A firming anti-aging composition and use thereof

Abstract

This invention relates to a firming and anti-aging composition and its application. The firming and anti-aging composition comprises the bioactive peptide Pal-KVK, a novel copper peptide, and a red algae extract, wherein the mass ratio of the bioactive peptide Pal-KVK, the novel copper peptide, and the red algae extract is 0.005–10:0.05–3.0:0.05–3.0. The small-molecule bioactive peptide Pal-KVK in this firming and anti-aging composition is safe and gentle, structurally stable, highly water-soluble, has a small molecular weight, and is easily absorbed. This firming and anti-aging composition utilizes a combination of a novel copper peptide and a red algae extract with the bioactive peptide, resulting in a synergistic effect of multiple active ingredients, leading to better anti-aging and deep hydration effects. Human testing results show that the face cream containing the firming and anti-aging composition has smoothing, wrinkle-reducing, and skin-rejuvenating effects.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a firming and anti-aging composition and its application. Background Technology

[0002] Currently, some anti-aging cosmetics use industrially produced ingredients with strong antioxidants to achieve immediate and long-lasting effects. While these ingredients provide noticeable immediate results, prolonged use poses significant health risks. Others add vitamin C and other vitamin-based antioxidants to achieve anti-aging effects, but these ingredients are unstable and easily oxidized. Furthermore, some anti-aging cosmetics use plant extracts with unclear active ingredients. Therefore, the development of cosmetics containing safe, stable, and clearly defined active ingredients is essential.

[0003] Active bioactive peptides are characterized by safety, stability, easy absorption, and small molecular weight. They also possess various biological activities such as free radical scavenging, antioxidant activity, and anti-aging properties. They can not only promote skin cell proliferation but also delay skin aging and promote skin wound repair, thus being widely used in skincare and anti-aging cosmetics. Small molecule peptides extracted from natural organisms or obtained through protease hydrolysis have drawbacks such as poor stability, short half-life, poor in vitro stability, and difficulty in improving purity, failing to meet the requirements of precision cosmetic development and manufacturing. In recent years, with the continuous advancement of research on small molecule peptides in cosmetics, artificial synthesis methods for small molecule peptides, such as chemically synthesized peptide libraries and biosynthesized molecular peptide libraries, have been continuously improved, leading to increasing demand.

[0004] For example, patent CN112080543A discloses a plant-derived polypeptide with anti-aging effects, a face cream containing it, and a preparation method thereof. The plant-derived polypeptide is a sage seed polypeptide. This patent states that the sage seed polypeptide can both promote the proliferation of human skin fibroblasts and promote the synthesis of collagen by human skin fibroblasts, thus having an anti-skin aging effect. Patent CN109223660A discloses a revitalizing and anti-aging peptide face cream with the following ingredients: water, 1,3-butanediol, methylparaben, panthenol, niacinamide, sodium hyaluronate, disodium EDTA; cetearyl glucoside, HE olive oil emulsifier, cetearyl alcohol, caprylyl glycol, propylene glycol, 1,3-propanediol, triethoxyoctylsilane, isohexadecane, caprylic / capric triglyceride, isononyl isononanoate, ascorbic acid, vitamin E acetate, polydimethylsiloxane; ethylhexylglycerin; carnosine, callus culture filtrate of *Eryngium sambac*, small molecule polypeptide nutritional essence, aluminum octenyl succinate, mica powder, *Myrothamnus flabellifolia* extract, capryloyl hydroxamic acid, phenoxyethanol, hydrolyzed collagen, licorice root extract, ubiquinone, lecithin; caramel color, and fragrance. The small molecule polypeptide nutritional essence is a mixture of oligopeptide-1, oligopeptide-2, oligopeptide-5, and acetyl hexapeptide-8.

[0005] However, small molecule peptides obtained by direct extraction from natural organisms or by protease hydrolysis have disadvantages such as poor stability, short half-life, and difficulty in improving purity, which cannot meet the requirements of precision development and manufacturing of cosmetics. Summary of the Invention

[0006] To address the issues of poor stability and short half-life of small molecule peptides used in existing cosmetics, this invention provides a firming and anti-aging composition and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a firming and anti-aging composition comprising the bioactive peptide Pal-KVK, a novel copper peptide, and an extract of red algae.

[0009] The mass ratio of the bioactive peptide Pal-KVK, the novel copper peptide, and the red algae extract is 0.005–10: 0.05–3.0: 0.05–3.0.

[0010] In one embodiment of the present invention, the chemical structure of the novel copper peptide is as follows:

[0011]

[0012] The novel copper peptide is Pal-GHK-Cu with the amino acid sequence H-Gly-His-Lys(Pal)-OH.(Cu), and its chemical formula is C. 30 H 52 CuN5O5.

