A composition with moisturizing, repairing, firming and anti-wrinkle effects and its application

Abstract

The present invention provides a composition having moisturizing, repairing, firming, and anti-wrinkle effects, and its use. The present invention belongs to the field of pharmaceuticals or personal care products and provides a pharmaceutical or personal care composition having moisturizing, repairing, firming, and anti-wrinkle effects, and its use. Specifically, the present invention provides an application of an annexin, which has been verified to have moisturizing, repairing, firming, and anti-wrinkle effects through cell and human efficacy tests. Human efficacy test results show that pharmaceutical or personal care compositions containing the above-mentioned protein have significant effects in moisturizing, repairing, firming, and anti-wrinkle.

Description

Technical Field

[0001] The present invention belongs to the field of medicine or personal care products, and specifically relates to a composition with moisturizing, repairing, firming, anti-wrinkle and other effects and its application. Background Art

[0002] With increasing demands for skin care, especially in modern life, the skin is prone to dryness, dehydration, sensitivity, and sagging due to a variety of factors, including environmental factors and unhealthy lifestyles. To address these issues, moisturizing, repairing, firming, and anti-wrinkle products are gaining widespread attention. The primary purpose of moisturizing and repairing is to provide the skin with adequate moisture and nutrients, maintaining its water-oil balance and keeping it healthy, smooth, and elastic. Moisturizing and repairing also helps strengthen the skin's barrier function, protecting it from external irritants and reducing skin damage and allergic reactions. Firming and anti-wrinkle products, on the other hand, combat wrinkles and sagging caused by natural aging and photoaging. Wrinkles are primarily caused by a decrease in collagen fibers, disrupted collagen fiber bundles, elastin denaturation, basal membrane damage, and angiogenesis, which in turn lead to water loss and wrinkles. In the pharmaceutical and personal care market, moisturizing, repairing, firming, and anti-wrinkle products are plentiful, including creams, lotions, serums, and facial masks. However, many raw materials only have a single function, so various components must be formulated to achieve the desired effect. Many components cannot be combined with other ingredients, resulting in limitations in the form of pharmaceutical or personal care compositions.

[0003] To overcome the limitations of existing technologies, researchers have begun to focus on the potential applications of annexins in skin care. Annexins are a class of calcium-dependent, phospholipid-binding proteins widely distributed throughout the body, playing important roles in cell signaling, inflammatory responses, cell growth, and differentiation. Annexin A5 (AnxA5) has attracted particular attention. Annexin A5 is a calcium-dependent, phospholipid-binding protein widely present in various cell types. It plays a crucial role in cell signaling, inflammatory responses, apoptosis, and cell differentiation. During apoptosis, Annexin A5 binds to phosphatidylserine (PS) exposed on the outside of the cell membrane, preventing phagocytes from recognizing apoptotic cells and thereby regulating the apoptotic process.

[0004] Annexin A5 has attracted considerable attention for its role in drug delivery. It significantly enhances the absorption and transmembrane transport of liposome-based drug delivery systems, enabling topical delivery of physiological concentrations of drugs and improving drug penetration in the skin (Davies et al., 2014). Annexin A5 also promotes rapid cell membrane repair by forming protective two-dimensional ribbon-like structures, exhibiting significant anti-inflammatory and anti-apoptotic effects, which are crucial for maintaining skin cell function (Bouter et al., 2015). Annexins interact with various intracellular biomolecules to exert their roles in signal transduction and cell protection. They can be incorporated into artificial liposomes or lipid vesicles, enabling better study and observation of their structure and function (Jeong et al., 2020). Application studies have shown that annexin A5 can maximize the protection of skin cells and maintain intact cellular function, demonstrating its potential in addressing skin dryness, allergies, and wrinkles caused by cellular dysfunction. Furthermore, peptide complexes, as derivatives of annexins, have been shown to promote the expression of basement membrane proteins, such as collagen XVII and laminin, in human epidermal keratinocytes, thereby improving the structural integrity and mechanical stability of the skin (Gomes et al., 2020). The application of these proteins in pharmaceuticals or personal care products, particularly through biotechnology extraction and utilization, such as fermentation processes or genetic engineering, can significantly enhance the anti-aging effects of pharmaceuticals or personal care products.

[0005] In summary, the application of annexins, particularly annexin A5, in pharmaceuticals and personal care products holds broad promise. Through advanced biotechnology and delivery systems, annexins and their related active ingredients play a significant role in enhancing the efficacy of pharmaceuticals and personal care products, simplifying formulation complexity, and meeting consumers' multifunctional skin care needs. The present invention aims to leverage the multiple benefits of annexin A5 to develop a pharmaceutical or personal care product with moisturizing, repairing, firming, and anti-wrinkle properties. This approach addresses the high demands of modern consumers for pharmaceuticals and personal care products, addresses skin problems such as dryness, allergies, sagging, and wrinkles, and provides consumers with a more comprehensive and effective skin care solution. Summary of the Invention

[0006] A first aspect of the present invention provides a use of an annexin in the preparation of a pharmaceutical composition or a personal care composition, wherein the pharmaceutical composition or the personal care composition has one or more of the following effects:

[0007] (a) Moisturizing;

[0008] (b) Repair;

[0009] (c) firming;

[0010] (d) Anti-wrinkle.

[0011] Annexins are a widely distributed class of calcium-dependent phospholipid-binding proteins. Based on molecular phylogenetic and comparative genomic analyses, this protein family has been uniformly designated as annexin (abbreviated as Anx), followed by capital letters representing the respective species: A, B, C, D, and E, representing vertebrates, invertebrates, fungi and myxomycetes, plants, and protists, respectively. Currently, the Anx A family has 12 members: A1-A11 and A13 (A12 is unassigned).

[0012] Annexin plays an important role in cell biology. It can bind to phosphatidylserine in the cell membrane and participate in various cellular processes, including membrane repair, signal transduction, cell division, inflammatory response, etc.

[0013] In one embodiment, the annexin protein of the present invention is annexin A (Anx A).

[0014] In one embodiment, the Annexin A is Annexin A5.

[0015] In one embodiment, the Annexin A5 is human Annexin A5.

[0016] In one embodiment, the human annexin A5 sequence is shown in SEQ ID No. 1:

[0017] SEQ ID No. 1:

[0018] AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTL

[0019] FGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTP

[0020] EELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDA

[0021] QALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQL

[0022] LLAVVKSIRSIPAYLAETLYYAMKGAGTDDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD.

[0023] Preferably, the annexin protein includes a wild type or a mutant type.

[0024] Annexin A5 (AnxA5) is widely distributed in various tissue cells of the body. In vitro studies have found that AnxA5 has multiple functions such as anti-phospholipase, anti-coagulation, and anti-kinase. It is currently mainly used as a reagent for detecting cell apoptosis.

[0025] During apoptosis, Annexin A5 recognizes and binds to phosphatidylserine exposed on the outer surface of the cell membrane, thereby preventing phagocytes from recognizing apoptotic cells and regulating the apoptotic process. As a marker of apoptosis, it is widely used in scientific research and medicine, and is particularly important in cancer research and treatment. Annexin A5 also plays a role in blood coagulation and anti-inflammatory responses, stabilizing cell membranes and preventing leakage of cellular contents, thereby reducing inflammation.

[0026] The applications of annexins are not limited to their anti-apoptotic and anti-inflammatory properties but also include their potential role in drug delivery systems. For example, annexins can be used to develop novel drug carriers to improve drug targeting and bioavailability. By binding annexins to drugs, drug trafficking and release within cells can be enhanced, thereby improving therapeutic efficacy.

[0027] In the field of medicine and personal care products, the present invention has studied in detail the effects of Annexin A5 as an active ingredient, which reduces skin damage caused by environmental factors and aging by enhancing the stability and repair capacity of cell membranes. It also promotes skin cell renewal and regeneration by regulating intercellular signaling, thereby achieving firming and anti-wrinkle effects. Compared to other skincare ingredients, Annexin A5 is multifunctional and highly effective, capable of simultaneously addressing multiple skin concerns, restoring skin to a healthy, smooth, and elastic state.

[0028] The present invention utilizes these biological properties of annexin A5 to develop pharmaceuticals or personal care products with moisturizing, repairing, firming, and anti-wrinkle properties. The annexin proteins useful in the present invention are not particularly limited and can be derived from any organism, preferably from mammals (such as primates), more preferably from humans, monkeys, or mice. Furthermore, it should be understood that "annexin" includes wild-type and mutant (including truncated) annexins, as long as the mutant annexin retains or maintains the detoxification activity of the wild-type annexin.

