically, the active ingredients of the blue copper peptide composition include blue copper peptide, mitochondrial energy peptide, SnMS protein, nicotinamide, and hyaluronic acid; preferably, the blue copper peptide composition further comprises a pharmaceutically acceptable carrier.

[0011] Specifically, the amino acid sequence of the mitochondrial energy peptide is shown in SEQ ID NO.1, and the amino acid sequence of the SnMS protein is shown in SEQ ID NO.2.

[0012] Specifically, by weight percentage, the proportions of each active ingredient in the blue copper peptide composition are as follows: blue copper peptide 0.01%-0.5%, mitochondrial energy peptide 0.001%-1%, SnMS protein 0.1%-10%, nicotinamide 0.1%-5%, and hyaluronic acid 0.01%-2%;

[0013] Preferably, the proportions of each active ingredient in the blue copper peptide composition are as follows: blue copper peptide 0.1%-0.3%, mitochondrial energy peptide 0.01%-0.1%, SnMS protein 1%-5%, nicotinamide 1%-3%, and hyaluronic acid 0.05%-1%;

[0014] More preferably, it contains 0.1% copper peptide, 0.02% mitochondrial energy peptide, 5% SnMS protein, 1% nicotinamide, and 0.05% hyaluronic acid.

[0015] Specifically, the blue copper peptide composition further contains panthenol, sodium 2-hydroxypyruvate, polyethylene glycol, glycerin, allantoin, betaine, and xanthan gum; preferably, by weight percentage, the proportions of each component are: panthenol 0.5%-2%, sodium 2-hydroxypyruvate 0.1%-0.5%, polyethylene glycol-8 1%-7%, glycerin 1%-5%, allantoin 0.1%-0.5%, betaine 0.01-0.5%, and xanthan gum 0.05-0.3%; more preferably, by weight percentage, the proportions of each component are: panthenol 1%-1.5%, sodium 2-hydroxypyruvate 0.1%-0.3%, polyethylene glycol-8 3%-5%, glycerin 3%-5%, allantoin 0.1%-0.3%, betaine 0.1-0.2%, and xanthan gum 0.1-0.15%.

[0016] Specifically, the blue copper peptide composition achieves anti-inflammatory effects by downregulating the expression of IL-6 and TNF-α inflammatory factors, inhibits melanin production by suppressing melanosome transport, and guides the orderly arrangement of newly formed collagen fibers by stimulating the synthesis of type I and type III collagen and core proteoglycans.

[0017] Specifically, the phototherapy is fractional laser, intense pulsed light, and / or radiofrequency microneedle-based minimally invasive phototherapy.

[0018] Specifically, the products include pharmaceuticals, skincare products, or medical dressings.

[0019] Specifically, the product is in the form of a gel, cream, lotion, or water, or a spray.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) Synergistic repair effect: The combination of blue copper peptide with mitochondrial energy peptide, SnMS protein and nicotinamide achieves a synergistic effect of 1+1>2 in collagen synthesis and anti-inflammation, which is far superior to single ingredients. It can significantly accelerate the healing of wounds after phototherapy microneedling and improve the quality of skin repair.

[0022] (2) Prevention of pigmentation at the source: Through the synergistic effect of anti-inflammatory regulation, inhibition of melanosome transport and antioxidant pathway at the gene level, post-inflammatory hyperpigmentation is inhibited from the root cause rather than after-the-fact remedy, which greatly reduces the incidence of postoperative PIH in Asian populations.

[0023] (3) It provides a brand-new postoperative care strategy: by regulating the complete chain of "gene reset-signal axis activation-matrix remodeling", the postoperative care is upgraded from passive moisturizing and anti-inflammatory to active regeneration guidance, which systematically solves the core problems such as slow postoperative barrier reconstruction, disordered collagen arrangement, and hyperpigmentation.

[0024] (4) Excellent safety: In vitro cell experiments have confirmed that different concentrations of the composition have no cytotoxicity to human keratinocytes, and have a safe basis for clinical application.

[0025] (5) Multi-target full-process repair: covering the entire process of postoperative inflammation control, barrier reconstruction, collagen remodeling and pigmentation prevention, forming an integrated synergistic repair network, which makes up for the shortcomings of the independent action pathway of existing products. Attached Figure Description

[0026] Figure 1 This is a schematic diagram showing the effects of copper peptide, mitochondrial energy peptide, and formulation composition on ROS content.