[0013] In one embodiment of the present invention, preferably, the mass ratio of the bioactive peptide Pal-KVK, the novel copper peptide, and the red algae extract is 0.005-5:0.1-3.0:0.1-3.0; more preferably, the mass ratio of the bioactive peptide Pal-KVK, the novel copper peptide, and the red algae extract is 0.005-3:0.5-3.0:0.5-3.0.

[0014] In one embodiment of the present invention, the bioactive peptide Pal-KVK, the novel copper peptide, and the red algae extract are placed in PPG-26-butanol polyether-26 and PEG-40 hydrogenated castor oil, and the mass ratio of the bioactive peptide Pal-KVK, the novel copper peptide, the red algae extract, PPG-26-butanol polyether-26, and PEG-40 hydrogenated castor oil is 0.005-10:0.05-3.0:0.05-3.0:0.01-0.5:0.04-2.0.

[0015] In one embodiment of the present invention, the weight ratio of PPG-26-butanol polyether-26 and PEG-40 hydrogenated castor oil is 1:4.

[0016] In one embodiment of the present invention, the method for preparing the novel copper peptide includes the following steps: mixing Pal-GHK with copper acetate in water to synthesize a novel copper peptide with the amino acid sequence H-Gly-His-Lys(Pal)-OH.(Cu).

[0017] In one embodiment of the present invention, a method for preparing a novel copper peptide includes the following steps:

[0018] I. Preparation of Pal-GHK

[0019] (1) Amine protection

[0020] Under stirring, Fmoc-Lys(Dde)-OH and amino acids containing 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butylcarbonyl (Boc) were added to the reactor and stirred at room temperature for 1-3 hours to protect the amino groups, thus obtaining GHK(Pal).

[0021] (2) Palmitic acid modification

[0022] With stirring, palmitic acid was added to the reactor and stirred at room temperature for 1-3 hours to carry out side chain modification.

[0023] The amino acid containing 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butylcarbonyl (Boc) is Fmoc-His(Trt)-OH or Boc-Gly-OH;

[0024] When the amino acid is Fmoc-His(Trt)-OH, the mass ratio of Fmoc-Lys(Dde)-OH, Fmoc-His(Trt)-OH, and palmitic acid is 2-4:2-4:0.5-2.

[0025] When the amino acid is Boc-Gly-OH, the mass ratio of Fmoc-Lys(Dde)-OH, Boc-Gly-OH and palmitic acid is 2-4:0.5-1:0.5-2.

[0026] (3) Deprotection

[0027] The product obtained in step (2) was eluted sequentially with eluent (20% piperidine or N,N-dimethylformamide and 2% hydrazine or N,N-dimethylformamide) (i.e., the product was first mixed with 20% piperidine or N,N-dimethylformamide and stirred for 1-3 hours, then centrifuged to remove the Fmoc group, and then mixed with 2% hydrazine or N,N-dimethylformamide and stirred for 1-3 hours, then centrifuged to remove the Dde group), removing the Fmoc group and the Dde group;

[0028] (4) Amino acid residue elongation

[0029] The product obtained in step (3) is added to an active reagent and reacted at room temperature for 1-3 hours to elongate the carbon chain of the amino acid residues. The active reagent includes N-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide, and the amount of active reagent is 1-3 times the molar amount of Fmoc-Lys(Dde)-OH.

[0030] (5) Separation and purification

[0031] The product obtained in step (4) was washed sequentially with dimethylformamide, dichloromethane and methanol, dried under vacuum, and then reacted with an aqueous solution containing trifluoroacetic acid and triisopropylsilane (a mixture of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 90:5:5). The product was separated from the resin, concentrated and centrifuged, and then purified and separated by reversed-phase high-performance liquid chromatography to obtain the Pal-GHK product.

[0032] II. Synthesis of Pal-GHK-Cu

[0033] Pal-GHK was mixed with copper acetate in water and reacted. The pH of the reaction solution was adjusted to 4-6 with sodium hydroxide. The precipitated sky-blue solid product was washed with water and dried under vacuum to obtain a copper complex with the amino acid sequence H-Gly-His-Lys(Pal)-OH.(Cu).

[0034] Secondly, the present invention provides a method for preparing a firming and anti-aging composition, comprising the following steps:

[0035] (1) Pal-KVK was mixed with PPG-26-butanol polyether-26 and PEG-40 hydrogenated castor oil to obtain a first mixture;

[0036] (2) Add the novel copper peptide to the first mixture, stir evenly, then add the red algae extract, mix and stir evenly to obtain the firming and anti-aging composition.