[0029] In summary, Annexin A5 has broad application prospects in medicines or personal care products. By combining advanced biotechnology and delivery systems, it can significantly enhance the efficacy of medicines or personal care products and meet consumers' growing demand for multifunctional skin care.

[0030] In one embodiment, the pharmaceutical or personal care compositions of the present invention comprise one or more auxiliary ingredients or excipients.

[0031] In one embodiment, the content of the annexin protein in the pharmaceutical composition or personal care composition is 0.01% to 1% by weight.

[0032] In one embodiment, the content of the annexin protein in the pharmaceutical composition or personal care composition is 0.01% to 1% by weight and volume.

[0033] In one embodiment, the content of the annexin protein in the pharmaceutical composition or personal care composition is 0.05% to 0.2% by weight or by volume.

[0034] In one embodiment, the content of the annexin protein of the present invention in the pharmaceutical composition or personal care composition is selected from 0.05% to 0.2% or 0.08% to 0.15% or 0.09 to 0.12%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% by weight or by volume.

[0035] In one embodiment, the content of the annexin protein of the present invention in the pharmaceutical composition or personal care composition is selected from 0.1% by weight or weight volume percentage;

[0036] In one embodiment, the weight ratio of the annexin to the auxiliary components is selected from 1:1 to 1:200.

[0037] In one embodiment, the weight ratio of the annexin to the auxiliary components is selected from 1:10 to 1:100.

[0038] In one embodiment, the weight ratio of the annexin to the excipient is selected from 1:10 to 1:500.

[0039] In one embodiment, the weight ratio of the annexin to the auxiliary components is selected from 1:50 to 1:200.

[0040] In one embodiment, in the composition of the present invention, the concentration of annexin is selected from 0.1 mg / mL to 10 mg / mL; more preferably, the concentration of annexin is selected from 0.5 mg / mL to 8 mg / mL, 0.8 mg / mL to 5 mg / mL or 1 mg / mL to 3 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL.

[0041] In the different types of pharmaceutical compositions or personal care compositions described in the present invention, annexin is used in a reasonable combination with a variety of auxiliary ingredients to ensure the efficacy and stability of the product.

[0042] In one embodiment, the auxiliary ingredient of the present invention is selected from one or more of preservatives, stabilizers, antioxidants, thickeners, emulsifiers, UV absorbers, penetration enhancers, plant extracts, moisturizers or emollients.

[0043] In one embodiment, the preservative is selected from one or more of phenoxyethanol, methylisothiazolinone or ethylhexylglycerin.

[0044] In one embodiment, the antioxidant is selected from one or more of vitamin E, vitamin C, or resveratrol.

[0045] In one embodiment, the ultraviolet absorber is selected from one or more of titanium dioxide, zinc oxide, avobenzone or octocrylene.

[0046] In one embodiment, the moisturizing agent is selected from one or more of hyaluronic acid, glycerin, propylene glycol, butylene glycol, hexylene glycol or urea.

[0047] In one embodiment, the thickener is selected from one or more of xanthan gum, carbomer, guar gum or sodium alginate.

[0048] In one embodiment, the emulsifier is selected from one or more of cetearyl alcohol, emulsifying wax, polysorbate or glyceryl stearate.

[0049] In one embodiment, the penetration enhancer is selected from one or more of propylene glycol, ethanol, ethylene glycol, or benzyl alcohol.

[0050] In one embodiment, the plant extract is selected from one or more of green tea extract, aloe vera extract, chamomile extract or calendula extract.

[0051] In one embodiment, the emollient is selected from one or more of mineral oil, jojoba oil, coconut oil, or squalane.

[0052] In one embodiment, the auxiliary material of the present invention is selected from one or more of a conditioner, a diluent, a filler, a binder, a wetting agent, a surfactant, a lubricant, a fragrance or a flavoring agent.

[0053] In one embodiment, the conditioning agent is selected from one or more of panthenol, glycerin, or lactic acid.

[0054] In one embodiment, the diluent is selected from one or more of water, ethanol or propylene glycol.

[0055] In one embodiment, the filler is selected from one or more of talc, silicon dioxide or microcrystalline cellulose.

[0056] In one embodiment, the binder is selected from one or more of polyethylene glycol, hydroxypropyl methylcellulose or carbomer.

[0057] In one embodiment, the humectant is selected from one or more of propylene glycol, glycerol or urea.

[0058] In one embodiment, the surfactant is selected from one or more of polysorbate, sodium lauryl sulfate, or cocamidopropyl betaine.

[0059] In one embodiment, the lubricant is selected from one or more of silicone oil, jojoba oil or mineral oil.

[0060] In one embodiment, the fragrance is selected from one or more of natural essential oils, synthetic fragrances or plant extracts.

[0061] In one embodiment, the flavoring agent is selected from one or more of licorice extract, mint extract, or vanilla extract.

[0062] In one embodiment, the pharmaceutical composition or personal care composition of the present invention is selected from one or more of a skin care composition and a hair care composition.

[0063] In one embodiment, the dosage form of the pharmaceutical composition or personal care composition is selected from a solution, a gel, an emulsion, a cream, a suspension, a film, a spray or a powder.

[0064] In one embodiment, the dosage form of the pharmaceutical composition or personal care composition is selected from a serum, lotion, cream or wax-based dosage product.

[0065] In one embodiment, the essence, lotion, or cream is selected from an O / W dosage form or a W / O dosage form.

[0066] In one embodiment, the pharmaceutical composition or personal care composition is in the form of a serum.

[0067] In one embodiment, the ratio of annexin to moisturizer is selected from 1:10 to 1:200. Moisturizers help maintain the skin's smoothness and plumpness, and increasing the moisturizer ratio can effectively enhance the product's moisturizing effect. Common moisturizers include glycerin, 1,3-butylene glycol, and hyaluronic acid. For example, in a serum, the ratios of glycerin and 1,3-butylene glycol are 3% and 4%, respectively.

[0068] In one embodiment, the ratio of annexin A5 to emulsifier is selected from 1:1 to 1:20. Emulsifiers help improve product texture and stability by ensuring uniform mixing of the aqueous and oil phases. Common emulsifiers include cetearyl alcohol and emulsifying wax. For example, in lotions and creams, emulsifiers may be used in amounts of 2% to 4%.

[0069] In one embodiment, the ratio of annexin A5 to thickener is selected from 1:1 to 1:50. The thickener helps increase the viscosity of the product, improving its feel and stability. Common thickeners include xanthan gum and carbomer. For example, in creams and wax-based dosage forms, the amount of xanthan gum used can reach 0.5%.

[0070] In one embodiment, the ratio of annexin A5 to preservative is selected from 1:2 to 1:10. An appropriate preservative concentration can effectively inhibit bacterial growth and ensure the microbial stability of the product. Common preservatives include phenoxyethanol, which is generally used at a concentration of 0.5% to 1%.

[0071] In one embodiment, the weight ratio of the annexin to the moisturizing agent is selected from 1:20 to 1:150, more preferably 1:50 to 1:100.

[0072] In one embodiment, the weight ratio of the annexin to the emulsifier is selected from 1:2 to 1:15, more preferably 1:5 to 1:10.

[0073] In one embodiment, the weight ratio of the annexin to the thickener is selected from 1:5 to 1:30, more preferably 1:10 to 1:20.

[0074] In one embodiment, the weight ratio of the annexin to the preservative is selected from 1:2 to 1:5, more preferably 1:3 to 1:8.

[0075] In one embodiment, the weight volume percentage of the annexin is selected from 0.01 to 1%, more preferably 0.1%.

[0076] In one embodiment, the humectant is selected from one or more of glycerin, 1,3-butylene glycol, or 1,3-hexanediol.

[0077] In one embodiment, the weight volume percentage of the glycerol is selected from 2-5%, more preferably 3%.

[0078] In one embodiment, the weight volume percentage of the 1,3-butanediol is selected from 2-5%, more preferably 4%.

[0079] In one embodiment, the weight volume percentage of the 1,3-hexanediol is selected from 1-2%, more preferably 1%.

[0080] In one embodiment, the diluent is selected from water.

[0081] In one embodiment, the weight volume percentage of water is 70-80%.

