Compositions and their use in the preparation of barrier repair products

Abstract

The present application relates to the field of cosmetics, in particular to a composition and its application in the preparation of barrier repair products. The present application provides a composition comprising: ganoderma extract, avocado oil unsaponifiable matter and ceramide; the mass ratio of the ganoderma extract, the avocado oil unsaponifiable matter and the ceramide is (1~5000):(1~50):(1~1000). The active composition in the present application is used for skin barrier repair in skin care products, promotes epidermal renewal, antioxidant, anti-inflammatory, anti-aging efficacy, compared with the commonly used barrier repair ingredients ceramide on the market, the active composition of our application increases ganoderma extract (ganoderma polysaccharide, etc.) and avocado extract (avocado oil unsaponifiable matter), and ceramide selects the combination of ceramide NP and ceramide EOP at a ratio of 10:1, because it is similar to the content ratio in healthy skin.

Description

Technical Field

[0001] This invention relates to the field of cosmetics, and more particularly to compositions and their use in the preparation of barrier repair products. Background Technology

[0002] The skin is the largest organ in the human body, and its outermost layer, the stratum corneum, forms the first line of defense between the body and the external environment—the skin's physical barrier. Currently, the academic community generally uses the "brick-and-mortar model" to explain the structure and function of the skin barrier: keratinocytes act like "bricks," arranged in an orderly fashion to form the skin's physical support framework; while intercellular lipids, filling the spaces between keratinocytes, act like "cement," tightly connecting the cells. When the skin barrier is damaged, the "brick-and-mortar model" is disrupted, leading to a significant loss of intercellular lipids, resulting in decreased skin hydration and weakened defense against external stimuli.

[0003] Intercellular lipids are mainly composed of ceramides, cholesterol, and free fatty acids, with ceramides accounting for approximately 50% of the lipid layer mass and serving as a core component for maintaining skin barrier function. Ceramides can tightly bind keratinocytes together, enhancing the skin's barrier function, reducing the entry of harmful substances into the skin through pores and sweat glands, while simultaneously locking in skin moisture and nutrients and reducing transepidermal water loss. Studies have shown that the barrier repair function of ceramides is closely related to its biological mechanisms, including promoting keratinocyte capsule formation, lipid synthesis, tight junctions of keratinocytes, and regulating keratinocyte proliferation and differentiation. Ceramides are not a single component in the skin but comprise multiple subtypes. Ceramide NP is the most abundant subtype, accounting for approximately 22.1% of the total ceramides in the stratum corneum. Ceramide EOP, a long-chain ceramide, is present in lower amounts but has a unique structure; its esterification structure can form a three-dimensional lipid network, significantly improving the hydration of the stratum corneum.

[0004] Besides ceramides, naturally derived skincare active ingredients have also received widespread attention in recent years. Ganoderma lucidum polysaccharides, one of the main active ingredients of the traditional Chinese medicine Ganoderma lucidum, possess significant antioxidant activity and can effectively scavenge free radicals. Studies have shown that Ganoderma lucidum polysaccharides can maintain skin moisture, increase the thickness of the epidermis and dermis, improve the microstructure of skin tissue, and reduce stress damage caused by keratinocyte oxidation. Ganoderma lucidum polysaccharides can also increase the activity of superoxide dismutase (SOD) in skin tissue and reduce the content of malondialdehyde (MDA), combating oxidative damage on multiple levels. Avocado oil unsaponifiables are the non-saponifiable active ingredients in avocado oil that nourish, strengthen, regenerate, and protect the skin, combating skin aging and restoring skin elasticity. Furthermore, avocado oil unsaponifiables also promote cell regeneration and repair skin tissue.

[0005] Currently, most skincare products on the market for skin barrier repair primarily focus on supplementing intercellular lipids such as ceramides, cholesterol, and fatty acids. However, existing technologies generally suffer from the following shortcomings: (1) Most products only focus on supplementing a single or a few lipid components, neglecting the fact that skin barrier repair is a complex system involving multiple biological processes such as cell proliferation, differentiation, anti-inflammation, and anti-oxidation; (2) Although some products add ceramides, they fail to scientifically combine them according to the natural proportions of different ceramide subtypes in healthy skin; (3) There is a lack of technical solutions for organically combining ceramides with natural plant active ingredients that have antioxidant and anti-inflammatory activities, making it difficult to achieve the leap from "supplementing lipids" to "comprehensively repairing barrier function". Summary of the Invention

[0006] In view of this, the present invention provides a composition and its application in the preparation of barrier repair products. The active ingredient composition of the present invention is used in skin care products to repair the skin barrier, promote epidermal renewal, and has antioxidant, anti-inflammatory, and anti-aging effects. Compared with ceramides, a commonly used barrier repair ingredient on the market, our active ingredient composition adds Ganoderma lucidum extract (Ganoderma lucidum polysaccharides, etc.) and avocado extract (avocado oil unsaponifiables). At the same time, the ceramides selected are a 10:1 ratio of ceramide NP and ceramide EOP, which is similar to the content ratio in healthy skin.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a composition comprising: Ganoderma lucidum extract, avocado oil unsaponifiables, and ceramides;

[0009] The mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, and the ceramide is (1~5000):(1~50):(1~1000).

[0010] In some embodiments of the present invention, the mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, and the ceramide in the above composition is: (1~4500):(1~50):(1~950) or (1~4000):(1~50):(1~900) or (1~3500):(1~50):(1~850) or (1~3000):(1~50):(1~800) or (1~2500):(1~50):(1~750) or (1~2000):(1~50):(1~700) or (1~1500):(1~50):(1~650) or (1~1000):(1~50):(1~50):(1~650) 1~600) or (1~500): (1~50): (1~550) or (1~450): (1~50): (1~500) or (1~400): (1~50): (1~450) or (1~350): (1~50): (1~400) or (1~300): (1~50): (1~350) or (1~250): (1~50): (1~300) or (1~200): (1~50): (1~250) or (1~150): (1~50): (1~200) or (1~100): (1~50): (1~150) or (1~50): (1~50): (1~100).

[0011] In some embodiments of the present invention, the ceramide in the above composition comprises ceramide NP and ceramide EOP.

[0012] In some embodiments of the present invention, the mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, the ceramide NP, and the ceramide EOP in the above composition is (1~50):(1~50):(1~50):(1~50); or

[0013] The mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, the ceramide NP, and the ceramide EOP is 5000:1:1000:1000.

[0014] In some embodiments of the present invention, the mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, the ceramide NP and the ceramide EOP in the above composition is 50:10:10:1 or 10:1:1:50 or 1:50:50:10 or 5000:1:1000:1000.

[0015] The present invention also provides the use of the above composition in the preparation of products for barrier repair, anti-oxidation, anti-inflammation and / or anti-aging.

[0016] In some embodiments of the present invention, the barrier repair described above includes upregulating the expression of the FLG gene and / or the LOR gene.

[0017] In some embodiments of the present invention, the above-described applications include: scavenging free radicals and / or reducing ROS content.

[0018] In some embodiments of the present invention, the anti-inflammatory effect described above includes downregulating the expression of the TNF-α gene and / or the NF-κB gene.

[0019] In some embodiments of the present invention, the anti-aging process described above includes: upregulating the expression of COL I, COL IV, COL VII and / or COL XVII genes; and / or downregulating the expression of MMP-1 gene.

[0020] The present invention also provides personal care products comprising the above-described composition.

[0021] In some embodiments of this application, the aforementioned products include, but are not limited to, personal care products and pharmaceuticals. Those skilled in the art will understand that, in addition to the compositions described in the above technical solutions, other excipients are also included, such as pharmaceutical excipients or excipients for personal care products.

[0022] In some embodiments of this application, the personal care products include: basic care products, hair care products and / or makeup products.