[0037] In one embodiment of the invention, the conditions for mixing Pal-KVK with PPG-26-butanol polyether-26 and PEG-40 hydrogenated castor oil can be selected as 15-35°C, for example, at room temperature (25°C).

[0038] In one embodiment of the present invention, the novel copper peptide is added to the first mixture and stirred evenly. The conditions for adding the red algae extract can be selected as 15-35°C, for example, it can be carried out at room temperature (25°C).

[0039] In one embodiment of the present invention, the weight ratio of PPG-26-butanol polyether-26 and PEG-40 hydrogenated castor oil is 1:4.

[0040] In the firming and anti-aging composition provided by this invention, the bioactive peptide Pal-KVK can release transforming growth factor β, thereby exerting anti-aging effects. A novel copper peptide promotes collagen production and restores the skin's repair ability, while red algae extract deeply replenishes skin moisture. The synergistic effect of multiple active ingredients enhances the anti-aging effect.

[0041] The bioactive peptide Pal-KVK in this invention, also known as palmitoyl tripeptide-5, CAS number: 623172-56-5, has the molecular formula C 37 H 67 F6N5O9. The bioactive peptide Pal-KVK can release transforming growth factor β. The transforming growth factor β (TGF-β) pathway is known to play a key role in the pathogenesis of skin anti-aging. TGF-β has a molecular weight of approximately 25 kDa and is a master regulator of the extracellular matrix (ECM). As a multifunctional cytokine, it regulates the growth and differentiation of various cell types. TGF-β can stimulate fibroblasts in the dermis to synthesize various proteins, such as collagen, which is a major component of the dermal ECM. TGF-β can trigger the synthesis of some components at the dermal-epidermal junction, such as laminin V, collagen IV, and collagen VII, while also inhibiting the expression of protein-degrading matrix metalloproteinases (MMPs), such as MMP-1, -2, -3, and -13. Therefore, the bioactive peptide Pal-KVK is added to the composition of this invention to release transforming growth factor β, thereby exerting an anti-aging effect.

[0042] The novel copper peptide in this invention is Pal-GHK-Cu with the amino acid sequence H-Gly-His-Lys(Pal)-OH.(Cu), and its chemical formula is C. 30 H 52CuN5O5 is an active peptide with skin repair properties. It can help the skin restore its self-repair function, promote collagen production, reduce wrinkle growth, and thus exert an anti-aging effect. Furthermore, this novel copper peptide can effectively improve the retention of copper peptides in the skin layer, enhance the bioavailability of copper peptides, and better exert its anti-aging effect.

[0043] The red algae extract in this invention contains various marine active substances, such as polysaccharides, polypeptides, phenols, and terpenes, which contribute to healthy skin. The red algae extract can effectively inhibit allergic reactions, suppress inflammatory factors IL-8, IL-6, and TNF-α, and reduce skin aging problems caused by allergic and inflammatory reactions.

[0044] Thirdly, the present invention provides an application of a firming and anti-aging composition, wherein the firming and anti-aging composition is used in cosmetics. The cosmetics include face creams, lotions, etc.

[0045] Fourthly, the present invention provides a cosmetic containing a firming and anti-aging composition, comprising a phase A component, a phase B component, and a phase C component, wherein the phase A component is an emollient and an emulsifier, the phase B component is a moisturizer and a chelating agent, and the phase C component is the firming and anti-aging composition, wherein the mass content of the firming and anti-aging composition in the cosmetic is 0.1-20.0%, more preferably 1.0-10.0%, and even more preferably 1.0-5.0%.

[0046] In one embodiment of the present invention, the A phase component, namely the softener and the emulsifier, is selected from one or a combination of several of the following: polydimethylsiloxane, triglyceride octanoate, hydrogenated polyisobutylene, cyclopentamethoxysiloxane, polydimethylsiloxane and C16-C18 alkyl polydimethylsiloxane crosslinked polymer, hydrogenated lecithin, and 2,6-di-tert-butyl-4-methylphenol;

[0047] The B-phase component, namely the humectant and chelating agent, is selected from one or more of water, glycerin, propylene glycol, betaine, carbomer, EDTA, or triethanolamine.

[0048] In the cosmetic containing the firming and anti-aging composition of the present invention, the emulsifier in phase A is used to promote better integration of Pal-KVK with novel copper peptides and red algae extracts, thereby promoting the formation of a stable system among the three and improving the stability of the system.

