Cosmetic composition containing triple polishing enzyme and preparation method thereof

Abstract

The invention relates to the field of cosmetic compositions, in particular to a cosmetic composition containing triple polishing enzyme and a preparation method of the cosmetic composition. The triple polishing enzyme comprises protease, protease K and superoxide dismutase, the weight part ratio of the protease to the protease K to the superoxide dismutase is 1: (0.5-2): (0.5-2), the total mass percent of the triple polishing enzyme in the composition is 0.0002%-0.001%, and the invention further discloses a preparation method of the cosmetic composition. A triple polishing enzyme synergistic system is formed through scientific compounding, the integrated skin care effect under low concentration is achieved, and the defects of a traditional chemical stripping agent and the shortages of a single enzyme preparation are overcome; meanwhile, by optimizing the proportion of formula components, the composition forms a liquid crystal structure, so that long-acting dual moisturizing of internal water locking and external water sealing is realized, triple polishing enzyme can be wrapped and protected, and the problems that the composition is easy to oxidize and inactivate and low in transdermal efficiency are solved.

Description

Technical Field

[0001] This invention relates to the field of cosmetic compositions, and more specifically to a cosmetic composition containing a triple polishing enzyme and a method for preparing the same. Background Technology

[0002] As people's demands for skin care continue to rise, the cosmetics industry is constantly exploring new technologies to meet consumer needs. Traditional cleansing cosmetics mainly rely on surfactants to achieve their cleansing function. These chemical ingredients can easily over-cleanse the skin, damage its natural barrier, and cause problems such as dryness, tightness, and sensitivity. Some are also skin irritants, easily causing discomfort such as redness and itching in people with sensitive skin. In recent years, the application of enzymes as natural biocatalysts in cleansing cosmetics has received much attention. Among them, superoxide dismutase (SOD) can effectively remove superoxide anion free radicals, reduce inflammation and cell damage caused by oxidative stress, and enzymes are less irritating and have better biocompatibility than traditional chemical cleansers. However, using SOD alone is difficult to meet the complex and diverse beauty needs, and it is also easy to be inactivated in cosmetics. Its stability and efficiency in complex skin environments also need to be improved. Therefore, researchers have begun to explore scientific integration. Bioactive ingredients with different mechanisms of action and targets aim to achieve integrated effects of "soothing and priming, gentle purification, precise renewal, and barrier strengthening" at low concentrations, providing a safe and efficient keratin management solution for sensitive skin. However, how to effectively combine SOD with other enzyme components and maintain its stability and efficiency in complex skin environments remains a key focus and challenge in current research. Overall, existing cosmetic technologies still have many shortcomings. Not only is it difficult to achieve effective combination and synergistic effects of multiple bioactive ingredients, but it is also difficult to achieve the aforementioned integrated effects at low concentrations. Existing antioxidant and after-sun repair products still need optimization in terms of formulation composition and synergistic mechanisms, failing to meet consumers' demand for highly efficient and stable antioxidant products. At the same time, the proportions and interactions of various components in current cosmetic formulations are not optimal, making it difficult to maximize the synergistic effects of each active ingredient while ensuring product stability.

[0003] Chinese invention patent CN113952250B discloses a stable anti-photoaging composition and its preparation method. The composition includes ascorbic acid, superoxide dismutase (SOD), triolein, squalene, crabapple seed oil, poloxamer 407, PPG-12 / SMDI copolymer, hexanediol, glycerin, and deionized water. This composition has the advantages of synergistic efficacy and synergistic stability, providing multi-pathway and comprehensive anti-photoaging benefits. The gel-release system allows for the slow release of active ingredients, thereby improving the product's gentleness and skin tolerance. However, this patent still has issues with preparation and anti-oxidative aging effects that are not ideal. Chinese patent application CN120837379A discloses a precisely targeted modified SOD pH-responsive nanoliposome and a synergistic anti-photoaging post-sun repair composition. This composition is obtained by modifying SOD nanoliposomes with hyaluronic acid, wherein the core layer is SOD and the shell layer is a ternary composite liposome. This invention, by constructing a pH-responsive nanodelivery system, can accurately identify the pH decrease characteristics in UV-damaged areas, triggering the controlled release of SOD at the target site and simultaneously scavenging O2. 2- It also contains H2O2, which blocks the chain reaction of oxidative stress. However, this patent still has the problem that its synergistic anti-photoaging and post-sun repair effects are not ideal.

[0004] Current cosmetic technologies have many shortcomings. Traditional cleansing cosmetics use surfactants as the core cleansing ingredient, which can easily over-cleanse the skin, damage the skin's natural barrier, and cause problems such as dryness, tightness, and sensitivity. Some products are also skin irritants, with particularly significant effects on people with sensitive skin. Using superoxide dismutase alone cannot meet diverse beauty needs, and it is easily deactivated, with its stability and efficacy in complex skin environments needing improvement. At the same time, current technologies cannot effectively combine and synergistically enhance multiple bioactive ingredients. At low concentrations, it is difficult to achieve the integrated effects of "soothing and priming, gentle purification, precise renewal, and barrier strengthening." Existing antioxidant and after-sun repair products still need optimization in terms of formulation composition and synergistic mechanisms, failing to meet consumers' demand for highly effective and stable antioxidant products. Furthermore, the proportions and interactions of various components in current cosmetic formulations are not optimal, making it difficult to maximize the synergistic effects of each active ingredient while ensuring product stability. Summary of the Invention

[0005] To address the aforementioned technical problems, the first aspect of this invention provides a cosmetic composition comprising a triple polishing enzyme, the raw materials of which include: an aqueous phase, an oil phase, a triple polishing enzyme, and an adjuvant; the raw materials of the aqueous phase include water, xanthan gum, glycerol, 1,2-pentanediol, and sodium phytate; the raw materials of the oil phase include caprylic / capric triglycerides, behenol, macadamia ternifera seed oil, and glycolipids; the raw materials of the triple polishing enzyme include protease, proteinase K, and oxidoreductase; the oxidoreductase includes superoxide dismutase.

