An e-jiao powder anti-aging composition, a preparation method thereof and application thereof

Abstract

This invention relates to an anti-aging composition of donkey-hide gelatin powder, its preparation method, and its application. The anti-aging composition of donkey-hide gelatin powder comprises, by mass percentage: 1-4% donkey-hide gelatin powder, 0.1-1% antioxidant and anti-inflammatory short peptides, 0.01-0.5% rare ginsenoside CK, 500-5000 ppm expression line relaxing short peptides, 1-3% solubilizer, and the balance water. By specifically setting the component content and the preparation method, a multi-level, multi-target three-dimensional anti-aging network is constructed, covering multiple key aging pathways such as nerve conduction, dermal matrix, cell metabolism, and moisturizing barrier. Furthermore, donkey oil is added to the anti-wrinkle cream used in its application, highlighting the role of donkey-hide gelatin in anti-aging in conjunction with the donkey-hide gelatin powder. The cream, serum, lotion, and lip balm formulas provided by this invention are stable, biocompatible, and possess good anti-aging and antioxidant effects, providing a safe and effective solution for addressing skin aging problems.

Description

Technical Field

[0001] This invention relates to the field of cosmetics, and more particularly to an anti-aging composition of donkey-hide gelatin powder, its preparation method, and its application. Background Technology

[0002] As the largest organ in the human body, the skin serves as a physical barrier against external environmental stimuli. The main factors inducing skin aging include aging itself and external stimuli. Aging leads to a decline in cell proliferation and significant changes in the composition of the dermis. Specifically, this manifests as a significant reduction in the content of key components such as collagen, elastin, and glycosaminoglycans in the extracellular matrix (ECM), resulting in thinning skin, fine lines, and other signs of aging. Simultaneously, with age, various endogenous factors such as mitochondrial damage and inflammatory responses lead to a large accumulation of reactive oxygen species. Furthermore, the decline in age-related cell repair capacity further exacerbates oxidative stress, ultimately accelerating the skin aging process.

[0003] In addition to wrinkles caused by natural skin aging, the accumulation of repetitive facial expressions over a long period can also lead to dynamic wrinkles (also known as expression lines). These wrinkles initially appear only when the face makes specific facial expressions. As we age or collagen continues to be lost, they can gradually transform into static wrinkles, commonly found in areas such as the corners of the eyes (crow's feet), forehead (forehead wrinkles), between the eyebrows (frown lines), and around the nasolabial folds (smile lines). The formation mechanism of expression lines is mainly related to a variety of factors, including frequent muscle contractions caused by excessive release of neurotransmitters, fixed daily facial expression habits, and physical pressure (such as improper sleeping posture). The core factor is skin aging.

[0004] Currently, most anti-aging products on the market use retinol, pro-xylane, and peptides as core active ingredients. Their mechanism of action focuses on accelerating the synthesis of collagen and hyaluronic acid to improve skin firmness and radiance, reduce fine lines, and simultaneously increase skin moisture content and enhance skin elasticity. However, existing anti-aging products generally suffer from the following technical deficiencies: First, they neglect the balanced regulation of "promoting collagen synthesis and inhibiting extracellular matrix degradation" and fail to effectively eliminate the damage caused by oxidative stress to the skin; second, they do not provide targeted management for expression lines caused by facial muscle movements; and third, they do not fully emphasize the synergistic anti-aging logic of combining dermal anti-aging with epidermal care, resulting in limited anti-aging effects.

[0005] Colla corii asini, also known as donkey-hide gelatin, is hailed as a "holy medicine for replenishing blood" and listed as a superior-grade herb in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). Its main components include collagen, short peptides, amino acids, and polysaccharides such as chondroitin sulfate. Current research has confirmed that donkey-hide gelatin possesses various pharmacological and health-promoting effects, including treating anemia, immune regulation, anti-oxidation, anti-fatigue, anti-tumor, and anti-aging. Notably, donkey-hide gelatin powder was approved as a new cosmetic ingredient in 2023. Further research has shown that donkey-hide gelatin powder exhibits excellent performance in promoting skin repair, anti-aging, anti-photoaging, and inhibiting ferroptosis, highlighting its enormous application potential in skin repair and anti-aging (anti-wrinkle) cosmetics. Researchers are gradually conducting studies on the application of donkey-hide gelatin in cosmetics.

[0006] Chinese invention patent publication CN119157813A discloses a method for preparing donkey-hide gelatin (Ejiao) for beauty and reducing age spots, achieving its beauty-enhancing effects through the synergistic effect of donkey skin, olive oil, and angelica. Chinese invention patent publication CN120898969A discloses the application of a combination of fish maw and Ejiao in the preparation of anti-aging products, achieving synergistic anti-aging through a triple mechanism. However, neither of these technical solutions fully utilizes the synergistic effect of Ejiao with other compounding substances.

[0007] Therefore, there is an urgent need to develop an anti-aging composition that can cover multiple key aging pathways, such as nerve conduction, dermal matrix, cell metabolism, and moisturizing barrier. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-aging composition of donkey-hide gelatin powder, its preparation method, and its applications (including anti-wrinkle cream and its preparation, as well as other applications). By comparing donkey-hide gelatin powder with representative anti-aging ingredients, the advantages of donkey-hide gelatin powder in anti-aging are explored. It is then combined with other anti-aging active ingredients, combining active anti-aging with passive defense, and dermal anti-aging with expression line management. This covers multiple key aging pathways such as nerve conduction, dermal matrix, cell metabolism, and moisturizing barrier. Based on donkey-hide gelatin powder, a comprehensive and advanced anti-aging ingredient combination is developed. This combination takes into account both dermal anti-aging and expression line management, active anti-aging and passive defense, addressing both the symptoms and the root cause, in order to simultaneously achieve the anti-aging goals of "rapid results" (smoothing wrinkles) and "long-term improvement" (firmness, plumpness, and elasticity).

[0009] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides an anti-aging composition of donkey-hide gelatin powder, wherein the raw materials of the anti-aging composition of donkey-hide gelatin powder include, by mass percentage: 1-4 wt.% donkey-hide gelatin powder, 0.1-1 wt.% antioxidant and anti-inflammatory short peptides, 0.01-0.5 wt.% rare ginsenoside CK, 500-5000 ppm expression line relaxing short peptides, 1-3 wt.% solubilizers, and the balance being water.

[0010] Preferably, the antioxidant and anti-inflammatory short peptide is selected from one or more of copper peptide, carnosine, or glutathione.

[0011] Preferably, the expression line relaxing short peptide is selected from one or more of acetyl hexapeptide-8, conopeptide, or snake venom peptide.

[0012] Further preferred, the antioxidant and anti-inflammatory short peptide is selected as copper peptide, and the expression line relaxing short peptide is selected as acetyl hexapeptide-8.

[0013] Preferably, the donkey-hide gelatin powder is pulverized into ultrafine powder with a particle size range of 100-800 mesh to facilitate dissolution and use.

[0014] Through comparative analysis of raw materials using cell experiments and 3D skin models, we found that donkey-hide gelatin powder exhibits good biocompatibility, promoting cell proliferation and migration, enhancing collagen and elastin expression, and demonstrating excellent overall performance with antioxidant and anti-inflammatory effects. However, at lower concentrations, copper peptide showed slightly better antioxidant effects than donkey-hide gelatin powder, while acetyl hexapeptide-8 was slightly superior in promoting collagen and elastin secretion and in water retention. Furthermore, acetyl hexapeptide-8 also showed efficacy in controlling expression lines. Therefore, it is essential to further enhance the anti-aging effects of donkey-hide gelatin powder compositions through ingredient combinations.

[0015] The efficacy of each component in the preferred combination is listed below: Donkey-hide gelatin powder: Donkey-hide gelatin is a traditional Chinese medicine made from donkey skin. Its main components are collagen and its hydrolyzed forms, including various amino acids and peptides. Its function is to "replenish raw materials and promote production," repairing and anti-aging from the dermis layer. It provides skin nutrition, promotes fibroblast activity, and has antioxidant effects.

[0016] Copper peptides: As a "wound healing" signal, copper peptides are naturally present in the human body, especially at the wound healing site. As a powerful signaling molecule, it can instruct skin cells (fibroblasts) to accelerate the synthesis of collagen, elastin, and glycosaminoglycans (such as hyaluronic acid), and has strong antioxidant and anti-inflammatory properties, inhibiting the degradation of the extracellular matrix.

[0017] Rare ginsenoside CK: activates autophagy, regulates the skin's biological clock, has anti-inflammatory and antioxidant properties, and promotes collagen synthesis.