[0027] Figure 2 This is a schematic diagram showing the effects of different samples on inflammatory factors. In the diagram, A represents the expression of IL-6 in samples with different treatments, and B represents the expression of TNFα in samples with different treatments.

[0028] Figure 3 This is a schematic diagram illustrating the inhibitory effect of nicotinamide and the formulation composition on melanin.

[0029] Figure 4 This is a schematic diagram showing the effect of different samples on collagen content. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Example 1: Preparation of the composition gel

[0034] A composition for post-phototherapy or microneedling care includes, as its active ingredients, blue copper peptide (GHK-Cu), mitochondrial energy peptide, SnMS protein, nicotinamide, and hyaluronic acid. The components, in a specific ratio, produce a synergistic effect, achieving high-quality wound healing and prevention of pigmentation through multi-target and multi-pathway linkage. The mitochondrial energy peptide is an active polypeptide with the amino acid sequence CLAGRRRRSV (as shown in SEQ ID NO.1) (derived from SA-LFP1 shown in SEQ ID NO.1 of patent CN117024518A), and the SnMS protein has the amino acid sequence shown in SEQ ID NO.2 (derived from the amino acid sequence shown in SEQ ID NO.1 of patent CN119161433A).

[0035] 1. Formula (by weight percentage)

[0036] Key ingredients: Blue copper peptide (GHK-Cu): 0.1%; Mitochondrial energy peptide: 0.02%; SnMS protein: 5%; Niacinamide: 1%; Hyaluronic acid: 0.05%;

[0037] Other components: Panthenol: 1%; Sodium 2-hydroxypyruvate: 0.3%; Polyethylene glycol-8: 3%; Glycerin: 3%; Allantoin: 0.3%; Betaine: 0.2%; Xanthan gum: 0.15%; Water: Balance;

[0038] 2. Configuration Method

[0039] (1) Weigh out the following ingredients according to the above formula: copper peptide, mitochondrial energy peptide, SnMS protein, nicotinamide, hyaluronic acid, sodium 2-hydroxypyruvate and panthenol. Heat and stir to about 80-85℃ and keep warm for 15 minutes before use.

[0040] (2) Weigh out polyethylene glycol-8, glycerin, allantoin, betaine and xanthan gum according to the above formula, then add the remaining water and mix well for later use.

[0041] (3) Stir the system in 1) and cool it down to 45-50℃, then add the sample from system 2) and disperse it evenly.

[0042] Example 2, Safety / Irritation Test - Cytotoxicity

[0043] 1. Cytotoxicity detection protocol

[0044] Human keratinocytes (HaCaT, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were seeded at a density of 1.0 × 10⁵ cells in 96-well plates. After 24 hours of stable culture, different concentrations of the formulated combination were added to the cells for treatment. The treatment group received 5% of the formulated combination; the control group received 0.05% of the formulated combination; and the blank control group received PBS. Cells were cultured at 37°C and 5% CO₂ for 24 hours. The culture medium was then discarded, and 90 μL of basal medium and 10 μL of CCK8 were added to each well. The mixture was incubated at 37°C for 40 minutes, and the absorbance was measured at 450 nm after incubation. Cell viability (%) = (OD of experimental group - OD of blank well) / (OD of blank control group - OD of blank well) 100%. Cell activity <70% indicates that the formulation composition is cytotoxic.

[0045] 2. Experimental Results

[0046] The cytotoxicity test results of different formulations are shown in Table 1, where + indicates: cytotoxicity; Representative: No cytotoxicity; results indicate that the formulation composition is non-toxic to HaCaT cells.

[0047] Table 1. Cytotoxicity Results of the Formulation Composition

[0048]

[0049] Example 3: Antioxidant Effect - ROS Content Test

[0050] ROS, or reactive oxygen species, damage biological membranes. They react with cellular membrane phospholipids to produce lipid peroxides, which can cause DNA damage and lead to aging. Furthermore, ROS can disrupt cell membrane integrity and the intracellular antioxidant system, resulting in cellular inflammation, apoptosis, and tumor formation. They also degrade elastin, collagen, and hyaluronic acid, leading to decreased skin elasticity, roughness, and wrinkles. Therefore, reducing the content of ROS in cells plays a crucial role in anti-oxidation and preventing aging. After AAPH treatment of cells, a large amount of ROS is produced. The relative fluorescence intensity is obtained by measuring the fluorescence absorbance at a specific wavelength and cell activity. Relative fluorescence intensity = ROS fluorescence intensity / cell activity.