[0082] Another aspect of the present invention provides a method for preparing a pharmaceutical composition or personal care composition having moisturizing, repairing, firming, and anti-wrinkle effects, comprising the following steps:

[0083] heating the aqueous phase ingredients;

[0084] Add active ingredients;

[0085] Cool and adjust pH;

[0086] Add preservatives;

[0087] Homogenization and filling.

[0088] In one embodiment, the cytotoxicity of the recombinant annexin A5 was evaluated against human dermal fibroblasts (hDF). The results showed that recombinant annexin A5 (AnxA5) had no significant cytotoxicity against hDF cells at concentrations below 1 mg / mL, ensuring its safety in pharmaceuticals, personal care products, and other applications.

[0089] In one embodiment, the present invention prepares an Annexin A5 personal care composition formulation using the Annexin A5 as an active ingredient according to the preparation method.

[0090] In one embodiment, the Annexin A5 personal care composition formulation exhibits significant moisturizing, repairing, firming, and anti-wrinkle effects on the skin. Human efficacy evaluations, combined with instrumental testing and subjective assessments, demonstrated that the personal care composition formulation significantly improved skin moisturizing, repairing, and firming properties, and effectively reduced wrinkles.

[0091] In one embodiment, the Annexin A5 personal care composition exhibits good safety during use by subjects. Subjects used the test product as directed within the prescribed timeframe and did not experience any adverse skin or systemic reactions, demonstrating the product's safety.

[0092] In one embodiment, the Annexin A5 personal care composition formulation significantly increases the moisture content of the stratum corneum of the subject's skin, has a significant moisturizing effect, and can effectively improve the hydration of the skin.

[0093] In one embodiment, the Annexin A5 personal care composition formulation significantly reduces transepidermal water loss of the skin, enhances the skin barrier function, reduces water loss, and significantly improves the moisturizing effect of the skin.

[0094] In one embodiment, the Annexin A5 personal care composition formulation significantly improves skin elasticity, can effectively enhance skin firmness, and make the skin more elastic and firm.

[0095] In one embodiment, the Annexin A5 personal care composition formulation significantly improves the firmness of the skin, can effectively improve the sagging condition of the skin, and make the skin firmer and smoother.

[0096] In one embodiment, the Annexin A5 personal care composition preparation has a significant effect on improving the skin melanin index, and can effectively reduce the melanin content of the skin, making the skin tone more even.

[0097] In one embodiment, the Annexin A5 personal care composition preparation has a significant effect on reducing the area of ​​under-eye wrinkles, can effectively reduce eye wrinkles, and improve the firmness of the eye skin.

[0098] In one embodiment, the Annexin A5 personal care composition formulation has a significant effect on reducing the area ratio of under-eye wrinkles, and can reduce the relative area of ​​under-eye wrinkles, further demonstrating its anti-wrinkle effect.

[0099] In one embodiment, the Annexin A5 personal care composition formulation has a significant effect on improving the L* value of skin color, can increase skin brightness, and contribute to whitening and even skin tone.

[0100] In one embodiment, the Annexin A5 personal care composition formulation has a significant effect on improving the ITA° value of skin color, and can improve the glossiness and evenness of the skin.

[0101] In one embodiment, the Annexin A5 personal care composition preparation has a significant effect on improving skin glossiness parameters, can significantly improve skin glossiness, and make the skin look healthier.

[0102] In one embodiment, the Annexin A5 personal care composition formulation does not significantly change the mean optical density of the spots, indicating that it is not significantly effective in reducing the optical density of the spots.

[0103] In one embodiment, the Annexin A5 personal care composition preparation has a significant effect on reducing the proportion of dark spots, and can effectively reduce the area of ​​dark spots and make the skin more even.

[0104] The annexin A5 of the present invention demonstrates significant progress and advantages in many aspects, especially in the application of medicine or personal care products. Its unique biological properties and multifunctionality make it stand out in the market.

[0105] 1. Multifunctional Applications: This invention provides the use of annexin in the preparation of pharmaceutical or personal care compositions, exhibiting multiple benefits, including moisturizing, repairing, firming, and anti-wrinkle. Annexin A5 significantly enhances the multifunctionality of these products by enhancing the stability and repair capacity of cell membranes, enabling them to address a variety of skin concerns and restore skin health, smoothness, and elasticity.

[0106] 2. Highly effective biological activity: Annexin A5 plays a crucial role in cell biology, binding to phosphatidylserine in cell membranes and participating in a variety of processes, including membrane repair, signal transduction, cell division, and inflammatory responses. Its application in anti-aging medicine or personal care products, by regulating intercellular signaling, promotes skin cell renewal and regeneration, significantly improving skin firmness and elasticity and reducing wrinkles.

[0107] 3. Optimized molecular properties and safety: Studies in the present invention have shown that recombinant annexin A5 has no significant cytotoxicity to human dermal fibroblasts (hDF) at concentrations below 1 mg / mL, ensuring its safety in pharmaceutical compositions, personal care compositions, or other applications.

[0108] 4. Significant Clinical Effects: Human efficacy evaluations have shown that personal care compositions containing Annexin A5 demonstrate significant benefits in moisturizing, repairing, firming, and anti-wrinkle properties. After using the personal care compositions, subjects experienced a significant increase in stratum corneum moisture content, a significant reduction in transepidermal water loss, improved skin elasticity and firmness, a significant reduction in wrinkles, and improved skin tone uniformity and brightness. These clinical trial results provide strong scientific support for the use of Annexin A5 in pharmaceutical or personal care compositions.

[0109] In summary, the membrane-binding protein A5 in the present invention has broad application prospects in pharmaceutical compositions or personal care compositions. By combining its advanced biological properties and delivery system, it can significantly enhance the efficacy of the product and meet the growing multifunctional needs of consumers.

[0110] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 The results of toxicity experiments on human dermal fibroblasts (hDF) are shown.

[0112] Figure 2 The results of the water content of the stratum corneum of the subjects' skin are shown, where "ns" means no statistical difference; "*" means 0.01≤P<0.05; "**" means 0.001≤P<0.01; "***" means <0.001, Figures 3 to 12 Same here.

[0113] Figure 3 The results of the transepidermal water loss values ​​of the subjects are shown.

[0114] Figure 4 Shown are the R2 results of the subjects' skin elasticity.

[0115] Figure 5 Shown are the results for subject skin firmness F4.

[0116] Figure 6 Shown are the results of the subjects' skin melanin index MI.

[0117] Figure 7 The VISIA-CR under-eye wrinkle area results for each subject are shown.

[0118] Figure 8 The results of the VISIA-CR under-eye wrinkle area percentage of the subjects are shown.

[0119] Figure 9 The VISIA-CR skin color L* value results for the subjects are shown.

[0120] Figure 10 The VISIA-CR skin color ITA° value results of the subjects are shown.

[0121] Figure 11 The results of the VISIA-CR skin glossiness parameter of the subjects are shown.

[0122] Figure 12The results of the VISIA-CR spot area percentage of the subjects are shown.

[0123] Related definitions

[0124] Unless defined otherwise, 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 belongs.

[0125] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0126] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0127] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0128] As used herein, the term "active ingredient" refers to the primary component in a pharmaceutical or personal care product that exerts a specific effect. In the present invention, the active ingredient includes Annexin A5. Active ingredients directly impact product performance and effectiveness and are typically added to formulations at a specific concentration to enhance skin moisturizing, repairing, firming, and anti-wrinkle effects.

[0129] As used herein, the term "composition" refers to a preparation containing Annexin A5 and possibly other auxiliary ingredients such as moisturizers, antioxidants or whitening ingredients, which work together to provide a more comprehensive skin care effect.

[0130] As used herein, the term "annexin" refers to a ubiquitous calcium-dependent phospholipid-binding protein. The term "annexin" is followed by a capital letter representing the species: A, B, C, D, and E represent vertebrates, invertebrates, fungi and myxomycetes, plants, and protists, respectively. Currently, the Anx A family has 12 members: A1-A11 and A13 (A12 is unassigned). Annexins play important roles in various cellular processes, including cell signaling, inflammatory responses, and cell division.

[0131] As used herein, the term "Annexin A5 (AnxA5)" refers to a member of the annexin family that is widely distributed in various tissue cells. Annexin A5 has demonstrated multiple functions in in vitro studies, including antiphospholipase, anticoagulant, and antikinase, and is currently used primarily as a reagent for detecting apoptosis. During apoptosis, Annexin A5 can recognize and bind to phosphatidylserine exposed on the outside of the cell membrane, thereby preventing phagocytes from recognizing apoptotic cells and regulating the process of apoptosis. Annexin A5 also plays a role in blood coagulation and anti-inflammatory responses, by stabilizing the cell membrane and preventing leakage of cell contents, thereby reducing inflammatory responses.