[0023] In some embodiments of this application, the basic care product includes, but is not limited to:

[0024] Facial cleanser / cleanser / facial lotion: Gently removes dirt, oil, and makeup residue from the face;

[0025] Cleansing oil / cleansing water / cleansing balm: specially designed to thoroughly remove makeup, especially waterproof cosmetics;

[0026] Toner / lotion: Used after cleansing, it can further cleanse the skin's surface of residue, while replenishing the skin's moisture, restoring the skin's pH balance, and laying a good foundation for the absorption of subsequent skin care products;

[0027] Serum: Contains a high concentration of active ingredients, providing deep nourishment and repair for specific skin problems (such as anti-aging, moisturizing, whitening, etc.);

[0028] Eye cream: Specially designed for the sensitive area around the eyes, it has the effects of reducing fine lines and dark circles and firming the skin around the eyes. The texture is usually light and easily absorbed.

[0029] Day / night lotion or cream: It has functions such as protection, repair and nourishment, and promotes cell regeneration. It can provide the skin with the necessary moisture and lock in the replenished moisture.

[0030] Sunscreen: Used for ultraviolet protection and prevention of photoaging, etc.

[0031] Face masks: provide extra nourishment and care for the skin, such as hydration, pore cleansing, or brightening of the complexion.

[0032] In some embodiments of the present invention, the washing and care products include, but are not limited to:

[0033] Scalp cleansing and care products: mainly used to clean the scalp and hair, and provide repair and moisturizing effects, including shampoos, hair creams and other cleansing products or conditioners, hair masks, hair oils and other care products;

[0034] Body cleansing and care: Cleansing and moisturizing of the entire body's skin to maintain healthy skin condition, including cleansing products such as shower gel, soap, and bath oil, or care products such as body lotion, moisturizer, and scrub;

[0035] Hand and other body part cleaning and care products: These are products for local cleaning and care, including hand sanitizers and other cleaning products, or hand creams and other care products.

[0036] In some embodiments of this application, the makeup products include, but are not limited to:

[0037] (1) Base makeup:

[0038] Foundation / cream: Used to even out skin tone and cover blemishes;

[0039] BB cream / CC cream: A lightweight base makeup product that combines skincare and makeup application;

[0040] Concealer / pen: Used to cover up specific areas such as pimples and dark circles;

[0041] Loose powder / setting powder: Sets makeup and reduces facial shine.

[0042] (2) Cheek makeup:

[0043] Blush: Adds natural color to the cheeks and improves complexion;

[0044] Contouring powder / stick: Use shading techniques to make facial contours more three-dimensional;

[0045] (3) Lip makeup:

[0046] Lipstick / lip gloss / lip tint: to change or emphasize the color of the lips;

[0047] Lip liner: Defines the clear outline of the lips and prevents lipstick from bleeding.

[0048] (4) Multifunctional cosmetics:

[0049] Highlighter sticks / liquids / powders: Highlight high points on the face (such as the bridge of the nose and cheekbones) to create a luminous effect.

[0050] In addition, there are products designed specifically for special occasions, such as long-lasting, non-fading lipsticks.

[0051] In some specific implementations, the composition described in the above technical solution is preferably mixed with a cosmetically acceptable carrier to prepare products including shampoo, shower gel, facial cleanser, serum, lotion, cream, gel, or mask, which can be used to prevent or repair skin problems caused by oxidative stress or to build a photoprotective barrier by thickening the epidermis to resist free radicals. It also has the effects of barrier repair, anti-aging, and whitening.

[0052] Addressing the issues of single-component formulation, unscientific ratios, ambiguous definitions of effective ingredients, and lack of synergistic mechanisms in existing technologies, this invention establishes a complete multi-pathway synergistic repair system for the skin barrier through the organic compounding of four specific components: Ganoderma lucidum extract (Ganoderma lucidum polysaccharides / peptides), avocado oil unsaponifiables (high content of unsaponifiables / avocado furan), ceramide NP, and ceramide EOP. Because this invention fundamentally changes the traditional approach of simply supplementing ceramides or piling up lipids, it integrates barrier repair, anti-oxidation, anti-inflammation, and epidermal regeneration into a synergistic whole. Therefore, this composition can achieve superior barrier repair effects while reasonably reducing the amount of ceramide added, while also providing comprehensive skincare benefits such as anti-inflammation, anti-oxidation, and anti-aging.

[0053] Furthermore, because this invention clearly distinguishes the effective components of Ganoderma lucidum extract (Ganoderma lucidum polysaccharides and Ganoderma lucidum polypeptides) from Ganoderma lucidum spore oil in the prior art, and precisely limits the content of unsaponifiable matter (≥80%) and avocado furan (≥70%) in avocado extract, the technical solution of this invention has clear reproducibility and industrial applicability. Simultaneously, because this invention verifies the synergistic effect of each component at different proportions using the Jin Zhengjun method (q values ​​> 1.15), and systematically completes the full-chain efficacy evaluation from barrier protein expression, inflammatory factor regulation, free radical scavenging to collagen synthesis and degradation, this invention not only fully supports its claimed multiple skincare effects, but also provides the cosmetics industry with a precise, efficient, and industrially scalable skin barrier repair solution. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0055] Figure 1 The fluorescence image of ROS detection in a 3D epidermal model after treatment with composition 17-22 is shown.

[0056] Figure 2 The effect of composition 17-22 on ROS fluorescence intensity in a UVB-induced 3D epidermal model is shown.

[0057] Figure 3 Fluorescence image of TNF-α protein expression in a 3D epidermal inflammation model after treatment with composition 22;

[0058] Figure 4 The effect of composition 22 on TNF-α protein expression in a UVB-induced 3D epidermal inflammation model was shown.

[0059] Figure 5 Fluorescence image of NF-κB protein expression in a 3D epidermal inflammation model after treatment with composition 17-22;

[0060] Figure 6 The effect of composition 17-22 on NF-κB protein expression in a UVB-induced 3D epidermal inflammation model was shown.

[0061] Figure 7 Fluorescence image of FLG protein expression in a 3D epidermal model after treatment with composition 22;

[0062] Figure 8 The effect of composition 22 on FLG protein expression in a UVB-induced 3D epidermal model is shown.

[0063] Figure 9 Fluorescence image of LOR protein expression in a 3D epidermal model after treatment with composition 22;

[0064] Figure 10 The effect of composition 22 on LOR protein expression in a UVB-induced 3D epidermal model is shown.

[0065] Figure 11 Fluorescence image of Ki67-positive cells in a 3D epidermal model after treatment with composition 17-21;

[0066] Figure 12 The effect of composition 17-21 on the rate of Ki67 positive cells in a UVB-induced 3D epidermal model was shown.

[0067] Figure 13 Image showing the results of cell scratching;

[0068] Figure 14 This demonstrates the effect of compositions 23-28 on the relative migration rate of keratinocytes;

[0069] Figure 15This demonstrates the effect of composition 28 on the relative viability of keratinocytes;

[0070] Figure 16 This demonstrates the effect of composition 28 on the expression of the COL IV gene in cells after UVB irradiation.

[0071] Figure 17 This demonstrates the effect of composition 28 on the expression of the COL VII gene in cells after UVB irradiation.

[0072] Figure 18 This study demonstrates the effect of compositions 23-28 on the expression of COL XVII genes in keratinocytes after UVB irradiation.

[0073] Figure 19 This study demonstrates the effect of composition 29-33 on the expression of the COL I gene in fibroblasts after UVA irradiation.

[0074] Figure 20 The effect of composition 29-33 on the expression of MMP-1 gene in fibroblasts after UVA irradiation was shown. Detailed Implementation

[0075] This invention discloses the composition and its use in the preparation of barrier repair products.

[0076] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0077] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0078] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0079] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0080] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0081] 1. This invention is the first to organically combine four specific components—Ganoderma lucidum extract (with Ganoderma lucidum polysaccharides and Ganoderma lucidum polypeptides as the main active ingredients), avocado oil unsaponifiables (with avocado furan content ≥70% in the unsaponifiables), ceramide NP, and ceramide EOP—to form a complete skin barrier repair composition.