[0049] In one embodiment of the present invention, the cosmetic is a face cream, with the following ingredients:

[0050]

[0051] In one embodiment of the present invention, the cosmetic is a face cream, with the following ingredients:

[0052]

[0053]

[0054] In one embodiment of the present invention, the cosmetic is a face cream, with the following ingredients:

[0055] In one embodiment of the present invention, the cosmetic is a face cream, with the following ingredients:

[0056]

[0057]

[0058] Fifthly, the present invention provides a method for preparing a cosmetic containing a firming and anti-aging composition, comprising the following steps: Weighing the corresponding mass of each substance according to the components of phase A. Stirring the substance slowly in a water bath at 75-80°C for approximately 30 minutes until completely dissolved, obtaining a uniform oily material A; Pre-dispersing the carbomer in phase B evenly in water before adding the other raw materials of phase B, then stirring the mixture at a constant speed in a water bath at 75-80°C for approximately 30 minutes until phase B is completely dissolved; Pouring mixture B into mixture A, stirring rapidly at 75-80°C for 25-30 minutes, then slowing down the temperature until phase C is added at 40-45°C. Continuing to cool to 30-35°C and stopping stirring, yielding a cosmetic containing a firming and anti-aging composition.

[0059] The bioactive peptide Pal-KVK used in this invention has the potential to activate TGF-β and has a well-defined structure. This invention also employs a novel copper peptide and red algae extract for compounding, promoting collagen growth and skin repair, synergistically enhancing the effects for better anti-aging and deep hydration. Furthermore, this invention includes an in vitro LAP-TGF-β activation assay and quantitative assays for cytokines and MMPs to clarify the activation of TGF-β by the Pal-KVK peptide and its inhibitory effects on the expression of TNF-α, IL-8, MMP-1, and MMP-3. In addition, this invention provides in vivo application studies, verifying that the proposed solution has a significant effect on smoothing crow's feet and wrinkles in clinical practice.

[0060] Compared with the prior art, the beneficial effects of the present invention include:

[0061] (1) The small molecule bioactive peptide Pal-KVK in the firming and anti-aging composition of this invention is safe and mild, structurally stable, highly water-soluble, has a small molecular weight, and is easily absorbed. The bioactive peptide in this invention is a small molecule bioactive peptide obtained through CADD (computer-aided drug design) and AIDD (AI-driven drug design), which can quickly and easily identify target compounds and specifically promote the release of TGF-β.

[0062] (2) The small molecule bioactive peptide Pal-KVK used in the firming and anti-aging composition of the present invention has a clear action pathway and significant effect.

[0063] (3) The firming and anti-aging composition of this invention uses a novel copper peptide and a red algae extract combined with bioactive peptides. The red algae extract contains up to 20 kinds of mineral elements, which can regulate the water balance of the internal and external environment of the skin's base and surface, promoting the movement of abundant skin moisture from the base to the skin surface, thus achieving skin hydration. Bioactive peptides can enhance cell activity and increase skin moisture content. The red algae extract can effectively inhibit allergic reactions, inhibit inflammatory factors IL-8, IL-6 and TNF-α, and reduce skin aging problems caused by allergic and inflammatory reactions.

[0064] (4) The three components work synergistically to address aging issues from within the skin through a multi-dimensional response. Human testing results show that the cream containing the firming and anti-aging composition has smoothing, wrinkle-reducing, and skin-rejuvenating effects.

[0065] (5) Copper peptides have very strong hydrophilicity and a small molecular structure, which can promote the absorption of other ingredients. Multiple active ingredients work synergistically to achieve better anti-aging and deep hydration effects. The novel copper peptide in this invention promotes collagen production and solves the problems of easy decomposition and low utilization rate of traditional copper peptides. Attached Figure Description

[0066] Figure 1 The HPLC chromatogram of Pal-GHK-Cu is shown below.

[0067] Figure 2 The mass spectrum of Pal-GHK-Cu is shown.

[0068] Figure 3 3D rendering of Pal-GHK-Cu;

[0069] Figure 4 ELISA was used to quantify the free TGF-β released from LAP-TGF-β.

[0070] Figure 5Twenty-four hours after PMA (0.1 μM)-induced stress signaling, the expression of TNF-α, IL-8, MMP-1, and MMP-3 in normal human keratinocytes (NHK) in 10 channels was detected at the protein level. TGF-β 12.5 ng / ml; pal-KVK 10 μM. (*P<0.01). Detailed Implementation

[0071] The present invention adopts the following technical solution:

[0072] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof. Other non-essential modifications and adjustments made according to the present invention still fall within the scope of protection of the present invention. Unless otherwise specified, the percentages (%) below are all by weight.

[0073] In the following examples, the chemical structure of the novel copper peptide is as follows:

[0074]

[0075] The novel copper peptide is Pal-GHK-Cu with the amino acid sequence H-Gly-His-Lys(Pal)-OH.(Cu), and its chemical formula is C. 30 H 52 CuN5O5.