[0018] Acetyl hexapeptide-8: Acetyl hexapeptide-8 is a classic, widely validated neurotransmitter blocking peptide, commonly known as "botox-like." It targets expression lines by competitively inhibiting the release of acetylcholine, thus weakening the signals generated by the contraction of facial expression muscles. It diminishes dynamic wrinkles and prevents static wrinkles. Its effects are precise and gentle. Unlike botox injections, it only "inhibits" rather than "blocks," making its effects gentler, reversible, and without toxic risks.

[0019] These four ingredients work together to create a synergistic effect, constructing a multi-layered, multi-target, three-dimensional anti-aging network. This ingredient combination comprehensively considers the combination of active repair and passive defense in the dermis, the combination of signaling pathways and cell metabolism, and the combination of dermal reconstruction and epidermal soothing. Moreover, the concentration ratio is scientifically formulated, enabling it to simultaneously achieve the anti-aging goals of "rapid results" (smoothing wrinkles) and "long-term improvement" (firmness, plumpness, and elasticity).

[0020] Further preferably, the donkey-hide gelatin powder anti-aging composition comprises the following components in weight percentages: 3 wt.% donkey-hide gelatin powder; 0.8 wt.% copper peptide; 0.2 wt.% rare ginsenoside CK; 5000 ppm acetyl hexapeptide-8; 2 wt.% solubilizer (using toxoxelam); and the balance being water. 3D skin model experiments revealed that when the components in the composition are used in the above-mentioned optimal amounts, the resulting composition exhibits the best anti-aging and moisturizing effects; however, when the component amounts exceed the limits defined in this invention, the anti-aging and moisturizing effects of the resulting composition decrease, and the cost-effectiveness is significantly reduced.

[0021] Preferably, the solubilizer is tococelen, namely vitamin E polyethylene glycol succinate, which can serve as the wall material of nanomicelles to encapsulate the poorly water-soluble rare ginsenoside CK, allowing it to be uniformly dispersed in the aqueous phase, and it also has the good antioxidant capacity of vitamin E.

[0022] Further preferably, the molecular weight of polyethylene glycol in the vitamin E polyethylene glycol succinate is controlled between 700 and 2000 Da, so that it can form stable nanomicelles without dissolving in water.

[0023] Further optimization was performed, with the mass ratio of tocosolvan to rare ginsenoside CK controlled at 2:1 to 300:1.

[0024] On the other hand, the present invention provides a method for preparing the aforementioned anti-aging composition of donkey-hide gelatin powder, comprising the following steps: (1) Prepare the ingredients according to the above-mentioned anti-aging composition of donkey-hide gelatin powder. Take the total amount of donkey-hide gelatin powder and 20-40 wt.% of water. Then slowly sprinkle the donkey-hide gelatin powder into the water. Heat the mixture to 55-65°C and keep it at this temperature while stirring for 0.5-2 hours. If a clear solution is obtained after stirring, proceed to the next step. If there are undissolved particles, pass the undissolved particles through an 80-120 mesh sieve and return the sieve material to the mixture and continue stirring until a clear solution is obtained, which is recorded as solution A.

[0025] (2) Take the full amount of antioxidant and anti-inflammatory short peptide (preferably blue copper peptide) and mix it with the remaining water, and then stir until transparent; then add a pH adjuster (preferably citric acid) to the solution to adjust the pH of the solution to 6.8~7.0 to obtain solution B.

[0026] (3) Mix solution A and solution B evenly to form the main system solution.

[0027] (4) Mix the total amount of rare ginsenoside CK with the total amount of solubilizer (such as 1,3-propanediol, tococelen) and heat in a water bath at 35~45℃ for 8~13 minutes until the mixture is homogeneous. Then slowly add the resulting mixture to the main system obtained in step (3) and stir.

[0028] (5) After stirring until the solution temperature drops to below 33~36℃ (more preferably below 35℃), add the full amount of expression line relaxing short peptide (such as acetyl hexapeptide-8), and stir at low speed for 8~13 minutes (more preferably 10 minutes) to avoid foaming, and obtain the donkey-hide gelatin powder anti-aging composition product.

[0029] Preferably, the water in step (1) is purified water.

[0030] Preferably, the donkey-hide gelatin powder in step (1) is an ultrafine powder that has been pulverized, with a particle size range of 100~800 mesh.

[0031] Preferably, the pH adjuster in step (2) is citric acid.

[0032] Preferably, the low-speed stirring in step (5) is at a speed of 100~10000 rpm.

[0033] In another aspect, the present invention provides an anti-wrinkle cream containing the aforementioned anti-aging composition of donkey-hide gelatin powder.

[0034] Preferably, the mass percentage of the donkey-hide gelatin powder anti-aging composition in the donkey-hide gelatin anti-wrinkle cream is 5-75%.

[0035] Further preferably, the anti-wrinkle cream containing donkey-hide gelatin powder also includes the following components in weight percentage: donkey oil: 1~10 wt.%, tocopheryl acetate: 0.2~2.0 wt.%, glyceryl stearate and PEG-100 stearate compound: 0.2~1.8 wt.%, glycerol: 1~4 wt.%, 1,3-propanediol: 2~6 wt.%, 1,2-hexanediol: 0.2~0.9 wt.%, p-hydroxyacetophenone: 0.3~0.8 wt.%, γ-polyglutamic acid: 0.1~2.5 wt.%, and the balance being water.

[0036] Preferably, the glycerol stearate and PEG-100 stearate compound is a compound in which glycerol stearate and PEG-100 stearate are compounded in a mass ratio of 1:1 to 1:2.

[0037] This invention also provides a method for preparing the aforementioned donkey-hide gelatin anti-wrinkle cream, comprising the following steps: I. Add the total amount of glycerol, 1,3-propanediol, 1,2-hexanediol, p-hydroxyacetophenone, γ-polyglutamic acid and water to an emulsifying pot, heat to 75~85℃, stir and mix evenly to obtain the aqueous phase.

[0038] II. Place the total amount of donkey oil, tocopheryl acetate, glyceryl stearate and PEG-100 stearate compound in an oil phase pot and heat to 75~85℃. Stir and mix evenly to obtain the oil phase.

[0039] III. Mix the oil phase obtained in step II and the aqueous phase obtained in step I, and stir homogenously to obtain a mixture.

[0040] IV. Cool the mixture from step III to 65-73°C (preferably 70°C), stir until homogeneous, then add the anti-aging composition of donkey-hide gelatin powder as described in claim 1 or 2, or the anti-aging composition of donkey-hide gelatin powder prepared by the method of claim 3 or 4, stir and mix evenly, and cool to 35-45°C (preferably 40°C) before discharging to obtain the donkey-hide gelatin anti-wrinkle cream product.

[0041] Finally, the donkey-hide gelatin powder anti-aging composition and donkey-hide gelatin powder anti-wrinkle cream described in this invention can be applied to anti-wrinkle treatment around the eyes, cheeks, and neck. The donkey-hide gelatin powder anti-aging composition can also be used as a core ingredient combination for preparing anti-wrinkle essences, anti-wrinkle lotions, lip balms, etc.

[0042] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects: 1) This invention achieves a combination of dermal reconstruction and epidermal soothing by specifically combining donkey-hide gelatin powder with antioxidant and anti-inflammatory short peptides, rare ginsenoside CK, expression line relaxing short peptides, and solubilizers. The antioxidant and anti-inflammatory short peptides (preferably blue copper peptides) + donkey-hide gelatin powder + ginsenoside CK form a "dermal repair iron triangle," acting on the dermis from different angles (signal transduction, raw material supply, and autophagy), strongly stimulating collagen regeneration, repairing the elastic network, and replenishing the matrix, fundamentally solving the problems of skin laxity, depressions, and wrinkles. The expression line management short peptides (preferably acetyl hexapeptide-8) are mainly responsible for "epidermal management," precisely targeting expression lines and minimizing the physical folding damage to the skin caused by muscle movement. In the overall formulation system of this invention, the raw materials do not act in isolation, but rather through multi-level and multi-pathway synergy and complementarity, jointly constructing a complete skin repair network covering signal initiation, raw material supply, cell renewal, and muscle regulation. Its synergistic effect is mainly reflected in the following three aspects: 1. The cascade synergy of the dermal repair "iron triangle": Blue copper peptide, as a signaling molecule, activates the collagen synthesis pathway and provides "production instructions"; donkey-hide gelatin powder is rich in collagen-characteristic amino acids and small molecule peptides, directly providing raw materials for synthesis. The two form a highly efficient coupling of "signal instructions + direct supply of raw materials", significantly improving the actual collagen output. Ginsenoside CK, by activating cell autophagy, clears aging organelles and regulates the collagen degradation balance, can enhance signal transduction efficiency in synergy with blue copper peptide, and in synergy with donkey-hide gelatin, it constructs a dual raw material guarantee of "endogenous + exogenous", jointly improving the sustainability of cell function and matrix remodeling. In addition, the anti-inflammatory and antioxidant properties of the three together construct a low-oxidation-low-micro-inflammation microenvironment, ensuring that the repair process is not disturbed and prolonging the stable period of the new matrix. 2. Dermal-epidermal bidirectional linkage: The dermal "iron triangle" provides solid support from the bottom layer by increasing collagen density and elastic network, improving static wrinkles and sagging; short peptides for relaxing expression lines reduce the formation of dynamic wrinkles by inhibiting neuromuscular contraction. Both work synergistically from two dimensions: "deep support" and "surface wrinkle reduction," achieving simultaneous improvement of both static and dynamic wrinkles. Simultaneously, dermal health provides better nutrition and signal support to the epidermis, while epidermal integrity contributes to a stable dermal microenvironment. Combined with solubilizers to promote the penetration of active ingredients, this achieves a cross-layer synergistic effect. Thirdly, complementary temporal and spatial effects: Temporally, acetyl hexapeptide-8 can quickly improve expression lines, providing immediate results; collagen remodeling in the dermal "iron triangle" requires a longer time to achieve fundamental structural repair, combining the two fast and slow approaches. Spatially, the formulation system focuses on extracellular matrix regeneration in the dermis and neuromuscular regulation in the epidermis, achieving precise regional action and avoiding functional overlap. This invention, through the above-mentioned specific formulation, constructs a three-dimensional, self-reinforcing skin repair system through complementary raw materials, cross-pathway interaction, hierarchical linkage, and temporal coordination.Each ingredient performs its specialty on its corresponding target and empowers each other through microenvironment regulation and metabolic support, ultimately achieving the dual goals of "repairing dermal structure + managing epidermal wrinkles", presenting a synergistic amplification effect of "1+1+1>3" in terms of efficacy.

