A skin protectant composition containing a biologically active material

Abstract

The application discloses a skin protective agent containing a bioactive composition, which comprises an A agent freeze-dried powder and a B agent solvent liquid, the A agent freeze-dried powder comprises a liposome-encapsulated animal umbilical cord extract, an active peptide composition and an excipient, wherein the liposome-encapsulated animal umbilical cord extract and the active peptide composition constitute the bioactive composition; and the B agent solvent liquid comprises a humectant, a soothing agent, a repairing agent and a stabilizer. The skin protective agent can efficiently stimulate the cell activity of the dermis layer in an endogenous manner, promote the regeneration of collagen and elastin, and increase the barrier; and can significantly reduce the transdermal water loss and build a moisturizing barrier and soothe and repair the damaged barrier in an exogenous manner. Tests show that the skin protective agent has excellent moisturizing, repairing, exfoliating and firming effects. All components of the skin protective agent are EWG all-green components, and are mild and non-irritating; all subjects agree that there is no adverse reaction, and the skin protective agent is particularly suitable for daily skin care, maintenance of damaged skin and post-medical beauty repair.

Description

Technical Field

[0001] This invention relates to the field of biomedicine and topical skin preparations, specifically a skin protectant containing a composition of bioactive ingredients. Background Technology

[0002] Skin protectants are important products in the field of skin health care, playing a significant role in preventing or alleviating skin problems such as dryness, chapping, damage, inflammation, and sagging. They are widely used in medical, industrial, and daily skincare settings. Existing technologies mainly fall into three categories: physical barrier protectants (medical device registration), chemically additive protectants (disinfectant registration), and functional ingredient protectants (cosmetic registration).

[0003] Physical barrier protectants, such as protective creams based on petrolatum or silicone oil, rely on occlusive oils to form a passive protective layer. They generally do not contain plant or bioactive ingredients (or are not mainly composed of plant or bioactive ingredients), do not have the function of actively repairing skin damage, have no anti-inflammatory or healing-promoting functions, and feel greasy when used, resulting in poor patient compliance.

[0004] Chemically-added protective agents contain antibacterial ingredients (such as triclosan and chlorhexidine) or anti-inflammatory ingredients (such as glucocorticoids) in their matrix to achieve antibacterial or anti-inflammatory effects. However, they have high safety risks, including potential problems such as drug resistance, skin atrophy, and systemic absorption. They also lack the mechanism to promote the skin's own repair. Furthermore, their application is limited, such as for the face, infants, and long-term care.

[0005] Functional ingredient-based protective agents are generally based on natural extracts (such as oats, centella asiatica, purslane, and sophora flavescens) or bioactive substances (such as hyaluronic acid, peptides, collagen, and animal umbilical cord protein). Dosage forms include water-based, emulsion, and cream-based formulations. The main problems currently are: reliance on empirical formulation design; bioactive ingredients are often designed in a "stacking" manner, lacking scientific validation of key issues such as the mechanism of action, incompatibilities, transdermal absorption enhancement, and stability assurance. This leads to the common phenomenon of "many ingredients but weak efficacy." Furthermore, the efficacy targets are numerous, the mechanisms of action are unclear, core active ingredients are not prominent or are simply superimposed. Because of the "stacking" design of bioactive ingredients, antagonistic effects may exist. Additionally, existing functional ingredient-based protective agents lack biomimetic design for skin barrier enhancement (cell activation and regeneration), synergistic effects are unverified, and problems such as poor permeability of the main active ingredients and poor protection of activity stability also exist.

[0006] In summary, existing skin protectants, from a functional perspective, fall into several categories: physical barrier protectants lack skin repair capabilities, chemically additive protectants focus on a single aspect like antibacterial or anti-inflammatory effects, and functional ingredient-based protectants, with their "stacking" formulations, lack prominent effects from their core bioactive components and exhibit unclear synergistic effects between components. Therefore, further optimization of skin protectants remains a crucial technical challenge in this field. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a skin protectant containing a composition of bioactive ingredients. Through the combined action of freeze-dried powder (agent A) and solvent (agent B), it regulates both endogenous and exogenous damaging factors of the skin, and significantly upregulates type I collagen and elastin, indicating a significant effect on strengthening the skin barrier. At the same time, subject tests show that it also has excellent moisturizing, repairing, firming, and exfoliating effects.

[0008] To solve the above-mentioned technical problems, the present invention provides a skin protectant containing a bioactive composition, comprising a lyophilized powder (A) and a solvent (B), wherein the mass-volume ratio of the lyophilized powder (A) and the solvent (B) is 1g:55-65mL. Agent A lyophilized powder comprises the following components in parts by weight: Liposome-encapsulated animal umbilical cord extract: 3-5 parts by weight; Active peptide composition: 0.071-0.443 parts by weight; Excipients: 3.2-5.5 parts by weight; The active peptide composition includes at least two of the following: arginine / lysine polypeptide, acetyl hexapeptide-8, tripeptide-10 citrulline, tripeptide-1, acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl tripeptide-5, palmitoyl tripeptide-1, and palmitoyl tripeptide-1 / palmitoyl hexapeptide-12. The liposome-encapsulated animal umbilical cord extract was obtained through the following method: S21. The umbilical cord tissue granules with Wharton's jelly retained were cultured. When the cells were harvested, the culture supernatant was collected and processed to obtain animal umbilical cord extract. S22. The animal umbilical cord extract obtained in S21 is mixed with liposome emulsifier at a mass ratio of 15-22:1, and then homogenized to obtain liposome-encapsulated animal umbilical cord extract. Solution B comprises the following components by mass: Moisturizer: 2.04-5.16 parts by weight; Soothing agent: 1.31-3.55 parts by weight; Repairing agent: 1.7-5.0 parts by weight; Stabilizer: 2.5-5.0 parts by weight.

[0009] The skin protectant of this invention uses freeze-drying technology to protect and stabilize the active ingredients. When used, it is reconstituted with agent B to obtain the skin protectant of this invention. Among them, the freeze-dried powder of agent A contains a bioactive ingredient composition, which is a liposome-encapsulated animal umbilical cord extract and active peptide composition. The two work synergistically to target cell activation and regeneration, enhance the vitality within the barrier, and play a role in endogenously activating cell regeneration and strengthening the barrier. The lyophilized powder (A) and solvent (B) work synergistically to effectively stimulate dermal cell vitality, promote the regeneration of type I collagen and elastin, and strengthen the skin barrier. Exogenously, it significantly reduces transepidermal water loss, strengthens the moisturizing barrier, and soothes and repairs barrier damage. By jointly regulating both endogenous and exogenous skin damage factors, the barrier-strengthening effect is significant. Furthermore, subject testing shows that it also has excellent moisturizing, repairing, firming, and exfoliating effects. When either the lyophilized powder (A) or solvent (B) acts alone, it cannot constitute a skin protectant and does not provide the endogenous and exogenous protective effects, resulting in a limited barrier effect and weaker skin protection. In addition, all components of the lyophilized powder (A) and solvent (B) of this invention are EWG (Environmentally, Working Group) green ingredients and do not contain preservatives, fragrances, pigments, or other potentially sensitizing ingredients. The formula is gentle and non-irritating, suitable for sensitive skin, and all subjects reported no adverse reactions. Therefore, the skin protectant containing a bioactive composition provided by this invention is particularly suitable for daily skin care, maintenance of damaged skin barriers, and post-medical aesthetic repair.

[0010] In this invention, the liposome-encapsulated animal umbilical cord extract is prepared by homogenizing the animal umbilical cord extract with a specific liposome emulsifier. Its cold and heat resistance tests show good stability. Furthermore, the liposome-encapsulated animal umbilical cord extract has a particle size of 0.1-1 μm, which is much smaller than the 30-50 μm diameter of human skin pores. Combined with the structure of the encapsulated lipophilic groups, this facilitates penetration into the skin. Increased stability and permeability promote transdermal or cellular absorption, prolong the active release time, and improve utilization efficiency. It should be noted that the animal umbilical cord extract in this invention is obtained by culturing umbilical cord tissue particles retaining Wharton's jelly. Upon cell harvesting, the culture supernatant is collected and processed to obtain the animal umbilical cord extract.