[0132] As used herein, the term "personal care product" refers to personal care products used to protect, cleanse, and improve the appearance of the skin. In the present invention, the personal care product comprises Annexin A5 to reduce skin damage caused by environmental factors and aging by enhancing the stability and repair capacity of cell membranes.

[0133] As used herein, the term "humectant" refers to an ingredient that helps the skin retain moisture, such as glycerin and 1,3-butylene glycol. These ingredients help maintain the skin's water and oil balance, keeping the skin moisturized and soft.

[0134] As used herein, the term "emulsifier" refers to compounds used to mix oil and water phase ingredients, such as cetearyl alcohol and emulsifying wax. These ingredients can help form a stable emulsion or cream, making the product texture uniform and easy to spread.

[0135] As used herein, the term "thickener" refers to ingredients added to a formulation to increase its viscosity, such as xanthan gum and carbomer. These ingredients help improve the feel and stability of the product, making it more suitable for application to the skin.

[0136] As used herein, the term "preservative" refers to a chemical substance, such as phenoxyethanol, that is used to inhibit the growth of microorganisms such as bacteria and mold. Preservatives ensure that a product remains stable and safe during storage and use.

[0137] As used herein, the term "oily ingredient" refers to oily substances used in pharmaceutical or personal care products to moisturize and nourish the skin, such as jojoba oil and shea butter. These ingredients can help repair the skin barrier and provide long-lasting lubrication and protection.

[0138] As used herein, the term "pH adjuster" refers to a chemical substance used to adjust the pH of a product, such as citric acid. An appropriate pH value helps ensure product stability and skin compatibility, avoiding irritation or damage to the skin.

[0139] As used herein, the term "human dermal fibroblasts (hDF)" refers to fibroblasts derived from human skin. These cells play a key role in the dermis, participating in skin repair, regeneration, and maintaining the integrity of the skin's structure. Fibroblasts synthesize and secrete various extracellular matrix components, such as collagen, elastin, and glycosaminoglycans, and play an important role in wound healing and tissue remodeling.

[0140] As used herein, the term "recombinant A5 protein" refers to annexin A5 produced by genetic engineering technology. The protein is purified and can be used in various biological experiments and applications.

[0141] As used herein, the term "w / v" refers to weight / volume percentage concentration and is used to describe the concentration of a solute in a solution. For example, 4% (w / v) means that there are 4 grams of solute per 100 milliliters of solution.

[0142] As used herein, the term "skin stratum corneum moisture content" refers to the amount of water contained in the stratum corneum of the cheek skin as measured by an instrument. This indicator reflects the degree of moisturization of the skin, with a higher value indicating more hydrated skin.

[0143] As used herein, the term "transepidermal water loss" refers to the rate of water evaporation from the cheek skin surface, as measured by an instrument. This indicator reflects the integrity of the skin's barrier function; lower values ​​indicate better skin barrier function and less water loss.

[0144] As used herein, the term "skin elasticity R2" refers to a parameter measured by an instrument to measure the elasticity of the skin on the cheeks. A higher R2 value indicates better skin elasticity, firmer skin, and more elastic and resilient the skin.

[0145] As used herein, the term "skin firmness F4" refers to a parameter for measuring the firmness of cheek skin using an instrument. A lower F4 value indicates firmer skin with less sagging.

[0146] As used herein, the term "skin melanin index (MI)" refers to the amount of melanin in the skin at a specific spot as measured by an instrument. A lower MI value indicates less melanin in the skin and a more even skin tone.

[0147] As used herein, the term "VISIA-CR" refers to a high-precision skin imaging and analysis system that is commonly used to assess skin health and the effectiveness of beauty products.

[0148] As used herein, the term "VISIA-CR Under-Eye Wrinkle Area" refers to the total area of ​​under-eye wrinkles derived from full-face photography analyzed using the VISIA-CR system. This metric reflects the degree of wrinkling in the eye area; a smaller area indicates fewer wrinkles.

[0149] As used herein, the term "VISIA-CR Under-Eye Wrinkle Area Ratio" refers to the ratio of under-eye wrinkle area to total facial area, as determined by full-face photography analysis using the VISIA-CR system. This metric reflects the distribution of under-eye wrinkles relative to the face; lower values ​​indicate a smaller under-eye wrinkle ratio.

[0150] As used herein, the term "VISIA-CR skin color L* value" refers to the skin brightness value of the cheek area determined by analyzing a full-face photograph taken by the VISIA-CR system. A higher L* value indicates lighter skin color and greater brightness.

[0151] As used herein, the term "VISIA-CR Skin Color ITA° Value" refers to the chromatic angle value of cheek skin color derived from full-face photography and analysis using the VISIA-CR system. A higher ITA° value indicates lighter and more even skin color.

[0152] As used herein, the term "VISIA-CR skin glossiness parameter" refers to the cheek skin glossiness parameter derived from full-face photography and analysis using the VISIA-CR system. A higher value indicates more radiant and healthier-looking skin.

[0153] As used herein, the term "VISIA-CR Spot Area Ratio" refers to the ratio of cheek spot area to total facial area, as determined by full-face photography analysis using the VISIA-CR system. A lower value indicates less spot coverage. DETAILED DESCRIPTION

[0154] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0155] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0156] Example 1. Cytotoxicity experiment

[0157] This study aimed to evaluate the cytotoxicity of recombinant annexin A5 (AnxA5) at various concentrations in human dermal fibroblasts (hDF). Cell viability was measured to determine the effects of AnxA5 at different concentrations on cell survival, thereby ensuring its safety in pharmaceutical, personal care, and other applications.

[0158] Reagents and materials:

[0159] Cell line: human dermal fibroblasts (hDF);

[0160] Reagents: DMEM (GIBCO), FBS (GIBCO), PBS (GIBCO), 0.25% trypsin (GIBCO), CCK8 (DOJINDO, Japan), etc.

[0161] The experiment included the following groups:

[0162] Blank control group (CON.): only an equal amount of PBS was added to the culture medium.

[0163] Experimental group: Different concentrations of AnxA5 were added to the culture medium, including 5 mg / mL, 1 mg / mL, 500 μg / mL, 50 μg / mL, 5 μg / mL, 1.6 μg / mL, and 500 ng / mL.

[0164] Experimental process:

[0165] Human skin fibroblasts (hDF) were cultured at a rate of 3*10 4 Cells were seeded into 96-well cell culture plates at a density of 100 cells / mL. After 24 hours, the culture medium was replaced with medium containing various concentrations of recombinant A5 protein. An equal volume of PBS was added to the blank control group and incubated for a further 72 hours. CCK8 was then added and incubated in a CO2 incubator for 2 hours. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated.

[0166] Cell viability (%) = (A1-A) / (A0-A) × 100%

[0167] Where A1 represents the absorbance of the sample group, A represents the absorbance of the cell-free CCK8 culture medium solution, and A0 represents the absorbance of the blank control group. Statistical analysis was performed using GraphPad Prism, and a sample-cell viability histogram was plotted.

[0168] Result analysis: Figure 1The figure shows the cell viability of different experimental groups. As shown, different concentrations of recombinant annexin A5 (AnxA5) have different effects on human dermal fibroblasts (hDF). A high concentration (5 mg / mL) of AnxA5 significantly reduced cell viability compared to the blank control (CON.), indicating cytotoxicity. Intermediate concentrations (1 mg / mL to 5 μg / mL) and low concentrations (1.6 μg / mL to 500 ng / mL) had little effect on cell viability or no significant toxicity. In particular, within the concentration range below 1 mg / mL, cell viability remained around or above 100%, indicating that AnxA5 has no significant cytotoxicity to cells at these concentrations.

[0169] This example demonstrates that recombinant annexin A5 has no significant cytotoxicity to human dermal fibroblasts (hDF) at concentrations below 1 mg / mL, providing a scientific basis for its safe use in pharmaceuticals, personal care products, and other biological applications.

[0170] Example 2. Preparation of Essence for Human Efficacy Testing

[0171] The serum was prepared according to the following formula: Annexin A5, water, 1,3-butylene glycol, glycerol, 1,3-hexanediol

[0172] (1) Materials:

[0173] Water (Aqua) (70-80%);

[0174] 1,3-Butylene Glycol: As a solvent and humectant, the usage amount is 4% (w / v) (2-5%);

[0175] Glycerin: As a moisturizer, the usage amount is 3% (w / v) (2-5%);

[0176] 1,3-Hexanediol: acts as a solvent and humectant, also acting as a preservative. The usage level is 1% (w / v) (1-2%).