[0043] 2) By specifically defining the preparation method and selecting parameters, a combination of active repair and passive defense of the dermis is achieved. By rationally distributing and mixing antioxidant and anti-inflammatory short peptides, ginsenoside CK, and donkey-hide gelatin powder, the "active repair" effect of these three components is achieved. They actively command or help skin cells repair damage and regenerate youthful substances. At the same time, the moisturizing and antioxidant and anti-inflammatory short peptides (preferably blue copper peptide) of donkey-hide gelatin powder and the antioxidant and anti-inflammatory properties of ginsenosides constitute a "passive defense system" to help the skin resist daily environmental damage. That is, the technical effect of combining active repair and passive defense is achieved through stepwise addition, thus achieving the effect of the composition from a comprehensive perspective.

[0044] 3) By adding appropriate amounts of antioxidant and anti-inflammatory short peptides and ginsenoside CK to the base of donkey-hide gelatin powder, a technical effect combining signaling pathways and cell metabolism is achieved. The antioxidant and anti-inflammatory short peptides (preferably copper peptides) focus on external signal transduction ("fast, start repair"); ginsenoside CK focuses on internal cell metabolic regulation ("clean up waste, restore vitality"). The mechanisms of action of the two are complementary, jointly activating the anti-aging program from inside and outside the cell, achieving a synergistic effect of 1+1>2.

[0045] 4) By carefully setting the concentration ratios of each component in the donkey-hide gelatin powder anti-aging composition, donkey-hide gelatin powder anti-wrinkle cream, and preparation method, both the effective concentration of each component and the prevention of toxicity due to excessive concentration are considered. Using the above technical solution, the donkey-hide gelatin powder anti-aging composition can be directly added to anti-aging skincare products, enabling the products to have anti-aging and anti-wrinkle effects. It is simple and convenient to use, has a wide range of applications, and thus helps improve the economic benefits of the donkey-hide gelatin powder composition, making it more suitable for market promotion. Attached Figure Description

[0046] Figure 1 Cell viability of HACAT cells after culturing for 24 hours with different concentrations of donkey-hide gelatin powder, acetyl hexapeptide-8, 50% retinol, copper peptide, and 95% psoralen.

[0047] Figure 2 Cell death / liveness staining images of HACAT cells cultured for 1 day, 3 days, and 5 days with 0.625 mg / mL copper peptide, 2.5 mg / mL acetyl hexapeptide-8, 0.01 mg / mL retinol, 0.0048 mg / mL psoralen, and 2.5 mg / mL donkey-hide gelatin powder, respectively.

[0048] Figure 3The cell viability of HACAT cells after culturing with 0.625 mg / mL copper peptide, 2.5 mg / mL acetyl hexapeptide-8, 0.01 mg / mL retinol, 0.0048 mg / mL psoralen, and 2.5 mg / mL donkey-hide gelatin powder for 1, 3, and 5 days was determined.

[0049] Figure 4 Microscopic images and live / dead staining images of HACAT cells cultured for 0, 24, and 48 hours after inducing scratch assays with 0.625 mg / mL copper peptide, 2.5 mg / mL acetyl hexapeptide-8, 0.01 mg / mL retinol, 0.0048 mg / mL psoralen, and 2.5 mg / mL donkey-hide gelatin powder.

[0050] Figure 5 For the reason Figure 4 The calculated changes in cell migration rate.

[0051] Figure 6 The results of skin hematoxylin-eosin (HE) staining after 3 days of UVA+UVB irradiation on a full-thickness 3D skin model treated with 2.5 mg / mL acetyl hexapeptide-8, 0.1 mg / mL retinol, 1 wt.% donkey-hide gelatin powder, 2 wt.% donkey-hide gelatin powder, 3 wt.% donkey-hide gelatin powder and 0.048 mg / mL psoralen.

[0052] Figure 7 for Figure 6 The pixel count results of the hematoxylin-eosin (HE) staining image.

[0053] Figure 8 Masson staining results of a full-thickness 3D skin model treated with 2.5 mg / mL acetyl hexapeptide-8, 0.1 mg / mL retinol, 1 wt.% donkey-hide gelatin powder, 2 wt.% donkey-hide gelatin powder, 3 wt.% donkey-hide gelatin powder and 0.048 mg / mL psoralen for 3 days after simultaneous irradiation with long-wave ultraviolet (UVA) and medium-wave ultraviolet (UVB).

[0054] Figure 9 for Figure 8 The average optical density (AOD) result is obtained from the Masson staining results.

[0055] Figure 10 The image shows the results of Van Geseman staining (EVG) of the skin elastic fibers after 3 days of simultaneous UVA and UVB irradiation on a full-thickness 3D skin model treated with 2.5 mg / mL acetyl hexapeptide-8, 0.1 mg / mL retinol, 1 wt.% donkey-hide gelatin powder, 2 wt.% donkey-hide gelatin powder, 3 wt.% donkey-hide gelatin powder and 0.048 mg / mL psoralen.

[0056] Figure 11 for Figure 10 The average optical density (AOD) results obtained from the EVG staining images are shown in the figure.

[0057] Figure 12 A comparison chart showing the transdermal moisture loss rate of 3D full-skin models after treatment with different raw materials.

[0058] Figure 13 This is a graph showing the changes in reactive oxygen species (ROS) content in a 3D full-skin model after treatment with different raw materials.

[0059] Figure 14 HE staining images of 3D whole skin models after treatment of the blank group (control), UVB group (UV), and anti-aging group (the anti-aging composition of donkey-hide gelatin powder obtained in Example 1).

[0060] Figure 15 Masson staining images of 3D full-skin models after treatment of the control group, UVB group (UV), and anti-aging group (the anti-aging composition of donkey-hide gelatin powder obtained in Example 1).

[0061] Figure 16 EVG staining images of 3D whole skin models after treatment of the blank group (control), UVB group (UV), and anti-aging group (the anti-aging composition of donkey-hide gelatin powder obtained in Example 1).

[0062] Figure 17 The graph shows the changes in ROS content in the 3D whole skin model after treatment of the blank group (control), UVB group (UV), and anti-aging group (the anti-aging composition of donkey-hide gelatin powder obtained in Example 1).

[0063] Figure 18 Transdermal moisture loss rate after treatment of the control group, UVB group (UV), and anti-aging group (the anti-aging composition of donkey-hide gelatin powder obtained in Example 1) in a 3D whole skin model.

[0064] Figure 19 Photos of the gelatin powder repair cream prepared in Examples 5-7 after undergoing a heating stability test. Detailed Implementation

[0065] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The specific implementation methods, structures, features, and effects of the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments. Those skilled in the art should understand that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0066] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0067] In the following content, all numbers disclosed herein, whether or not they use the words "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0068] Unless otherwise specified, all raw materials used in this invention are commercially available. The donkey-hide gelatin powder described in this invention is a product of Dong-E-E-Jiao Health Industry Technology Co., Ltd.

[0069] This embodiment illustrates a method for preparing an anti-aging composition using donkey-hide gelatin powder. The specific steps are as follows: (1) Treatment of donkey-hide gelatin: Weigh the donkey-hide gelatin powder and slowly sprinkle it into 60℃ purified water (accounting for 30% of the total water volume), and stir until it is completely swollen (about 1 hour). If any undissolved particles are found, they can be sieved through a 100-mesh sieve and then re-dissolved.

