A layered anti-aging composition and its application

Abstract

This invention discloses a layered anti-aging composition and its application. It comprises 4-10 parts of *Vigna angularis* fruit extract, 0.01-0.2 parts of soluble collagen, and 10-20 parts of *Fucus vesiculosus* extract, all parts by weight. The composition of this invention employs a layered anti-aging skincare strategy ("layered" generally refers to different skin areas, i.e., "cellular layer-dermis-epidermal layer"). This composition can promote the expression of LOR (lower retinal occlusion) and the relative expression level of TGM5 (epidermis), increase the content of type I procollagen C-terminal peptide and elastin (dermis), enhance mitochondrial activity, and increase ATP content (cellular layer). That is, it works progressively from the epidermis to the dermis to the cellular layer, having a relevant effect on each layer of skin, and possessing the functions of enhancing skin elasticity, firming, anti-aging, and repairing the skin barrier.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics, specifically relating to a layered anti-aging composition and its application. Background Technology

[0002] As we age, the accumulation of endogenous stress and exogenous stimuli gradually affects skin health. Internal causes: Primarily due to the passage of time, also known as natural aging. Genes are the main cause of natural skin aging, determining its speed and extent. However, other endogenous factors, including nutrition, endocrine function, and immunity, also influence skin aging through the body's overall processes. External causes: Primarily caused by ultraviolet radiation from the sun, also known as photoaging. Ultraviolet radiation is the most important external factor causing skin aging, damaging the elastic and collagen fibers in the dermis, accelerating skin aging, sagging, wrinkles, and age spots.

[0003] The skin is composed of the epidermis and dermis. Because different layers of skin face different stimuli, they exhibit different characteristics and problems during the aging process. Epidermal aging manifests as dryness, fine lines, and dullness; dermal aging manifests as loss of elasticity, decreased plumpness, and wrinkles. Skin aging is not only about changes in the epidermis and dermis; at its root, it is the aging of the skin's constituent units—the cells. In 2023, *Cell* published twelve markers of aging: genomic instability, telomere wear, epigenetic alterations, loss of protein homeostasis, macroautophagy inactivation, nutrient sensing dysregulation, mitochondrial dysfunction, cellular senescence, stem cell depletion, altered intercellular communication, chronic inflammation, and gut microbiota dysbiosis.

[0004] It is evident that skin aging is caused by multiple factors, and a single treatment may not be able to improve the multi-layered aging problems.

[0005] Flat-leaved vanilla is known for its captivating and unique aroma.

[0006] Collagen is a structural protein that accounts for 30% of the total protein in the human body. As the most important component of the extracellular matrix, collagen accounts for more than 80% of the dermis.

[0007] Fucus vesiculosus is a cold-temperate perennial seaweed that is widely distributed along the Atlantic and Pacific coasts and is rich in a variety of bioactive components. Summary of the Invention

[0008] To comprehensively address the problem of aging, this invention provides a layered anti-aging composition and its application. The composition employs a layered anti-aging skincare strategy, progressing from the epidermis to the dermis and then to the cells, enhancing mitochondrial activity, increasing ATP levels, boosting collagen and elastin content, and promoting the expression of acetylcholine, thereby enhancing skin elasticity, combating aging, and repairing the skin barrier.

[0009] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0010] The present invention provides a layered anti-aging composition comprising 4-10 parts of flat-leaved vanilla fruit extract, 0.01-0.2 parts of soluble collagen and 10-20 parts of fucus vesiculosus extract, all of which are parts by weight.

[0011] In this invention, the composition preferably consists of 4-10 parts of the flat-leaved vanilla fruit extract, 0.01-0.2 parts of the soluble collagen, and 10-20 parts of the fucus vesiculosus extract.

[0012] In this invention, the preferred mass ratio of the soluble collagen and the Fucus vesiculosus extract is 6:0.05:12, 8:0.05:12, 10:0.05:12, 8:0.01:12, 8:0.08:12, 8:1:12, 8:0.05:10, 8:0.05:13 or 8:0.05:15, and more preferably (6-10):(0.01-0.08):(10-15).

[0013] In this invention, the amount of the flat-leaved vanilla fruit extract can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts, preferably 6-10 parts.

[0014] In this invention, the extract of flat-leaved vanilla fruit can be prepared by conventional methods in the art, preferably by the following method: soaking flat-leaved vanilla fruit in a solvent, microfiltering the filtrate obtained by solid-liquid separation, and obtaining flat-leaved vanilla fruit extract.

[0015] The flat-leaved vanilla pods generally refer to the pods of the vanilla plant.