[0006] As an implementable example, the mass ratio of the protease, proteinase K and superoxide dismutase is 1:(0.5-2):(0.5-2).

[0007] Existing cleansing products often cause over-cleansing, damaging the skin's natural barrier and leading to discomfort such as dryness, tightness, sensitivity, and even redness, swelling, and itching in sensitive skin. This solution uses a triple polishing enzyme composed of protease, proteinase K, and superoxide dismutase as its core active ingredient, replacing the dominant role of traditional chemical cleansers. Leveraging the natural biocatalytic properties of enzymes, it achieves gentle enzymatic cleansing with low irritation and high biocompatibility, fundamentally avoiding the over-cleansing problem of surfactants and protecting the skin's natural barrier. Simultaneously, it addresses the limitations in efficacy and poor stability of using single superoxide dismutase (SOD) products. Existing technologies using only SOD... SOD is difficult to meet diverse beauty needs, and it is easily inactivated in cosmetic systems and complex skin environments, resulting in insufficient stability and efficiency. This application scientifically combines SOD with protease and proteinase K to form a triple polishing enzyme system. Through the complementary and synergistic effects of the mechanisms of action of each enzyme, it not only retains the core functions of SOD in anti-oxidation and free radical scavenging, but also achieves precise keratin management with the help of protease and proteinase K, solving the problem of the one-sided efficacy of single enzyme preparations. At the same time, the stable system constructed by the reasonable ratio of oil phase and water phase in the formula can reduce the probability of SOD inactivation in the formula and improve its stability and efficiency in complex skin environments.

[0008] Existing technologies struggle to integrate bioactive ingredients with different mechanisms of action, failing to achieve the integrated effects of "soothing, purifying, renewing, and strengthening the skin barrier" at low concentrations. Furthermore, active ingredients tend to aggregate and exhibit poor synergy. This application addresses these challenges by using a precise ratio of protease: proteinase K: SOD = 1:(0.5-2):(0.5-2) to achieve the orderly synergistic effect of the three enzymes. First, SOD scavenges free radicals and inhibits inflammation, creating a gentle environment for subsequent enzymatic reactions. Then, protease and proteinase K precisely and controllably cleave the intercellular junction proteins of keratinocytes, mimicking physiological keratin exfoliation. This forms a synergistic mechanism of "soothing-purifying-precise exfoliation," achieving integrated skincare efficacy at low concentrations and overcoming the technical difficulties of binding and poor synergy of active ingredients.

[0009] As an example of implementation, the triple polishing enzyme is present in the cosmetic composition at a mass percentage of 0.0002-0.001%.

[0010] As an feasible example, the mass ratio of the oil phase to the water phase is 1:(5-10).

[0011] As an example of implementation, the raw materials for preparing the triple polishing enzyme also include an enzyme protectant.

[0012] Furthermore, the enzyme protectant includes one of mannitol, xylitol, trehalose, sodium hyaluronate, or polyethylene glycol.

[0013] Furthermore, the mass ratio of mannitol to superoxide dismutase is 1:(10-30).

[0014] Mannitol is a classic bioactive ingredient protectant among polyols. It can form a hydration protective film on the surface of enzyme molecules by interacting with the protease, proteinase K, and superoxide dismutase molecules in triple polishing enzymes. This reduces conformational changes in enzyme molecules caused by environmental changes, such as temperature, pH fluctuations, and interactions with other components in the formulation. It effectively prevents enzyme denaturation and inactivation, improves the storage stability of triple polishing enzymes in cosmetic formulation systems, and ensures that it can maintain high bioactivity in the complex skin microenvironment after contact with the skin, thus fully exerting the efficacy of enzymatic reactions.

[0015] As an example of implementation, the additives include humectants and surfactants.

[0016] Furthermore, the moisturizer includes one of caprylyl glycol, 1,2-pentanediol, 1,6-hexanediol, ethylhexylglycerin, sorbitol, or polyglycerol-3.

[0017] Furthermore, the surfactant comprises one of sodium polyacrylamide dimethyl taurate, sodium acrylate / sodium acrylamide dimethyl taurate copolymer, carbomer, sodium lauroyl glutamate, sucrose stearate, or polyquaternium-73.

[0018] A second aspect of the present invention provides a method for preparing a cosmetic composition comprising a triple polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 50-100℃; S2. Mix the oil phase raw materials and stir to dissolve at 50-100℃. Then add the aqueous phase to the oil phase and stir evenly at 4000-8000 rpm. S3. Add the triple polishing enzyme and auxiliary agent, stir and mix evenly, degas, and filter to obtain a cosmetic composition containing the triple polishing enzyme.

[0019] Beneficial effects (i) This invention scientifically integrates protease, SOD and proteinase K to form a triple polishing enzyme system with synergistic effect, realizing the synergistic mechanism of "soothing-purifying-precise peeling-efficient removal". It can achieve the integrated effect of "soothing base, gentle purification, precise renewal and barrier strengthening" at low concentration, effectively solving the problems of one-sided efficacy and insufficient stability of single enzyme preparations.

[0020] (ii) In this invention, superoxide dismutase, as the core of the redox enzyme, can quickly remove excess reactive oxygen species (ROS) in the skin microenvironment, inhibit the activation of inflammatory pathways, reduce the risk of the skin being in a "hypersensitive" state, and create a milder, low-inflammatory environment for subsequent enzymatic reactions, effectively overcoming the shortcomings of traditional chemical peeling agents that are highly irritating and highly pH dependent.

[0021] (III) In this invention, the synergistic effect of protease and proteinase K enables precise and controllable cutting of the loosened intercellular junction proteins, mimicking part of the physiological shedding mechanism, achieving "layer-by-layer" rather than "brutal" peeling, and effectively solving the problem of chemical peeling agents over-cleansing the skin and damaging the skin barrier.