[0177] Annexin A5: As an active ingredient, the usage amount is 0.1% (w / v) (0.01-1%).

[0178] (2) Preparation steps:

[0179] Step 1: Prepare and heat the water phase ingredients

[0180] In a clean container, combine water, 1,3-butylene glycol, glycerin, and 1,3-hexanediol. Heat the mixture to 75°C to 80°C to ensure all ingredients are completely dissolved.

[0181] Step 2: Add active ingredients

[0182] The mixture is cooled to 40°C and annexin A5 is added. This step needs to be performed at a lower temperature to prevent the active ingredient from being denatured by heat.

[0183] Step 3: Homogenization and filling

[0184] Use a high shear homogenizer to process the mixture to ensure that all ingredients are evenly mixed to form a uniform serum. Fill the serum into sterile containers to prevent contamination. If necessary, pH adjusters (such as citric acid) and preservatives can be added to the formula to make it more suitable for skin use.

[0185] Example 3. Human efficacy evaluation experiment

[0186] This example aimed to evaluate the moisturizing, repairing, firming, and anti-wrinkle effects of a serum containing recombinant annexin A5 (AnxA5) on the skin, as well as its effects on improving skin color and pigmentation, through human efficacy evaluation. A comprehensive evaluation of the serum's moisturizing, repairing, firming, and anti-wrinkle effects, as well as its effects on improving skin color and pigmentation, was conducted using test products in subjects, combined with instrumental testing and subjective evaluation.

[0187] 3.1 Test sample: Essence of Example 2

[0188] 3.2 Reference for the scheme:

[0189] T / CAB 0152-2022: Test Methods for Cosmetics' Anti-Wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing, and Soothing Efficacy; T / GDCA 009-2022: Human Body Evaluation Methods for Cosmetics' Repairing Efficacy;

[0190] T / TDCA 003-2021: Test Method for Firming Effect of Cosmetics;

[0191] Liu Weiyi, Zhou Lin, Zhao Hua. "Evaluation of Cosmetic Efficacy (ⅩⅢ)-Consumer Usage Test" [J]. Surfactant Detergent & Cosmetics Industry, 2021, 51(06): 485-490.

[0192] 3.3 Purpose of testing: To have subjects use the test product, and to conduct a comprehensive evaluation of the product's moisturizing, repairing, firming, anti-wrinkle effects, and skin color and spot improvement effects through instrument testing and subjective evaluation.

[0193] 3.4 Testing Methods: 33 healthy Chinese male and female subjects aged 25 to 60 years with facial skin characterized by dryness, lack of moisture, poor barrier function, sagging, lack of elasticity, visible pigmentation, prominent facial lines, fine lines or wrinkles, and under-eye wrinkles meeting the "SKIN AGING ATLAS" scale of 2 to 5 were selected. Using a before-and-after comparison method, the subjects' stratum corneum moisture content, transepidermal water loss (TEWL), skin elasticity, skin firmness, and melanin index were measured. Subjective assessments were performed, and full-face photographs were taken. The results were compared using statistical tests to determine if any statistical differences were found.

[0194] 3.5 Detection location

[0195] (1) Skin stratum corneum moisture content - cheeks

[0196] (2) Transepidermal water loss value - cheek

[0197] (3) Skin elasticity R2—cheek

[0198] (4) Skin firmness F4—cheeks

[0199] (5) Skin Melanin Index MI - designated spots

[0200] (6) VISIA-CR Under-eye Wrinkle Area - Full-face photo analysis

[0201] (7)VISIA-CR Under-eye Wrinkle Area Ratio - Full-face Photo Analysis

[0202] (8) VISIA-CR skin color L* value - full face photo analysis of cheeks

[0203] (9) VISIA-CR skin color ITA° value - full face photo analysis of cheeks

[0204] (10) VISIA-CR skin glossiness parameter - full face photo analysis of cheeks

[0205] (11)VISIA-CR spot area ratio - full face photo analysis cheek

[0206] Table 1. Detection indicators and specific detection methods

[0207]

[0208]

[0209] Note: D0 is the 0th day from the start of the test, D14 is the 14th day from the start of the test, and the same below.

[0210] 3.6 Statistical Methods: SPSS statistical analysis software was used. If the values ​​showed a normal distribution, the T-test was used for statistical analysis; if the values ​​showed a non-normal distribution, the rank sum test was used for statistical analysis. Two-tailed tests were used, with an α level of 0.05.

[0211] 3.7 Questionnaire Result Statistical Method and Safety Evaluation Method

[0212] 3.7.1 Statistical method for questionnaire results: The percentage of people with a score ≥ 4 was calculated; the binomial distribution method was used for statistical analysis, with the expected value set at 0.5 and the significance level at α = 0.05. If the P value was less than 0.05, it indicated a significant difference.

[0213] 3.7.2 Safety evaluation adopts statistical descriptive method: analyzing the degree and duration of adverse events case by case.

[0214] The formula for calculating the rate of change is as follows:

[0215] Δ(difference) after using the test product for 14 days = D14 - D0

[0216] △ (difference) after using the test product for 28 days = D28 - D0

[0217] △ (difference) after using the test product for 56 days = D56 - D0

[0218]

[0219]

[0220]

[0221] In the formula, D0 is the basic value of skin parameters before using the test product in the test area.

[0222] D14 - Skin parameter values ​​after the test area has been treated with the test product for 14 days.

[0223] D28 - Skin parameter values ​​after the test area has used the test product for 28 days.

[0224] D56 - Skin parameter values ​​after the test area has used the test product for 56 days.

[0225] 3.8 Test results

[0226] 3.8.1 Sample Completion: 33 subjects were enrolled and ultimately counted, including 3 males and 30 females, aged 28 to 53 years, with an average age of 45.82 ± 0.93 years. Test Product: Essence prepared in Example 2. Instructions: Apply an appropriate amount to the face, gently massaging until absorbed, once daily, morning and evening.

[0227] Example 3-1. Safety evaluation

[0228] Each subject used the test product as required within the prescribed time. Questioning, examination, and recording of any skin reactions or systemic adverse reactions during the trial were performed, including the manifestation, time of occurrence, treatment measures, and outcome of the adverse events. A judgment was also made on the relationship between the adverse events and the test product used. Details are shown in Table 2 below:

[0229] Table 2. Skin reactions in human trials

[0230]

[0231]

[0232] As shown in Table 2 , no adverse skin reactions were observed in the 33 subjects throughout the trial.

[0233] Example 3-2. Water content of the stratum corneum

[0234] Table 3 shows the moisture content of the stratum corneum of the subjects' skin, and Table 4 shows the improvement rate of the moisture content of the stratum corneum of the subjects' skin. Figure 2 The bar graph of the test results of the moisture content of the stratum corneum of the subjects' skin is shown in Table 3, Table 4 and Figure 2 As shown, when the subjects used the test product essence for 14 consecutive days, the moisture content of the skin stratum corneum increased significantly compared with the baseline value (P<0.001), with an increase rate of 25.41%; when the subjects used the test product for 28 consecutive days, the moisture content of the skin stratum corneum increased significantly compared with the baseline value (P<0.001), with an increase rate of 48.46%; when the subjects used the test product for 56 consecutive days, the moisture content of the skin stratum corneum increased significantly compared with the baseline value (P<0.001), with an increase rate of 68.35%. The results show that the essence of Example 2 has a significant moisturizing effect.

[0235] Measurement value: The higher the moisture content of the skin's stratum corneum, the more hydrated the skin.

[0236] Table 3. Test results of moisture content of skin stratum corneum (au, relative moisture content)

[0237]

[0238] Note: The data in the table are mean ± standard error.

[0239] Table 4. Improvement rate of moisture content in the stratum corneum

[0240]

[0241] Example 3-3. Transepidermal water loss value

[0242] Table 5 shows the test results of the subjects' transepidermal water loss values, and Table 6 shows the improvement rates of the subjects' transepidermal water loss values. Figure 3 The bar graph of the transepidermal water loss values ​​of the subjects is shown in Table 5, Table 6 and Figure 3 As shown, when the subjects used the test product essence for 14 consecutive days, the transepidermal water loss value of the skin was significantly reduced compared with the baseline value (P<0.001), with a decrease rate of 6.26%; when the subjects used the test product essence for 28 consecutive days, the transepidermal water loss value of the skin was significantly reduced compared with the baseline value (P<0.001), with a decrease rate of 20.24%; when the subjects used the test product essence for 56 consecutive days, the transepidermal water loss value of the skin was significantly reduced compared with the baseline value (P<0.001), with a decrease rate of 28.95%. The results show that the essence of Example 2 significantly enhanced the skin barrier function and reduced water loss.