[0011] In one specific scheme, in S21, the method for processing the culture supernatant is as follows: first centrifugation, then ultrafiltration concentration, and then filtration to obtain animal umbilical cord extract; preferably, the centrifugation speed is 1000-3000 rpm, the ultrafiltration concentration factor is at least 10 times, and the interception of components with a molecular weight of not less than 3 kDa; In S22, the homogenization process includes first homogenizing under normal pressure to obtain crude liposomes, pre-cooling them to 4-8°C, and then homogenizing them under high pressure at 130-180MPa to obtain liposome-encapsulated animal umbilical cord extract; preferably, the particle size of the liposome-encapsulated animal umbilical cord extract is 0.1-1μm.

[0012] In this specific scheme, umbilical cord tissue granules with Wharton's jelly are obtained, for example, by the following method: fresh umbilical cords from animals that have given birth naturally (without the use of drugs during the delivery process) are collected, rinsed repeatedly to remove blood clots, oozing tissue, etc., and then the cleaned tissue is cut into 2-4 cm long segments, the blood vessels (3 blood vessels) are removed, Wharton's jelly is retained, and then it is cut into 1-3 square millimeter tissue granules to obtain umbilical cord tissue granules with Wharton's jelly retained.

[0013] In this specific method, umbilical cord tissue granules are cultured, for example, by transferring them into a culture flask and directly adding culture medium. During the culture process, the medium is changed every 3-5 days according to the growth of the umbilical cord cells. Once the cells have grown and covered the bottom of the culture flask, the cells are harvested and then cultured or cryopreserved as needed. Each time cells are harvested, the culture supernatant is collected. This invention does not have special requirements for the cell culture medium; any culture medium used for umbilical cord cell culture is suitable for this invention.

[0014] In this specific scheme, centrifugation is used to remove cell debris and other substances. Then, ultrafiltration is used to concentrate and enrich active ingredients and desalinate small molecules. Finally, filtration is performed, for example, using a 0.22μm filter membrane, to remove bacteria, fungi, and other large microorganisms.

[0015] In this specific scheme, the mass ratio of animal umbilical cord extract to liposome emulsifier is, for example, 15:1, 16:1, 19:1, or 22:1, preferably 19:1. The amount of liposome emulsifier is sufficient to encapsulate the animal umbilical cord extract. Atmospheric homogenization is performed, for example, at room temperature and a low rotation speed of 1500-2000 rpm, to obtain crude liposomes with a particle size of 10-100 μm. It should be noted that high-pressure homogenization is performed, for example, in a high-pressure microfluidic homogenizer, and can be performed multiple times, such as 2, 3, or 4 times, to control the particle size of the liposome-encapsulated animal umbilical cord extract between 0.1-1 μm.

[0016] In one specific embodiment, the liposome emulsifier in S22 is PRO-LIPO NEO liposome emulsifier. PRO-LIPO NEO liposome emulsifier is a commercially available product containing 24.95 parts by weight of lecithin, 0.02 parts by weight of sunflower seed oil, 0.03 parts by weight of tocopherol, and 75 parts by weight of 1,3-propanediol. It should be noted that the animal umbilical cord extract obtained from the culture supernatant in this invention exhibits better stability only after being encapsulated with PRO-LIPO NEO liposome emulsifier. Encapsulation with other liposome emulsifiers results in poor stability, failing heat resistance and / or cold resistance tests.

[0017] In one specific embodiment, the active peptide composition of the lyophilized powder A comprises the following components by weight: Arginine / lysine polypeptide: 0.008-0.015 parts by weight; Acetyl hexapeptide-1: 0.048-0.055 parts by weight; Tripeptide-10 Citrulline: 0.008-0.015 parts by weight Palmitoyl tripeptide-5: 0.0008-0.0015 parts by weight; The excipients in the freeze-dried powder of Agent A include one or more of mannitol, trehalose, soluble collagen, and pullulanose.

[0018] In this specific formulation, the active peptide composition primarily functions synergistically with liposome-encapsulated animal umbilical cord extract to endogenously revitalize cells and strengthen the skin barrier. The selection of the active peptide composition mainly considers three indicators: collagen-boosting capacity, MMP-1-decreasing capacity, and DEJ protein-boosting capacity of the basement membrane. In this invention, the active peptide composition is preferably composed of arginine / lysine polypeptide, acetyl hexapeptide-1, tripeptide-10 (citrulline), and palmitoyl tripeptide-5, which exhibits better synergistic effects with the liposome-encapsulated animal umbilical cord extract of this invention. The main function of the arginine / lysine peptide is to send signals to skin cells, stimulating the synthesis of collagen and elastin, enhancing the skin barrier function, and reducing moisture loss, thereby improving wrinkles and skin elasticity. The main mechanism of action of acetyl hexapeptide-1 is similar to that of botulinum toxin, inhibiting excessive release of neurotransmitters, relaxing facial muscle contractions, and reducing dynamic wrinkles (such as crow's feet and forehead wrinkles). The main mechanism of action of tripeptide-10 citrulline is to act as a carrier to help other ingredients penetrate, while promoting collagen regeneration and repairing damaged protein structures, enhancing skin hydration, and combating static wrinkles and sagging. The main mechanism of action of palmitoyl tripeptide-5 is to activate transforming growth factor-β (TGF-β), promoting the production of collagen, fibronectin, and glycosaminoglycans, repairing the damaged dermis, and significantly improving deep wrinkles and skin firmness. All peptides used in this invention can be synthesized chemically and are commercially available. Furthermore, in this specific embodiment, the excipient is preferably a combination of mannitol and soluble collagen, both of which are commercially available.

[0019] In one specific formulation, the humectants in the solvent of agent B include one or more of the following: glycerin, sodium hyaluronate, hydrolyzed hyaluronic acid, tremella polysaccharide, hydrolyzed sugars, Alcaligenes polysaccharide, and hydrolyzed pectin. The soothing agents in the solvent for Agent B include one or more of D-panthenol, β-glucan, and hydrolyzed royal jelly protein. The repair agents in the solvent for Agent B include one or more of vitamin B3, oxyphenamine, and tetrahydromethylpyrimidine carboxylic acid; The stabilizers in the solvent of agent B include one or more of 1,2-pentanediol, 1,2-hexanediol, and p-hydroxyacetophenone.

[0020] In this specific formulation, the moisturizer is preferably composed of four ingredients: glycerin, sodium hyaluronate, hydrolyzed hyaluronic acid, and tremella polysaccharide. Glycerin forms hydrogen bonds with water through three hydroxyl groups, continuously capturing environmental moisture and maintaining the hydration of the stratum corneum; it is preferably present in 2-5 parts by weight. Sodium hyaluronate is a high-molecular-weight sodium hyaluronate, preferably with a molecular weight of not less than 1.8. MDa (methyl methacrylate) can form a three-dimensional mesh-like hydrating film on the skin surface, reducing TEWL (transient endothelial wound healing) and providing immediate smoothness; preferably, it is 0.02-0.06 parts by weight. Hydrolyzed hyaluronic acid (enzyme-cleaved oligomeric sodium hyaluronate) is a small-molecule hyaluronic acid, preferably with a molecular weight not exceeding 10 kDa. It can penetrate the stratum corneum, carrying moisture to fill intercellular spaces and increasing endogenous water capacity. Combined with large-molecule sodium hyaluronate, it can significantly improve the skin's water-locking and water-retaining capabilities, providing long-lasting moisturizing effects; preferably, it is 0.01-0.05 parts by weight. Tremella fuciformis polysaccharide is a highly branched-chain acidic polysaccharide with high hydrogen bond water-locking ability, forming a soft film and enhancing the skin barrier's water tightness; preferably, it is 0.01-0.05 parts by weight. When the moisturizer is composed of these four components, they work synergistically to significantly increase the moisture content of the stratum corneum, strengthen water absorption, improve skin elasticity, and enhance skin softness. It should be noted that all four components are commercially available and the raw materials are readily available.