[0076] A method for preparing novel copper peptides includes the following steps:

[0077] I. Preparation of Pal-GHK

[0078] (1) Amine protection

[0079] Fmoc-Lys(Dde)-OH resin and Boc-Gly-OH were added to the reactor under stirring and stirred at room temperature for 2.0 h to protect the amino groups and obtain GHK(Pal);

[0080] (2) Palmitic acid modification

[0081] Palmitic acid was added to the reactor under stirring, and the reaction was carried out at room temperature for 2.0 h to perform side chain modification.

[0082] The ratio of Fmoc-Lys(Dde)-OH, Boc-Gly-OH and palmitic acid is 3.37:1:1.46 by mass.

[0083] (3) Deprotection

[0084] The product obtained in step (2) was eluted sequentially with eluent (20% piperidine and 2% N,N-dimethylformamide) (i.e., the product was first mixed with 20% piperidine and stirred for 2.0 h and then centrifuged to remove the Fmoc group; then mixed with 2.0% N,N-dimethylformamide and stirred for 2.0 h and centrifuged to remove the Dde group), thus removing the Fmoc group and the Dde group;

[0085] (4) Amino acid residue elongation

[0086] The product obtained in step (3) was added to the active reagents N-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide and reacted at room temperature for 2.0 h to elongate the carbon chain of the amino acid residues; the amount of active reagent was 2.0 times the molar amount of Fmoc-Lys(Dde)-OH resin.

[0087] (5) Separation and purification

[0088] The product obtained in step (4) was washed 6 times with dimethylformamide, 6 times with dichloromethane, and 2 times with methanol, and then dried under vacuum. The resin with the product attached was then reacted with an aqueous solution containing trifluoroacetic acid and triisopropylsilane (a mixture of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 90:5:5). The product was separated from the resin, concentrated to a residue, precipitated with cold diethyl ether and centrifuged. The solid was then washed 3 times with cold diethyl ether and centrifuged, dried under vacuum, and then analyzed using reversed-phase high-performance liquid chromatography (column: Unisil, C18, 10μm). The peptide was purified and separated using a 250x50mm filter to obtain the Pal-GHK product.

[0089] II. Synthesis of Pal-GHK-Cu

[0090] Pal-GHK was mixed with copper acetate in water and reacted. The pH of the reaction solution was adjusted to 4-6 with sodium hydroxide. The precipitated sky-blue solid product was washed three times with water and dried under vacuum to obtain a novel copper complex with the amino acid sequence H-Gly-His-Lys(Pal)-OH.(Cu).

[0091] (6) The prepared Pal-GHK-Cu was characterized by high-performance liquid chromatography (Agilent, 1100) and mass spectrometry (Agilent, 6120), such as... Figure 1-2 As shown in the figure, under the analytical conditions, the prepared Pal-GHK-Cu exhibits good separation, high UV absorbance, relatively symmetrical peak shape, retention time of approximately 12 minutes, and a molecular weight of 640.33 g / mol.

[0092] The 3D rendering of Pal-GHK-Cu is as follows Figure 3As shown. Safety testing of the novel copper peptide.

[0093] The skin application safety of the novel copper peptide was evaluated according to Sections 6.4, 6.5, and 6.6 of the "Cosmetic Safety Technical Specifications" (2015 edition). See Tables 1 and 2 for details.

[0094] Table 1. Results of cell proliferation assay (human keratinocytes)

[0095]

[0096] Table 2. Results of cell proliferation assay (human fibroblasts)

[0097]

[0098] In the table above, SC represents: negative control;

[0099] SD stands for: Relative Mobility;

[0100] PC indicates: positive control;

[0101] It was found that after treatment with the novel copper peptide at 25 μg / L for 24 hours, the proliferation of keratinocytes increased by nearly 200%, while the proliferation of copper peptide-treated cells increased by nearly 20%. Further analysis indicates that the novel copper peptide has better epidermal repair capabilities. Table 3 shows that after treatment with the novel copper peptide at 25 μg / L for 24 hours, the proliferation rate of fibroblasts increased by more than 300%, which is superior to the proliferation rate after 24 hours of copper peptide treatment. This novel copper peptide can maintain the proliferative activity of fibroblasts and has potential effects on dermal repair.

[0102] Table 3 Cell migration results

[0103]

[0104] It was found that after treatment with the novel copper peptide at 25 μg / L for 24 h, the relative migration rate of fibroblasts significantly increased (p<0.01). Compared with copper peptide, the novel copper peptide exhibited better cell migration ability, further demonstrating its superior skin repair capabilities.