[0070] (2) Dissolve the antioxidant and anti-inflammatory short peptide (blue copper peptide in this example) in the remaining purified water, stir until transparent, and adjust the pH to 6.9 with pH adjuster (citric acid in this example).

[0071] (3) The two are mixed to form the main system.

[0072] (4) Premix rare ginsenoside CK with solubilizer (tocoxexyl in this example), heat in a water bath at 35-45°C for 10 minutes until homogeneous, and slowly add to the main system while stirring; (5) When the system temperature drops below 35°C, add a short peptide that is sensitive to facial wrinkles (acetyl hexapeptide-8 in this example), and stir at low speed for 10 minutes to avoid foaming.

[0073] After the above four steps, the anti-aging composition of donkey-hide gelatin powder is obtained.

[0074] Examples 2, 3, and 4 are the same as Example 1 in other settings, except for the selection and specific proportions of raw materials. The specific raw material proportions are shown in Table 1, which is a table of raw material proportions for the anti-aging compositions of donkey-hide gelatin powder in Examples 1-4.

[0075] Table 1 Comparative Example 1 This comparative example demonstrates the adverse effects of omitting the expression line relaxing peptide. The other settings in this comparative example are the same as in the previous example, except that the expression line relaxing peptide was removed. The findings revealed that: 1) Dynamic wrinkles could not be specifically inhibited, resulting in an incomplete overall anti-wrinkle effect; 2) The dermal-epidermal "two-way linkage" mechanism was disrupted, and the newly formed collagen in the dermis was continuously compressed by facial muscles, potentially damaging its long-term stability and orderly arrangement, thus weakening the maintenance effect of deep repair; 3) The product lost its rapid epidermal improvement ability, leading to a decrease in user experience and onset speed. This proves that the absence of this ingredient downgrades the formula from "three-dimensional synergistic repair" to "single-layer repair," failing to achieve the synergistic amplification effect of "1+1+1>3," clearly demonstrating the indispensable role of the expression line relaxing peptide in achieving comprehensive anti-wrinkle effects.

[0076] Comparative Example 2 This comparative example demonstrates the adverse effects of not containing antioxidant and anti-inflammatory short peptides and expression line relaxing short peptides. The other settings in this comparative example are the same as in the previous example, except that both antioxidant and anti-inflammatory short peptides and expression line relaxing short peptides were removed. The findings are as follows: First, this results in the "dermal repair triangle" lacking a crucial signal initiation link (copper peptide). Collagen and elastin synthesis lacks efficient "instructions," leading to insufficient power. Even with the retention of raw material supply (donkey-hide gelatin) and autophagy regulation (ginsenoside CK), the fundamental goal of dermal matrix reconstruction is difficult to achieve effectively. Second, the complete "dermal-epidermal bidirectional linkage" mechanism is completely disrupted. Dermal repair power weakens, while epidermal facial muscle contraction becomes uncontrolled, and the formula completely loses its three-dimensional anti-wrinkle capabilities of "deep support" and "surface wrinkle reduction." Simultaneously, the stability of the repair microenvironment decreases due to the lack of anti-inflammatory and antioxidant regulation by copper peptides, impairing the durability of the newly formed matrix. Third, the overall efficacy was severely compromised, and the product degenerated into a basic formula with only partial nutritional and maintenance functions. It could neither rebuild the dermal structure nor manage dynamic wrinkles in the epidermis, thus failing to achieve the dual core objectives of "repair + management." This result confirms that copper peptide and acetyl hexapeptide-8 each have irreplaceable strategic functions in the system. Their simultaneous absence caused the collapse of the carefully designed multi-level synergistic network of "signal-raw material-cell-muscle," ultimately resulting in the complete disappearance of the synergistic amplification effect of "1+1+1>3."

[0077] Comparative Example 3 This comparative example demonstrates the adverse effects of omitting antioxidant and anti-inflammatory short peptides, expression line relaxing short peptides, and rare ginsenoside CK. The other settings in this comparative example are the same as in the previous example, except that the antioxidant and anti-inflammatory short peptides, expression line relaxing short peptides, and rare ginsenoside CK were simultaneously removed. The findings are as follows: First, the active repair capability is completely lost, and the system degenerates into a passive supply mechanism. Only donkey-hide gelatin powder is retained in the formula as an amino acid raw material and a solubilizer for delivery assistance, while the key "signaling instructions" (blue copper peptide) driving collagen synthesis and the "regulators" (ginsenoside CK) for clearing and optimizing cell function are missing. This results in the loss of all active regulation and core driving force for dermal regeneration and remodeling, making it impossible to initiate and complete any fundamental structural repair. Second, the carefully constructed synergistic network completely disintegrates. With the removal of the three functional components, both the "dermal repair iron triangle" mechanism and the "dermal-epidermal bidirectional linkage" mechanism cease to exist. The product can neither stimulate matrix regeneration from deep within nor inhibit the formation of dynamic wrinkles on the surface. All synergistic effects based on the precise coordination between ingredients, such as "signal-raw material coupling," "cell cleansing-signal enhancement," and "deep support-surface relaxation," are completely negated, reducing the formula to a simple, passive raw material supply system. Third, the overall efficacy of the product experiences a precipitous drop, failing to achieve any of its intended goals. This comparative formula has completely lost the complete skin repair function designed in the example. It fails to achieve the fundamental goal of "repairing the dermal structure" and completely abandons the goal of "managing epidermal wrinkles," its efficacy relegated to basic nutrient supply and penetration assistance, fundamentally different from the original design's "systemic active repair" positioning. The results of this extreme comparative example demonstrate that copper peptide, ginsenoside CK, and acetyl hexapeptide-8 are the three indispensable functional pillars for this formula to achieve active, synergistic, and three-dimensional repair. Their simultaneous absence signifies the complete collapse of the entire multi-layered repair network architecture, thus powerfully confirming the scientific validity, completeness, and necessity of each core ingredient in the original formula design.

[0078] Comparative Example 4 This comparative example illustrates the adverse consequences of replacing donkey-hide gelatin powder with other substances. The other settings in this comparative example are the same as in the previous example, except that sodium hyaluronate was used instead of donkey-hide gelatin powder. The findings reveal that the core consequence is the loss of a key pillar of the "dermal repair triangle." The core function of donkey-hide gelatin powder is to provide specific characteristic amino acids and small molecule peptides for collagen synthesis, acting as the "raw material supply." While sodium hyaluronate offers superior moisturizing and immediate filling properties, it cannot provide the direct raw materials required for collagen biosynthesis. This results in a lack of substrate support for the synthetic "signaling instructions" issued by copper peptides, leading to inefficient or even "idling" signaling pathways. The cell functions optimized by ginsenoside CK cannot be fully utilized, causing the "iron triangle" to degenerate from a highly efficient synergy of "signal-raw material-regulation" to a dysfunction of "signal-filling-regulation," severely weakening structural regeneration capacity. Secondly, the product's repair properties undergo a fundamental shift. Donkey-hide gelatin powder aims to promote substantial biosynthesis of collagen fibers and durable matrix remodeling. Sodium hyaluronate, on the other hand, primarily produces a reversible, temporary feeling of fullness through physical water absorption and swelling. This replacement shifts the product's core mechanism from "fundamental dermal reconstruction" to "surface moisturizing and filling," failing to address structural issues such as skin laxity and wrinkles at their root, making long-term repair goals difficult to achieve.

[0079] Finally, the integrity and long-term effectiveness of the synergistic network are compromised. The active ingredients in donkey-hide gelatin powder play a crucial role in supporting tissue repair, such as promoting fibroblast migration and adhesion—a function that sodium hyaluronate lacks. After replacement, the formula suffers a functional gap in promoting cell behavior to support long-term repair, leading to a decline in the long-term efficacy and integrity of the entire repair system. This comparative example demonstrates that sodium hyaluronate cannot functionally replace donkey-hide gelatin powder within the system designed in this invention. While the formula achieves immediate moisturizing and filling effects after replacement, it loses the core raw material supply and cell support capabilities that drive substantial collagen regeneration and matrix remodeling, resulting in the failure of the synergistic repair network. The product degenerates from an "active repair type" to a "moisturizing and filling type," completely deviating from the fundamental anti-aging and repair goals designed in this invention. This conversely confirms the irreplaceable role of donkey-hide gelatin powder as a source of specific amino acids and active peptides in this system.