[0021] In this specific formulation, the soothing agent is preferably composed of three components: D-panthenol, β-glucan, and hydrolyzed royal jelly protein. D-panthenol, commonly known as provitamin B5, can be converted into pantothenic acid in the skin, synthesizing coenzyme A, accelerating barrier lipid synthesis, and reducing erythema and stinging. It is preferably present in 0.3-0.5 parts by weight. β-glucan binds to TLR-2 / Dectin-1 in keratinocytes, inhibiting the NF-κB pathway, reducing IL-6 / IL-8 release, and rapidly reducing redness. It is preferably present in 0.009-0.05 parts by weight. Hydrolyzed royal jelly protein, obtained from the hydrolysis of royal jelly, is a complex composed of 2.25% hydrolyzed royal jelly protein, 25% butylene glycol, and 72.75% water. Its function is to effectively block TRPV1 signaling, reducing neurogenic stinging and heat sensitivity. It is preferably present in 1-3 parts by weight. It should be noted that all three components are commercially available, and the raw materials are readily available.

[0022] In this specific formulation, the repair agent is preferably composed of a combination of vitamin B3, oxyphenamine, and tetrahydromethylpyrimidine carboxylic acid, with vitamin B3 used to enhance NAD+. +The first component, containing 1-3 parts by weight, enhances DNA repair enzyme activity, promotes ceramide synthesis, and tightens the skin barrier. The second component, containing 10% Lactobacillus / soybean fermentation product filtrate, 10% butylene glycol, 2% 1,2-pentanediol, and 78% water, is a repairing composition. Its main function is to strengthen keratinocyte differentiation and restore microecological balance. The third component, containing 0.5-1.5 parts by weight, is a white crystalline powder prepared by fermentation of an extreme halophilic bacterium. It effectively protects the skin, resists UV / heat / oxidative stress, and rapidly repairs the integrity of the keratinized capsule, earning it the title of "skin cell shield." The fourth component, containing 0.2-0.5 parts by weight, is also present. It should be noted that all four components are commercially available and the raw materials are readily available.

[0023] In this specific formulation, the stabilizer is preferably composed of a combination of 1,2-pentanediol, 1,2-hexanediol, and p-hydroxyacetophenone. 1,2-pentanediol possesses dihydroxy water-binding and moisturizing properties, reduces TEWL (transient volatile organic compounds), and has antibacterial and preservative effects, and is preferably present in 1.5-3 parts by weight. 1,2-hexanediol possesses broad-spectrum antibacterial effects and is preferably present in 0.5-1 parts by weight. p-Hydroxyacetophenone possesses highly effective antibacterial and antioxidant effects and is preferably present in 0.5-1 parts by weight. The three components work synergistically to effectively inhibit bacteria and prevent spoilage, moisturize, and provide antioxidant effects without the addition of preservatives, thereby stabilizing the system. It should be noted that all four components are commercially available, and the raw materials are readily available.

[0024] In one specific embodiment, the lyophilized powder of agent A is obtained by the following method: After adding solvent to a vacuum homogenizing emulsifier, under a pressure of -0.04 to -0.06 MPa and stirring conditions, an active peptide composition, excipients, and liposome-encapsulated animal umbilical cord extract are added sequentially. The mixture is then homogenized at 1500-2000 rpm. After homogenization, the mixture is filtered through a 200-600 mesh filter cloth at a temperature not exceeding 30°C to obtain a lyophilized solution. The lyophilized solution is then filled into containers, partially stoppered, and placed in a vacuum freeze dryer for lyophilization. Preferably, during the preparation of the lyophilized powder of agent A, the system temperature after adding the active peptide composition is adjusted to 40°C to 45°C, and the system temperature when adding the liposome-encapsulated animal umbilical cord extract is adjusted to 30°C to 35°C.

[0025] In one specific embodiment, the solvent solution for agent B is obtained by the following method: After adding solvent to a vacuum homogenizing emulsifier, a humectant, a soothing agent, a repairing agent, and a stabilizer are added sequentially at 70°C to 75°C, -0.04 to -0.06 MPa, and under stirring conditions. The mixture is then filtered through a 200-600 mesh filter cloth at 30°C to obtain the solvent solution for agent B, which can then be filled into containers. Preferably, during the preparation of the solvent solution for agent B, after adding the humectant, homogenization is performed at 2000-2500 rpm for 3-4 minutes; after adding the soothing agent, homogenization is performed at 2000-2500 rpm for 3-4 minutes.

[0026] The main beneficial effects of this invention include: 1) The freeze-dried powder of Agent A works synergistically with the solvent of Agent B to regulate both endogenous and exogenous skin damage factors. It significantly upregulates the levels of type I collagen and elastin, indicating a significant effect on strengthening the skin barrier. In addition, subject tests show that it also has excellent moisturizing, repairing, firming, and exfoliating effects.

[0027] 2) In the freeze-dried powder of Agent A, the core bioactive components are a combination of animal umbilical cord extract and active peptides encapsulated in liposomes. The two work synergistically to target cell activation and regeneration, enhance the vitality within the barrier, and play a role in endogenously activating cell regeneration and strengthening the barrier.

[0028] 3) Liposome-encapsulated animal umbilical cord extract: The animal umbilical cord extract is homogenized with a specific emulsifier and culture supernatant to encapsulate the animal umbilical cord extract. The liposome-encapsulated animal umbilical cord extract has good stability and permeability, which is conducive to promoting transdermal or cellular absorption, prolonging the active release time and improving utilization efficiency.

[0029] 4) The skin protectant provided by this invention is an EWG all-green ingredient and does not contain preservatives, fragrances, pigments or other potential sensitizing ingredients. All subjects agreed that there were no adverse reactions. Attached Figure Description

[0030] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a particle size distribution diagram of the animal umbilical cord extract encapsulated in PRO-LIPO NEO liposomes before and after high-pressure homogenization in Example 1. Figure 2 This is a product image of the two-in-one dual-compartment skin protectant in Test Example 2, which includes lyophilized powder (A) and solvent (B). Figure 3 This is a schematic diagram illustrating the application method of the skin protectant in Test Example 2; Figure 4 This is the result of the change in skin stratum corneum moisture content before and after the test in Test Example 2; Figure 5 This is the result of the change in the transepidermal water loss (TEWL) value of the skin before and after the test in Example 2; Figure 6 This is the result of the change in the skin firmness parameter R0 value before and after the test in Test Example 2; Figure 7 This is the result of the change in the DI value of the scaling index before and after the test in Test Example 2; Figure 8 These are photos of the scales from subjects D0 and D28 in test case 2; Figure 9 These are photos of the scales from subjects D0 and D28 in test case 2; Figure 10 These are photos of the scales from subjects D0 and D28 in test case 29; Figure 11 This is the result of the change in skin wrinkle area before and after the test in Example 2; Figure 12 This is the grading standard for crow's feet wrinkles in Test Example 2; Figure 13 These are photos of crow's feet from test subject D0 and D28 in test case 2. The two images on the left are of subject D0, and the two images on the right are of subject D28. Figure 14 These are photos of crow's feet from test subject D0 and D28 in test case 2. The two images on the left are of subject D0, and the two images on the right are of subject D28. Figure 15 These are photos of crow's feet from test subject D0 and D28 in test case 2. The two images on the left are of subject D0, and the two images on the right are of subject D28. Detailed Implementation

[0032] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Preparation of liposome-encapsulated animal umbilical cord extract.