[0022] (iv) The synergistic effect of the triple polishing enzyme raw materials in this invention enables each component to remain stable in the complex skin environment, which not only ensures the stability of the product, but also maximizes the synergistic effect of each active ingredient, overcoming the problems of active ingredients being difficult to bind effectively and being easily deactivated in the prior art.

[0023] (v) The cosmetic composition of the present invention optimizes the formulation ratio and interaction by rationally proportioning each component, which not only ensures the stability of the product, but also maximizes the synergistic effect of each active ingredient, effectively solving the problem that the proportion and interaction of each component in the prior art have not yet reached the optimal level.

[0024] (vi) The cosmetic composition provided by this invention, by adjusting the ratio of oil phase, water phase and surfactant, forms a liquid crystal state. The molecular arrangement of the liquid crystal structure has both hydrophilic and lipophilic sites, which can simultaneously bind water molecules in the water phase and lipid molecules in the oil phase: the inner layer can lock in a large amount of water, reducing transepidermal water loss (TEWL) of the skin epidermis; the lipid components of the outer layer form a sealing film, further blocking water evaporation, achieving a dual moisturizing effect of "internal water locking + external water sealing", and the moisturizing time is far superior to that of ordinary moisturizers used alone. In addition, the molecular gaps and mesh cavities of the liquid crystal structure can encapsulate triple polishing enzymes, making it a highly efficient "active ingredient carrier", solving the pain points of easy oxidation, easy inactivation and low transdermal efficiency of active ingredients in cosmetics. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the fluorescence intensity of the target protein.

[0026] Figure 2 This is a graph showing the relative expression levels of AQP3 protein.

[0027] Figure 3 A visualization of the fluorescence signal distribution.

[0028] Figure 4 This is a semi-quantitative analysis diagram of total fluorescence intensity in a three-dimensional full-thickness skin organ-on-a-chip model.

[0029] Figure 5 This is a depth-fluorescence intensity curve in a three-dimensional full-thickness skin organ-on-a-chip model. Detailed Implementation

[0030] Example 1 The first aspect of this example provides a cosmetic composition containing a triple polishing enzyme, the raw materials of which, by weight, comprise: 89.3997 parts of an aqueous phase, 10 parts of an oil phase, 0.0003 parts of a triple polishing enzyme, and 0.6 parts of an adjuvant; the raw materials of the aqueous phase, by weight, comprise: 82.3197 parts of water, 0.03 parts of xanthan gum, 5 parts of glycerol, 2 parts of 1,2-pentanediol, and 0.05 parts of sodium phytate; the raw materials of the oil phase, by weight, comprise: 5 parts of caprylic / capric triglyceride, 2 parts of behenol, 1.95 parts of macadamia seed oil, and 1.05 parts of glycolipid (the glycolipid belongs to the 2-O-pyranorhamnosyl-pyranorhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate type, CAS: 4348-76-9, model number Bio201). The bio-glycolipids were purchased from Shanghai Yusheng Industrial Co., Ltd.; the raw materials of the triple polishing enzyme include protease (model: uppsazyme, purchased from Jiangyin Beiruisen Biochemical Technology Co., Ltd.), SOD enzyme (model: Solis-SOD, purchased from Ximei Chemical Technology Co., Ltd.), proteinase K (model: proteinase K, purchased from Jinpu Nuoan Biotechnology Co., Ltd.), and mannitol; the mass ratio of protease, SOD, proteinase K, and mannitol is 1:1:1:0.05; the auxiliary agents, by mass parts, include: 0.3 parts octyl glycol and 0.3 parts sodium polyacrylamide dimethyl taurate.

[0031] The second aspect of this example provides a method for preparing a cosmetic composition containing a triple polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 75°C; S2. Mix the oil phase raw materials and stir at 75°C until the system is transparent. Then add the aqueous phase to the oil phase and stir at 6000 rpm until homogeneous. S3. Add sodium polyacrylamide dimethyl taurate, stir at 6000 rpm for 3 min, cool the system to 45°C, add triple polishing enzyme, stir at 6000 rpm to mix evenly, degas and restore to room temperature of 25°C, filter, and the cosmetic composition containing triple polishing enzyme is obtained; denoted as composition 1.

[0032] In this example, the total mass percentage of the triple polishing enzyme in composition 1 is 0.0003%.

[0033] Example 2 The first aspect of this example provides a cosmetic composition containing a triple polishing enzyme, the raw materials of which, by weight, comprise: 87.38 parts of an aqueous phase, 12 parts of an oil phase, 0.0005 parts of a triple polishing enzyme, and 0.6 parts of an auxiliary agent; the raw materials of the aqueous phase, by weight, comprise: 80.3 parts of water, 0.03 parts of xanthan gum, 5 parts of glycerol, 2 parts of 1,2-pentanediol, and 0.05 parts of sodium phytate; the raw materials of the oil phase, by weight, comprise: 6 parts of caprylic / capric triglycerides, 3 parts of behenol, 1.95 parts of macadamia seed oil, and 1.05 parts of glycolipid; the raw materials of the triple polishing enzyme comprise: protease, SOD, proteinase K, and mannitol; the mass ratio of protease, SOD, proteinase K, and mannitol is 1:0.5:0.5:0.03; the auxiliary agent, by weight, comprises: 0.3 parts of caprylyl glycol and 0.3 parts of sodium polyacrylamide dimethyl taurate.

[0034] The second aspect of this example provides a method for preparing a cosmetic composition containing a triple polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 50°C; S2. Mix the oil phase raw materials and stir at 50°C until the system is transparent. Then add the aqueous phase to the oil phase and stir at 4000 rpm until homogeneous. S3. Add sodium polyacrylamide dimethyl taurate, stir at 4000 rpm for 3 min, cool the system to 30°C, add triple polishing enzyme, stir at 4000 rpm to mix evenly, degas and restore to room temperature of 25°C, filter, and the cosmetic composition containing triple polishing enzyme is obtained; denoted as composition 2.