[0082] The core innovation of this composition lies in breaking through the traditional technical paradigm of "single ceramide supplementation" or "simple stacking of multiple lipids," and establishing a multi-pathway synergistic barrier repair mechanism of "supplementing key lipids + anti-oxidation + anti-inflammation + anti-aging." Through the synergistic effect of the four components, a better barrier repair effect is achieved while reasonably reducing the amount of ceramide added.

[0083] 2. This invention precisely selects and clearly defines the active ingredients from natural sources:

[0084] (1) Ganoderma lucidum extract: Ganoderma lucidum polysaccharides and Ganoderma lucidum polypeptides are the main active ingredients. This is fundamentally different from the use of Ganoderma lucidum spore oil (mainly containing fat-soluble components such as unsaturated fatty acids and triterpenoid ganoderic acids) in the existing technology - the two have different chemical compositions, different solubilities, and different pathways of action, and cannot be substituted for each other.

[0085] (2) Avocado oil unsaponifiables: content ≥80%, and the unsaponifiables are mainly composed of avocado furan, with avocado furan content accounting for ≥70% of the unsaponifiables. This is significantly different from the existing technology that uses "avocado oil" in general or "avocado extract" without specifying the content of active ingredients. By accurately defining the chemical composition of key active ingredients, the reproducibility and industrial applicability of the technical solution are ensured.

[0086] 3. This invention, through systematic DPPH free radical scavenging experiments and Jin Zhengjun method synergistic effect evaluation, determined the preferred compound ratio range of four components: Ganoderma lucidum extract (calculated as polysaccharide), avocado extract (calculated as unsaponifiable matter), ceramide NP, and ceramide EOP.

[0087] 4. This invention, through a complete in vitro efficacy evaluation system, verified the multiple effects of the above composition in the following aspects:

[0088] (1) Barrier repair function

[0089] Upregulate the expression of FLG (figrin) and LOR (lobeline) in a 3D epidermal skin model (after UVB irradiation).

[0090] Promotes the proliferation (MTT assay) and migration (scratch assay) of keratinocytes.

[0091] Reduced the expression of UVB-induced inflammatory cytokine TNF-α and NF-κB nuclear translocation in a 3D epidermal skin model.

[0092] (2) Antioxidant effects

[0093] Scavenging DPPH free radicals;

[0094] Reduce UVB-induced ROS levels in 3D epidermal skin models.

[0095] (3) Firming and anti-wrinkle effects

[0096] Upregulates COL I gene expression in UVA-irradiated fibroblasts;

[0097] Downregulates MMP-1 gene expression in UVA-irradiated fibroblasts;

[0098] Upregulates the expression of COL IV, COL VII, and COL XVII genes in UVB-irradiated keratinocytes.

[0099] The Ganoderma lucidum extract of this invention was purchased from Draco Natural Products.

[0100] The avocado oil unsaponifiables of this invention were purchased from Laboratoires Expanscience.

[0101] The ceramide NP of this invention was purchased from Croda Inc.

[0102] The ceramide EOP of this invention was purchased from Shenzhen Dickman Technology Development Co., Ltd.

[0103] The Ganoderma lucidum spore oil of this invention was purchased from Guangzhou Yue Microbial Technology Co., Ltd.

[0104] In the embodiments, comparative examples and verification examples of this invention, all raw materials and reagents used are commercially available.

[0105] The present invention will be further illustrated below with reference to the embodiments:

[0106] Examples 1 to 4 and Comparative Examples 1 to 12

[0107] The amount and ratio of the compositions added in each embodiment and its comparative examples (Table 1).

[0108] Table 1. Information on the amount and proportion of compositions added in each example and its comparative examples.

[0109]

[0110] Example 1: Evaluation of antioxidant efficacy (DPPH free radical scavenging experiment)

[0111] Technical Principle: Free radicals are oxygen free radicals produced during the aging process, which can cause oxidative damage to cells. Scavenging free radicals can effectively repair cell damage, delay aging, and enhance the body's vitality. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable nitrogen-centered free radical. Its ethanol solution is deep purple and has a characteristic maximum absorption peak at 517 nm. When a free radical scavenger is present in the system, the single electrons of DPPH are paired, causing its color to lighten and its absorbance at 517 nm to decrease. Therefore, the ability of the analyte to scavenge DPPH free radicals can be evaluated by measuring the degree of decrease in absorbance.

[0112] The present invention uses the DPPH free radical scavenging experiment to evaluate the in vitro antioxidant activity of the compositions described in Examples 1-4, and uses the Jin Zhengjun method (q-value method) to evaluate the synergistic effect of each component in the composition.

[0113] 1. Experimental Design

[0114] 1) The amount and ratio of the compositions added in each embodiment and its comparative example (Table 1);

[0115] 2) Preparation of DPPH standard solution: Accurately weigh 7.8 mg of DPPH powder and place it in a 100 mL brown volumetric flask. Add an appropriate amount of anhydrous ethanol solvent and dilute to the mark. Mix thoroughly to prepare a 0.2 mmol / L DPPH working solution. After preparation, wrap the volumetric flask with aluminum foil and store it at 4℃ protected from light for later use.

[0116] 3) Preparation of sample solution: Using DMSO as solvent, prepare a composition solution of the corresponding concentration (mass percentage).

[0117] 4) Experimental grouping: The experiment was set up with a blank group, a control group, and a sample group, with 3 parallel tubes in each group. The total reaction volume of a single tube was strictly controlled to be 4 mL. The specific sample addition scheme is shown in Table 2.

[0118] Table 2 DPPH Experimental Grouping and Sample Addition Scheme

[0119]

[0120] 5) DPPH reaction and absorbance detection: The reaction was allowed to proceed at room temperature in the dark for 30 min. After the reaction, the absorbance of each tube was measured at 517 nm using a UV spectrophotometer.

[0121] 6) Calculation of DPPH free radical scavenging rate: Scavenging rate (%) = [1-(A1-A2) / A0]×100;

[0122] definition:

[0123] A0: Average absorbance of the control group (DPPH working solution + DMSO, no sample);

[0124] A1: Average absorbance of the sample group (DPPH working solution + sample solution).

[0125] A2: Average absorbance of the blank group (anhydrous ethanol + sample solution, after subtracting the sample's own color background);

[0126] 7) Evaluation of synergistic effect: The q-value was calculated using the Jin Zhengjun method to evaluate the synergistic effect of each component in the composition.

[0127] q = C / (A + B - A × B);

[0128] in:

[0129] A: DPPH free radical scavenging rate of components a+b (Ganoderma lucidum extract + avocado oil unsaponifiables);

[0130] B: DPPH free radical scavenging rate of component c+d (ceramide NP+ceramide EOP);

[0131] C: DPPH free radical scavenging rate of components a+b+c+d (intact composition).

[0132] Criteria for determining q-value:

[0133] q > 1.15: Has a synergistic effect;

[0134] q = 0.85~1.15: This function performs simple addition;

[0135] q < 0.85: There is an antagonistic effect.

[0136] 2. Experimental Results

[0137] (1) DPPH free radical scavenging activity

[0138] Table 3 shows the DPPH free radical scavenging rate and synergistic effect evaluation (q-value) of each embodiment and comparative example. The DPPH free radical scavenging rates of Examples 1-3 were 51.33%, 32.33%, and 77.30%, respectively. All examples showed significant DPPH free radical scavenging activity, with Example 3 (a:b:c:d = 1:50:50:10) achieving the best scavenging rate of 77.30%. Example 4 further expanded the combination ratio to 5000:1:1000:1000, and its DPPH free radical scavenging rate still reached 75.47%, which was basically equivalent to the scavenging effect of Example 3. This indicates that even within a significantly expanded ratio range, the composition still maintains stable and excellent free radical scavenging activity. The above results show that when the four components of this invention—Ganoderma lucidum extract, avocado oil unsaponifiables, ceramide NP, and ceramide EOP—are combined within a specific ratio range, they can effectively scavenge free radicals. Although the scavenging activity varies at different ratios, it remains at a high level.