[0034] S1. Preparation of animal umbilical cord extract: S11. Umbilical Cord Collection: Fresh umbilical cords were collected from domestic pigs that had given birth naturally (the sows were Topke Landrace pigs, originating in China, and free from any infectious diseases). No drugs were used during the farrowing process. The entire collection process was pig-friendly and in accordance with animal welfare and ethics. The fresh pig umbilical cords were repeatedly rinsed to remove blood clots and oozing tissue. The cleaned tissue was cut into 2-4 cm long segments, blood vessels (3 in total) were removed, Wharton's jelly was retained, and the segments were further cut into 1-3 mm² tissue particles. The tissue particles were transferred to culture flasks and cultured in a-MEM medium. The medium was changed every 3-5 days depending on the growth of the umbilical cord cells. Once the cells had covered the bottom of the culture flask, they were harvested and then cultured or cryopreserved as needed. The culture supernatant was collected each time cells were harvested.

[0035] S12. Perform centrifugation at 2000 rpm (to remove cell debris and other substances), ultrafiltration concentration (10-fold concentration, molecular weight cutoff of 3 kDa and above), and sterilization via 0.22-micron filtration membrane to obtain animal umbilical cord extract (pig umbilical cord extract). It is a colorless to pale yellow clear liquid with a slight characteristic animal protein odor. The pH value (25℃) is 6.0–8.5. Microbiological and harmful heavy metal indicators comply with the "Cosmetic Safety Technical Specifications" (2015 edition). The main components are active protein and water, with a typical content of 2% active protein and 98% water. Storage conditions: Sealed, protected from light, frozen at -15 to -20℃.

[0036] S2, Liposome Emulsifiers: Three commercially available liposome emulsifiers were selected: Picocare SE, PRO-LIPONEO, and PrimeLipid PI. The components of each liposome emulsifier are detailed in Table 1. Table 1: Formulation of liposome emulsifiers

[0037] S3. Preparation of liposome-encapsulated animal umbilical cord extract: S31. At room temperature, the animal umbilical cord extract obtained in S12 is mixed with the three liposome emulsifiers in S2 at a mass ratio of 19:1 and stirred evenly. The mixture is then homogenized at 1500-2000 rpm under normal pressure to obtain crude liposomes with a particle size of 10-100 micrometers (μm).

[0038] S32. The crude liposomes from S31 are processed by a high-pressure microfluidic homogenizer at a homogenization pressure of 130-180 MPa and homogenized three times to obtain fine liposomes, i.e., liposomes encapsulating animal umbilical cord extract.

[0039] S4. Stability testing was performed on the obtained liposome-encapsulated animal umbilical cord extract: Heat resistance test: The test conditions are 40±1℃ for 24 hours. If precipitation occurs, it indicates instability. Cold resistance test: The test conditions are to place it at -10±1℃ for 24 hours. If precipitation occurs, it is considered unstable. Test results showed that the animal umbilical cord extract encapsulated in PRO-LIPO NEO did not precipitate in either the heat or cold resistance tests, while the animal umbilical cord extract encapsulated in Picocare SE precipitated in both heat and cold resistance tests. The animal umbilical cord extract encapsulated in PrimeLipid PI did not precipitate in the heat resistance test, but showed slight precipitation in the cold resistance test. Therefore, the stability results indicate that the animal umbilical cord extract encapsulated in PRO-LIPO NEO exhibited the best stability.

[0040] Particle size tests before and after high-pressure homogenization: The crude liposomes of animal umbilical cord extract encapsulated with PRO-LIPO NEO before high-pressure homogenization had a particle size of 10-100 μm, and the particle size after high-pressure homogenization was 0.1-1 μm; the crude liposomes of animal umbilical cord extract encapsulated with Picocare SE before high-pressure homogenization had a particle size of 50-200 μm, and the particle size after high-pressure homogenization was 5-10 μm; the crude liposomes of animal umbilical cord extract encapsulated with PrimeLipidPI before high-pressure homogenization had a particle size of 50-200 μm, and the particle size after high-pressure homogenization was 0.1-10 μm. Regarding the particle size after high-pressure homogenization, the particle size of animal umbilical cord extract encapsulated with PRO-LIPO NEO was 0.1-1 μm, which is much smaller than the diameter of human skin pores (30-50 μm). Combined with the lipophilic group structure after encapsulation, this facilitates penetration into the skin. Figure 1 The particle size distribution of the PRO-LIPO NEO-encapsulated animal umbilical cord extract before and after high-pressure homogenization is shown.

[0041] In summary, due to the good stability and particle size distribution of the animal umbilical cord extract encapsulated with PRO-LIPO NEO, it will be used as a liposome-encapsulated animal umbilical cord extract in the future.

[0042] Example 2: Preparation of skin protectant.

[0043] 1) Raw materials: Mannitol (trade name: mannitol, commercially available); Soluble collagen (trade name: recombinant human collagen (BSS) v01, commercially available); Liposome-encapsulated animal umbilical cord extract (the animal umbilical cord extract encapsulated with PRO-LIPO NEO in Example 1). Arginine / Lysine peptide (trade name: Arginine / Lysine peptide, commercially available); Acetyl hexapeptide-1 (trade name JYMed® ACHEX-1 AP, commercially available); Palmitoyl tripeptide-5 (trade name: palmitoyl tripeptide-5, commercially available); Tripeptide-10 citrulline (trade name: SpecPed®CIT TR10P, commercially available); Glycerin (trade name: glycerin, commercially available); Sodium hyaluronate (trade name Hybloom® High Molecular Weight Sodium Hyaluronate (HA-THM), commercially available); Hydrolyzed hyaluronic acid (trade name NanoHA® Enzymatically hydrolyzed oligomeric sodium hyaluronate, commercially available); Tremella polysaccharide (trade name: Tremella fuciformis fruiting body extract, commercially available) D-Panthenol (trade name: D-Panthenol, commercially available); β-glucan (trade name: Celec® β-glucan, commercially available); Hydrolyzed royal jelly protein (trade name ROYALBIOCYTE BG, commercially available); Vitamin B3 (trade name: Vitamin B3, available commercially); Ottim (product name: Ottim OPTIMEALTH 10W, available in stores); Tetrahydromethylpyrimidine carboxylic acid (trade name: SYTOIN® Ectoin, commercially available); p-Hydroxyacetophenone (trade name SymSave® H, commercially available); 1,2-Pentanediol (trade name: Hydrolite® 5 green, commercially available); 1,2-Hexanediol (trade name: Hydrolite® 6O, commercially available).

[0044] 2) Preparation method: Preparation of S1 and A agent lyophilized powder: S11. With the scraper stirring (20 rpm) on, add arginine / lysine peptide, acetyl hexapeptide-1, tripeptide-10 citrulline, and palmitoyl tripeptide-5 (i.e., active peptide composition, the order of addition is not limited) sequentially to the vacuum homogenizing emulsifier (deionized water is directly weighed into the emulsifier). Vacuum the emulsifier to -0.06 MPa, heat to 45°C while stirring, stir for 10 minutes, add mannitol and soluble collagen (i.e., excipients, the order of addition is not limited), and continue stirring for 10 minutes until completely dissolved and homogeneous. Cool to 35°C, add liposome-encapsulated animal umbilical cord extract, stir at 35 rpm for 5 minutes, then homogenize at 2000 rpm for 4 minutes. After that, cool to below 30°C and filter through a 400-mesh filter cloth to obtain the lyophilized liquid. S12. Fill the lyophilized liquid into 5ml vials of a powder + liquid dual-compartment bottle, with a net content of 2.6ml / vial. Place the filled vials on a tray, partially stopper them, and put them into a vacuum freeze dryer. Set the freeze-drying process parameters: Pre-freezing: temperature -45℃, duration 4 hours; First drying: first stage temperature -15℃, vacuum degree -0.01mbar, duration 7 hours; second stage temperature -10℃, vacuum degree -0.01mbar, duration 10 hours; Desorption drying: first stage temperature 15℃, vacuum degree -0.01mbar, duration 5 hours; second stage temperature 25℃, vacuum degree -0.015mbar, duration 5 hours. After setting, start the freeze dryer, and the freeze-drying will be completed automatically. Then, purge with nitrogen, stopper the vial, and remove the freeze-dried powder of agent A, with a net content of 0.10g / vial.