[0035] In this example, the total mass percentage of the triple polishing enzyme in composition 2 is 0.0005%.

[0036] Example 3 The first aspect of this example provides a cosmetic composition containing a triple polishing enzyme, the raw materials of which, by weight, comprise: 84.38 parts of an aqueous phase, 15 parts of an oil phase, 0.0007 parts of a triple polishing enzyme, and 0.6 parts of an auxiliary agent; the raw materials of the aqueous phase, by weight, comprise: 77.3 parts of water, 0.03 parts of xanthan gum, 5 parts of glycerol, 2 parts of 1,2-pentanediol, and 0.05 parts of sodium phytate; the raw materials of the oil phase, by weight, comprise: 6 parts of caprylic / capric triglycerides, 3 parts of behenol, 1.95 parts of macadamia seed oil, and 1.05 parts of glycolipid; the raw materials of the triple polishing enzyme comprise: protease, SOD, proteinase K, and mannitol; the mass ratio of protease, SOD, proteinase K, and mannitol is 1:2:2:0.1; the auxiliary agent, by weight, comprises: 0.3 parts of caprylyl glycol and 0.3 parts of sodium polyacrylamide dimethyl taurate.

[0037] The second aspect of this example provides a method for preparing a cosmetic composition containing a triple polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 100°C; S2. Mix the oil phase raw materials and stir at 100°C until the system is transparent. Then add the aqueous phase to the oil phase and stir evenly at 8000 rpm. S3. Add sodium polyacrylamide dimethyl taurate, stir at 8000 rpm for 3 min, cool the system to 60°C, add triple polishing enzyme, stir at 8000 rpm to mix evenly, degas and restore to room temperature of 25°C, filter, and the cosmetic composition containing triple polishing enzyme is obtained; denoted as composition 3.

[0038] In this example, the total mass percentage of the triple polishing enzyme in composition 3 is 0.0007%.

[0039] Comparative Example 1 The first aspect of this example provides a cosmetic composition containing a polishing enzyme, the raw materials of which, by weight, comprise: 89.3997 parts of an aqueous phase, 10 parts of an oil phase, 0.0003 parts of polishing enzyme, and 0.6 parts of an auxiliary agent; the raw materials of the aqueous phase, by weight, comprise: 82.3197 parts of water, 0.03 parts of xanthan gum, 5 parts of glycerol, 2 parts of 1,2-pentanediol, and 0.05 parts of sodium phytate; the raw materials of the oil phase, by weight, comprise: 5 parts of caprylic / capric triglycerides, 2 parts of behenol, 1.95 parts of macadamia seed oil, and 1.05 parts of glycolipid; the raw materials of the polishing enzyme comprise: SOD, proteinase K, and mannitol; the mass ratio of proteinase, SOD, proteinase K, and mannitol is 0:1:2:0.05; the auxiliary agent, by weight, comprises: 0.3 parts of caprylyl glycol and 0.3 parts of sodium polyacrylamide dimethyl taurate.

[0040] The second aspect of this example provides a method for preparing a cosmetic composition containing a polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 75°C; S2. Mix the oil phase raw materials and stir at 75°C until the system is transparent. Then add the aqueous phase to the oil phase and stir at 6000 rpm until homogeneous. S3. Add sodium polyacrylamide dimethyl taurate, stir at 6000 rpm for 3 min, cool the system to 45°C, add polishing enzyme, stir at 6000 rpm to mix evenly, degas and restore to room temperature (25°C), filter, and obtain the cosmetic composition containing polishing enzyme; denoted as composition 4.

[0041] In this example, the total mass percentage of polishing enzyme in composition 4 is 0.0003%.

[0042] Comparative Example 2 The first aspect of this example provides a cosmetic composition containing a polishing enzyme, the raw materials of which, by mass parts, comprise: 89.3997 parts of an aqueous phase, 10 parts of an oil phase, 0.0003 parts of polishing enzyme, and 0.6 parts of an auxiliary agent; the raw materials of the aqueous phase, by mass parts, comprise: 82.3197 parts of water, 0.03 parts of xanthan gum, 5 parts of glycerol, 2 parts of 1,2-pentanediol, and 0.05 parts of sodium phytate; the raw materials of the oil phase, by mass parts, comprise: 5 parts of caprylic / capric triglycerides, 2 parts of behenol, 1.95 parts of macadamia seed oil, and 1.05 parts of glycolipid; the raw materials of the polishing enzyme comprise: protease, SOD, and mannitol; the mass ratio of protease, SOD, proteinase K, and mannitol is 2:1:0:0.05; the auxiliary agent, by mass parts, comprises: 0.3 parts of caprylyl glycol and 0.3 parts of sodium polyacrylamide dimethyl taurate.

[0043] The second aspect of this example provides a method for preparing a cosmetic composition containing a polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 75°C; S2. Mix the oil phase raw materials and stir at 75°C until the system is transparent. Then add the aqueous phase to the oil phase and stir at 6000 rpm until homogeneous. S3. Add sodium polyacrylamide dimethyl taurate, stir at 6000 rpm for 3 min, cool the system to 45°C, add polishing enzyme, stir at 6000 rpm to mix evenly, degas and restore to room temperature (25°C), filter, and the cosmetic composition containing polishing enzyme is obtained; denoted as composition 5.

[0044] In this example, the total mass percentage of polishing enzyme in composition 5 is 0.0003%.