[0016] Before soaking, the flat-leaved vanilla fruit is generally pulverized. After pulverization, the flat-leaved vanilla fruit powder has a mesh size of about 10 mesh.

[0017] The mass ratio of the flat-leaved fragrant fruit to the solvent can be 1:(5-10), for example, 1:5 or 1:10.

[0018] The solvent is generally an alcohol solvent, preferably ethanol.

[0019] The soaking is generally performed at room temperature. The soaking time is conventional in the art, preferably 2-5 hours, for example 3 hours.

[0020] The solid-liquid separation method can be conventional in the art, such as filtration. The filtration is generally gauze filtration. The mesh size of the gauze can be 600-200 mesh.

[0021] The filtrate obtained from the solid-liquid separation is typically concentrated. This concentration is usually performed under reduced pressure.

[0022] The microfiltration typically employs a microfiltration membrane. The pore size of the microfiltration membrane is preferably 2.5–10 μm, for example, 2.5 μm, 6 μm, 8 μm, or 10 μm.

[0023] In a preferred embodiment, the extract of flat-leaved vanilla fruit is prepared by the following method: flat-leaved vanilla fruit is pulverized, soaked in ethanol at room temperature for 3 hours (the mass ratio of flat-leaved vanilla fruit to ethanol is 1:10), filtered through 200-mesh gauze to obtain the supernatant, the supernatant is concentrated under reduced pressure to obtain flat-leaved vanilla fruit concentrate, and filtered through a microfiltration membrane to obtain the flat-leaved vanilla fruit extract.

[0024] In this invention, the amount of soluble collagen can be 0.02 parts, 0.03 parts, 0.04 parts, 0.05 parts, 0.06 parts, 0.08 parts, 0.09 parts, 0.1 parts, 0.12 parts or 0.15 parts, preferably 0.01-0.1 parts.

[0025] In this invention, the soluble collagen can be conventional recombinant collagen in the art, and can be one or more of recombinant type I, III, V, VII and XVII, such as recombinant type III collagen.

[0026] In this invention, the amount of the Fucus vesiculosus extract can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, preferably 10-15 parts.

[0027] In this invention, the extract of *Fucus vesiculosus* can be prepared by conventional methods in the art, preferably by the following method: heating a mixture containing *Fucus vesiculosus*, ultrafiltration of the filtrate obtained from solid-liquid separation, and obtaining the extract of *Fucus vesiculosus*.

[0028] Before use, the *Fucus vesiculosus* is generally rinsed repeatedly with water to remove mud, sand, and salt, and then dried. The drying temperature can be 40-60℃.

[0029] The *Fucus vesiculosus* is generally pulverized before use. After pulverization, the *Fucus vesiculosus* powder has a mesh size of 20-50 mesh.

[0030] The solvent used in the mixture containing *Fucus vesiculosus* is generally water. The water is generally deionized water.

[0031] In the mixture containing Fucus vesiculosus, the mass ratio of Fucus vesiculosus to solvent can be 1:(10-15), preferably 1:15.

[0032] The pH value of the mixture containing Fucus vesiculosus can be 3-3.5, for example, 3. Hydrochloric acid is generally used to adjust the pH value of the system.

[0033] The heating time can be 2-5 hours, for example, 2 hours.

[0034] The heating temperature can be 60-80℃, for example, 80℃.

[0035] The solid-liquid separation method can be conventional in the art, such as filtration. The filtration is generally gauze filtration. The mesh size of the gauze can be 600-200 mesh.

[0036] During ultrafiltration, the ultrafiltration membrane preferably has a rejection rate of 10 kDa.

[0037] In the ultrafiltration process, the pore size of the ultrafiltration membrane is preferably 0.45-0.8 μm, for example, 0.45 μm or 0.6 μm.

[0038] In a preferred embodiment, the extract of *Fucus vesiculosus* is prepared by the following method: *Fucus vesiculosus* is washed with water, dried at 40°C, and pulverized to obtain *Fucus vesiculosus* powder with a mesh size of approximately 20-50 mesh; the pH of the mixture of *Fucus vesiculosus* powder and water is adjusted to 3 using hydrochloric acid solution, the mass ratio of *Fucus vesiculosus* powder to water is 1:15, and the mixture is heated at 80°C for 2 hours; after cooling, the mixture is filtered through 200-mesh gauze to obtain a filtrate, which is then ultrafiltered through a 0.45 μm ultrafiltration membrane with a rejection rate of 10 kDa to obtain the *Fucus vesiculosus* extract.

[0039] In this invention, the composition preferably further contains a preservative.

[0040] The preservative can be of conventional types in the art, and is preferably one or more of 1,3-butanediol, 1,2-propanediol, dipropylene glycol, and glycerol.