[0045] Preparation of solvent solutions for agents S2 and B: S21. With the scraper stirring (25 rpm) on, heat the deionized water in the vacuum homogenizing emulsifier to 75°C. Then, add glycerin, sodium hyaluronate, hydrolyzed hyaluronic acid, and tremella polysaccharide (i.e., moisturizer, the order of addition is not limited) in sequence. After adding, stir for 10 minutes to disperse evenly, then homogenize at 2500 rpm for 3 minutes, and continue stirring at 30 rpm for 5 minutes. Then, add D-panthenol, β-glucan, and hydrolyzed royal jelly protein (i.e., soothing agent, the order of addition is not limited) in sequence. After adding, stir for 5 minutes, then homogenize at 2000 rpm for 4 minutes. Cool to 50°C. Then, add vitamin B3, oxyphenamine, tetrahydromethylpyrimidine carboxylic acid, 1,2-pentanediol, 1,2-hexanediol, and p-hydroxyacetophenone (i.e., repair agent and stabilizer) in sequence. Continue to cool to 35°C with slow stirring at 25 rpm. Filter the material through a 400-mesh filter cloth to obtain the solvent material. S22. Fill the solvent contents of S21 into the solvent bottle of the powder + liquid two-combination double-compartment bottle, and fill the net content of 6.0ml / bottle to obtain solvent solution B. S3. Assembly of lyophilized powder A and solvent B: Assemble the lyophilized powder A and solvent B into a powder + liquid two-compartment bottle and cap it to obtain the facial skin protectant of an (animal) umbilical cord extract in Example 1: Net content (0.10g lyophilized powder + 6.0ml solvent) / bottle. Figure 2 As shown.

[0046] The material components of this embodiment are detailed in Tables 2-13: Table 2: Formulation of Lyophilized Powder A in Experimental Group 1

[0047] Table 3: Formulation of solvent B in experimental group 1

[0048] Table 4: Formulation of Lyophilized Powder A for Experimental Group 2

[0049] Table 5: Formulation of solvent B in experimental group 2

[0050] Table 6: Formulation of Lyophilized Powder A in Experimental Group 3

[0051] Table 7: Formulation of solvent B in experimental group 3

[0052] Table 8: Formulation of Lyophilized Powder A for Experimental Group 4

[0053] Table 9: Formulation of solvent B in experimental group 4

[0054] Table 10: Formulation of Lyophilized Powder A for Experimental Group 5

[0055] Table 11: Formulation of solvent solution B for experimental group 5

[0056] Table 12: Formulation of Lyophilized Powder A for Experimental Group 6

[0057] Table 13: Formulation of solvent solution B for experimental group 6

[0058] Comparative Example 1: Compared with experimental group 1 in Example 2, the difference is that in this comparative example, the animal umbilical cord extract is in a non-liposome encapsulated form and does not contain an active peptide composition. The preparation method of the lyophilized powder of agent A is as follows: Preparation of S1 and A agent lyophilized powder: S11. With the wall-scraping agitator on (20 rpm), add mannitol and soluble collagen (i.e., excipients, the order of addition is not limited) to the vacuum homogenizing emulsifier (deionized water is directly weighed into the emulsifier), and continue stirring for 10 minutes until completely dissolved and homogeneous; cool to 35°C, add the animal umbilical cord extract obtained in S12 of Example 1, stir at 35 rpm for 5 minutes, then homogenize at 2000 rpm for 4 minutes, then cool to below 30°C, and filter through a 400-mesh filter cloth to obtain the freeze-dried liquid; S12, the same as step S12 in Example 2; In this comparative example, the preparation processes of solvent B and skin protectant are the same as in Example 2.

[0059] The formulation of the lyophilized powder A in this comparative example is shown in Table 14 (the formulation of the solvent B is the same as that of experimental group 1 in Example 2): Table 14: Formulation of Lyophilized Powder A for Comparative Example 1

[0060] Comparative Example 2 Compared with experimental group 1 in Example 2, the difference is that in this comparative example, the animal umbilical cord extract is in a non-liposome encapsulated form, and the preparation method of the lyophilized powder of agent A is as follows: Preparation of S1 and A agent lyophilized powder: S11. With the wall-scraping agitator on (20 rpm), add arginine / lysine peptide, acetyl hexapeptide-1, tripeptide-10 citrulline, and palmitoyl tripeptide-5 (i.e., active peptide composition, the order of addition is not limited) sequentially to the vacuum homogenizing emulsifier (deionized water is directly weighed into the emulsifier). Vacuum the emulsifier to -0.06 MPa, heat to 45°C while stirring, stir for 10 minutes, add mannitol and soluble collagen (i.e., excipients, the order of addition is not limited), and continue stirring for 10 minutes until completely dissolved and homogeneous. Cool to 35°C, add the animal umbilical cord extract obtained in S12 of Example 1, stir at 35 rpm for 5 minutes, then homogenize at 2000 rpm for 4 minutes, then cool to below 30°C, and filter through a 400-mesh filter cloth to obtain the lyophilized liquid. S12, the same as step S12 in Example 2; In this comparative example, the preparation processes of solvent B and skin protectant are the same as in Example 2.

[0061] The formulation of the lyophilized powder A in this comparative example is shown in Table 15 (the formulation of the solvent B is the same as that of experimental group 1 in Example 2): Table 15: Formulation of Lyophilized Powder A for Comparative Example 2

[0062] Comparative Example 3: Commercially available "Chongyuan Collagen Soothing Repair Ampoule", production date 2024120102.

[0063] Test Example 1: Efficacy Test.

[0064] 1. Test samples: Skin protectant of experimental group 1 in Example 2 (hereinafter referred to as Example 1 group. According to the analysis of the proportion of the main active ingredients in the formula, among experimental groups 1-6 in Example 2, the worst effect is experimental group 1. Therefore, experimental group 1 will be used as the test sample in the future. The test results of other experimental groups will be better than those of experimental group 1), and skin protectants of comparative examples 1 to 3 (the skin protectant of comparative example 1 is referred to as comparative example 1 group, the skin protectant of comparative example 2 is referred to as comparative example 2 group, and the skin protectant of comparative example 3 is referred to as comparative example 3 group). 2. Raw materials and equipment: 2.1 Cells: Human dermal fibroblasts (Beina Biotechnology, BNCC356201); 2.2 Reagents: DMEM (Jining Industrial Co., Ltd., JN-CC2736), FBS (Avantor Seradigm, 76294-180), Penicillin / Streptomycin (Gibco, 15070-063), Trypsin (Gibco, 25300-062), PBS (Gibco, 20012027), VC (Solebio, SV8120), DMSO (Solebio, D8371), MTT (Solebio, M8180), Human Col IELISA KIT (Boson, bsk11046); 2.3 Instruments: CO2 cell incubator (Eppendorf, C170), thermometer and hygrometer (Medex, JB914F), fluorescence inverted microscope (Olympus, CKX53), microplate reader (PerkinElmer, HH34940001), constant temperature water bath (Shanghai Lichen Bangxi, HH-2), centrifuge (Shanghai Baju Industrial Co., Ltd., NMDTW-E026), pipettes (Eppendorf, 1-10ul / 1000ul / 10-100ul), cell counter (Thermo Fisher Scientific, Counterss3), refrigerator (Haier), clean bench (Shanghai Shangjing Purification Equipment Co., Ltd., CA-1390-2), analytical balance (Ohaus, PWN224ZH); 2.4 Culture conditions for samples and control groups Control group: DMEM complete medium (basal medium: serum: antibiotics = 89:10:1); Negative control group (NC): Human dermal fibroblasts + DMEM complete medium (basal medium: serum: antibiotics = 89:10:1, cells resuspended in complete medium). Positive control group PC: human dermal fibroblasts + DMEM complete medium containing 10 μg / mL VC (basal medium: serum: antibiotics = 89:10:1, VC diluted to 10 μg / mL with complete medium). Sample group: human dermal fibroblasts + DMEM complete medium containing the sample concentration (basal medium: serum: antibiotics = 89:10:1, dilute the sample to the target concentration with complete medium). 2.5 Experimental Design and Methods 2.5.1 Cell viability assay: Collect cells in the logarithmic growth phase and adjust the cell concentration to 2.0 × 10⁻⁶. 5 Cells were seeded at a concentration of 100 μL / mL into 96-well plates, with each well containing 100 μL of culture medium. After culturing in a CO2 cell incubator for 24 h, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, the sample groups were administered the drug at concentration gradients of 5% (V / V), 2.5% (V / V), 1.25% (V / V), 0.625% (V / V), and 0.313% (V / V). The blank group and negative control group were given normal culture medium, while the positive control group was given DMEM complete culture medium containing 10 μg / mL VC. After culturing for another 24 h, the MTT assay was performed, and the absorbance (OD) value was read at 490 nm to calculate cell viability.