[0045] Comparative Example 3 The first aspect of this example provides a cosmetic composition containing a polishing enzyme, the raw materials of which, by weight, comprise: 87.38 parts of an aqueous phase, 12 parts of an oil phase, 0.0005 parts of polishing enzyme, and 0.6 parts of an auxiliary agent; the raw materials of the aqueous phase, by weight, comprise: 80.3 parts of water, 0.03 parts of xanthan gum, 5 parts of glycerol, 2 parts of 1,2-pentanediol, and 0.05 parts of sodium phytate; the raw materials of the oil phase, by weight, comprise: 6 parts of caprylic / capric triglycerides, 3 parts of behenol, 1.95 parts of macadamia seed oil, and 1.05 parts of glycolipid; the raw materials of the polishing enzyme comprise: protease, SOD, proteinase K, and mannitol; the mass ratio of protease, SOD, proteinase K, and mannitol is 1:0.4:0.6:0.03; the auxiliary agent, by weight, comprises: 0.3 parts of caprylyl glycol and 0.3 parts of sodium polyacrylamide dimethyl taurate.

[0046] The second aspect of this example provides a method for preparing a cosmetic composition containing a polishing enzyme, comprising the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 50°C; S2. Mix the oil phase raw materials and stir at 50°C until the system is transparent. Then add the aqueous phase to the oil phase and stir at 4000 rpm until homogeneous. S3. Add sodium polyacrylamide dimethyl taurate, stir at 4000 rpm for 3 min, cool the system to 30°C, add polishing enzyme, stir at 4000 rpm to mix evenly, degas and restore to room temperature (25°C), filter, and the cosmetic composition containing polishing enzyme is obtained; denoted as composition 6.

[0047] In this example, the total mass percentage of polishing enzyme in composition 6 is 0.0005%.

[0048] Performance testing The cosmetic compositions corresponding to Examples 1-3 and Comparative Examples 1-3 were used for performance testing to test moisturizing, antioxidant, exfoliating, and transdermal penetration effects.

[0049] 1. Moisturizing effect test Composition 1 was selected for moisturizing test in this experiment.

[0050] Intradermal hydration is a key physiological mechanism for maintaining the moisture content of the stratum corneum, which relies heavily on the dermis's function as a "water reservoir." The dermis continuously delivers moisture to the stratum corneum through specialized water-carrying channels formed by aquaporin-3 (AQP3), ensuring the stability of the skin barrier and maintaining moisture balance. This process is self-regulating; when the stratum corneum sends a dehydration signal, it can quickly respond and replenish moisture, ensuring the skin is in an optimal hydrated state. This experiment evaluates the moisturizing effect of the tested substance by detecting its ability to promote the expression of aquaporin-3 after application.

[0051] This experiment evaluates the moisturizing effect of the tested substance by detecting its ability to promote the expression of aquaporin-3 after application.

[0052] 1.1 Cell Culture: Resuscitate cells, change the medium the next day, and culture in a CO2 cell incubator.

[0053] 1.2 Experimental Grouping: See Table 1 for details of the grouping.

[0054] Table 1

[0055] 1.3 Sample Safe Concentration Screening: Cells were first cultured, and the cell density was adjusted for plate formation. Based on the cytotoxicity assay of immortalized keratinocytes (HaCaT), the MTT assay was used to detect the toxic effects of different sample concentrations on HaCaT, and the safe working concentration range of the samples was screened for subsequent efficacy evaluation experiments. Seven concentrations were selected for MTT assay, and then sample concentrations with cell viability ≥90% were selected for subsequent testing.

[0056] Sample processing method: Take the triple polishing enzyme sample and dilute it with DMEM complete medium to 0.125 μg / mL, and then dilute it in a 2-fold gradient to a total of 7 sample concentrations as shown in Table 2.

[0057] Table 2

[0058] Based on the cell viability assay results, a sample with a concentration of 0.002 μg / mL was selected for the next step of the test. See the detailed test results below. Figure 1 In the diagram, blue indicates the cell nucleus, and red indicates the target protein. Stronger red fluorescence indicates higher expression of the target protein. The relative expression levels of AQP3 protein are shown in Table 3, and the data analysis graph for the relative expression levels of AQP3 protein is shown below. Figure 2 As shown.

[0059] Table 3

[0060] The relative expression level (%) of AQP3 protein was calculated as: (integrated optical density value of the sample group / integrated optical density value of the negative control group) × 100%; the enhancement rate (%) was calculated as: (mean value of the sample group - mean value of the negative control group) / mean value of the negative control group × 100%. Compared with the negative control group, "ns" indicates p ≥ 0.05, "*" indicates 0.01 ≤ p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001.

[0061] Based on the above test results, after 72 hours of treatment, the relative expression level of AQP3 protein in the positive control group was 167.54±4.51%, which was 67.54% higher than that in the negative control group (100.00±12.98%), and the difference was statistically significant (p<0.05). This indicates that EGCG in the positive control group promoted the relative expression of AQP3 protein in HaCaT cells. The relative expression level of AQP3 protein in the sample group with a concentration of 0.002 μg / mL was 149.10±4.31%, which was 49.10% higher than that in the negative control group (100.00±12.98%), and the difference was statistically significant (p<0.05). This indicates that the sample with a concentration of 0.002 μg / mL promoted the relative expression of AQP3 protein in HaCaT cells.

[0062] The results showed that, using human immortalized keratinocytes (HaCaT) as a model, the relative expression level of AQP3 protein was 149.10±4.31% after 72 hours of treatment with the sample triple polishing enzyme at a concentration of 0.002 μg / mL, which was 49.10% higher than that of the negative control group and showed a significant difference (p<0.05). This indicates that the sample triple polishing enzyme at a concentration of 0.002 μg / mL can promote the expression of AQP3 protein and has a moisturizing effect under this experimental system.

[0063] 2. Antioxidant test This experiment selected Composition 2 and Composition 6 for antioxidant testing.

[0064] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical. Its ethanol solution is deep purple and exhibits strong absorption around 517 nm. In the presence of a free radical scavenger, the light absorption of the DPPH ethanol solution decreases due to the pairing of unpaired electrons with the DPPH. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts, thus allowing evaluation of the test sample's ability to scavenge free radicals.

[0065] Test steps: 2.1 Sample preparation: Take a sample diluted to a concentration of 0.1 wt% for testing.