[0080] Comparative Example 5 This comparative example illustrates the adverse consequences of replacing donkey-hide gelatin powder with other substances. The setup of this comparative example is the same as the previous example, except that polyethylene glycol 600 was used instead of donkey-hide gelatin powder. The findings are as follows: First, the material basis for dermal repair is completely removed. Donkey-hide gelatin powder, as a specialized source of amino acids and active peptides for collagen biosynthesis, has its function replaced by polyethylene glycol 600, an inert solvent that completely lacks the ability to supply biological raw materials. This causes the "dermal repair iron triangle" to collapse due to the lack of a "raw material supply" pillar. The signaling instructions of copper peptide and the cell regulatory function of ginsenoside CK cannot achieve collagen production due to the lack of substrate support, resulting in a complete loss of structural regeneration capacity. Second, the mechanism of action of the formulation undergoes a fundamental transformation. The product is downgraded from a biological repair system based on the synergy of active ingredients to a physical solvent carrier with only dissolving and delivery functions, making no direct contribution to skin repair.

[0081] Ultimately, the entire synergistic repair network completely broke down. Within the system established in this invention, polyethylene glycol 600 could not form any functional coupling with the other active ingredients, and the original auxiliary functions of donkey-hide gelatin powder, such as promoting cell migration, also disappeared. This completely failed to achieve the design goal of dermal reconstruction through multi-level synergy, and the formula degenerated into a system with incomplete efficacy. This comparative example demonstrates that, within the system established in this invention, replacing the core biological raw materials with inactive solvents will cause the formula to degenerate from "active repair" to "basic carrier," thus confirming the irreplaceable role of donkey-hide gelatin powder as the core active substance and synergistic cornerstone in this invention.

[0082] The ingredients and proportions of each comparative example are shown in Table 2. Table 2 is the ingredient and proportion table for comparative examples 1 to 5.

[0083] Table 2 Example 5 This embodiment illustrates a method for preparing donkey-hide gelatin repair cream, specifically including the following steps: The mass ratio of glycerol stearate to PEG-100 stearate in the glycerol stearate and PEG-100 stearate compound is 1:1.

[0084] The preparation method includes the following steps: I. Add glycerol, 1,3-propanediol, 1,2-hexanediol, p-hydroxyacetophenone, γ-polyglutamic acid and water to an emulsifying pot, heat to 75-85℃, stir and mix evenly to obtain the aqueous phase.

[0085] II. Place donkey oil, tocopheryl acetate, glyceryl stearate and PEG-100 stearate compound in an oil phase pot and heat to 75-85℃, stir and mix evenly to obtain the oil phase.

[0086] III. Mix the oil phase and water phase and stir homogenously.

[0087] IV. Cool the mixture from step (3) to 70°C, stir to homogenize, then add the donkey-hide gelatin powder anti-aging composition, stir to mix, cool to 40°C and discharge to obtain the donkey-hide gelatin anti-wrinkle cream.

[0088] Example 6 The other settings in this embodiment are the same as in embodiment 5, except that the raw material ratio is different, as shown in Table 3.

[0089] Example 7 The other settings in this embodiment are the same as in embodiment 5, except that the raw material ratio is different, as shown in Table 3.

[0090] Comparative Example 6 This comparative example is used to demonstrate the adverse effects of not setting up the anti-aging compositions of donkey oil and donkey-hide gelatin powder. This comparative example does not contain donkey oil, tocopheryl acetate, glycerin, 1,2-hexanediol, or the donkey-hide gelatin powder anti-aging composition of Example 1 (replaced with 20 wt.% commercially available collagen solution), as shown in Table 3. Findings: First, the fundamental properties of the product have changed, downgrading from "active repair type" to "basic moisturizing type." The core "donkey-hide gelatin powder anti-aging composition" has been removed, meaning that all the key active ingredients that achieve the "dermal repair iron triangle" (blue copper peptide, donkey-hide gelatin, ginsenoside CK) and the "dermal-epidermal bidirectional linkage" (acetyl hexapeptide-8) in the formula are no longer present. Therefore, the product completely loses its ability to actively stimulate collagen regeneration, regulate cell function, and precisely manage expression lines. Its claimed fundamental anti-wrinkle and firming goals cannot be achieved, ultimately degenerating into a common skincare product with only basic moisturizing and temporary filling effects.

[0091] Secondly, the basic physicochemical system and stability of the formula are severely compromised, and the physical basis for synergistic effects is undermined. The absence of donkey oil (the core oil phase) and tocopheryl acetate (an oil-soluble antioxidant) not only degrades the product's texture, feel, and moisturizing properties but also weakens the system's stability, the capacity to carry oil-soluble components, and overall antioxidant protection. The removal of glycerin and 1,2-hexanediol (key moisturizers and solvents) directly leads to a significant decrease in the product's immediate and long-lasting moisturizing performance and may affect the stability of the aqueous phase and the solubility of components, disrupting the basic physicochemical balance of the formula and making it difficult for even some active ingredients to function effectively. Finally, a sophisticated mechanism of action is replaced by a low-level one, resulting in a fundamental difference in efficacy. Replacing the entire "donkey-hide gelatin powder anti-aging composition" with "20wt.% commercially available collagen solution" is a crude substitution of a sophisticated "bio-repair network" composed of small-molecule active peptides, saponins, and specialized amino acids with a single large-molecule moisturizing filler. This reduces the mechanism of action from a multi-target, proactive "signal initiation-raw material supply-cellular regulation-muscle modulation" to a passive "surface film formation and physical filling," creating an insurmountable generational gap in efficacy depth, persistence, and fundamental effectiveness. This comparative example demonstrates that the original formulation is an indivisible organic whole. Its basic framework components and core active composition together constitute the necessary conditions for achieving "synergistic biorepair." Any deficiency would lead to a complete collapse of the product from its design concept to its actual efficacy, thus powerfully confirming the integrity, necessity, and scientific validity of the original formulation design.

[0092] Comparative Example 7 This comparative example illustrates the adverse effects of using a very small amount of the donkey-hide gelatin powder anti-aging composition and omitting other components. This comparative example does not contain tocopheryl acetate, glyceryl stearate and PEG-100 stearate complex, glycerin, 1,3-propanediol, 1,2-hexanediol, γ-polyglutamic acid, and the donkey-hide gelatin powder anti-aging composition of Example 1 is set to 0.1 wt%, as shown in Table 3. Findings: First, the basic physical system of the formulation completely collapses, and the product loses its basic functionality. Removing the core emulsifier (glyceryl stearate and PEG-100 stearate complex) directly leads to emulsification failure, preventing the product from forming a stable paste, potentially resulting in oil-water separation and a rough texture. Simultaneously, removing the key polyol moisturizers (glycerin, 1,3-propanediol, 1,2-hexanediol) and the highly effective water-locking polymer (γ-polyglutamic acid) completely disintegrates the product's moisturizing system, rendering its basic hydrating and water-locking functions virtually zero after application, and potentially even causing dry and uncomfortable skin. Secondly, the core repair efficacy is completely unattainable due to severely insufficient concentration of active ingredients. Reducing the content of the donkey-hide gelatin powder anti-aging composition to 0.1 wt% means that the concentrations of key components such as copper peptide, ginsenoside CK, and acetyl hexapeptide-8 are far below their respective effective action thresholds. Therefore, the carefully designed "dermal repair triangle" and "dermal-epidermal bidirectional linkage" mechanism cannot be activated at all, and the product completely loses any biological activity in stimulating collagen regeneration, regulating cell function, and improving expression lines. Finally, the stability and protective properties of the formulation are impaired. Removing the oil-soluble antioxidant tocopheryl acetate not only reduces the antioxidant stability of the formulation itself during storage but also weakens the protection of the skin's lipid layer, which is detrimental to maintaining the healthy microenvironment required for repair. This comparative result shows that sufficient core active ingredients and a complete and stable formulation matrix (including emulsification system, moisturizing architecture, and antioxidant protection) are two indispensable pillars of an effective product. Both are indispensable and together ensure the usability, stability, and ultimate efficacy of the product, thus verifying the rigor and necessity of the formulation design in the original embodiment.