[0139] (2) Evaluation of synergistic effect

[0140] The Jin Zhengjun method was further used to calculate q-values ​​to evaluate the interactions between the components. The results showed that the q-values ​​for Examples 1-4 were 1.18, 1.46, 1.18, and 1.98, respectively, all greater than the synergistic effect threshold of 1.15. This indicates that when the four components are combined within the specified ratio range, their free radical scavenging effect is not a simple summation of the individual component effects, but rather exhibits a clear super-additive synergistic effect. Example 4 had the highest q-value (1.98), demonstrating the most prominent synergistic effect. These results confirm that the antioxidant activity of the composition of this invention depends on the synergistic combination of the four components—Ganoderma lucidum extract, avocado oil unsaponifiables, ceramide NP, and ceramide EOP—within a specific ratio range.

[0141] (3) Comparison between Ganoderma lucidum extract and Ganoderma lucidum spore oil

[0142] This experiment further included comparative examples 9-12. The concentrations of each component in comparative examples 9-12 were consistent with those in examples 1-4, with the only difference being that the Ganoderma lucidum extract was replaced with an equal amount of Ganoderma lucidum spore oil (i.e., Ganoderma lucidum spore oil was used to replace Ganoderma lucidum polysaccharides / peptides as the active ingredients derived from Ganoderma lucidum). The other components (avocado oil unsaponifiables, ceramide NP, ceramide EOP) and their proportions remained unchanged.

[0143] The results showed that the DPPH free radical scavenging rates of Comparative Examples 9-12 were 34.76%, 27.54%, 57.37%, and 46.84%, respectively, all lower than those of Examples 1-4 (scavenging rates of 51.33%, 32.33%, 77.30%, and 75.47%, respectively), with reductions of 32.28%, 14.82%, 25.78%, and 37.93%, respectively. These results indicate that, under the same compounding ratio, replacing Ganoderma lucidum extract with Ganoderma lucidum spore oil significantly weakens the free radical scavenging activity of the composition, demonstrating that the contribution of Ganoderma lucidum extract to free radical scavenging cannot be replaced by Ganoderma lucidum spore oil.

[0144] Table 3 DPPH free radical scavenging detection results

[0145]

[0146] Examples 5 to 21

[0147] 1. The amount and ratio of the compositions added in each embodiment (Table 4).

[0148] Table 4. Information on the amount and proportion of compositions added in each embodiment.

[0149]

[0150] Example 2: Evaluation of antioxidant efficacy (ROS fluorescent probe experiment)

[0151] Technical principle:

[0152] This experiment used the DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) fluorescent probe method to detect the level of intracellular reactive oxygen species (ROS) in a 3D epidermal skin model. DCFH-DA is a lipophilic substance that is itself non-fluorescent and can freely cross the cell membrane to enter the cell. Once inside the cell, DCFH-DA is hydrolyzed by intracellular esterases to generate DCFH (dichlorodihydrofluorescein), which is more polar and cannot freely cross the cell membrane. This allows the probe to be labeled and retained within the cell. DCFH itself is also non-fluorescent, but when ROS are present in the cell, DCFH is oxidized to DCF (dichlorofluorescein), which exhibits strong green fluorescence. The intensity of the green fluorescence of DCF is directly proportional to the level of intracellular ROS. By detecting the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, the intracellular ROS content can be quantitatively reflected.

[0153] Based on the above principles, this experiment first used UVB irradiation to create a 3D epidermal skin model to induce the production of excessive intracellular ROS. Subsequently, the model was labeled with a DCFH-DA probe, and fluorescence images were observed and acquired using a fluorescence microscope. The fluorescence intensity reflected the level of reactive oxygen species in each group of model tissues. The antioxidant efficacy of each composition (Composition 17-Composition 22) was comprehensively evaluated by assessing its ability to scavenge excess reactive oxygen species induced by UVB.

[0154] 1. Experimental Design

[0155] 1) Grouped administration: The 3D epidermal model (EpiKutis) was administered to the target group. ® The samples were transferred to 6-well plates and randomly divided into blank control group (BC), negative control group (NC), positive control group (PC), and sample group, with 3 replicates per group. 0.9 mL of LEpiGrowth medium was added to each well in each group. In the PC group, 7 μg / mL vitamin E (VE) was applied to the model surface, and in the sample group, working solutions (compositions 17-22) were evenly applied to the model surface. After drug administration, the 6-well plates were incubated in a CO2 incubator (37°C, 5% CO2) for 24 h.

[0156] 2) UVB irradiation: After incubation, all groups except the blank control group underwent UVB irradiation at a dose of 600 mJ / cm². 2 After irradiation, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0157] 3) Cleaning: After incubation, clean the remaining test sample with sterile PBS and wipe away the residual liquid with sterile cotton swabs.

[0158] 4) ROS probe incubation: After incubation, add 20 μM of probe working solution to each well. Incubate in a CO2 incubator (37℃, 5% CO2) for 1 h, shaking the plate once every 20 min to ensure uniform model incubation.

[0159] 5) Sample collection: After incubation, cut off the model, place it in a 1.5 mL centrifuge tube, add 1 mL of 4% paraformaldehyde fixative to the tube, wrap it with aluminum foil, and send it for frozen sectioning.

[0160] 6) Cleaning and mounting: Clean the cut frozen sections with PBS for 8 min. After cleaning, wipe the surrounding liquid clean with absorbent paper, add 20 μL of fluorescent anti-quenching mounting medium, and cover with a coverslip.

[0161] 7) Photographs: Observe with a fluorescence microscope, take fluorescence images, and analyze and calculate the cumulative optical density (IOD) using ImageJ software.

[0162] 8) Inhibition rate calculation: Inhibition rate (%) = (average relative IOD of NC group - average relative IOD of sample group) / average relative IOD of NC group × 100%.

[0163] 9) Statistical Analysis: GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0164] Table 5. Experimental grouping of ROS fluorescent probes in 3D epidermal models

[0165]

[0166] 2. Experimental Results

[0167] The results of the reactive oxygen species (ROS) content detection are shown in Table 6. Figure 1 and Figure 2 Compared with the blank control group (BC), the ROS fluorescence intensity of the negative control group (NC, UVB irradiation group) was significantly increased (P < 0.01), indicating that UVB irradiation successfully induced excessive production of reactive oxygen species in the 3D epidermal skin model, and the oxidative damage stimulation conditions in this experiment were effective. Compared with the negative control group, the ROS fluorescence intensity of the positive control group was significantly decreased (P < 0.01), indicating that the positive control was effective and verifying the reliability of the experimental system.

[0168] Compared with the negative control group, the ROS content of sample combinations 17-22 of the present invention was significantly reduced (P<0.01), with inhibition rates of 74.92%, 72.67%, 54.34%, 68.81%, 48.87% and 80.39%, respectively. This indicates that each composition can remove excess reactive oxygen species induced by UVB irradiation to varying degrees and reduce oxidative stress damage, with sample combination 22 showing the best inhibitory effect.

[0169] Table 6. Results of ROS content detection in 3D epidermal models after treatment with compositions 17-22

[0170]

[0171] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # P-value < 0.01 ## Compared with the NC group, the significance was as follows: * This means that a p-value < 0.05 indicates that... *A p-value < 0.01 indicates that... ** .