[0051] HaCaT cells in the logarithmic growth phase were digested with 0.25% trypsin to detach adherent cells, and the cells were counted at 1–4 × 10^6 cells / mL to prepare a cell suspension. Cells were seeded at a density of 1 × 10^5 cells / mL in 96-well plates. An oxidative damage model (AAPH injury) was established when the cells reached approximately 80% confluence. The blank control group was not subjected to AAPH injury and was treated with an equal volume of PBS. The model group and the drug group were treated with complete culture medium containing a final concentration of 85 mM AAPH for 2 hours. After treatment, the drug treatment groups were treated with the drug diluted in complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin) to ensure the final concentrations met were: 0.1% copper peptide (GHK-Cu); 0.02% mitochondrial energy peptide; 5% SnMS protein; 1% nicotinamide; 0.05% hyaluronic acid), 0.1% copper peptide, and 0.02% mitochondrial energy peptide. The model group was treated with an equal volume of PBS and complete culture medium. The blank control group, model group, and drug-treated group were incubated at 37°C and 5% CO2 for 24 hours. After the culture was completed, the culture medium was discarded, the cells were washed with PBS, DMEM culture medium and 10 μM DCFH probe solution were added, and the cells were incubated at 37°C for 20 min. After washing the cells twice with DMEM culture medium, the OD values ​​of each well were measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Then, the cells were replaced with basal medium containing 10% CCK8 and incubated for 45 min. The OD values ​​of each well were then measured at 450 nm.

[0052] The results are as follows Figure 1 As shown, after treatment with 0.1% copper peptide, the ROS content of cells decreased by 13%, and after treatment with 0.02% mitochondrial energy peptide, the ROS content of cells decreased by 28%. The experimental results show that both copper peptide and mitochondrial energy peptide can slightly reduce the ROS content. Compared with the control group, the formulation composition can significantly reduce the ROS content of cells after treatment, with a reduction rate of 48%, and the effect is significantly better than using 0.1% copper peptide or 0.02% mitochondrial energy peptide alone.

[0053] Example 4: Anti-inflammatory effect

[0054] Lipopolysaccharide (LPS) binds to cell surface receptors, activating the expression of inflammatory cytokine genes and inducing an inflammatory response. Cells then secrete a series of pro-inflammatory factors, known as senescence-associated secretory phenotypes (SASPs), which promote chronic inflammation and drive normal cells into the senescence process. Therefore, inhibiting the expression of inflammatory factors and reducing the inflammatory response can help prevent cells from entering the senescence process. This experiment used a LPS-induced inflammatory cell model treated with test samples to detect the levels of IL-6 and TNFα in the corresponding cells to determine whether the composition of this invention can inhibit the expression of the inflammatory factors IL-6 and TNFα.

[0055] Mouse macrophages in logarithmic growth phase (Raw264.7 cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were collected and digested with 0.25% trypsin to detach the adherent cells and prepare a cell suspension. Cells were seeded at a density of 5 x 10^5 cells / mL in 96-well plates and incubated until approximately 80% confluence. The blank control group received PBS; the treatment group received 1 μg / mL LPS and the formulated combination (blue copper peptide (GHK-Cu): 0.1%; mitochondrial energy peptide: 0.02%; SnMS protein: 5%; nicotinamide: 1%; hyaluronic acid: 0.05%) and 0.02% mitochondrial energy peptide, followed by incubation at 37°C and 5% CO2 for 24 h. After incubation, the cell culture medium was collected to obtain sample solutions, and the IL-6 and TNFα ELISA procedures were performed according to the instructions. The OD values ​​of each well were measured sequentially at 450 nm using a microplate reader within 15 min.

[0056] The results are as follows Figure 2 As shown, compared with the blank control group, the model group IL-6 ( Figure 2 As shown in Figure A), TNFα concentration ( Figure 2 As shown in Figure B, all IL-6 and TNFα concentrations increased significantly, indicating that the inflammation model was successfully induced. After treatment with 0.02% mitochondrial energy peptide, IL-6 concentration decreased by 13% and TNFα concentration decreased by 17%. After treatment with the formulated composition, both IL-6 and TNFα concentrations decreased by 29%, and the concentrations of inflammatory factors decreased, indicating that both 0.02% mitochondrial energy peptide and the formulated composition have anti-inflammatory effects. Compared with 0.02% mitochondrial energy peptide, the reduction rate of inflammatory factor content after treatment with the formulated composition was nearly twice that after treatment with mitochondrial energy peptide, indicating that the composition of this invention has a better anti-inflammatory effect through the synergistic effect of multiple components.