[0065] Calculation method: Cell viability (%) = OD value of the group to be calculated / OD value of the negative control group * 100% The calculation results are shown in Table 16: Table 16: Cell Viability Results in Efficacy Testing - Type I Collagen Test Cases

[0066] As can be seen from Table 16, cell viability is greater when the concentration of skin protectant is lower. Based on the effect of skin protectant concentration on cell viability, all sample groups were tested for the upregulation rate of type I collagen and elastin at concentrations of 0.625% and 0.313%.

[0067] 2.5.2 Type I collagen upregulation rate test: The cell concentration was adjusted to 1.0 × 10⁻⁶. 5 / mL, re-coated into 6-well plates, and cultured for 24 h, with each well containing 100 μL of culture medium. After culturing in a CO2 cell incubator for 24 h, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, the sample groups were administered drugs at concentration gradients of 0.625% (V / V) and 0.313% (V / V) (Example 1 group was given an additional 0.156% concentration, while Controls 1-3 did not have this concentration; however, the cell viability of the 0.625% concentration in Comparative Example 2 group was low, therefore, the type I collagen upregulation rate test at this concentration was not performed in Comparative Example 2 group). The blank group and negative control group were given normal culture medium, and the positive control group was given DMEM complete culture medium containing 10 μg / mL VC. Each group was set up in 3 replicates. After culturing for another 24 h, the supernatant was collected, stored at -80℃, and the type I collagen upregulation rate was tested using an ELISA kit. The test results are shown in Table 17. Table 17: Results of Type I Collagen Upregulation Rate

[0068] Note: In Table 17, P<0.001 indicates a highly significant difference; 0.001≤P<0.01 indicates a very significant difference; 0.01≤P<0.05 indicates a significant difference; and P≥0.05 indicates no significant difference.

[0069] As shown in Table 17, the upregulation rate of type I collagen in Example 1 group showed a highly significant difference, and the upregulation rate of type I collagen in Comparative Example 2 group also showed a highly significant difference; while Comparative Example 1 group without the addition of active peptide composition was listed below. Overall, the upregulation rate of Comparative Example 3 group was the worst.

[0070] 2.5.3 Elastin Upregulation Test: The cell concentration was adjusted to 1.0 × 10⁻⁶. 5 / mL, re-coated into 6-well plates, and cultured for 24 h, with each well containing 100 μL of culture medium. After culturing in a CO2 cell incubator for 24 h, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, the sample groups were administered drugs at concentration gradients of 0.625% (V / V) and 0.313% (V / V) (Example 1 group was given an additional 0.156% concentration, while Controls 1-3 did not have this concentration; however, the cell viability of the 0.625% concentration in Comparative Example 2 group was low, therefore, the elastin upregulation rate test at this concentration was not performed in Comparative Example 2 group). The blank group and negative control group were given normal culture medium, and the positive control group was given DMEM complete culture medium containing 10 μg / mL VC. Each group was set up in 3 replicates. After culturing for another 24 h, the supernatant was collected, stored at -80℃, and the elastin upregulation rate was tested using an ELISA kit. The test results are shown in Table 18. Table 18: Results of Elastin Upregulation

[0071] Note: In Table 18, P<0.001 indicates a highly significant difference; 0.001≤P<0.01 indicates a very significant difference; 0.01≤P<0.05 indicates a significant difference; and P≥0.05 indicates no significant difference.

[0072] As can be seen from Table 18, the elastin upregulation rate in Example 1 group showed extremely significant differences, while the 0.313% concentration in Comparative Example 1 group showed extremely significant differences, and the 0.625% concentration showed very significant differences. The effects of Comparative Example 2 and Comparative Example 3 groups were worse than those of Example 1 group and Comparative Example 1 group.

[0073] Based on a comprehensive comparison of the upregulation rates of type I collagen and elastin, the efficacy of group 1 in Example 1 was the best. Therefore, it can be concluded that experimental groups 2-6 in Example 2 would have better effects.

[0074] Test Example 2: Human Benefits - Moisturizing, Repairing, Firming, Exfoliating, Anti-wrinkle, Gentle and Non-irritating, Suitable for Sensitive Skin.

[0075] 1. Test sample: Experimental group 1 of Example 2 (hereinafter referred to as Example 1 group); 2. Test Objective: This test measures the skin's stratum corneum moisture content and the baseline transepidermal water loss rate (>15g / h / m) of 31 participants with sensitive skin (lactic acid stinging ≥3 points, positive). 2 Female or male subjects with loose skin and 1-4 grade outer corner wrinkles around the eyes were included in the study. Changes in skin stratum corneum moisture content, transepidermal water loss (TEWL) value, skin firmness parameter (R0) value, scaling index (DI) value, skin wrinkle area, and subject self-assessment were evaluated before and after using the test sample to verify the efficacy of the test sample in moisturizing, repairing, firming, exfoliating, and anti-wrinkle effects, as well as its gentle, non-irritating, and sensitive skin-friendly properties.

[0076] 3. Subjects 3.1 Subject criteria: Healthy women and men aged 30-60; Sensitive skin (those with a positive lactic acid stinging score of ≥3). Skin stratum corneum moisture content, baseline value of transepidermal water loss rate >15g / h / m 2 Loose skin; wrinkles at the outer corners of the eyes, grade 1-4; They can cooperate well with the test subjects and maintain a regular lifestyle during the research period; Able to read and understand all contents of the informed consent form, and willing to sign the informed consent form; During the testing period, participants agree not to use any cosmetics, drugs, or health products that may affect the results. Products with the same efficacy as the test samples must not be used during the testing period; During the testing period, no other cosmetics or personal care products may be used except for the test sample; Currently, I am not involved in, and will not be involved in, any other research involving the test area during the research period; No cosmetic procedures may be performed during the testing period.

[0077] 3.2 Exclusion criteria: Those with skin conditions that may affect their interpretation of test results; People with a high risk of allergies; Women who are pregnant, breastfeeding, or planning to become pregnant during the testing period; Those with severe heart, liver, or kidney dysfunction and severe immunodeficiency; Those with mental illness, severe endocrine disorders, or those taking oral contraceptives; Those who participated in drug clinical trials or other tests within the past 30 days, or those who have systematically used drugs that may affect test results within the past 2 weeks; or those who have taken orally or used topical beauty products that may affect test results within the past 2 weeks. Unable to cooperate with the tester; The researchers deemed the individual unsuitable to participate in this study.

[0078] 3.3 Withdrawal Criteria: Patients may withdraw from the clinical study due to complications, adverse events or other reasons during the study period, but the reason for withdrawal should be stated.

[0079] 3.4 Enrollment details: A total of 32 participants were included, aged 41-59 years. One participant did not complete the test (due to personal reasons and withdrew midway through the test); 31 participants provided valid data.