[0172] Verification Example 3: Evaluation of Anti-inflammatory Efficacy (Immunofluorescence Experiment)

[0173] Technical Principle: The inflammatory response is a defensive response of the skin after exposure to external stimuli such as UVB radiation. After UVB irradiation, the skin is first induced to produce excessive reactive oxygen species (ROS) by keratinocytes, which then activate multiple intracellular signaling pathways. Among these, the activation of the nuclear factor-kappa B (NF-κB) signaling pathway is the core of the inflammatory response. NF-κB is a widely distributed nuclear transcription factor in cells. In its resting state, NF-κB binds to its repressor protein IκB to form a complex, existing in an inactive state in the cytoplasm. When cells are exposed to external stimuli such as UVB radiation, IκB is phosphorylated and degraded via the ubiquitin-proteasome pathway, releasing NF-κB. The released NF-κB then translocates from the cytoplasm into the nucleus, binds to the NF-κB binding site in the promoter region of downstream target genes, and initiates the transcriptional expression of various inflammation-related genes. These target genes include various pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Therefore, the level of NF-κB in the cell nucleus can directly reflect the activation level of inflammatory signaling pathways and is a key upstream indicator for evaluating anti-inflammatory efficacy. Tumor necrosis factor-α (TNF-α) is one of the most important downstream pro-inflammatory cytokines in the above-mentioned inflammatory cascade. Under normal physiological conditions, the expression level of TNF-α in the skin is low. After UVB irradiation, TNF-α is a key early response factor produced by keratinocytes, and its mRNA can be induced to express approximately 1.5 hours after UVB irradiation. As an inflammatory signaling molecule, TNF-α can be secreted extracellularly, further activating the NF-κB signaling pathway in neighboring cells, forming a positive feedback amplification effect of inflammatory signals, ultimately leading to inflammatory responses such as erythema and edema in the skin. Therefore, the reduction of TNF-α content is a key downstream indicator for evaluating the anti-inflammatory efficacy of the test substance.

[0174] Based on the above principles, this experiment uses a UVB irradiation 3D epidermal skin model (EpiKutis). ® This invention aims to activate the NF-κB signaling pathway and induce excessive secretion of TNF-α. The inhibitory effects of the various compositions (compositions 17-22) on UVB-induced NF-κB activation and excessive TNF-α secretion were evaluated using immunofluorescence, thereby comprehensively assessing their anti-inflammatory efficacy.

[0175] 1. Experimental Design

[0176] 1) Grouped administration: The 3D epidermal model (EpiKutis) was administered to the target group. ® The samples were transferred to 6-well plates and randomly assigned to blank control group (BC), negative control group (NC), positive control group (PC), and sample group, with three replicates per group. 0.9 mL LEpiGrowth medium was added to each well in each group. In the PC group, 0.01% dexamethasone was applied to the model surface, and in the sample group, the working sample solution was evenly applied to the model surface. After drug administration, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0177] 2) UVB irradiation: After incubation, all groups except the blank control group underwent UVB irradiation at a dose of 600 mJ / cm². 2 After irradiation, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0178] 3) Cleaning: After incubation, clean the remaining test sample with sterile PBS and wipe away the residual liquid with sterile cotton swabs.

[0179] 4) Immunofluorescence detection: The sample was fixed with 4% paraformaldehyde for 24 h. Immunofluorescence detection was performed using antibodies against NF-κB and TNF-α. The sample was photographed under a microscope, and the cumulative optical density (IOD) was analyzed and calculated using ImageJ software.

[0180] 5) Inhibition rate calculation: Inhibition rate (%) = (average relative IOD of NC group - average relative IOD of sample group) / average relative IOD of NC group × 100%.

[0181] 6) Statistical Analysis: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0182] Table 7 Grouping of Immunofluorescence Detection in 3D Epidermal Inflammation Model

[0183]

[0184] 2. Experimental Results

[0185] 2.1 Effect of Composition 22 on the expression of inflammatory factor TNF-α in a UVB irradiation-induced 3D epidermal model

[0186] The results of immunofluorescence assays are shown in Table 8. Figure 3 and Figure 4Compared with the BC group, the TNF-α protein expression level in the NC group (UVB irradiation group) was significantly increased (P < 0.01), indicating that UVB irradiation successfully activated the inflammatory response in the 3D epidermal skin model and promoted the secretion of the pro-inflammatory factor TNF-α, demonstrating the effectiveness of the stimulation conditions in this experiment. Compared with the NC group, the TNF-α content in the PC group was significantly decreased (P < 0.01), indicating the effectiveness of the positive control and verifying the reliability of the experimental system. Compared with the NC group, the TNF-α protein expression level in sample combination 22 of this invention was significantly decreased (P < 0.01), with an inhibition rate of 59.77%, indicating that sample combination 22 can effectively alleviate the skin inflammatory response induced by UVB irradiation and has good anti-inflammatory effects.

[0187] Table 8. Results of TNF-α protein expression detection in the 3D epidermal inflammation model after treatment with Composition 22

[0188]

[0189] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # P-value < 0.01 ## Compared with the NC group, the significance was as follows: * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0190] 2.2 Effect of Composition 17-22 on NF-κB protein expression in a UVB irradiation-induced 3D epidermal model

[0191] The results of immunofluorescence assays are shown in Table 9. Figure 5 and Figure 6 Compared with the BC group, the NF-κB protein expression level in the NC group was significantly increased after UVB irradiation (P < 0.01), indicating that the NF-κB-related inflammatory signaling pathway was successfully activated. Compared with the NC group, the NF-κB expression level in the PC group was significantly decreased (P < 0.01), verifying the effectiveness of the experimental system. Compared with the NC group, the NF-κB protein expression levels in sample combinations 1-6 were all significantly decreased (P < 0.01), with inhibition rates of 50.00%, 32.26%, 30.11%, 34.95%, 27.96%, and 54.30%, respectively, among which combination 22 showed the best effect. The above results suggest that the composition of the present invention can exert anti-inflammatory effects by inhibiting NF-κB.

[0192] Table 9. Results of NF-κB protein expression detection in 3D epidermal models after treatment with composition 17-22

[0193]

[0194] Note: When performing statistical analysis using the t-test method, significance compared to group BC is indicated by #, and P-value < 0.05 is indicated as... # P-value < 0.01 ## Compared with the NC group, the significance was as follows: * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0195] Verification Example 4: Evaluation of Barrier Repair Efficacy

[0196] Technical Principle: The skin barrier is mainly composed of the stratum corneum, and the integrity of its structure and function depends on the dynamic life activities of keratinocytes. Keratinocytes proliferate continuously from the basal layer of the epidermis, with newly formed cells gradually migrating upwards. After undergoing a series of complex programmed differentiation processes, they eventually form anucleate keratinocytes and are orderly shed. This cycle of "proliferation-migration-differentiation-shedding" ensures the continuous renewal and integrity of the barrier, forming the cellular basis of the dynamic balance of the skin barrier. In the repair process after barrier damage, each of the above-mentioned cellular life activities plays an irreplaceable role. Proliferative capacity determines the efficiency of new cell supply, migration capacity determines whether new cells can cover the damaged area in time, and the normal expression of terminal differentiation proteins during differentiation directly determines the structural and functional integrity of the newly formed stratum corneum. Among them, filaggrin (FLG) is a major precursor of natural moisturizing factors, participating in the structural assembly of the keratinized capsule and playing a crucial role in maintaining hydration and barrier integrity of the stratum corneum; lobelin (LOR) is the most abundant structural protein in the keratinized capsule, accounting for approximately 80% of the total capsule proteins, and plays a central role in reinforcing the physical barrier of the stratum corneum; nuclear proliferation-associated antigen (Ki67) is a key biological marker characterizing cell proliferation capacity, and its expression level directly reflects the proliferative activity of epidermal basal cells. Changes in the expression levels of these three proteins are direct indicators for evaluating whether the skin's physical barrier structure has been repaired.