[0243] Measurement value: The lower the TEWL value, the better the skin barrier function.

[0244] Table 5. Transepidermal water loss values ​​(g / m 2 / h)Test results

[0245]

[0246]

[0247] Note: The data in the table are mean ± standard error.

[0248] Table 6. Improvement rate of transepidermal water loss

[0249]

[0250] Example 3-4. Skin elasticity R2

[0251] Table 7 shows the test results of the skin elasticity R2 of the subjects, and Table 8 shows the improvement rate of the skin elasticity R2 of the subjects. Figure 4 The bar graphs of the R2 results of the skin elasticity of the subjects are shown in Tables 7, 8 and Figure 4As shown, when the subjects used the test product essence for 14 consecutive days, the skin elasticity R2 increased significantly compared with the baseline value (P<0.001), with an increase rate of 6.00%; when the subjects used the test product essence for 28 consecutive days, the skin elasticity R2 increased significantly compared with the baseline value (P<0.001), with an increase rate of 18.00%; when the subjects used the test product essence for 56 consecutive days, the skin elasticity R2 increased significantly compared with the baseline value (P<0.001), with an increase rate of 30.00%. The results show that the essence of Example 2 significantly improves skin elasticity.

[0252] Measurement: Skin elasticity R2 is a unitless elastic coefficient, measured using a skin elasticity meter. It reflects the skin's ability to recover after mechanical stimulation. Higher values ​​indicate greater skin elasticity.

[0253] Table 7. Skin elasticity R2 test results

[0254]

[0255] Note: The data in the table are mean ± standard error.

[0256] Table 8. Skin elasticity R2 improvement rate

[0257]

[0258] Example 3-5. Skin firmness F4

[0259] Table 9 shows the test results of the skin firmness F4 of the subjects, and Table 10 shows the improvement rate of the skin firmness F4 of the subjects. Figure 5 The bar graph of the skin firmness F4 results of the subjects is shown in Tables 9, 10 and Figure 5 As shown, when the subjects used the test product essence for 14 consecutive days, the skin firmness F4 decreased significantly compared with the baseline value (P<0.001), with a decrease rate of 3.57%; when the subjects used the test product essence for 28 consecutive days, the skin firmness F4 decreased significantly compared with the baseline value (P<0.001), with a decrease rate of 9.42%; when the subjects used the test product essence for 56 consecutive days, the skin firmness F4 decreased significantly compared with the baseline value (P<0.001), with a decrease rate of 16.56%. The results show that the essence of Example 2 significantly improves skin firmness.

[0260] Measurement: Skin firmness F4 is a unitless firmness coefficient. The lower the value, the firmer the skin.

[0261] Table 9. Skin firmness F4 test results

[0262]

[0263] Note: The data in the table are mean ± standard error.

[0264] Table 10. Skin firmness F4 improvement rate

[0265]

[0266] Example 3-6. Skin Melanin Index MI

[0267] Table 11 shows the test results of the melanin index MI of the subjects' skin, and Table 12 shows the improvement rate of the melanin index MI of the subjects' skin. Figure 6 The results of the melanin index MI of the subjects are shown in the bar graphs in Tables 11, 12 and Figure 6 As shown, when the subjects used the test product essence for 14 consecutive days, the skin melanin index MI decreased significantly compared with the baseline value (P<0.001), and the decrease rate was 7.37%; when the subjects used the test product essence for 28 consecutive days, the skin melanin index MI decreased significantly compared with the baseline value (P<0.001), and the decrease rate was 10.18%; when the subjects used the test product essence for 56 consecutive days, the skin melanin index MI decreased significantly compared with the baseline value (P<0.001), and the decrease rate was 12.96%.

[0268] Measurement value: The skin melanin index MI is measured by a colorimeter and is a unitless index value. The lower the value, the less melanin content in the skin.

[0269] Table 11. Skin melanin index MI test results

[0270]

[0271] Note: The data in the table are mean ± standard error.

[0272] Table 12. Skin Melanin Index MI Improvement Rate

[0273]

[0274] Example 3-7. VISIA-CR Under-eye Wrinkle Area

[0275] Table 13 shows the results of the VISIA-CR under-eye wrinkle area test for the subjects, and Table 14 shows the improvement rates of the VISIA-CR under-eye wrinkle area for the subjects. Figure 7 The bar graphs showing the results of the VISIA-CR under-eye wrinkle area of ​​the subjects are shown in Tables 13, 14 and Figure 7As shown, when the subjects used the test product essence for 14 consecutive days, the area of ​​VISIA-CR under-eye wrinkles decreased significantly compared to the baseline value (P<0.001), with a decrease rate of 22.57%. When the subjects used the test product essence for 28 consecutive days, the area of ​​VISIA-CR under-eye wrinkles decreased significantly compared to the baseline value (0.001≤P<0.01), with a decrease rate of 21.33%. When the subjects used the test product essence for 56 consecutive days, the area of ​​VISIA-CR under-eye wrinkles decreased significantly compared to the baseline value (P<0.001), with a decrease rate of 42.94%. The results show that the essence of Example 2 significantly reduced the area of ​​under-eye wrinkles and had a significant anti-wrinkle effect.

[0276] Measurement value: VISIA-CR Under-eye Wrinkle Area indicates the total area of ​​wrinkles in the under-eye area. The lower the value, the smaller the under-eye wrinkle area.

[0277] Table 13.VISIA-CR Under-eye Wrinkle Area (mm 2 )Test results

[0278]

[0279] Note: The data in the table are mean ± standard error.

[0280] Table 14. Improvement rate of under-eye wrinkle area of ​​VISIA-CR

[0281]

[0282] Example 3-8. VISIA-CR Under-eye Wrinkle Area Ratio

[0283] Table 15 shows the test results of the subject's VISIA-CR under-eye wrinkle area ratio, and Table 16 shows the improvement rate of the subject's VISIA-CR under-eye wrinkle area ratio. Figure 8 The bar graphs showing the percentage of wrinkle area under the eyes of the subjects in VISIA-CR are shown in Tables 15, 16 and Figure 8 As shown, when the subjects used the test product essence for 14 consecutive days, the VISIA-CR under-eye wrinkle area ratio decreased significantly compared to the baseline value (0.01≤P<0.05), with a decrease rate of 14.43%; when the subjects used the test product essence for 28 consecutive days, the VISIA-CR under-eye wrinkle area ratio decreased significantly compared to the baseline value (0.01≤P<0.05), with a decrease rate of 12.91%; and when the subjects used the test product essence for 56 consecutive days, the VISIA-CR under-eye wrinkle area ratio decreased significantly compared to the baseline value (0.001≤P<0.01), with a decrease rate of 28.10%. The results show that the essence of Example 2 significantly reduced the under-eye wrinkle area ratio, further demonstrating its anti-wrinkle effect.

[0284] Measurement value: The lower the VISIA-CR under-eye wrinkle area ratio value, the smaller the under-eye wrinkle area.

[0285] Table 15. VISIA-CR under-eye wrinkle area ratio test results

[0286]

[0287]

[0288] Note: The data in the table are mean ± standard error.

[0289] Table 16. VISIA-CR under-eye wrinkle area improvement rate

[0290]

[0291] Example 3-9. VISIA-CR Skin Color L* Value

[0292] Table 17 shows the test results of the VISIA-CR skin color L value of the subjects, and Table 18 shows the improvement rate of the VISIA-CR skin color L value of the subjects. Figure 9 The bar graphs showing the VISIA-CR skin color L value results for the subjects are shown in Tables 17, 18 and Figure 9 As shown, when the subjects used the test product essence for 14 consecutive days, the VISIA-CR skin color L* value increased significantly compared with the baseline value (0.001≤P<0.01), and the increase rate was 0.82%; when the subjects used the test product essence for 28 consecutive days, the VISIA-CR skin color L* value increased significantly compared with the baseline value (P<0.001), and the increase rate was 2.88%; when the subjects used the test product essence for 56 consecutive days, the VISIA-CR skin color L* value increased significantly compared with the baseline value (P<0.001), and the increase rate was 3.69%.