[0093] Comparative Example 8 This comparative example illustrates the adverse effects of omitting the donkey-hide gelatin powder anti-aging composition and omitting other components. This comparative example does not include 1,2-hexanediol, p-hydroxyacetophenone, γ-polyglutamic acid, and replaces the donkey-hide gelatin powder anti-aging composition of Example 1 with 15 wt% commercially available sodium hyaluronate solution, as shown in Table 3. Findings: First, the core efficacy undergoes a fundamental change. Replacing the complete "donkey-hide gelatin powder anti-aging composition" with "15 wt% commercially available sodium hyaluronate solution" means the complete removal of all active ingredients, including copper peptide, ginsenoside CK, and acetyl hexapeptide-8. The product therefore completely loses its ability to achieve structural regeneration through the "dermal repair triangle" and improve dynamic wrinkles through "epidermal layer management." Although sodium hyaluronate possesses excellent moisturizing and immediate filling properties, its physical mechanism of action cannot replace the original composition's biological repair functions at the levels of signal transduction and cell regulation. This downgrades the product's fundamental goal from active anti-aging repair to basic moisturizing and temporary wrinkle reduction. Second, the basic performance and stability of the formulation exhibit shortcomings. Removing 1,2-hexanediol (which functions as both a moisturizing and preservative agent) and p-hydroxyacetophenone (a mild preservative) may weaken the preservative system and increase the risk of microbial contamination. Simultaneously, removing the highly effective water-locking polymer γ-polyglutamic acid not only weakens its own moisturizing ability but also prevents it from achieving its potential synergistic moisturizing effect with sodium hyaluronate, resulting in an incomplete long-lasting water-locking network. Ultimately, the precise synergistic foundation is completely lost. The original formula was an "active repair network" where the functions of each component were coupled, but this modification not only removed the core "active action node" but also the crucial "support node." This reduces the formula to a simple physical mixture of ingredients, lacking biological-level functional synergy between components, and failing to achieve the systemic synergistic effect of "1+1+1>3." This comparative example demonstrates that replacing the multi-target active composition specifically designed in this invention with a single moisturizing ingredient and weakening the auxiliary system will completely downgrade the product from its design concept to its efficacy, thus reversing the indispensability of the core composition and complete structure in the original formula.

[0094] Table 3 shows the formulations of the donkey-hide gelatin anti-wrinkle creams of Examples 5-7, Comparative Examples 6-8, and the ordinary anti-wrinkle creams of the blank examples.

[0095] Table 3 Test case Experimental Example 1 1. Cytotoxicity experiment comparing donkey-hide gelatin powder with anti-aging raw materials: Human immortalized keratinocytes (HACAT) were selected as a cell model. HACAT cells were cultured for 24 hours with a series of different concentrations of donkey-hide gelatin powder and four comparative raw materials, including acetyl hexapeptide-8, retinol (50%), copper peptide, and psoralen (95%). Cell viability was then determined using the CCK-8 assay to investigate the cytotoxicity of these materials to HACAT. The experimental results are shown below. Figure 1 .

[0096] 2. Cell proliferation experiment comparing donkey-hide gelatin powder with anti-aging raw materials: HACAT was selected as a cell model. Cell proliferation experiments were conducted using 0.625 mg / mL copper peptide, 2.5 mg / mL acetyl hexapeptide-8, 0.01 mg / mL retinol, 0.0048 mg / mL psoralen, and 2.5 mg / mL donkey-hide gelatin powder. The effects of different raw materials on the proliferation activity of HACAT cells were investigated by live / dead cell staining and CCK-8 assay. The experimental results are shown below. Figure 2 and Figure 3 .

[0097] 3. Cell migration experiment comparing donkey-hide gelatin powder with anti-aging raw materials: HACAT was selected as a cell model. Scratch experiments were performed using 0.625 mg / mL copper peptide, 2.5 mg / mL acetyl hexapeptide-8, 0.01 mg / mL retinol, 0.0048 mg / mL psoralen, and 2.5 mg / mL donkey-hide gelatin powder. The effects of different raw materials on the migration rate of HACAT cells were evaluated by changes in cell migration rate after 24 and 48 hours of scratching (qualitative analysis using microscopic images and quantitative analysis of cell migration rate) and by live / dead staining. The experimental results are shown below. Figure 4 and Figure 5 .

[0098] 4. Cross-sectional study of the anti-aging efficacy of raw materials based on a full-skin model (3D skin): A. Comparative Study Methods for the Anti-aging Efficacy of Raw Materials Based on Skin's In Vitro Artificial Full-Thickness 3D Skin Model: After culturing the artificial full-thickness 3D skin in vitro for 24 hours, cell culture medium containing 1%, 2%, and 3% donkey-hide gelatin powder, 2.5 mg / mL acetyl hexapeptide-8, 0.1 mg / mL retinol, 0.048 mg / mL psoralen, and 6.25 mg / mL copper peptide was used as the experimental group. The blank group received no drug treatment and no UVA+UVB mixed ultraviolet light irradiation. The modeling group (no drug treatment, with normal cell culture medium) and the experimental group received UVA+UVB mixed ultraviolet light irradiation for 3 days in addition to the culture medium intervention, with an irradiation intensity of 75 mJ / cm². 2 UVB+ 10J / / cm 2UVA was applied, with medium changes performed every 24 hours during the culturing period. Twenty-four hours after the last administration and irradiation, sections were fixed and embedded. After dewaxing and rehydration, sections were stained with HE, Masson, and Verhoeff's Van Gieson (EVG) staining, respectively. Stained sections were observed under a bright-field microscope (20× resolution), and statistical analysis was performed using ImageJ software. Transdermal water loss after treatment with each raw material was also measured, and the ROS content in the skin was detected using a tissue reactive oxygen species (ROS) detection kit (DHE fluorescence method). Experimental results are shown below. Figures 6-13 .

[0099] like Figure 1 The results show that donkey-hide gelatin powder is non-toxic to HACAT cells at concentrations of 20 mg / mL and below; acetyl hexapeptide-8 is non-toxic to HACAT cells at concentrations of 5 mg / mL and below, with slight toxicity at concentrations of 10-20 mg / mL; copper peptide is non-toxic below 0.3125 mg / mL; retinol is non-toxic below 0.0195 mg / mL; and psoralen is non-toxic below 0.0049 mg / mL. This indicates that, except for acetyl hexapeptide-8, anti-aging components containing polyphenol structures or copper ions have high toxicity, greatly limiting their upper concentration limits. This also provides a basis for the concentration and ratio of anti-aging raw materials in compound formulations and further confirms the good biocompatibility of donkey-hide gelatin powder.

[0100] pass Figure 2 and Figure 3 The results showed that at 2.5 mg / mL, donkey-hide gelatin powder significantly promoted HACAT cell proliferation, while acetyl hexapeptide-8 had no significant effect on cell proliferation. Copper sulfate at 0.625 mg / mL, retinol at 0.01 mg / mL, and psoralen at 0.0048 mg / mL all significantly inhibited HACAT cell proliferation. This indicates that even though copper sulfate, retinol, and psoralen did not exhibit cytotoxicity at the tested concentrations on day 1, they will show cytotoxicity during longer culture periods. This further validates the excellent biocompatibility of donkey-hide gelatin powder and clarifies the upper limit of the concentration of several anti-aging ingredients, providing a basis for ingredient compounding.

[0101] pass Figure 4 and Figure 5The results showed that at 2.5 mg / mL, both donkey-hide gelatin powder and acetyl hexapeptide-8 significantly promoted HaCaT cell migration, while 0.625 mg / mL of copper peptide, 0.01 mg / mL of retinol, and 0.0048 mg / mL of psoralen had almost no effect on promoting cell migration. This result further validates the excellent biocompatibility and bioactivity of donkey-hide gelatin powder and demonstrates that acetyl hexapeptide-8 also possesses good biocompatibility and bioactivity, providing important experimental evidence for subsequent raw material compounding.

[0102] pass Figure 6 and Figure 7 The results showed that in the control group (normal skin), the epidermal structure was clear, with approximately 1-2 layers of keratinocytes arranged neatly. The dermis contained abundant fibrous tissue with uniform fiber thickness and distribution, without obvious breakage or disordered arrangement. In the UV-treated group, the cell layer was significantly damaged, showing cavities and disordered arrangement. The presence of some nucleated cells in the stratum corneum indicated incomplete keratinization, and the connections became loose, presenting obvious symptoms of aging and damage. Compared with the UV-induced modeling group, the cell layer thickness of the 1% donkey-hide gelatin powder group increased by 41%, and nucleated cells were present in the stratum corneum, indicating less damage than the UV group. Compared with the UV group, the cell layer thickness of the 2% donkey-hide gelatin powder group increased by 87%, with clearly visible basal cells, indicating less damage than the UV group and the 1% donkey-hide gelatin powder group. Compared with the UV group, the cell layer thickness of the 3% donkey-hide gelatin powder group increased by 83%, with a relatively intact cell layer, indicating less damage than the UV group and the 1% donkey-hide gelatin powder group, and similar to the 2% donkey-hide gelatin powder group. Compared with the UV group, the acetyl hexapeptide-8 group increased by 103%, with a relatively intact cell layer and less damage. Compared with the UV group, the retinol group increased by 27.33%, with significant cell layer damage and some nucleated cells present in the stratum corneum, indicating more severe damage than the donkey-hide gelatin group. Compared with the UV group, the cell layer thickness of the psoralen group increased by 43%, with nucleated cells present in the stratum corneum, and the degree of damage was similar to the 1% donkey-hide gelatin powder group. This indicates that the 2% donkey-hide gelatin powder and 0.25% acetyl hexapeptide-8 groups showed the best effects.