[0197] To systematically evaluate the barrier repair efficacy of the compositions of this invention, this module conducts research at two levels. First, a 3D epidermal skin model was irradiated with UVB, and the expression levels of FLG, LOR, and Ki67 were detected by immunofluorescence to directly evaluate the repair effect of the test substances on the skin's physical barrier structure. Based on this, the migration ability of keratinocytes was further detected by a cell scratch assay, and the proliferation ability of keratinocytes was detected by an MTT assay, exploring the intrinsic driving force of barrier repair at the cellular behavioral level. These two levels of detection complement each other, together constituting a systematic evaluation system for the barrier repair efficacy of this invention.

[0198] Effects of the compositions (combinations 17-22) on a UVB irradiation-induced 3D epidermal barrier damage model (immunofluorescence assay).

[0199] 1. Experimental Design

[0200] 1) Grouped administration: The 3D epidermal model (EpiKutis) was administered to the target group. ® The samples were transferred to 6-well plates and randomly divided into a blank control group (BC), a negative control group (NC), a positive control group (PC), and a sample group, with three replicates per group. 0.9 mM LEpiGrowth medium was added to each well in each group. For the PC group, 50 μM pirinic acid (WY14643) was applied to the model surface, while for the sample group, the working sample solution was evenly applied to the model surface. After drug administration, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0201] 2) UVB irradiation: After incubation, all groups except the blank control group underwent UVB irradiation at a dose of 600 mJ / cm². 2 After irradiation, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0202] 3) Cleaning: After incubation, clean the remaining test sample with sterile PBS and wipe away the residual liquid with sterile cotton swabs.

[0203] 4) Immunofluorescence detection: The sample was fixed with 4% paraformaldehyde for 24 h. Immunofluorescence detection was performed using antibodies against FLG, LOR and Ki67. The sample was photographed under a microscope, and the cumulative optical density (IOD) was analyzed and calculated using ImageJ software.

[0204] 5) Calculation of improvement rate: Improvement rate (%) = (average relative IOD of sample group - average relative IOD of NC group) / average relative IOD of NC group × 100%.

[0205] 6) Statistical Analysis: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0206] Table 10 Grouping of Immunofluorescence Detection in 3D Epidermal Barrier Injury Model

[0207]

[0208] 2. Experimental Results

[0209] 2.1 Effect of Composition 22 on UVB-induced decrease in expression of 3D epidermal barrier structural proteins

[0210] The results of immunofluorescence assays are shown in Table 11. Figure 7 and Figure 8 Compared with the blank control group (BC), the negative control group (NC) showed a significant decrease in FLG protein expression after UVB irradiation (P < 0.01), indicating successful model establishment. Compared with the NC group, the PC group showed a significant increase in FLG expression (P < 0.01), verifying the effectiveness of the experimental system. Compared with the NC group, the sample combination 22 group showed a significant increase in FLG protein expression (P < 0.01), with an increase rate of 190.00%, indicating that sample combination 22 can effectively reverse the FLG expression inhibition caused by UVB irradiation.

[0211] Table 11 Results of FLG protein expression detection in 3D epidermal models after treatment with composition 22

[0212]

[0213] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # P-value < 0.01 ## Compared with the NC group, the significance was as follows: * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 is represented as **.

[0214] Further analysis of LOR protein expression revealed the following results (Table 12). Figure 9 and Figure 10Compared with the BC group, the LOR protein expression level in the NC group (UVB irradiation group) was significantly reduced (P < 0.01), indicating that UVB irradiation also successfully inhibited LOR protein expression. Compared with the negative control group, the LOR protein expression level in the positive control group was significantly increased (P < 0.01), further verifying the reliability of the experimental system. Compared with the negative control group, the LOR protein expression level in sample combination 22 was significantly increased (P < 0.01), with an increase rate of 115.38%, indicating that sample combination 22 could also effectively reverse the LOR expression inhibition caused by UVB irradiation.

[0215] The above results suggest that the sample combination 22 described in this invention can effectively repair skin physical barrier damage caused by UVB irradiation by simultaneously promoting the expression of two key barrier structural proteins, FLG and LOR, and has a good barrier repair effect.

[0216] Table 12 Results of LOR protein expression detection in 3D epidermal models after treatment with composition 22

[0217]

[0218] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # P-value < 0.01 ## Compared with the NC group, the significance was as follows: * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0219] 2.2 Effects of Compositions 17-21 on UVB-induced 3D epidermal cell proliferation

[0220] The results of Ki67 immunofluorescence assay showed (Table 13); Figure 11 and Figure 12 Compared with the BC group, the Ki67 positive cell rate in the NC group decreased significantly after UVB irradiation (P < 0.01), indicating successful modeling. Compared with the NC group, the Ki67 positive cell rate in the PC group increased significantly (P < 0.01), verifying the effectiveness of the experimental system. Compared with the NC group, the Ki67 positive cell rates in sample compositions 17 to 21 all increased significantly (P < 0.01), with increases of 257.81%, 172.77%, 101.56%, 156.03%, and 92.19%, respectively, with composition 17 showing the best effect. These results suggest that the compositions of the present invention can promote epidermal regeneration and accelerate barrier structure reconstruction by activating the proliferative activity of keratinocytes in the basal layer of the epidermis, thereby achieving skin repair effects.

[0221] Table 13. Detection results of Ki67 positive cells in the 3D epidermal model after treatment with composition 17-21.

[0222]

[0223] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # P-value < 0.01 ## Compared with the NC group, the significance was as follows: * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0224] Verification Example 5: Effect of Compositions 23-28 on Keratinocyte Migration (Cell Scratch Assay)

[0225] 1. Experimental Design

[0226] 1) Cell seeding: After resuscitating human primary keratinocytes, when the plating rate reaches about 60%, seed the cells into 6-well plates and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0227] 2) Grouping and Drug Administration: When the cell deposition rate in the 6-well plates reached approximately 40%–60%, drug administration was performed according to the test groups (Table 14), with 3 replicates per group. The blank control group (BC) received 2 mL of KcGrowth culture medium per well, the positive control group (PC) received 2 mL of KcGrowth culture medium containing 1 ng / mL epidermal growth factor (EGF) per well, and the sample group received 2 mL of KcGrowth culture medium containing the corresponding test sample per well. After drug administration, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0228] 3) Scraping and culture: After incubation, scratching was performed. After scratching, the cells were washed three times with PBS, and cell culture medium was added. The cells were then cultured in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0229] 4) Photography and Measurement: The initial scratch width was photographed under a 4x microscope 0 h after the scratch was applied. 24 h after the scratch, the area was cleaned once with PBS, and photographed again under a 4x microscope. The scratch area at each time point was measured using ImageJ software.

[0230] 5) Calculation of migration rate and lift rate:

[0231] Migration rate (%) = (Initial scratch area − 24 h scratch area) / Initial scratch area × 100%;

[0232] Improvement rate (%) = (Sample group migration rate - Blank control group migration rate) / Blank control group migration rate × 100%;

[0233] 6) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0234] Table 14 Grouping of Human Primary Keratinocyte Scratch Test

[0235]

[0236] 2. Experimental Results

[0237] The results of the cell scratch assay are shown in Table 15. Figure 13 and Figure 14 The relative cell migration rate of the positive control group (PC) was significantly higher than that of the blank control group (P < 0.01), confirming the effectiveness of the experimental system and the reliability of the test results. Compared with the blank control group (BC), the relative cell migration rates of sample combinations 23-28 described in this invention were all significantly increased (P < 0.01), with increases of 47.00%, 42.00%, 36.00%, 39.00%, 23.00%, and 56.00%, respectively, indicating that each composition has biological activity that promotes keratinocyte migration, and combination 28 has the strongest promoting effect. The above results suggest that the compositions described in this invention can exert skin barrier repair effects by enhancing the migration ability of keratinocytes.