[0066] 2.2 Preparation of 0.12 mg / mL DPPH ethanol solution: Weigh 1.2 mg of 1,1-diphenyl-2-trinitrophenylhydrazine and add it to 10 mL of 95% ethanol in a 15 mL centrifuge tube.

[0067] 2.3 The positive control (VC) was diluted with water to a concentration of 0.02 mg / mL.

[0068] 2.4. Add ethanol solution to the negative control.

[0069] The experiment was conducted in a 96-well plate, with sample / positive control wells (T), sample / positive control background wells (T0), negative control DPPH wells (C), and negative control background wells (C0) (C and C0 were prepared with 95 wt% ethanol solvent). Three parallel wells were set up for each sample well (T) for each tested concentration, and three parallel wells were also set up for the negative control DPPH wells (C). 0.2 mL of the same solution was added to each of the sample tubes (T) and the sample background wells (T0). After incubation at room temperature for 5 min, the absorbance was measured at 517 nm using a microplate reader.

[0070] Calculation formula:

[0071] Where: T—absorbance value (OD value) of sample well / positive control well; T0—Absorbance (OD value) of sample well / positive control background well; C—Negative control DPPH well absorbance (OD value); C0—Absorbance value (OD value) of the background well for negative control.

[0072] The test results for the above tests are detailed in Table 4.

[0073] Table 4

[0074] Under the conditions of this experiment, the DPPH scavenging rate of sample composition 2 at a concentration of 0.1 wt% was 9.27 ± 1.56%, which was significantly different from the negative control group (p < 0.05), indicating that the 0.1 wt% concentration of the triple polishing enzyme had an antioxidant effect. The DPPH scavenging rate of sample composition 6 (protease, SOD, proteinase K, mannitol in a mass ratio of 1:0.4:0.6:0.03) at a concentration of 0.1 wt% was 3.22 ± 0.42%. Reducing the content of one enzyme in the composition significantly decreased its antioxidant effect.

[0075] 3. Stimulation test Composition 3 was selected for irritation testing in this experiment.

[0076] The testing principle is based on the fact that the chorioallantoic membrane is a respiratory membrane surrounding the chicken embryo. Taking advantage of the intact, clear, and transparent vascular system of the chorioallantoic membrane in the mid-stage of hatching, a certain amount of the test substance is directly exposed to the chorioallantoic membrane. After a period of time, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and vascularization) are observed, and then a score is obtained by combining the results.

[0077] 3.1 Preparation of CAM (Chicken Embryo Allantoic Membrane) Membrane 3.1.1.0-day-old chicken embryos, let stand for more than 2 hours, wash the chicken embryos with 37℃ grade III water, and incubate in an incubator for 9 days; 3.1.2. When the chicken embryos are incubated for 8 days, perform candling inspection and mark the location of the air cell on the eggshell surface; 3.1.3 When the eggs are incubated for 9 days, use a polisher to make a hole in the eggshell, and then use dental serrated curved tweezers to peel off the marked part of the eggshell to expose the white egg membrane. 3.1.4. Moisten the egg membrane with 0.9wt% NaCl solution, then pour off the excess 0.9wt% NaCl solution; 3.1.5. Use tweezers to remove the intima, ensuring that the vascular membrane is not damaged.

[0078] 3.2. Stimulation by adding a sample Sample preparation: The sample was diluted with 0.9wt% NaCl solution to a test concentration of 0.1wt%. The 0.1wt% test sample, negative control, positive control, and matrix control (the baseline control is mainly used to demonstrate the effectiveness of the test method, especially to check the reactivity of each batch of chicken embryos) were all transparent liquids, and the reaction time method was used. Reaction time method: 0.3 mL of the transparent test sample was directly added to the CAM surface, and the CAM reaction was observed. Photos and videos were taken to record the time of occurrence of each toxic effect within 5 minutes of the reaction.

[0079] 3.3 Calculation Formula The irritations score (IS) is used in experiments conducted using the reaction time method. The irritations score (IS) is calculated using the following formula, and the result is rounded to two decimal places:

[0080] Where: sec H (hemorrhage time): the average time at which bleeding begins to occur as observed on the CAM membrane, in seconds (s); sec L (vessellysis time): The average time, in seconds (s), at which vascularization begins to occur as observed on the CAM membrane. sec C (coagulation time): The average time for the onset of clotting observed on the CAM membrane, measured in seconds (s).

[0081] The eye irritation of the test substances was classified based on the calculated IS values. Negative and positive controls were included in the experiment. The negative control had an IS value of 0.00, while the positive control had an IS value ranging from 10 to 19, which was considered acceptable. The evaluation results of the stimulation rating method are shown in Table 5.

[0082] Table 5

[0083] The results were evaluated according to the stimulus rating method in Table 5. The negative control stimulus rating IS value was 0.00, and IS < 1, indicating no irritation; the positive control stimulus rating IS value was 15.92, and IS > 10, indicating strong irritation / corrosiveness; the 0.1 wt% concentration sample was a transparent test substance, and the reaction time method was used for testing. The results were evaluated according to the reaction time method in the table above, and the stimulus rating IS value was 0.13, IS < 1, indicating no irritation.

[0084] Under the conditions of this test, the irritation of the samples was evaluated using the chicken embryo chorioallantoic membrane test (HEM-CAM) method, referring to the SN / T 2329-2009 (2017) standard.

[0085] Experimental results: The IS score of the 0.1 wt% concentration of the test sample composition was 0.13, and since IS < 1, it was judged to be non-irritating. It is predicted that the 0.1 wt% concentration of the triple polishing enzyme sample is mild and non-irritating.

[0086] 4. Exfoliation test This experiment selected composition 1, composition 4, and composition 5 for exfoliation testing.

[0087] Skin cells are constantly generated, matured, and transformed from the basal layer. The cells that reach the stratum corneum are flat, dead cells, also known as "keratinocytes." Millions of dead keratinocytes are produced in the skin every day. The stratum corneum is easily worn down in daily life, turning into imperceptible scales that fall off. After they fall off, they are replaced by new keratinocytes, serving as an important barrier to protect the skin.