[0103] pass Figure 8 and Figure 9Masson staining results showed that in the control group (normal skin), the collagen fibers in the dermis were wavy, evenly distributed, and appeared blue. Compared with the control group, the UV group showed degradation, denaturation, and breakage of dermal collagen fibers, with a 12.1% decrease in dermal collagen fiber content. Compared with the UV group, the 1% donkey-hide gelatin powder group, 2% donkey-hide gelatin powder group, 3% donkey-hide gelatin powder group, 2.5 mg / mL acetyl hexapeptide-8 group, and 0.048 mg / mL psoralen group showed denser and more regular collagen fibers in the dermis, with collagen fiber content increasing by 21%, 49%, 31%, 57%, and 83%, respectively. The retinol group had a similar collagen fiber content to the UV group, but showed some degree of degradation in dermal collagen fibers. Overall, in terms of promoting collagen production alone, the acetyl hexapeptide-8 and psoralen groups were the most effective, followed by the 2% donkey-hide gelatin powder group.

[0104] pass Figure 10 and Figure 11 The EVG staining results showed that: in the blank group (normal), the elastic fiber structure of the skin was clear, appearing purplish-black, slightly curved and wavy, with no breakage or fragmentation, and the overall arrangement was uniform. Compared with the Blank group, the elastic fibers in the UV group were disordered, with fiber breakage and curling, indicating denaturation or degradation, and a decreased ability to bind with dye, resulting in significantly lighter staining. Compared with the UV group, the 3% donkey-hide gelatin powder group and the 2.5 mg / mL acetyl hexapeptide-8 group showed significantly less denaturation, a relatively clear fiber structure, and less damage, with elastic fiber content increasing by 17% and 19%, respectively; compared with the UV group, the 0.1 mg / mL retinol group showed significant fiber breakage, more severe damage, and a 16% decrease in elastic fiber content. There was no significant difference in skin structure and elastic fiber content between the 1% donkey-hide gelatin powder and 0.048 mg / mL psoralen groups and the UV group.

[0105] pass Figure 12 It can be seen that, compared with the UV group, the transdermal water loss rate (water loss rate) of the 1% donkey-hide gelatin powder, 2% donkey-hide gelatin powder, 3% donkey-hide gelatin powder, 6.25 mg / mL copper peptide, 2.5 mg / mL acetyl hexapeptide-8, and 0.048 mg / mL retinol groups decreased by 30.41%, 42.11%, 50.35%, 19.30%, 56.61%, and 24.56%, respectively, after drug administration. Among them, 3% donkey-hide gelatin powder and 2.5 mg / mL acetyl hexapeptide-8 had the best water retention effect.

[0106] pass Figure 13The results showed that the ROS content in the UV group increased 2.13 times compared to the control group, indicating that UV irradiation increases the ROS content in skin tissue and causes skin damage. Compared with the UV group, the ROS content in the 1% donkey-hide gelatin powder, 2% donkey-hide gelatin powder, 3% donkey-hide gelatin powder, and 0.048 mg / mL psoralen groups decreased by 35%, 49%, 55%, and 12%, respectively, with the 3% donkey-hide gelatin powder group showing the best effect. There was no significant difference in ROS content between the 2.5 mg / mL acetyl hexapeptide-8 group and the 0.1 mg / mL retinol group and the UV group.

[0107] The comparative results of the efficacy of anti-aging raw materials show that: 1) Donkey-hide gelatin powder at 20 mg / mL (2%) and acetyl hexapeptide-8 at 5 mg / mL (0.5%) showed no cytotoxicity, while copper peptide (<0.31 mg / mL, non-toxic), retinol (<0.020 mg / mL, non-toxic) and psoralen (<0.0049 mg / mL, non-toxic) all showed high cytotoxicity.

[0108] 2) The results of the horizontal comparison of five raw materials on the 3D skin model showed that 3% donkey-hide gelatin powder and 2.5mg / mL acetyl hexapeptide-8 both have good effects in promoting collagen and elastin synthesis and retaining moisture. However, 3% donkey-hide gelatin powder has a higher cost performance. At the same time, the antioxidant effect of 3% donkey-hide gelatin powder is significantly better than that of 2.5mg / mL acetyl hexapeptide-8.

[0109] 3) Among the five ingredients, 3% donkey-hide gelatin powder has the best antioxidant effect, followed by blue copper peptide.

[0110] 4) 3% donkey-hide gelatin and acetyl hexapeptide-8 had the lowest transdermal water loss rate.

[0111] 5) Overall, among the five ingredients, donkey-hide gelatin powder has the most comprehensive anti-aging effects and the highest cost performance.

[0112] Experimental Example 2 Validation of the anti-aging efficacy of donkey-hide gelatin powder anti-aging composition based on Skin in vitro artificial full-thickness 3D skin model: A skin photodamage model was established after culturing artificial full-thickness 3D skin in vitro for 24 hours. The experimental groups were as follows: the blank group received no drug treatment and no UVA+UVB mixed ultraviolet light irradiation; the modeling group (no drug treatment) received UVA+UVB mixed ultraviolet light irradiation for 3 days; and the anti-aging group (treated with donkey-hide gelatin powder anti-aging composition) received UVA+UVB mixed ultraviolet light irradiation for 3 days. The irradiation intensity was 75 mJ / cm². 2 UVB+ 10J / / cm 2UVA was applied topically every 12 hours, with a dosage of 500 μL each time. Twenty-four hours after the last irradiation, water loss was measured using a transdermal water loss analyzer. The treated sections were then fixed, embedded, dewaxed, rehydrated, and stained with HE, Masson's method, and EVG. The stained sections were observed under a bright-field microscope (×20 resolution), and statistical analysis was performed using ImageJ software. Simultaneously, the ROS content in the skin was detected using a tissue reactive oxygen species (ROS) detection kit (DHE fluorescence method). The experimental results of Example 1 are shown below. Figures 14-19 .

[0113] pass Figure 14 It can be seen that the epidermal structure of the blank group (normal) is clear, with multiple layers of keratinocytes, and the cells are arranged neatly. The dermis contains abundant fibrous tissue, with fibers of uniform thickness and distribution, and no obvious breakage or disordered arrangement. Compared with the blank group, the epidermal layer of the UV group is severely damaged, with fiber degradation and breakage, and scattered cells. Compared with the UV group, the epidermal structure of the anti-aging group is relatively intact, the cells are arranged more orderly and neatly, and the overall condition is greatly improved.

[0114] pass Figure 15 It can be concluded that the epidermis of the UV group was severely damaged, and collagen was shrunken and accumulated. In contrast, the epidermis of the anti-aging group was more normal, and the collagen content was close to that of the blank group (normal group).

[0115] pass Figure 16 It can be concluded that the elastic fiber structure of the skin in the blank group is clear, slightly curved and wavy, with no breakage or fragmentation, and the overall distribution is uniform. Compared with the blank group, the epidermis of the UV group is severely damaged, and no obvious elastic fibers are seen. Compared with the UV group, the overall condition of the elastic fibers in the anti-aging group is greatly improved, with most fibers having clear structures and normal morphology.

[0116] pass Figure 17 It can be concluded that the ROS content in the UV group was 2.7 times higher than that in the control group, indicating that UV irradiation caused significant damage to skin tissue. Compared with the UV group, the ROS content in the anti-aging group was reduced by 61.24%, which is close to that in the control group.

[0117] pass Figure 18 The results show that the UV group had a 1.06-fold higher water loss rate compared to the control group, indicating that UV irradiation affected the skin barrier function and caused skin damage. The anti-aging group, compared to the UV group, had a 32.81% lower water loss rate. These 3D skin model experimental results demonstrate that our designed donkey-hide gelatin powder anti-aging composition has a good anti-aging effect.

[0118] Example of effect Example of effect 1 1. Skin irritation test Test Method: Twenty volunteers were selected and divided into four groups of five. The anti-aging compositions of donkey-hide gelatin powder from Examples 1 to 4 were tested. Each participant used the inner sides of their left and right arms as controls. 0.3 ml of the composition prepared in Examples 1 to 4 was applied to the inner side of the left arm each time, while the right arm was left untreated as a control. The treatment was performed twice daily, and skin reactions were recorded after 24 hours and 72 hours.

[0119] The results showed that no skin redness or erythema or other allergic reactions were observed 24 hours and 72 hours after applying the product of this invention, indicating that the donkey-hide gelatin anti-aging composition of this invention is safe to use in cosmetics without adverse effects.

[0120] 2. Stability assessment Stability testing was conducted in accordance with the "Standard for Stability Testing of Cosmetics" T / GDCQMA 00-2023.