[0238] Table 15. Cell scratch test results of compositions 23-28

[0239]

[0240] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0241] Validation Example 6: Effect of Composition 28 on Keratinocyte Proliferation (MTT assay)

[0242] 1. Experimental Design

[0243] 1) Cell seeding: After resuscitating human primary keratinocytes, when the plating rate reaches about 60%, seed the cells into 96-well plates and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0244] 2) Grouping and Drug Administration: When the cell deposition rate in the 96-well plates reached approximately 20%–30%, drug administration was performed according to the test groups (Table 16), with 200 μL of sample added to each well, and three replicates per group. The blank control group received 200 μL of KcGrowth medium per well, the positive control group received 200 μL of KcGrowth medium containing 1 ng / mL epidermal growth factor (EGF) per well, and the sample group received 200 μL of KcGrowth medium containing the corresponding concentration of the test sample per well. After drug administration, the 96-well plates were placed in a CO2 incubator (37℃, 5% CO2) for 24 h, 48 h, and 72 h, respectively. For groups requiring 48 h and 72 h of incubation, the medium was changed halfway daily.

[0245] 3) MTT assay: After cells were incubated for 24 h, 48 h, and 72 h, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The cells were incubated at 37°C in the dark for 4 h. After incubation, the supernatant was discarded, and 150 µL of DMSO was added to each well. The OD value was read at 490 nm.

[0246] 4) Calculation of relative cell viability: Relative cell viability (%) = (sample well OD − zeroing well OD) / (solvent control well OD − zeroing well OD) × 100%.

[0247] 5) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0248] Table 16 Grouping of Human Primary Keratinocyte Proliferation Assay (MTT Method)

[0249]

[0250] 2. Experimental Results

[0251] Cell viability assay results (Table 17) showed that, compared with the blank control group (BC), the positive control group (PC) significantly promoted the proliferation of keratinocytes at 24 h, 48 h, and 72 h after drug administration (P < 0.01), indicating that the experimental system and positive control were effective and the assay results were reliable. Compared with the blank control group, the sample combination 28 described in this invention also significantly promoted the proliferation of keratinocytes at the same time points (24 h, 48 h, and 72 h) (P 24 h < 0.05, P 48 h, 72 h < 0.01), and the promoting effect showed a time-dependent increasing trend. Figure 15 The above results indicate that sample combination 28 possesses biological activity that promotes keratinocyte proliferation, suggesting that it can exert a repairing effect by enhancing the cell regeneration capacity of the epidermal basal layer and accelerating the repair of the damaged skin barrier.

[0252] Table 17 Cell viability test results of composition 28

[0253]

[0254] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0255] Verification Example 7: Evaluation of Firming and Anti-wrinkle Efficacy

[0256] Effects of compositions (combinations 23-28) on UVB irradiation-induced inhibition of collagen gene expression in keratinocytes (RT-qPCR detection).

[0257] Technical Principle: Collagen IV, Collagen VII, and Collagen XVII are key collagens in the dermal-epidermal junction (DEJ). Collagen XVII is a transmembrane collagen specifically expressed by basal keratinocytes and is an important component of hemidesmosomes, crucial for epidermal cell regeneration and maintaining the integrity of the dermal-epidermal junction. UVB (medium-wave ultraviolet radiation, wavelength 280-320 nm) irradiation inhibits the gene expression of these collagens, leading to weakened dermal-epidermal junction and skin laxity. This module constructs a damage model by irradiating keratinocytes with UVB and uses real-time quantitative PCR (RT-qPCR) to detect changes in the gene expression levels of COL IV, COL VII, and COL XVII, evaluating the pro-expression effects of each composition on related collagens in the DEJ region.

[0258] 1. Experimental Design

[0259] 1) Cell seeding: After resuscitating human primary keratinocytes, when the plating rate reaches about 60%, seed the cells into 6-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0260] 2) Grouping and Drug Administration: When the cell deposition rate in the 6-well plates reached 30%–50%, drugs were administered to the test groups (Table 18), with 3 replicates per group. 2 mL of KcGrowth medium was added to each well of the blank control group (BC) and negative control group (NC); 2 mL of KcGrowth medium containing 100 ng / mL transforming growth factor (TGF-β1) was added to each well of the positive control group (PC); and 2 mL of KcGrowth medium containing the corresponding test sample was added to each well of the sample group. After drug administration, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0261] 3) UVB irradiation stimulation: After the culture period, except for the blank control group, all other groups were subjected to UVB irradiation at a dose of 300 mJ / cm². 2 After irradiation, the samples were placed in a CO2 incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0262] 4) Gene expression detection: After incubation, the cells were washed twice with PBS, and 1 mL of AG RNAex ProReagent was added to each well. After cell lysis by pipetting, the samples were collected. RNA was extracted, reverse transcribed into cDNA, and then subjected to real-time PCR. The relative gene expression levels of COL Ⅳ, COL VII, and COL ⅩⅦ were calculated using the 2^(-ΔΔCt) method.

[0263] 5) Calculation of gene expression upregulation rate: Upregulation rate (%) = (sample group − negative control group) / negative control group × 100%.

[0264] 6) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0265] Table 18 Grouping of UVB irradiation-induced collagen gene expression detection in keratinocytes

[0266]

[0267] 2. Experimental Results

[0268] 2.1 Effect of Composition 28 on the inhibition of COL IV and COL VII gene expression in UVB irradiation-induced keratinocytes

[0269] Gene expression detection results showed that, compared with the blank control group (BC), the expression levels of COL IV and COL VII genes in the negative control group (NC, UVB irradiation group) were significantly downregulated (PCOL IV < 0.01, PCOL VII < 0.05), indicating that UVB irradiation successfully constructed a cell model of impaired collagen expression in the dermal junction region (DEJ region). Compared with the negative control group, the expression levels of COL IV and COL VII genes in the positive control group (PC) were significantly upregulated (P < 0.01), indicating that the positive control was effective and verifying the effectiveness of the experimental system. Compared with the negative control group, the expression levels of COL IV (Table 19, ...) in the sample composition 28 of this invention were significantly higher. Figure 16 ), COL VII (Table 20, Figure 17 The expression levels of both genes were significantly upregulated (P < 0.01), with upregulation rates of 101.47% and 184.09%, respectively. This indicates that sample composition 28 can effectively reverse the inhibition of COL Ⅳ and COL VII gene expression caused by UVB irradiation, suggesting that it can enhance the tight connection between the dermis and epidermis, improve the structural support of the skin, and thus exert firming and anti-wrinkle effects.

[0270] Table 19 Results of COL IV gene expression detection after treatment with composition 28

[0271]

[0272] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # A p-value < 0.01 indicates that... ## Compared with the NC group, the significance was... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0273] Table 20 Results of COL VII gene expression detection after treatment with composition 28

[0274]

[0275] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # A p-value < 0.01 indicates that... ##Compared with the NC group, the significance was... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0276] 2.2 Effect of Compositions 23-28 on the Inhibition of COL XVII Gene Expression in Keratinocytes Induced by UVB Irradiation

[0277] The results of COL XVII gene expression detection showed (Table 21, Figure 18 Compared with the blank control group (BC), the expression level of COL ⅩⅦ gene in the negative control group (NC, UVB irradiation group) was significantly downregulated (P < 0.01), indicating that UVB irradiation successfully constructed a cell model of impaired collagen expression in the dermal junction region (DEJ). Compared with the negative control group, the expression level of COL ⅩⅦ gene in the positive control group (PC) was significantly upregulated (P < 0.01), indicating that the positive control was effective and verifying the effectiveness of the experimental system. Compared with the negative control group (NC), the expression of COL ⅩⅦ gene in sample combinations 23-28 of this invention was significantly upregulated (P < 0.01), with upregulation rates of 84.31%, 66.67%, 47.06%, 62.75%, 31.37%, and 107.84%, respectively, indicating that each composition could reverse the inhibition of COL ⅩⅦ gene expression caused by UVB irradiation to varying degrees, with combination 28 showing the best effect. The above results suggest that the compositions 23-28 of the present invention can improve the skin's dermal-epidermal junction by upregulating the expression of COL XVII gene, promoting the adhesion and regeneration of epidermal stem cells / basal cells, enhancing the structural stability of the DEJ region, and thus achieving skin-firming and anti-wrinkle effects.