[0088] Under alkaline conditions, divalent copper ions can be reduced to monovalent copper ions by proteins. These monovalent copper ions then interact with a unique BCA Solution A (containing BCA) to produce a sensitive color reaction. Two molecules of BCA chelate one copper ion, forming a purple reaction complex. This water-soluble complex exhibits strong absorbance at 562 nm, and the absorbance shows a good linear relationship with protein concentration over a wide range; the protein concentration can be calculated from the absorbance value. Therefore, the exfoliating ability of a sample can be evaluated by assessing the total amount of keratinized protein exfoliated from isolated skin.

[0089] Test steps 4.1 Determination of total keratin content Sample group: Rinse pigskin with water for 15 seconds, apply 1 mL of 0.1 wt% sample to the experimental area of ​​pigskin, gently rub for 3 minutes; rinse off the sample with water, massage for another 3 minutes, and then rinse thoroughly with water. Control group: Treat pigskin with pure water instead of the sample.

[0090] 4.2 Using a keratin exfoliating membrane, apply pressure to the isolated pig skin and peel it off by hand. Repeat this process 20 times. Remove the membrane, cut it into small pieces with alcohol-sterilized scissors, and place it into a microcentrifuge tube containing 2 mL of cell lysis buffer. Take 3 pieces from each site and centrifuge at 15000 r / min for 15 min at 4℃. Use a Bradford protein assay kit to determine the total amount of keratin in the supernatant after centrifugation. Perform 3 replicates per group.

[0091] Result determination The total amount of keratin in the sample should be expressed as: mean ± standard deviation (SD). Compared with the negative control group, the total amount of keratin in the sample group decreased. Using T-test one-tailed analysis, there was a statistically significant difference (p<0.05), indicating that the substance has an exfoliating effect. The data results are shown in Table 6.

[0092] Table 6

[0093] Note: Compared with the negative control group: "ns" means p≥0.05, "*" means 0.01≤p<0.05, "**" means p<0.01, "***" means p<0.001.

[0094] Experimental Conclusions: Under the conditions of this experiment, using pig skin as an in vitro model, after treatment with a 0.1 wt% concentration of triple polishing enzyme, the total keratin content was 911.46 ± 2.72 μg / mL, which was 18.01% lower than that of the negative control group (1111.62 ± 57.16 μg / mL), and the difference was statistically significant (p<0.05), indicating that the 0.1 wt% concentration of triple polishing enzyme has an exfoliating effect. After treatment with a 0.1 wt% concentration of polishing enzyme composition 4 (protease, SOD, proteinase K, mannitol in a mass ratio of 0:1:2:0.05), the total keratin content was 1095.25 ± 23.23 μg / mL, which was 1.4% lower than that of the negative control group (1111.62 ± 57.16 μg / mL), but the difference was not statistically significant (p<0.05). After treatment with 0.1 wt% of the polishing enzyme composition 5 (protease, SOD, proteinase K, and mannitol in a mass ratio of 2:1:0:0.05), the total keratin content was 1095.25 ± 23.23 μg / mL, which was 1.4% lower than that of the negative control group (1086.21 ± 35.50 μg / mL). The difference was not statistically significant (p<0.05), indicating that in the synergistic effect of multiple enzymes, the absence of a certain enzyme can reduce the effect or prevent the desired effect from being achieved.

[0095] 5. Transdermal penetration test This experiment used composition 1 prepared in Example 1 for testing.

[0096] Based on the principle of transdermal absorption, an in vitro three-dimensional full-thickness skin organ-on-a-chip model was used to simulate the transdermal process of substances. The active ingredients from different raw materials were labeled with the fluorescent dye FITC and coated onto the surface of the full-thickness skin model, then treated at 37°C and 5% CO2 for 24 hours. Subsequently, the model was fixed, sectioned, and the distribution of FITC fluorescence signals in different skin layers was observed using a laser confocal microscope. Semi-quantitative analysis was performed to compare the differences in penetration depth and intensity between the two methods.

[0097] Test steps: 5.1 Model Construction: Construct a three-dimensional full-layer skin organ chip model and complete quality inspection.

[0098] 5.2 Setting up groups: Set up a 0.1wt% triple polishing enzyme (before coating) group and a 0.1wt% triple polishing enzyme (after coating) group, with 3 parallel skin models in each group.

[0099] 5.3 Sample preparation and fluorescent labeling: 0.1 wt% triple polishing enzyme (before coating) and 0.1 wt% triple polishing enzyme (after coating) were respectively bound to FITC for fluorescent labeling.

[0100] 5.4 Experimental Treatment: 200 μL of 0.1 wt% triple polishing enzyme (before coating) and 0.1 wt% triple polishing enzyme (after coating) were evenly coated onto the surface of each model. 800 μL of culture medium was added to the lower chamber. The mixture was incubated at 37°C and 5% CO2 for 24 hours, followed by gentle rinsing three times with pre-warmed DPBS to remove any residual substances.

[0101] 5.5 Fixation: The model tissue was fixed for 2 hours at 4°C using 4% paraformaldehyde (PFA).

[0102] 5.6 Dehydration, embedding and sectioning: The model was sequentially dehydrated in 10wt%, 20wt%, and 30wt% sucrose solutions until it sank to the bottom of the 30% sucrose solution, indicating that dehydration was complete. The dehydrated skin model was then embedded in OCT embedding agent and frozen sectioned with a section thickness of 10μm.

[0103] 5.7 Observation: The distribution of FITC fluorescence signal in the sections was observed using a laser confocal microscope to evaluate the skin penetration and retention effect of the fluorescent marker.

[0104] Data processing: Fluorescence signals were acquired and semi-quantitatively analyzed using the built-in software of the laser confocal microscope and image processing software such as ImageJ. Excel and GraphPad Prism 9.0 software were used for data processing and graphing. Independent samples t-tests were used to compare the differences between the control and experimental groups. Significance criteria were defined as follows: "ns" indicates p ≥ 0.05, "*" indicates 0.01 ≤ p < 0.05, "**" indicates p < 0.01, and "***" indicates p < 0.001. Data are expressed as mean ± SEM.