[0057] Example 5: Inhibition of melanin formation

[0058] Melanin is not typically secreted directly outside the cell in free form; instead, its functional distribution is achieved through specific intercellular transport mechanisms. After synthesis by melanosomes within melanocytes, melanin is transferred to neighboring keratinocytes via dendritic processes. This process relies on intercellular membrane fusion or exocytosis (e.g., transport mediated by Rab GTPases and SNARE proteins). In the skin, melanosomes are ultimately taken up by keratinocytes and stored in their cytoplasm, forming skin pigment deposits. Therefore, melanin can be obtained by disrupting cells for detection.

[0059] Specific experimental steps:

[0060] (1) Cells were collected after 24 hours of treatment with different sample solutions (drug administration group: formulation composition (blue copper peptide (GHK-Cu): 0.1%; mitochondrial energy peptide: 0.02%; SnMS protein: 5%; nicotinamide: 1%; hyaluronic acid: 0.05%), 1% nicotinamide; positive control group: 1mM kojic acid).

[0061] (2) Centrifuge at low speed for 1000 rpm for 5 min, discard the supernatant, add 1 mL PBS to wash the cells, gently pipette the suspension to resuspend the cells, and repeat this step twice.

[0062] (3) Add 650 μL of 1M NaOH solution containing 10% DMSO, gently pipette to resuspend the cells, and place in a water bath at 80°C for 2 hours.

[0063] (4) After the water bath, 200 μL of each well is transferred into a clean 96-well plate. Three parallel replicates are made for each group, and the absorbance at 405 nm is measured using an ELISA reader.

[0064] (5) Based on the OD values ​​of each experimental group, calculate the melanin content inhibition rate of mouse melanoma cells B16-F10 cells. The formula is: melanin content inhibition rate (%) = (1 - (OD value of experimental group / OD value of control group)). 100%.

[0065] (6) Evaluate the effect of the sample on melanin based on the calculated inhibition rate, and evaluate the whitening effect of the sample.

[0066] The results are as follows Figure 3 As shown, compared with the positive control group, the formulated composition and 1% niacinamide both showed melanin removal effects. The positive control group (1mM kojic acid) had a melanin removal rate of 11.69%, niacinamide had a melanin removal rate of 23.84%, and the formulated composition had a melanin removal rate of 37.66%. These data indicate that both the formulated composition and 1% niacinamide can reduce melanin production and have a certain whitening effect; the combined effect is significantly better than using niacinamide alone.

[0067] Example 6: Promoting Collagen Synthesis - Collagen Content Test

[0068] Collagen is the most abundant protein found in connective tissue. Collagenase, synthesized and secreted by fibroblasts, degrades collagen in the skin, leading to skin aging. Therefore, inhibiting the expression of collagenase in cells and increasing collagen content plays an important role in preventing aging and increasing skin plumpness and firmness. In environments with excessive ultraviolet radiation, the activities of collagenase and elastase increase significantly, elastin is hydrolyzed, and collagen synthesis is inhibited. In this experiment, test samples were used to treat cells after ultraviolet radiation, and the collagen I content in the corresponding cells was detected to determine whether the composition of the present invention can promote collagen production.

[0069] Human skin fibroblasts (HSF cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) in the logarithmic growth phase were taken, and 0.25% trypsin digestion solution was added to digest and detach the adherent cells. The cells were counted at 1-4 × 10^6 cells / mL to prepare a cell suspension. The cells were seeded at a density of 1 × 10^5 cells / mL in 6-well plates, and a UV photoaging model was established when the cells reached a confluence of about 80%. The blank control group was given an equal volume of PBS, and the culture medium was added to a final volume of 2 mL. No UV irradiation was performed. The UV group was given an equal volume of PBS, and the culture medium was added to a final volume of 2 mL. UV irradiation was then performed. The drug treatment groups were formulated with the following compositions: (GHK-Cu: 0.1%; Mitochondrial Energy Peptide: 0.02%; SnMS Protein: 5%; Nicotinamide: 1%; Hyaluronic Acid: 0.05%), 1% Nicotinamide, 0.1% GHK-Cu, and 5% SnMS Protein. After repeated washing with PBS until colorless, 200 μL of PBS was added, and the mixture was irradiated under an 80 mJ / cm² UV lamp with a 15 cm distance between the lamp and the culture flask. After irradiation, the PBS was discarded. The UV group was given PBS solution and complete culture medium, while the drug treatment groups were given serially diluted drug and complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin). The blank control group, UV group, and drug treatment groups were incubated at 37°C in a 5% CO₂ incubator for 48 h. After culture, cells in well 1 were digested, counted, and diluted to 0.5 × 10^6 cells / mL. Cells from the remaining wells were scraped off, resuspended in 500 μL, and then 50 μL of each well was sonicated for 30 seconds. Total protein was measured using the BCA method. The cell suspensions in the other wells were diluted according to the protein concentration of well 1 to ensure a total cell suspension concentration of 0.5 × 10^6 cells / mL. The adjusted cell suspensions were sonicated for 30 seconds, centrifuged at 1500g for 15 min, and the cell supernatant was collected to obtain the sample solution. The procedure was performed according to the collagen I ELISA instructions. The OD values ​​of each well were measured sequentially at 450 nm using a microplate reader within 15 min.