[0278] Table 21. Results of COL XVII gene expression detection after treatment with compositions 23-28

[0279]

[0280] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # A p-value < 0.01 indicates that... ## Compared with the NC group, the significance was... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0281] Validation Example 8: Effect of Compositions 29-33 on the expression of collagen COL I and matrix metalloproteinase MMP-1 genes in UVA-irradiated fibroblasts (RT-qPCR detection)

[0282] Technical Principle: Type I collagen (Collagen I) accounts for approximately 80% of the total protein in the dermis and is a key structural protein for maintaining skin plumpness. Matrix metalloproteinase 1 (MMP-1) is a collagenase that can degrade extracellular matrix components such as Collagen I, Collagen III, and Collagen V. UVA (long-wave ultraviolet radiation, wavelength 320-400 nm) irradiation induces upregulation of MMP-1 expression in fibroblasts while inhibiting Collagen I expression, leading to collagen degradation, loss of skin's mechanical support, and thus wrinkles. This module constructs a photoaging model by irradiating fibroblasts with UVA, detects changes in the gene expression of COL I and MMP-1, and comprehensively evaluates the firming and anti-wrinkle effects of the samples.

[0283] 1. Experimental Design

[0284] 1) Cell seeding: After cell resuscitation, when the plate-laying rate reaches about 60%, human primary fibroblast cells are seeded into 6-well plates and incubated overnight in a CO2 incubator (37℃, 5% CO2).

[0285] 2) Grouping and Drug Administration: When the cell deposition rate in the 6-well plates reached 30%–50%, drug administration was performed according to the test groups (Table 22), with 3 replicates per group. 2 mL of FbGrowth medium was added to each well of the blank control group (BC) and negative control group (NC), 2 mL of FbGrowth medium containing 100 ng / mL transforming growth factor (TGF-β1) was added to each well of the positive control group (PC), and 2 mL of FbGrowth medium containing the corresponding concentration of the test sample (combinations 29-33) was added to each well of the sample group. After drug administration, the 6-well plates were placed in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0286] 3) UVA irradiation: After incubation, all groups except the blank control group underwent UVA irradiation with a dose of 30 J / cm². 2 After irradiation, the samples were placed in a CO2 incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0287] 4) Gene expression detection: After incubation, wash twice with PBS, add 1 mL of AG RNAex ProReagent to each well, lyse the cells by pipetting, and collect the samples. Extract RNA, reverse transcribe it into cDNA, and perform real-time PCR detection. Calculate the relative expression levels of COL I and MMP-1 genes using the 2^(-ΔΔCt) method.

[0288] 5) Calculation of gene expression upregulation and downregulation rates:

[0289] COL I gene expression upregulation rate (%) = (sample group − negative control group) / negative control group × 100%.

[0290] MMP-1 gene expression downregulation rate (%) = (negative control group − sample group) / negative control group × 100%.

[0291] 6) Statistical Analysis of Results: GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0292] Table 22 Grouping of UVA irradiation-induced collagen and matrix metalloproteinase gene expression detection in fibroblasts

[0293]

[0294] 2. Experimental Results

[0295] 2.1 Effect of compositions 29-33 on UVA irradiation-induced inhibition of COL I gene expression in fibroblasts

[0296] The results of COL I gene expression detection showed (Table 23, ...). Figure 19 Compared with the blank control group (BC), the expression level of COL I gene in the negative control group (NC, UVA irradiation group) was significantly downregulated (P < 0.01), indicating that UVA irradiation successfully constructed a fibroblast photoaging damage model. Compared with the negative control group (NC), the expression level of COL I gene in the positive control group (PC) was significantly upregulated (P < 0.01), indicating that the positive control was effective and verifying the reliability of the experimental system. Compared with the negative control group (NC), the expression of COL I gene in sample combinations 29-33 of this invention was significantly upregulated (P for combination 29-32 < 0.01, P for combination 33 < 0.05), with upregulation rates of 86.57%, 68.66%, 47.76%, 70.15%, and 32.84%, respectively, indicating that each composition can effectively reverse the inhibition of COL I expression caused by UVA irradiation, with combination 29 showing the best effect.

[0297] Table 23. Results of COL I gene expression detection after treatment with compositions 29-33

[0298]

[0299] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # A p-value < 0.01 indicates that... ## Compared with the NC group, the significance was... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0300] 2.2 Effects of compositions 29-33 on MMP-1 gene expression in UVA-irradiated fibroblasts

[0301] MMP-1 gene expression detection results showed (Table 24, ...). Figure 20 Compared with the blank control group (BC), the expression level of MMP-1 in the negative control group (NC, UVA irradiation group) was significantly upregulated (P < 0.01), indicating that UVA irradiation successfully induced high expression of MMP-1 in the fibroblast photoaging damage model. Compared with the negative control group (NC), the expression level of MMP-1 in the positive control group (PC) was significantly downregulated (P < 0.01), indicating that the positive control was effective and verifying the reliability of the experimental system. Compared with the negative control group (NC), the expression of MMP-1 in sample combinations 29-33 of this invention was significantly downregulated (P for combination 29-32 < 0.01, P for combination 33 < 0.05), with downregulation rates of 55.19%, 42.62%, 30.05%, 43.72%, and 19.67%, respectively, indicating that each composition effectively reversed the upregulation of MMP-1 expression caused by UVA irradiation, with combination 29 showing the best effect.

[0302] Combining the aforementioned results of COL I gene upregulation (Table 23, Figure 19 The composition of this invention can effectively repair UVA-induced photoaging damage and protect the integrity of the extracellular matrix of the dermal layer through a dual pathway of "promoting collagen synthesis and inhibiting collagen protein hydrolysis", thereby restoring the skin's mechanical support and achieving firming and anti-wrinkle effects.

[0303] Table 24 Results of MMP-1 gene expression detection after treatment with compositions 29-33

[0304]

[0305] Note: When performing statistical analysis using the t-test method, the significance level compared to group BC is determined by... # This means that a p-value < 0.05 indicates that... # A p-value < 0.01 indicates that... ## Compared with the NC group, the significance was... * This means that a p-value < 0.05 indicates that... * A p-value < 0.01 indicates that... ** .

[0306] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, include: Reishi mushroom extract, avocado oil unsaponifiables, and ceramides; The mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, and the ceramide is (1~5000):(1~50):(1~1000).

2. The composition according to claim 1, characterized in that, The mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables and the ceramide is (1~3000):(1~50):(1~500).

3. The composition according to claim 1 or 2, characterized in that, The ceramides include: ceramide NP and ceramide EOP.

4. The composition according to claim 3, characterized in that, The mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, the ceramide NP, and the ceramide EOP is (1~50):(1~50):(1~50):(1~50); or The mass ratio of the Ganoderma lucidum extract, the avocado oil unsaponifiables, the ceramide NP, and the ceramide EOP is 5000:1:1000:1000.

5. The use of the composition according to any one of claims 1 to 4 in the preparation of products for barrier repair, anti-oxidation, anti-inflammation and / or anti-aging.

6. The application as described in claim 5, characterized in that, The barrier repair includes upregulating the expression of FLG and / or LOR genes.

7. The application as described in claim 5 or 6, characterized in that, The antioxidants include: scavenging free radicals and / or reducing ROS levels.

8. The application as described in any one of claims 5 to 7, characterized in that, The anti-inflammatory effects include downregulating the expression of the TNF-α gene and / or the NF-κB gene.

9. The application as described in any one of claims 5 to 8, characterized in that, The anti-aging measures include: upregulating the expression of COL I, COL IV, COL VII and / or COL XVII genes; and / or downregulating the expression of MMP-1 gene.

10. Personal care products, characterized in that, include: The composition according to any one of claims 1 to 4.

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