[0105] The following results were obtained through observation and semi-quantitative analysis of FITC fluorescence signals in skin model sections using laser confocal microscopy: The visual distribution of fluorescence signals is shown below. Figure 3 As shown: Control group (before wrapping), Figure 3 (Left) The fluorescence signal is mainly concentrated in the outermost layer (stratum corneum) of the skin model, with limited penetration depth and weak signal intensity; the experimental group (after wrapping) Figure 3 (Right) The fluorescence signal not only has a significantly enhanced intensity, but also successfully penetrates to deeper skin layers, including the deep epidermis and even the dermis, with a wider and more uniform distribution.

[0106] The results of the semi-quantitative analysis of total fluorescence intensity are as follows: Figure 4As shown in Table 7, quantitative and statistical analysis of the total fluorescence intensity revealed that the total fluorescence intensity of the sample before wrapping was 3769792966±20469723, while the total fluorescence intensity of the sample after wrapping was 20852870992±379309648. The total fluorescence intensity of the wrapped sample within the skin model was approximately 5.5 times that before wrapping, and this difference was highly significant (p<0.001).

[0107] Table 7

[0108] Penetration depth analysis such as Figure 5 As shown, the fluorescence signal in the control group was concentrated on the model surface (221 μm), while the fluorescence signal deepened after encapsulation with 0.1 wt% triple polishing enzyme (796 μm), a depth 3.6 times that of the control group. The skin model depth-fluorescence intensity curve clearly shows that 0.1 wt% triple polishing enzyme (after encapsulation) maintained a high fluorescence intensity throughout the entire detection depth range, especially in the dermal region at depths greater than 400 μm, where a significant signal was still detectable. In contrast, the fluorescence signal of 0.1 wt% triple polishing enzyme (before encapsulation) rapidly decreased with increasing depth. This directly demonstrates that the encapsulation technology effectively promotes the penetration of the active ingredient through the skin barrier, achieving deeper penetration.

[0109] This experiment, based on a three-dimensional full-thickness skin organ-on-a-chip model, used fluorescent labeling combined with laser confocal microscopy to systematically evaluate the percutaneous penetration ability of triple polishing enzyme before and after encapsulation. The conclusions are as follows: (1) Significantly improved penetration efficiency: Compared with before encapsulation, the total retention of active ingredient in the skin model of the 0.1wt% concentration of composition 1 after encapsulation (in terms of total fluorescence intensity) was significantly increased (approximately 5.5 times), demonstrating that the encapsulation technology significantly improved the transdermal penetration efficiency of the active ingredient.

[0110] (2) Significantly enhanced penetration depth: The encapsulation process enables the active ingredients to overcome the skin barrier and penetrate from the surface to deeper layers of the skin, achieving deeper targeted delivery.

[0111] The results fully validated the effectiveness and superiority of the encapsulation technology used in promoting the transdermal absorption of active ingredients.

Claims

1. A cosmetic composition comprising a triple polishing enzyme, characterized in that, The raw materials for preparation include: aqueous phase, oil phase, triple polishing enzyme and auxiliary agents; The raw materials for the aqueous phase include water, xanthan gum, glycerol, 1,2-pentanediol and sodium phytate; The raw materials for the oil phase include caprylic / capric triglycerides, behenol, macadamia seed oil, and glycolipids; The raw materials for the triple polishing enzyme include protease, proteinase K, and oxidoreductase. The oxidoreductase includes superoxide dismutase; The mass ratio of the protease, proteinase K and superoxide dismutase is 1:(0.5-2):(0.5-2).

2. The cosmetic composition comprising a triple polishing enzyme according to claim 1, characterized in that, The triple polishing enzyme is present in the cosmetic composition at a mass percentage of 0.0002-0.001%.

3. The cosmetic composition comprising a triple polishing enzyme according to claim 1, characterized in that, The mass ratio of the oil phase to the water phase is 1:(5-10).

4. The cosmetic composition comprising a triple polishing enzyme according to claim 1, characterized in that, The raw materials for the triple polishing enzyme also include an enzyme protectant.

5. The cosmetic composition comprising a triple polishing enzyme according to claim 4, wherein the enzyme protectant comprises one of mannitol, xylitol, trehalose, sodium hyaluronate, or polyethylene glycol.

6. The cosmetic composition comprising a triple polishing enzyme according to claim 5, characterized in that, The mass ratio of mannitol to superoxide dismutase is 1:(10-30).

7. The cosmetic composition comprising a triple polishing enzyme according to claim 1, characterized in that, The additives include humectants and surfactants.

8. The cosmetic composition comprising a triple polishing enzyme according to claim 7, characterized in that, The moisturizers mentioned include one of caprylyl glycol, 1,2-pentanediol, 1,6-hexanediol, ethylhexylglycerin, sorbitol, or polyglycerol-3.

9. The cosmetic composition comprising a triple polishing enzyme according to claim 7, characterized in that, The surfactants mentioned include one of sodium polyacrylamide dimethyl taurate, sodium acrylate / sodium acrylamide dimethyl taurate copolymer, carbomer, sodium lauroyl glutamate, sucrose stearate, or polyquaternium-73.

10. A method for preparing a cosmetic composition comprising a triple polishing enzyme according to any one of claims 1-9, characterized in that, Includes the following steps: S1. Mix the aqueous raw materials and stir to dissolve at 50-100℃; S2. Mix the oil phase raw materials and stir to dissolve at 50-100℃. Then add the aqueous phase to the oil phase and stir evenly at 4000-8000 rpm. S3. Add the triple polishing enzyme and auxiliary agent, stir and mix evenly, degas, and filter to obtain a cosmetic composition containing the triple polishing enzyme.

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