[0293] Measurement value: VISIA-CR Skin Color L* value. The higher the value, the lighter the skin color.

[0294] Table 17. VISIA-CR skin color L* value test results

[0295]

[0296] Note: The data in the table are mean ± standard error.

[0297] Table 18. Improvement rate of VISIA-CR skin color L* value

[0298]

[0299] Example 3-10. VISIA-CR Skin Color ITA° Value

[0300] Table 19 shows the test results of the VISIA-CR skin color ITA° values ​​of the subjects, and Table 20 shows the improvement rates of the VISIA-CR skin color ITA° values ​​of the subjects. Figure 10 The bar graphs showing the results of the VISIA-CR skin color ITA° values ​​of the subjects are shown in Tables 19, 20 and Figure 10 As shown, when the subjects used the test product essence for 14 consecutive days, the VISIA-CR skin color ITA° value increased significantly compared with the baseline value (0.01≤P<0.05), with an increase rate of 1.56%; when the subjects used the test product essence for 28 consecutive days, the VISIA-CR skin color ITA° value increased significantly compared with the baseline value (0.01≤P<0.05), with an increase rate of 1.33%; when the subjects used the test product essence for 56 consecutive days, the VISIA-CR skin color ITA° value increased significantly compared with the baseline value (P<0.001), with an increase rate of 5.48%. The results show that the essence of Example 2 significantly improved the glossiness and evenness of the skin.

[0301] Measurement: The higher the VISIA-CR Skin Color ITA° value, the lighter the skin color.

[0302] Table 19. VISIA-CR skin color ITA° value test results

[0303]

[0304] Note: The data in the table are mean ± standard error.

[0305] Table 20. Improvement rate of VISIA-CR skin color ITA° value

[0306]

[0307] Example 3-11. VISIA-CR Skin Glossiness Parameters

[0308] Table 21 shows the test results of the VISIA-CR skin glossiness parameters of the subjects, and Table 22 shows the improvement rate of the VISIA-CR skin glossiness parameters of the subjects. Figure 11 The bar graphs showing the results of the VISIA-CR skin glossiness parameters of the subjects are shown in Tables 21, 22 and Figure 11As shown, when the subjects used the test product essence for 14 consecutive days, the VISIA-CR skin glossiness parameter increased significantly compared with the baseline value (0.01≤P<0.05), with an increase rate of 12.69%; when the subjects used the test product essence for 28 consecutive days, the VISIA-CR skin glossiness parameter increased significantly compared with the baseline value (0.001≤P<0.01), with an increase rate of 20.90%; when the subjects used the test product essence for 56 consecutive days, the VISIA-CR skin glossiness parameter did not change significantly compared with the baseline value (P≥0.05). The results show that the essence of Example 2 can significantly improve the glossiness of the skin. Measurement value: VISIA-CR skin glossiness represents the reflected light on the skin surface. The higher the parameter value, the more lustrous the skin.

[0309] Table 21. VISIA-CR skin gloss parameter test results

[0310]

[0311] Note: The data in the table are mean ± standard error.

[0312] Table 22. Improvement rate of VISIA-CR skin glossiness parameters

[0313]

[0314] Example 3-12. VISIA-CR spot area ratio

[0315] Table 23 shows the results of the VISIA-CR spot area ratio test for the subjects, and Table 24 shows the improvement rate of the VISIA-CR spot area ratio for the subjects. Figure 12 The bar graphs showing the mean optical density results of the VISIA-CR spots of the subjects are shown in Tables 25, 26 and Figure 12 As shown, when the subjects used the test product essence for 14 consecutive days, the VISIA-CR spot area ratio decreased significantly compared with the baseline value (0.001≤P<0.01), and the decrease rate was 11.88%; when the subjects used the test product essence for 28 consecutive days, the VISIA-CR spot area ratio decreased significantly compared with the baseline value (P<0.001), and the decrease rate was 12.77%; when the subjects used the test product essence for 56 consecutive days, the VISIA-CR spot area ratio decreased significantly compared with the baseline value (0.001≤P<0.01), and the decrease rate was 15.39%.

[0316] Measurement value: The lower the VISIA-CR spot area ratio, the smaller the spot range.

[0317] Table 23. VISIA-CR spot area ratio test results

[0318]

[0319] Note: The data in the table are mean ± standard error.

[0320] Table 24. Improvement rate of VISIA-CR spot area

[0321]

[0322] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0323] Preparation Example 1: Essence Preparation Plan

[0324] 1.1 Ingredients and recommended ratios

[0325] Water (Aqua): 70-80%;

[0326] 1,3-Butylene Glycol: 4%;

[0327] Glycerin: 3%;

[0328] 1,3-Hexanediol: 1%;

[0329] Annexin A5: 0.15%;

[0330] Preservative (Phenoxyethanol): 0.5%-1%;

[0331] pH adjuster (such as citric acid): appropriate amount.

[0332] 1.2 Preparation steps

[0333] Prepare and heat the aqueous phase: In a clean container, combine water, 1,3-butylene glycol, glycerin, and 1,3-hexanediol. Heat to 75°C to 80°C, ensuring all ingredients are completely dissolved.

[0334] Add active ingredient: When the mixture cools to 40°C, add Annexin A5 and stir well.

[0335] Cool and adjust pH: Continue cooling to room temperature. Check and adjust pH to between 4.5 and 6.5, gradually adding citric acid to achieve the desired range.

[0336] Add preservatives: Add the preservative phenoxyethanol as needed and in the recommended amount to ensure the stability and safety of the product during storage.

[0337] Homogenization and filling: Use a high shear homogenizer to process the mixture to ensure that all ingredients are evenly mixed to form a uniform serum, and then fill the serum into sterile containers.

[0338] Preparation Example 2: Emulsion Preparation Scheme

[0339] 2.1 Ingredients and recommended ratios

[0340] Water (Aqua): 60-70%

[0341] 1,3-Butylene Glycol: 5%;

[0342] Glycerin: 4%;

[0343] 1,3-Hexanediol: 2%;

[0344] Annexin A5: 0.12%;

[0345] Oily ingredients (such as jojoba oil): 5%;

[0346] Emulsifier (such as Cetearyl Alcohol): 2%;

[0347] Preservatives (such as phenoxyethanol): 0.5%-1%;

[0348] pH adjuster (such as citric acid): appropriate amount.

[0349] 2.2 Preparation steps

[0350] Prepare and heat the aqueous phase: In a clean container, combine water, 1,3-butylene glycol, glycerin, and 1,3-hexanediol. Heat to 75°C to 80°C, ensuring all ingredients are completely dissolved.

[0351] Heat the oily ingredients: Combine jojoba oil and cetearyl alcohol and heat to 75°C to 80°C, ensuring complete dissolution and uniform mixing.

[0352] Emulsification process: Slowly add the oil phase to the water phase under continuous stirring, using a homogenizer to ensure good emulsification.

[0353] Cool and add active ingredient: Cool the mixture to about 40°C, add annexin A5 and stir well.

[0354] Adjust pH: Test and adjust pH to between 4.5 and 6.5, gradually adding citric acid to reach the desired range.

[0355] Added preservative: Add preservative phenoxyethanol as needed and in the recommended amounts.

[0356] Homogenization and filling: The mixture is processed using a high shear homogenizer and the emulsion is filled into sterile containers.

[0357] Preparation Example 3: Cream Preparation Scheme

[0358] 3.1 Ingredients and recommended ratios

[0359] Water (Aqua): 50-60%;

[0360] 1,3-Butylene Glycol: 4%;

[0361] Glycerin: 5%;

[0362] 1,3-Hexanediol: 2%;

[0363] Annexin A5: 0.08%;

[0364] Oily ingredients (such as shea butter): 10%;

[0365] Emulsifier (such as emulsifying wax): 4%;

[0366] Thickener (such as xanthan gum): 0.5%;

[0367] Preservatives (such as phenoxyethanol): 0.5%-1%;

[0368] pH adjuster (such as citric acid): appropriate amount.

[0369] 3.2 Preparation steps

[0370] Prepare and heat the aqueous phase: In a clean container, combine water, 1,3-butylene glycol, glycerin, and 1,3-hexanediol. Heat to 75°C to 80°C, ensuring all ingredients are completely dissolved.

[0371] Heat the oily ingredients: Combine the shea butter and emulsifying wax and heat to 75°C to 80°C, ensuring they are completely dissolved and evenly mixed.