[0105] Examples 1-3 and Comparative Examples 1-5

[0106] The raw material composition of Examples 1-3 and Comparative Examples 1-5 is shown in Table 4.

[0107] Table 4. Raw material composition ratios for Examples 1-3 and Comparative Examples 1-5

[0108]

[0109]

[0110]

[0111] The preparation process flow of the face creams in Examples 1-3 of this invention is as follows:

[0112] (1) Preparation of Phase A: According to the components of Phase A in Table 4, weigh out the corresponding mass of each substance. Place them in a water bath at 78℃ and stir slowly for about 30 minutes until they are completely dissolved to obtain a uniform oily material A.

[0113] (2) Preparation of phase B: Carbomer in phase B is pre-dispersed evenly in water before adding other raw materials of phase B. Then, it is placed in a water bath at 78°C and stirred at a constant speed for about 30 minutes until phase B is completely dissolved.

[0114] (3) Pour mixture B into mixture A and stir rapidly at 78°C for 26 minutes. Then, reduce the speed and cool down until 42°C, at which point phase C is added. Continue cooling down to 32°C and stop stirring to obtain the face cream.

[0115] The preparation process flow of the face creams of Comparative Examples 1-5 of the present invention is as follows:

[0116] (1) Preparation of Phase A: According to the components of Phase A in Table 4, weigh out the corresponding mass of each substance. Place them in a water bath at 78℃ and stir slowly for about 30 minutes until they are completely dissolved to obtain a uniform oily material A.

[0117] (2) Preparation of phase B: Carbomer in phase B is pre-dispersed evenly in water before adding other raw materials of phase B. Then, it is placed in a water bath at 78°C and stirred at a constant speed for about 30 minutes until phase B is completely dissolved.

[0118] (3) Pour mixture B into mixture A and stir rapidly at 78°C for 26 minutes. Then, reduce the speed and cool down until 42°C, at which point phase C is added. Continue cooling down to 32°C and stop stirring to obtain the face cream.

[0119] 1. Activation of LAP-TGF-β in vitro

[0120] Spread anti-LAP monoclonal antibody (R&D Systems) (Shanghai Yubo Biotechnology Co., Ltd., MAA536Hu22) onto the plate and incubate overnight at 4°C. After washing, add 5.25 ng / well of LAP-TGF-β (R&D Systems) (Shanghai Hushi Pharmaceutical Technology Co., Ltd., HK4234). Then add Pal-KVK (sequence Pal-Lys-Val-Lys-OH) or placebo saline (25 μM) and incubate for 1 h to 24 h (37°C). Subsequently, follow the manufacturer's instructions to use TGF-β. An immunoassay system (Promega) is used to test the samples. Enzyme-linked immunosorbent assay (ELISA) is used for the specific detection of bioactive TGF-β.

[0121] As is well known, the transforming growth factor β (TGF-β) pathway plays a crucial role in skin aging, serving as one of the main switches for these cells. This invention designs an in vitro LAP-TGF-β activation assay to identify peptides that can react with TGF-β from LAP-TGF-β. Figure 4 This shows the results of in vitro TGF-β release from the LAP-TGF-β complex after culture with placebo saline and Pal-KVK (25 μM). Figure 4 The ordinate represents the release level of TGF-β. Pal-KVK induced the release of active TGF-β over time, while this was not the case in the negative control (i.e., the placebo-added) where Pal-KVK was not added.

[0122] 2. Inhibits pro-inflammatory factors in vitro

[0123] (1) Pal-KVK peptide in vitro assay, including the following steps:

[0124] Normal human keratinocytes (NHK) from the foreskin (Shanghai Yanzun Biotechnology Co., Ltd.) were cultured in CnT-07 medium (CELLnTEC, Switzerland) at 37°C for two days. Afterward, 0.1 μM PMA (myristate-13-acetic acid phorbol ester) was added. Immediately after PMA stimulation, the test substances TGF-β 12.5 ng / ml and pal-KVK 10 μM were added. Cytokines and matrix metalloproteinases (MMPs) in the cell culture supernatant were quantified using the Procarta Cytokine Assay Kit (Panomics, Fremont, USA) via a multiplex bead array system (Luminex 100xMAP).

[0125] (2) Since the bioactive peptide Pal-KVK exhibits time-related TGF-β activation, this invention aims to evaluate the inhibitory function of Pal-KVK on pro-inflammatory factors. Therefore, this invention designed an in vitro assay to compare the inhibitory effects of TGF-β and the bioactive peptide Pal-KVK on pro-inflammatory factors in normal human keratinocytes. Keratinocytes were exposed to 0.1 μM of phorbol 12-myristate-13-acetate (PMA) as a chemical stress signal to induce a pro-inflammatory response. Both TGF-β and pal-KVK strongly inhibited the upregulation of MMP-1, MMP-3, and pro-inflammatory cytokines (TNF-α and IL-8), as shown in the results. Figure 5 As shown.