[0121] High-temperature stability test: The donkey-hide gelatin repair creams from Examples 5-7, Comparative Examples 6-8, and the blank example were placed in sealed containers and subjected to a 30-day stability test at high temperature (50 ± 2 ℃). The appearance, odor, and skin feel of each example or comparative example were measured after 0 days and 30 days at high temperature (50 ± 2 ℃). The results are shown in Table 4 and... Figure 19 .

[0122] Table 4 shows the results of the examination of appearance, odor, and skin feel stability of Examples 5-7 and Comparative Examples 6-8.

[0123] Table 4 Through Table 4 and Figure 19 It can be seen that the anti-wrinkle cream sample prepared by this invention has good high-temperature stability and no abnormalities were found.

[0124] 3. Evaluation of long-lasting anti-aging effects Using the donkey-hide gelatin anti-wrinkle creams prepared in Examples 5-7, Comparative Examples 6-8, and the blank example as samples, adult female volunteers used the test samples continuously for 14 days under normal conditions (applied to the entire face after cleansing, twice a day, morning and evening) to evaluate the anti-aging efficacy of the test samples. 350 female subjects with dull facial skin tone, expression lines, and fine lines, aged 35-60 years, were selected and divided into 7 groups of 50 each. They used the donkey-hide gelatin anti-wrinkle creams prepared in Examples 5-7, Comparative Examples 6-8, and the blank example, respectively. The creams were applied evenly after cleansing, twice a day, morning and evening, for four and eight weeks. A questionnaire survey was used to understand the subjects' evaluation of the improvement in wrinkles after using the product for four weeks. The evaluation indicators were mainly: wrinkle reduction effect and redness reduction. The results were categorized as: no obvious effect, moderate effect, relatively obvious effect, and very obvious effect. The evaluation results are shown in Table 5.

[0125] Table 5 As shown in Table 5, the higher the proportion of the added donkey-hide gelatin powder anti-aging composition, the better the anti-wrinkle / aging effect. Furthermore, using donkey oil instead of traditional triglycerides significantly enhances the anti-wrinkle ability, with a much better effect than Comparative Example 6, which contains collagen. Conversely, Comparative Examples 7 and 8, containing only small amounts of active ingredients, show very poor anti-aging effects. This experiment further demonstrates that the donkey-hide gelatin powder anti-aging composition designed in this invention and the donkey-hide gelatin powder anti-wrinkle cream prepared using it have firming and anti-wrinkle effects, exhibiting excellent anti-wrinkle / anti-aging results.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A donkey-hide gelatin powder anti-aging composition, characterized in that, The raw materials of the anti-aging composition of donkey-hide gelatin powder include, by mass percentage: 1-4 wt.% donkey-hide gelatin powder, 0.1-1 wt.% antioxidant and anti-inflammatory short peptides, 0.01-0.5 wt.% rare ginsenoside CK, 500-5000 ppm expression line relaxing short peptides, 1-3 wt.% solubilizers, and the balance being water; The antioxidant and anti-inflammatory short peptides are selected from one or more of copper peptides, carnosine, or glutathione. The expression line relaxing short peptide is selected from one or more of acetyl hexapeptide-8, conopodeptide, or snake venom peptide.

2. The anti-aging composition of donkey-hide gelatin powder according to claim 1, characterized in that, The solubilizer is selected from one or more of poloxamer, 1,3-propanediol, or toxoxalum and a mixture thereof.

3. A method for preparing an anti-aging composition of donkey-hide gelatin powder, characterized in that, The donkey-hide gelatin powder anti-aging composition is the donkey-hide gelatin powder anti-aging composition according to claim 1 or 2, and the preparation method includes the following steps: (1) Prepare the raw materials of the anti-aging composition of donkey-hide gelatin powder according to claim 1 or 2, take the total amount of donkey-hide gelatin powder and 20-40 wt.% of water, then slowly sprinkle the donkey-hide gelatin powder into the water, heat the mixture to 55-65°C, and maintain the temperature at this temperature for 0.5-2 hours. If a clear solution is obtained after stirring, proceed to the next step; if there are undissolved particles, pass the undissolved particles through an 80-120 mesh sieve, and return the sieve material to the mixture and continue stirring until a clear solution is obtained, which is recorded as solution A; (2) Take the full amount of antioxidant and anti-inflammatory short peptides and mix them with the remaining water, then stir until transparent; then add a pH adjuster to adjust the pH of the solution to 6.8~7.0 to obtain solution B; (3) Mix solution A and solution B thoroughly to form the main system solution; (4) Mix the total amount of rare ginsenoside CK with the total amount of solubilizer, and heat in a water bath at 35~45℃ for 8~13 minutes until the mixture is homogeneous. Then slowly add the resulting mixture to the main system obtained in step (3) and stir. (5) After stirring until the solution temperature drops to below 33~36℃, add the full amount of expression line relaxation short peptides, and stir at low speed for 8~13 minutes to avoid foaming, and obtain the donkey-hide gelatin powder anti-aging composition product.

4. The preparation method according to claim 3, characterized in that, The water mentioned in step (1) is purified water; The donkey-hide gelatin powder mentioned in step (1) is an ultrafine powder that has been pulverized, with a particle size range of 100~800 mesh; The pH adjuster mentioned in step (2) is citric acid; The low-speed stirring in step (5) is 100~10000 rpm.

5. A donkey-hide gelatin powder anti-wrinkle cream, characterized in that, The anti-wrinkle cream containing donkey-hide gelatin powder contains the anti-aging composition of donkey-hide gelatin powder according to any one of claims 1-3, and the mass percentage of the anti-aging composition of donkey-hide gelatin powder in the anti-wrinkle cream is 5-75 wt.%.

6. The anti-wrinkle cream made from donkey-hide gelatin powder according to claim 5, characterized in that, The donkey-hide gelatin powder anti-wrinkle cream is an eye cream or face cream.

7. The anti-wrinkle cream made from donkey-hide gelatin powder according to claim 5, characterized in that, The anti-wrinkle cream containing donkey-hide gelatin powder also includes the following components in weight percentage: donkey oil: 1~10 wt.%, tocopheryl acetate: 0.2~2.0 wt.%, glyceryl stearate and PEG-100 stearate compound: 0.2~1.8 wt.%, glycerin: 1~4 wt.%, 1,3-propanediol: 2~6 wt.%, 1,2-hexanediol: 0.2~0.9 wt.%, p-hydroxyacetophenone: 0.3~0.8 wt.%, γ-polyglutamic acid: 0.1~2.5 wt.%, and the balance being water.

8. A method for preparing an anti-wrinkle cream made from donkey-hide gelatin powder, characterized in that, The donkey-hide gelatin powder anti-wrinkle cream is the donkey-hide gelatin powder anti-wrinkle cream according to any one of claims 5 to 7, and the preparation method includes the following steps: I. Add the total amount of glycerol, 1,3-propanediol, 1,2-hexanediol, p-hydroxyacetophenone, γ-polyglutamic acid and water to an emulsifying pot, heat to 75~85℃, stir and mix evenly to obtain the aqueous phase; II. Place the total amount of donkey oil, tocopheryl acetate, glyceryl stearate and PEG-100 stearate compound in an oil phase pot and heat to 75~85℃. Stir and mix evenly to obtain the oil phase. III. Mix the oil phase obtained in step II and the aqueous phase obtained in step I, and stir homogenously to obtain a mixture; IV. Cool the mixture from step III to 65-73°C, stir until homogeneous, and then add the donkey-hide gelatin powder as described in claim 1 or 2. The anti-aging composition or the donkey-hide gelatin powder anti-aging composition prepared by the method of claim 3 or 4 is stirred and mixed evenly and then cooled to 35~45℃ before being discharged to obtain the donkey-hide gelatin anti-wrinkle cream product.

9. The application of a donkey-hide gelatin powder anti-aging composition and a donkey-hide gelatin powder anti-wrinkle cream in anti-wrinkle treatment around the eyes, cheeks, and neck, characterized in that... The anti-aging composition of donkey-hide gelatin powder is the donkey-hide gelatin powder according to claim 1 or 2. The anti-aging composition or the donkey-hide gelatin powder anti-aging composition prepared by the method of claim 3 or 4, wherein the donkey-hide gelatin powder anti-wrinkle cream is the donkey-hide gelatin powder anti-wrinkle cream according to any one of claims 5 to 7 or the donkey-hide gelatin powder anti-wrinkle cream prepared by the method of claim 8.

10. The application of a donkey-hide gelatin powder anti-aging composition in the preparation of anti-wrinkle essences, anti-wrinkle lotions, and lip balms, characterized in that, The anti-aging composition of donkey-hide gelatin powder is the donkey-hide gelatin powder according to claim 1 or 2. The anti-aging composition or the donkey-hide gelatin powder anti-aging composition prepared by the method of claim 3 or 4, wherein the donkey-hide gelatin powder anti-aging composition is used as the core raw material of anti-wrinkle essence, anti-wrinkle lotion, and lip balm.

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