[0372] Emulsification process: Slowly add the oil phase to the water phase under continuous stirring, using a homogenizer to ensure good emulsification.

[0373] Cool and add active ingredient: Cool the mixture to about 40°C, add annexin A5 and stir well.

[0374] Adjust pH: Test and adjust pH to between 4.5 and 6.5, gradually adding citric acid to reach the desired range.

[0375] Added preservative: Add preservative phenoxyethanol as needed and in the recommended amounts.

[0376] Add thickener: Add xanthan gum and stir well to ensure it is completely dissolved.

[0377] Homogenization and filling: The mixture is processed using a high shear homogenizer and the cream is filled into sterile containers.

Claims

1. A use of annexin in preparing a personal care composition, characterized in that: The personal care composition has one or more of the following effects: (a) Moisturizing; (b) repair; (c) firmness; (d) anti-wrinkle; The annexin is human annexin A5, and the amino acid sequence of the human annexin A5 is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The personal care compositions comprise one or more adjunct ingredients.

3. The use according to claim 2, characterized in that The auxiliary ingredients include one or more auxiliary materials.

4. The use according to claim 2 or 3, characterized in that The content of the annexin in the personal care composition is 0.01% to 1% by weight or by volume.

5. The use according to claim 4, characterized in that The content of the annexin in the personal care composition is 0.01% to 0.2% by weight or by volume.

6. The use according to claim 4, characterized in that The content of the annexin in the personal care composition is 0.05% to 0.15% by weight or by volume.

7. The use according to claim 4, characterized in that The content of the annexin in the personal care composition is 0.08% to 0.12% by weight or by volume.

8. The use according to claim 4, characterized in that The content of the annexin in the personal care composition is 0.1% by weight or by volume.

9. The use according to claim 4, characterized in that The concentration of the membrane-binding protein is 0.1 mg / mL to 1 mg / mL.

10. The use according to claim 4, characterized in that The concentration of the membrane-binding protein is 0.5 mg / mL to 1 mg / mL.

11. The use according to claim 4, characterized in that The concentration of the membrane-binding protein is 0.8 mg / mL to 1 mg / mL.

12. The use according to claim 2, characterized in that The auxiliary ingredients are selected from one or more of preservatives, stabilizers, antioxidants, thickeners, emulsifiers, ultraviolet absorbers, penetration enhancers, plant extracts, moisturizers or emollients.

13. The use according to claim 12, characterized in that The preservative is selected from one or more of phenoxyethanol, methylisothiazolinone or ethylhexylglycerin.

14. The use according to claim 12, characterized in that The antioxidant is selected from one or more of vitamin E, vitamin C or resveratrol.

15. The use according to claim 12, characterized in that The thickener is selected from one or more of xanthan gum, carbomer, guar gum or sodium alginate.

16. The use according to claim 12, characterized in that The emulsifier is selected from one or more of cetearyl alcohol, emulsifying wax, polysorbate or glyceryl stearate.

17. The use according to claim 12, wherein: The ultraviolet absorber is selected from one or more of titanium dioxide, zinc oxide, avobenzone or octocrylene.

18. The use according to claim 12, wherein: The penetration enhancer is selected from one or more of propylene glycol, ethanol, ethylene glycol or benzyl alcohol.

19. The use according to claim 12, wherein: The plant extract is selected from one or more of green tea extract, aloe extract, chamomile extract or calendula extract.

20. The use according to claim 12, wherein: The moisturizing agent is selected from one or more of hyaluronic acid, glycerin, propylene glycol, butylene glycol, hexylene glycol or urea.

21. The use according to claim 12, wherein: The emollient is selected from one or more of mineral oil, jojoba oil, coconut oil or squalane.

22. The use according to claim 3, characterized in that The auxiliary material is selected from one or more of a conditioner, a diluent, a filler, a binder, a wetting agent, a surfactant, a lubricant, a fragrance or a flavoring agent.

23. The use according to claim 22, characterized in that The conditioning agent is selected from one or more of panthenol, glycerin or lactic acid.

24. The use according to claim 22, characterized in that The diluent is selected from one or more of water, ethanol or propylene glycol.

25. The use according to claim 22, characterized in that The filler is selected from one or more of talc, silicon dioxide or microcrystalline cellulose.

26. The use according to claim 22, characterized in that The binder is selected from one or more of polyethylene glycol, hydroxypropyl methylcellulose or carbomer.

27. The use according to claim 22, characterized in that The wetting agent is selected from one or more of propylene glycol, glycerol or urea.

28. The use according to claim 22, characterized in that The surfactant is selected from one or more of polysorbate, sodium lauryl sulfate or cocamidopropyl betaine.

29. The use according to claim 22, characterized in that The lubricant is selected from one or more of silicone oil, jojoba oil or mineral oil.

30. The use according to claim 22, wherein The fragrance is selected from one or more of natural essential oils, synthetic essences or plant extracts.

31. The use according to claim 22, wherein The flavoring agent is selected from one or more of licorice extract, mint extract or vanilla extract.

32. The use according to claim 20, wherein The weight ratio of the annexin to the moisturizing agent is selected from 1:10 to 1:

200.

33. The use according to claim 20 or 32, characterized in that The weight ratio of the annexin to the moisturizing agent is selected from 1:20 to 1:

150.

34. The use according to claim 20 or 32, characterized in that The weight ratio of the annexin to the moisturizing agent is selected from 1:50 to 1:

100.

35. The use according to claim 16, wherein The weight ratio of the annexin to the emulsifier is selected from 1:1 to 1:

20.

36. The use according to claim 16 or 35, characterized in that The weight ratio of the annexin to the emulsifier is selected from 1:2 to 1:

15.

37. The use according to claim 16 or 35, characterized in that The weight ratio of the annexin to the emulsifier is selected from 1:5 to 1:

10.

38. The use according to claim 15, wherein The weight ratio of the annexin to the thickener is selected from 1:1 to 1:

50.

39. The use according to claim 15 or 38, characterized in that The weight ratio of the annexin to the thickener is selected from 1:5 to 1:

30.

40. The use according to claim 15 or 38, wherein The weight ratio of the annexin to the thickener is selected from 1:10 to 1:

20.

41. The use according to claim 13, wherein The weight ratio of the annexin to the preservative is selected from 1:2 to 1:

10.

42. The use according to claim 13 or 41, characterized in that The weight ratio of the annexin to the preservative is selected from 1:2 to 1:

5.

43. The use according to claim 13 or 41, characterized in that The weight ratio of the annexin to the preservative is selected from 1:3 to 1:

8.

44. The use according to claim 12, wherein The moisturizing agent is selected from one or more of glycerol, 1,3-butylene glycol or 1,3-hexanediol.

45. The use according to claim 44, characterized in that The weight volume percentage of the glycerol is 2-5%.

46. ​​The use according to claim 44 or 45, characterized in that The weight volume percentage of the glycerol is 3%.

47. The use according to claim 44, wherein The weight volume percentage of the 1,3-butanediol is 2-5%.

48. The use according to claim 44 or 47, wherein The weight volume percentage of the 1,3-butanediol is 4%.

49. The use according to claim 44, wherein The weight volume percentage of the 1,3-hexanediol is 1-2%.

50. The use according to claim 44 or 49, characterized in that The weight volume percentage of the 1,3-hexanediol is 1%.

51. The use according to claim 22 or 24, characterized in that The diluent is water.

52. The use according to claim 51, wherein The weight volume percentage of the water is 70-80%.

53. The use according to claim 1, wherein The personal care composition is selected from one or more of a skin care composition and a hair care composition.

54. The use according to claim 1, wherein The dosage form of the personal care composition is selected from one of water, gel, emulsion, cream, suspension, film, spray or powder.

55. The use according to claim 54, wherein The dosage form of the personal care composition is selected from one of essence, emulsion, cream or wax-based dosage form products.

56. The use according to claim 55, wherein The essence, lotion and cream are selected from O / W dosage form or W / O dosage form.

57. The use according to claim 54, wherein The dosage form of the personal care composition is a serum.

58. A method for preparing a personal care composition having moisturizing, repairing, firming and anti-wrinkle effects, characterized in that: The following steps are involved: heating the aqueous phase ingredients; Add active ingredients; Mixing and filling; The active ingredient is human annexin A5, and the amino acid sequence of the human annexin A5 is shown in SEQ ID No.

1.

59. The method of claim 58, wherein The method may further comprise the steps of cooling, adjusting pH, and / or adding a preservative.

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