[0126] Figure 5In the figure, the vertical axis represents the fold change in the expression level of the same gene without treatment. Taking MMP-1 as an example, the change was large when PMA stimulation was used, with a change value exceeding 10; the change was significantly smaller when TGF-β was used; and the change value was negative when Pal-KVK peptide was used, indicating that it inhibited the upregulation of MMP-1 expression. * indicates P < 0.01, and the data is valid. Compared with direct use of TGF-β, Pal-KVK is more effective in inhibiting the upregulation of MMP-1, MMP-3, and pro-inflammatory cytokines (TNF-α and IL-8). One reason is that TGF-β has a relatively large molecular weight, making it difficult to directly penetrate the skin.

[0127] 3. Evaluation of in vivo anti-wrinkle effect

[0128] An in vivo anti-wrinkle evaluation experiment was conducted using the face creams from Examples 1-3 and Control Examples 1-5. The study included 320 healthy female subjects aged 35 to 55 years (mean age 48 years). Subjects exhibited crow's feet / fine lines, under-eye wrinkles / fine lines, dark circles / eye bags, and ptosis. Subjects provided informed written consent. Subjects agreed to cooperate and understand the necessity and duration of the control in order to adhere to the protocol established by the clinical trial center. A half-face test was performed, using the cream twice daily (morning and evening) for 28 consecutive days. (EOTECH-France) directly studied changes in skin topography parameters (mean roughness Ra, maximum topographic relief Rt, and mean relief Rz) for crow's feet and under-eye wrinkles in vivo. Subjects were also asked to conduct self-subjective evaluations.

[0129] (1) 320 subjects were divided into 8 groups of 40 people each. Each group used face cream of Examples 1-3 and Control Examples 1-5 on half of their face and did not use face cream on the other half of their face.

[0130] Under these research conditions, after 28 days of twice-daily use, different research parameters confirmed the following effects: compared with the initial state, it has a smoothing and anti-wrinkle effect on crow's feet. Specific data are shown in Table 5 (mean values ​​of 10 subjects in each group).

[0131] Table 5. Variations of average roughness Ra, maximum topographic relief Rt, and average relief Rz

[0132]

[0133] In the experimental group using Sample Example 1, the average roughness (Ra) decreased significantly by 10% on day 7 compared to the initial state (before applying face cream), and by 12% and 14% on days 14 and 28, respectively. The Ra values ​​were measured directly from the testing instrument.

[0134] In the experimental group using Sample Example 1, compared to the initial state (before applying the cream), it caused a significant decrease in the average fluctuation (Rz), with D7, D14, and D28 decreasing by 9%, 12%, and 15%, respectively. The Rz values ​​were measured directly from the testing instrument.

[0135] In the experimental group using Sample Example 1, compared to the initial state (before applying face cream), the terrain undulation (Rt) decreased significantly by 11% on D7, and by 14% and 15% on D14 and D28, respectively. The Rt values ​​were measured directly from the testing instrument.

[0136] The true three-dimensional micro-topography of the crow's feet area was acquired using the DermaTOP (EOTECH-France) 3D measurement device. It was observed that subjects showed significant smoothing and anti-wrinkle effects after 28 days of treatment with samples 1-3. In the DermaTOP analysis images, the half-face treated with samples 1-3 showed a significant reduction in crow's feet.

[0137] (2) Subjective self-evaluation

[0138] After 28 days of treatment with Example 1, subjective evaluations from self-assessment questionnaires and skin conduction responses were analyzed. Many participants were satisfied with the product's sensory characteristics and efficacy, particularly its effectiveness in reducing wrinkles.

[0139] Self-evaluation showed that after 28 days of use, up to 83% of the participants were satisfied with the immediate skin moisturizing effect of the Formula of Example 1, up to 93% of the participants were satisfied with the immediate skin softening effect of the Formula of Example 1, up to 90% of the participants reported a reduction in under-eye wrinkles, 93% of the participants reported a reduction in crow's feet, 95% of the participants reported a reduction in puffiness, 93% of the participants reported a reduction in eye bags, and 95% of the participants reported a reduction in periorbital wrinkles and dark circles.