[0080] 4. Sample Usage Instructions: 4.1 Application area: Face; 4.2 Frequency of use and cycle of use: Use once in the morning or evening, 1.5 to 2 ml each time, for 28 consecutive days.

[0081] 4.3 Usage: Take one bottle of Bottle A (lyophilized powder) and one bottle of Bottle B (essence solvent). (In this test example, the lyophilized powder (A) and the solvent (B) were packaged separately.) Figure 2 The dual-compartment bottle shown contains two components: bottle A (containing lyophilized powder A) and bottle B (containing solvent B). Open both bottles, pour the solvent B from bottle B into the lyophilized powder A from bottle A, replace the stopper, and shake bottle A to completely dissolve the lyophilized powder. Remove the stopper and attach the dropper. Take a drop into your palm, apply it to your face with your fingers, and gently massage until fully absorbed. See detailed instructions for use. Figure 3 .

[0082] Notes: 1. Environmental conditions throughout the testing process: Temperature 21±1℃, Humidity: 50±10%RH. 2. Before testing, laboratory technicians blinded the samples and removed vendor-related information from the sample packaging to avoid affecting the test results. After testing, all test samples were collected.

[0083] 5 Test Items 5.1 Lactic acid stinging test: 50 μL of 10% lactic acid solution was dropped onto a single layer of non-woven fabric with a diameter of 0.8 cm. This non-woven fabric was applied to the nasolabial fold on one side of the subject, with distilled water used as a control on the other side. Researchers asked the subjects about their stinging sensation at 2.5 min and 5 min, and scored them using the "4-point method" in the table below. Subjects with a cumulative stinging sensation of ≥3 points on the lactic acid side relative to the distilled water side were considered to have lactic acid stinging and could be identified as having sensitive skin.

[0084] The lactic acid stinging rating scale is shown in Table 19: Table 19: Lactic acid stinging rating scale

[0085] 5.2 Instrument Testing Items For details on instrument testing items and instructions, please refer to Table 20: Table 20: Instrument Test Items and Test Instructions

[0086] 6. Self-assessment by the participants A questionnaire survey was conducted 28 days after the sample was used to evaluate the user's experience. The evaluation indicators are detailed in Table 21. Table 21: Evaluation Indicators

[0087] 7. Test Procedure: See Table 22 for details. Table 22: Test Procedures

[0088] 8. Test Results and Analysis 8.1 Descriptive Statistics The statistical analysis software SPSS 26.0 was used to perform statistical analysis on the data, calculating the mean, standard deviation, and rate of change of each parameter at each time point. The calculation formulas are as follows: Rate of change = [(Value after use - Value before use) / Value before use] × 100% 8.2 Normality Test If the normality test of the above data is performed and the asymptotic significance (two-tailed) value is >0.05, then the data series follows a normal distribution.

[0089] 8.3 Difference Analysis 8.3.1 Comparison before and after using samples: When both sets of data are normally distributed, use the paired T-test to analyze the difference between the two sets of data; when the two sets of data are not normally distributed, use the rank-sum test to analyze the difference between the two sets of data.

[0090] 8.3.2 Analysis of the self-assessment results of the participants: The binomial distribution method was used for statistical analysis. The expected value of the recognition level was set at 60% (the proportion of people with a score of ≥4). The statistical significance of the survey questions was tested using a 95% confidence interval.

[0091] 8.4 Criteria for Result Judgment 8.4.1 Moisturizing efficacy evaluation indicators: If the skin stratum corneum moisture content increases significantly (p<0.05) or the transepidermal water loss (TEWL) value decreases significantly (p<0.05) before and after using the sample, the test sample is considered to have moisturizing effect.

[0092] 8.4.2 Indicators for judging repair efficacy: If the transepidermal water loss (TEWL) value of the skin significantly decreased (p<0.05) and the moisture content of the stratum corneum of the skin significantly increased (p<0.05) before and after using the sample, the test sample is considered to have repairing effects.

[0093] 8.4.3 Criteria for determining firming efficacy: If the skin firmness parameter values ​​show a significant improvement before and after using the sample (p<0.05), then the test sample is considered to have a firming effect.

[0094] 8.4.4 Indicators for judging anti-wrinkle efficacy: If the wrinkle area decreased significantly before and after using the sample (p<0.05), the test sample is considered to have anti-wrinkle effect.

[0095] 8.4.5 Indicators for judging exfoliating efficacy: If the scaly index (DI) value decreased significantly before and after using the sample (p<0.05), then the test sample is considered to have exfoliating efficacy.

[0096] 8.4.6 Criteria for determining mildness and non-irritation: Human trials involving no fewer than 30 participants showed no adverse reactions during the entire process of using the sample.

[0097] 8.4.7 Criteria for determining suitability for sensitive skin: Human trials involving no fewer than 30 subjects with sensitive skin showed no adverse reactions during the entire process of using the sample.

[0098] 8.4.8 Questionnaire Conclusion: When the distribution of respondents' agreement with the self-reported questionnaires on moisturizing, firming, exfoliation, and anti-wrinkle issues was significantly higher than the expected value of 60% (p<0.05), it supported the efficacy of the sample in moisturizing, firming, exfoliation, and anti-wrinkle.

[0099] 8.5 Test Results 8.5.1 Skin stratum corneum moisture content (Corneometer) The test results of the skin stratum corneum moisture content are shown in Table 23, and Figure 4 : Table 23: Results of Skin Stratum Corneum Moisture Content Test

[0100] Table 23 and Figure 4 As can be seen, compared with before using the sample, there was a significant difference in the skin stratum corneum moisture content of the 31 subjects after 28 days of sample use, with a change rate of 44.85% compared with the baseline value. This indicates that the test sample has a moisturizing effect after 28 days of use. 8.5.2 Transepidermal water loss (TEWL) value (Tewameter) The results of the transepidermal water loss (TEWL) value test are shown in Table 24, and Figure 5 : Table 24: Test results of transepidermal water loss (TEWL) value of skin

[0101] From Table 24 and Figure 5 It can be seen that, compared with before the use of the sample, the transepidermal water loss (TEWL) values ​​of the 31 subjects after 28 days of sample use were significantly different, with a change rate of -12.04% compared with the baseline value. Combined with the results of the skin stratum corneum moisture content test, this indicates that the test sample has a repairing effect after 28 days of use.

[0102] 8.5.3 Skin firmness parameter R0 value (Cutometer) The test results for the skin firmness parameter R0 are detailed in Table 25, and... Figure 6 : Table 25: Results of Skin Tightness Parameter R0 Value Test

[0103] Table 25 and Figure 6As can be seen, compared with before using the sample, the skin firming parameter R0 value of the 31 subjects was significantly different after using the sample for 28 days, with a change rate of -15.29% compared with the baseline value. This indicates that the test sample has a firming effect after 28 days of use.

[0104] 8.5.4 Scaling Index (DI) value (VC20 + Corneofix) The test results for the scaly index (DI) value are detailed in Table 26, and... Figure 7 : Table 26: Test results of the scaling index (DI) value

[0105] Table 26 and Figure 7 As can be seen, compared with before using the sample, the scaling index (DI) values ​​of the 31 subjects were significantly different after using the sample for 28 days, with a change rate of -17.47% compared with the baseline value. This indicates that the test sample has an exfoliating effect after 28 days of use. Figure 8-10 Images of scales from subjects 8, 18, and 29 are shown. Figure 8 This is a picture of subject number 8. Figure 9 This is a picture of subject number 18. Figure 10 This is a picture of subject number 29.

[0106] 8.5.5 Skin wrinkle area (PRIMOS-CR) The results of the skin wrinkle area test are detailed in Table 27, and Figure 11 : Table 27: Results of Skin Wrinkle Area Test

[0107] Table 27 and Figure 11 As can be seen, compared with before using the sample, there was a significant difference in the area of ​​skin wrinkles in the 31 subjects after 28 days of use, with a change rate of -10.29% compared with the baseline value. This indicates that the test sample has an anti-wrinkle effect after 28 days of use.