[0070] The results are as follows Figure 4 As shown, compared with the blank control group, the collagen content in the UV group was significantly reduced, indicating that the photoaging model was successfully established. Compared with the UV group, after cells were treated with different samples, the collagen content of the formulation composition increased by 33%, the collagen content of 1% nicotinamide increased by 16%, the collagen content of 0.1% copper peptide increased by 13%, and the collagen content of 5% SnMS protein increased by 10%. The results show that each sample can increase the collagen content; and the formulation composition of the present invention can significantly increase the collagen content, with its collagen content increasing by more than twice that of other samples, demonstrating excellent collagen production effect.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of the blue copper peptide composition in the preparation of products for preventing pigmentation after phototherapy microneedling.

2. Application of the blue copper peptide composition in the preparation of products for accelerating wound healing after phototherapy microneedling.

3. The application according to any one of claims 1-2, characterized in that, The active ingredients of the copper peptide composition include copper peptide, mitochondrial energy peptide, SnMS protein, nicotinamide, and hyaluronic acid; preferably, the copper peptide composition further comprises a pharmaceutically acceptable carrier.

4. The application according to claim 3, characterized in that, The amino acid sequence of the mitochondrial energy peptide is shown in SEQ ID NO.1, and the amino acid sequence of the SnMS protein is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, The active ingredients in the blue copper peptide composition, by weight percentage, are: blue copper peptide 0.01%-0.5%, mitochondrial energy peptide 0.001%-1%, SnMS protein 0.1%-10%, nicotinamide 0.1%-5%, and hyaluronic acid 0.01%-2%; preferably, the active ingredients in the blue copper peptide composition are: blue copper peptide 0.1%-0.3%, mitochondrial energy peptide 0.01%-0.1%, SnMS protein 1%-5%, nicotinamide 1%-3%, and hyaluronic acid 0.05%-1%; more preferably, the composition is: blue copper peptide 0.1%, mitochondrial energy peptide 0.02%, SnMS protein 5%, nicotinamide 1%, and hyaluronic acid 0.05%.

6. The application according to claim 5, wherein the blue copper peptide composition further comprises panthenol, sodium 2-hydroxypyruvate, polyethylene glycol, glycerin, allantoin, betaine, and xanthan gum; preferably, the proportions of each component by weight percentage are: panthenol 0.5%-2%, sodium 2-hydroxypyruvate 0.1%-0.5%, polyethylene glycol-8 1%-7%, glycerin 1%-5%, allantoin 0.1%-0.5%, betaine 0.01-0.5%, and xanthan gum 0.05-0.3%; more preferably, the proportions of each component by weight percentage are: panthenol 1%-1.5%, sodium 2-hydroxypyruvate 0.1%-0.3%, polyethylene glycol-8 3%-5%, glycerin 3%-5%, allantoin 0.1%-0.3%, betaine 0.1-0.2%, and xanthan gum 0.1-0.15%.

7. The application according to any one of claims 1-2, characterized in that, The blue copper peptide composition achieves anti-inflammatory effects by downregulating the expression of IL-6 and TNF-α inflammatory factors, inhibits melanin production by suppressing melanosome transport, and guides the orderly arrangement of newly formed collagen fibers by stimulating the synthesis of type I and type III collagen and core proteoglycans.

8. The application according to any one of claims 1-2, characterized in that, The phototherapy is a minimally invasive phototherapy such as fractional laser, intense pulsed light, and / or radiofrequency microneedles.

9. The application according to any one of claims 1-2, characterized in that, The products include pharmaceuticals, skincare products, or medical dressings.

10. The application according to claim 8, characterized in that, The product is in the form of gel, cream, emulsion, water, or spray.

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