[0140] Table 6 Self-evaluation of participant satisfaction

[0141] Evaluation Project 0 points 1 point 2 points 3 points 4 points Satisfaction Instantly moisturizes skin 0 people 0 people 3 people 20 people 17 people 93％ Instantly softens skin 0 people 0 people 4 people 19 people 18 people 90％ Reduce under-eye wrinkles 0 people 0 people 4 people 18 people 19 people 90％ Reduce crow's feet wrinkles 0 people 0 people 3 people 19 people 18 people 93％ Reduced swelling 0 people 0 people 2 people 18 people 20 people 95％ Reduce eye bags 0 people 0 people 3 people 21 people 16 people 93％ Dark circles reduced 0 people 0 people 2 people 22 people 16 people 95％

[0142] Note: 0 points indicates dissatisfaction, 1 point indicates relative dissatisfaction, 2 points indicates neutrality, 3 points indicates satisfaction, and 4 points indicates satisfaction. Satisfaction rate = (Number of participants with a score greater than 2) ÷ (Total number of participants) × 100%

[0143] (3) Security Testing

[0144] Human patch experiments were conducted in accordance with the *Cosmetic Safety Technical Specifications*, GB17149.1-1997 *Diagnostic Criteria and Treatment Principles for Cosmetic Skin Diseases*, and GB17149.2-1997 *Diagnostic Criteria and Treatment Principles for Cosmetic Contact Dermatitis*. This study recruited 160 volunteers. Inclusion criteria for volunteers were: 1) age between 15 and 60 years old, both male and female; 2) no serious diseases, no immunodeficiency or autoimmune diseases, and no prior skin treatment or cosmetic procedures on the test site; 3) no active allergic diseases; 4) no use of any anti-inflammatory drugs on the test site within the past two months; 5) no use of antihistamines within the past week or immunosuppressants within the past month; 6) no patch testing within the past month. Informed consent was obtained from the volunteers. All patch applicators were numbered on the back and mixed with equal amounts of Examples 1-3. The treated patch applicators were applied to the flexor aspect of the subjects' forearms, and skin condition was observed after 24 and 48 hours. The grading of skin reaction after patch application is shown in Table 7.

[0145] Table 7 Grading of Skin Patch Reaction Severity

[0146]

[0147] The results showed that the products in Examples 1-3 all scored 0 at 24 hours and 48 hours, and no adverse skin reactions were observed, indicating that the composition and cream products involved in this invention are safe and non-irritating to the skin.

[0148] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A firming and anti-aging composition, characterized in that, It is composed of bioactive peptide Pal-KVK, novel copper peptide, red algae extract, PPG-26-butanol polyether-26, and PEG-40 hydrogenated castor oil, wherein the mass ratio of the bioactive peptide Pal-KVK, novel copper peptide, red algae extract, PPG-26-butanol polyether-26, and PEG-40 hydrogenated castor oil is 0.005~10 : 0.05~3.0 : 0.05~3.0 : 0.01~0.5 : 0.04~2.0; The chemical structure of the novel copper peptide is as follows: 。 2. The method for preparing the firming and anti-aging composition according to claim 1, characterized in that, Includes the following steps: Pal-KVK was mixed with PPG-26-butanol polyether-26 and PEG-40 hydrogenated castor oil to obtain the first mixture; The novel copper peptide was added to the first mixture and stirred evenly. Then, the extract of red algae was added and stirred evenly to obtain a firming and anti-aging composition.

3. The application of the firming and anti-aging composition according to claim 1, characterized in that, The firming and anti-aging composition is used in the preparation of cosmetics.

4. A cosmetic product comprising the firming and anti-aging composition of claim 1, characterized in that, It is composed of phase A, phase B, phase C, and phase D. Phase A consists of emollients and emulsifiers, Phase B consists of moisturizers and chelating agents, Phase C consists of preservatives, and Phase D consists of the firming and anti-aging composition. The firming and anti-aging composition has a mass content of 0.1-20.0% in the cosmetic.

5. The cosmetic product according to claim 4, characterized in that, The A phase component is selected from one or more of the following: polydimethylsiloxane, triglyceride octanoate, hydrogenated polyisobutylene, cyclopentadimethylsiloxane, polydimethylsiloxane and C16~C18 alkyl polydimethylsiloxane cross-linked polymer, hydrogenated lecithin, and 2,6-di-tert-butyl-4-methylphenol. The B-phase component, namely the humectant and chelating agent, is selected from one or a combination of several of the following: water, glycerin, propylene glycol, betaine, carbomer, EDTA, or triethanolamine. The C-phase component, i.e. the preservative, is selected from a mixture of 1,2-octanediol and 2-phenoxyethanol and water.

6. The cosmetic product according to claim 5, characterized in that, The cosmetic product is a face cream, and its ingredients are as follows:

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