[0108] 8.5.6 Subjects' self-assessment of their acceptance of the results The results of the survey on the participants' self-assessment and acceptance of their findings are detailed in Table 28. Table 28: Results of the Survey on Subjects' Self-Assessment and Acceptance of Results

[0109] Table 28 shows the self-assessment results. After 28 days of continuous use of the tested product, 100.00% of the participants agreed that it helped improve skin hydration; 100.00% of the participants agreed that the sample had a moisturizing effect; 93.55% of the participants agreed that the sample had an exfoliating effect; 100.00% of the participants agreed that it helped repair the skin; 100.00% of the participants agreed that it helped improve skin elasticity; 100.00% of the participants agreed that the sample had a firming effect; 100.00% of the participants agreed that it helped improve fine lines and wrinkles; 96.77% of the participants agreed that the sample had an anti-wrinkle effect; 100.00% of the participants agreed that the sample was generally satisfactory; 100.00% of the participants agreed that it was gentle (non-irritating); 100.00% of the participants agreed that it was suitable for sensitive skin; and 100.00% of the participants agreed that there were no adverse reactions.

[0110] 8.5.7 Assessment of Crow's Feet Before enrollment, the level of crow's feet (outer corner wrinkles) around the eyes was assessed. The evaluation criteria are detailed in [link to evaluation criteria]. Figure 12 Simultaneously, images of crow's feet wrinkles before and after the test were recorded for subjects #3, #22, and #28. Figure 13 This is a picture of subject number 3. Figure 14 This is a picture of subject number 22. Figure 15 This is a picture of subject number 28.

[0111] 8.6 Test Conclusion Under the test conditions, the test sample showed moisturizing, repairing, firming, exfoliating, and anti-wrinkle effects after 28 days of use. In a study involving 31 subjects aged 41-59 with sensitive skin, who used the sample once daily (morning or evening) for 28 consecutive days under normal conditions, no adverse reactions were observed in any of the 31 subjects. Based on these results, the sample is gentle, non-irritating, and suitable for sensitive skin.

[0112] Subject conclusion: Under the test conditions, based on the results of this consumer test, the sample is mild and non-irritating, and suitable for sensitive skin.

[0113] Adverse reaction assessment: No adverse reactions occurred in any of the 31 subjects throughout the test.

[0114] Detailed information about the subjects is shown in Table 29: Table 29: Subject Details

[0115] Test period: from May 12, 2025 to June 9, 2025, a total of 28 days.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A skin protectant containing a bioactive composition, characterized in that, It includes freeze-dried powder (A agent) and solvent (B agent), with a mass-to-volume ratio of 1g:55-65mL. Agent A lyophilized powder comprises the following components in parts by weight: Liposome-encapsulated animal umbilical cord extract: 3-5 parts by weight; Active peptide composition: 0.071-0.443 parts by weight; Excipients: 3.2-5.5 parts by weight; The active peptide composition includes at least two of the following: arginine / lysine polypeptide, acetyl hexapeptide-8, tripeptide-10 citrulline, tripeptide-1, acetyl hexapeptide-1, acetyl tetrapeptide-11, palmitoyl tripeptide-5, palmitoyl tripeptide-1, and palmitoyl tripeptide-1 / palmitoyl hexapeptide-12. The liposome-encapsulated animal umbilical cord extract was obtained through the following method: S21. The umbilical cord tissue granules with Wharton's jelly retained were cultured. When the cells were harvested, the culture supernatant was collected and processed to obtain animal umbilical cord extract. S22. The animal umbilical cord extract obtained in S21 is mixed with liposome emulsifier at a mass ratio of 15-22:1, and then homogenized to obtain liposome-encapsulated animal umbilical cord extract. Solution B comprises the following components by mass: Moisturizer: 2.04-5.16 parts by weight; Soothing agent: 1.31-3.55 parts by weight; Repairing agent: 1.7-5.0 parts by weight; Stabilizer: 2.5-5.0 parts by weight.

2. The skin protectant containing a bioactive composition as described in claim 1, characterized in that, In S21, the method for processing the culture supernatant is as follows: first centrifuge, then concentrate by ultrafiltration, and then filter to obtain animal umbilical cord extract; In S22, the homogenization process includes first homogenizing at normal pressure to obtain crude liposomes, pre-cooling them to 4-8℃, and then homogenizing them at high pressure at 130-180MPa to obtain liposome-encapsulated animal umbilical cord extract.

3. The skin protectant containing a bioactive composition as described in claim 2, characterized in that, In S21, the centrifugation speed is 1000-3000 rpm, the ultrafiltration concentration factor is at least 10 times, and the interception of components with a molecular weight of not less than 3 kDa; In S22, the particle size of the liposome-encapsulated animal umbilical cord extract is 0.1-1 μm.

4. The skin protectant containing a bioactive composition as described in claim 1, characterized in that, The liposome emulsifier in S22 is PRO-LIPO NEO liposome emulsifier.

5. The skin protectant containing a bioactive composition as described in claim 1, characterized in that, The active peptide composition of Agent A lyophilized powder comprises the following components by weight: Arginine / lysine polypeptide: 0.008-0.015 parts by weight; Acetyl hexapeptide-1: 0.048-0.055 parts by weight; Tripeptide-10 Citrulline: 0.008-0.015 parts by weight Palmitoyl tripeptide-5: 0.0008-0.0015 parts by weight; The excipients in the freeze-dried powder of Agent A include one or more of mannitol, trehalose, soluble collagen, and pullulanose.

6. The skin protectant containing a bioactive composition as described in claim 1, characterized in that, The humectants in the solvent of Agent B include one or more of the following: glycerin, sodium hyaluronate, hydrolyzed hyaluronic acid, tremella polysaccharide, hydrolyzed sugars, Alcaligenes polysaccharide, and hydrolyzed pectin. The soothing agents in the solvent for Agent B include one or more of D-panthenol, β-glucan, and hydrolyzed royal jelly protein. The repair agents in the solvent for Agent B include one or more of vitamin B3, oxyphenamine, and tetrahydromethylpyrimidine carboxylic acid; The stabilizers in the solvent of agent B include one or more of 1,2-pentanediol, 1,2-hexanediol, and p-hydroxyacetophenone.

7. The skin protectant containing a bioactive composition as described in claim 1, characterized in that, Agent A lyophilized powder is obtained by the following method: After adding solvent to a vacuum homogenizing emulsifier, under a pressure of -0.04 to -0.06 MPa and stirring conditions, an active peptide composition, excipients, and liposome-encapsulated animal umbilical cord extract are added sequentially. Then, homogenization is carried out at a speed of 1500-2000 rpm. After completion, the mixture is filtered through a 200-600 mesh filter cloth at a temperature not exceeding 30°C to obtain a lyophilized liquid. After the lyophilized liquid is filled, it is partially stoppered and placed in a vacuum freeze dryer for lyophilization.

8. The skin protectant containing a bioactive composition as described in claim 7, characterized in that, During the preparation of Agent A lyophilized powder, the system temperature after adding the active peptide composition is adjusted to 40℃ to 45℃, and the system temperature when adding the liposome-encapsulated animal umbilical cord extract is adjusted to 30℃ to 35℃.

9. The skin protectant containing a bioactive composition as described in claim 1, characterized in that, The solvent solution for agent B is obtained by the following method: After adding solvent to a vacuum homogenizing emulsifier, humectant, soothing agent, repairing agent and stabilizer are added sequentially under the conditions of 70℃ to 75℃, -0.04 to -0.06MPa and stirring. The solution is then filtered through a 200-600 mesh filter cloth at 30℃ to 35℃ to obtain the solvent solution for agent B, which can then be filled into containers.

10. The skin protectant containing a bioactive composition as described in claim 9, characterized in that, During the preparation of solvent solution B, after adding the humectant, homogenize at 2000-2500 rpm for 3-4 minutes; after adding the soothing agent, homogenize at 2000-2500 rpm for 3-4 minutes.

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