[0041] The amount of the preservative can be conventional in the art, for example, 70-90%, where the percentage is its mass percentage of the total composition.

[0042] In this invention, the composition may have layered anti-aging effects, and is preferably used in cosmetics as an ingredient that enhances skin elasticity, firms, resists aging, or repairs the skin barrier.

[0043] In this invention, the composition preferably has the effects of promoting the expression of LOR protein, the relative expression level of TGM5, increasing the content of type I procollagen C-terminal peptide and collagen, increasing the content of elastin, enhancing mitochondrial activity, or increasing ATP content.

[0044] In this invention, the composition can be used in cosmetics after dilution. The dilution can be performed using distilled water. The preferred concentration after dilution is 2-10%, where the percentage is the mass of the composition relative to the total mass of the diluted liquid.

[0045] The present invention provides a method for preparing the layered anti-aging composition, which includes the following steps: mixing the components of the composition evenly.

[0046] The mixture is generally homogenized by stirring at room temperature in a homogenizer for 20 minutes.

[0047] The present invention also provides an application of the layered anti-aging composition in the field of cosmetics.

[0048] In this invention, the composition may have layered anti-aging effects, and is preferably used in cosmetics as an ingredient that enhances skin elasticity, firms, resists aging, or repairs the skin barrier.

[0049] In this invention, the composition preferably has the effects of promoting the expression of LOR protein, the relative expression level of TGM5, increasing the content of type I procollagen C-terminal peptide and collagen, increasing the content of elastin, enhancing mitochondrial activity, or increasing ATP content.

[0050] In this invention, the form of the cosmetic is not limited, including but not limited to water, emulsion, spray, essence, cream, mask, facial cleanser, foundation or cushion, etc.

[0051] In this invention, the composition can be used in cosmetics after dilution. The dilution can be performed using distilled water. The preferred concentration after dilution is 2-10%, where the percentage is the mass of the composition relative to the total mass of the diluted liquid.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] The reagents and raw materials used in this invention are all commercially available.

[0054] The positive and progressive effects of this invention are as follows:

[0055] The composition of this invention has a layered anti-aging skin care strategy ("layered" generally refers to different skin areas, namely "cell layer-dermis-epidermis"). This composition can promote the expression of LOR protein, the relative expression level of TGM5 (epidermis), increase the content of type I procollagen C-terminal peptide and elastin (dermis), enhance mitochondrial activity, and increase ATP content (cell layer). That is, it has a progressive effect on each layer of skin from epidermis to dermis to cell layer, and has the effects of enhancing skin elasticity, firming, anti-aging and repairing the skin barrier.

[0056] In some preferred embodiments, the present invention demonstrates its anti-aging effect in the epidermis by promoting the expression of LOR (Large Inch Reduction) and the relative expression level of TGM5; it demonstrates its anti-aging effect in the dermis by increasing the content of type I procollagen C-terminal peptide and elastin; and it demonstrates its anti-aging effect in the cellular layer by enhancing mitochondrial activity and increasing ATP content. The composition of the present invention can be customized layer by layer according to the different causes of aging in each layer of skin, providing layered anti-aging solutions and addressing aging problems from superficial to deep. Attached Figure Description

[0057] Figure 1 The effects of the blank control group, negative control group, positive control group, and the composition of Example 2 on ATP content in HDF cells were compared with the negative control group. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0058] Figure 2 The effects of the blank control group, positive control group, and the composition of Example 2 on the elastin content in HDF cells were compared with the blank control group. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0059] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0060] Unless otherwise specified, the raw materials and equipment used in the embodiments are all commonly used raw materials and equipment in the field and are all commercially available products.

[0061] Flat-leaved fragrant fruit and bladderwort are both commercially available products.

[0062] Soluble collagen was purchased from Jiangsu Chuangjian Medical Technology Co., Ltd., and it was recombinant type III collagen.

[0063] Examples 1-9 and Comparative Examples 1-3

[0064] The method for preparing the composition includes the following steps:

[0065] (1) Flat-leaved vanilla fruit extract: Flat-leaved vanilla fruit was crushed to a particle size of about 10 mesh, and soaked in ethanol at room temperature for 3 hours (ethanol: saponin fruit = 5:1). The supernatant was obtained by filtering through 200 mesh gauze. The supernatant was concentrated under reduced pressure to obtain a concentrate, which was then filtered through an 8 μm microfiltration membrane to obtain flat-leaved vanilla fruit extract.

[0066] (2) Fucus vesiculosus extract: Fucus vesiculosus was repeatedly rinsed with tap water to remove mud and salt, dried (40℃) and then pulverized to obtain Fucus vesiculosus powder with a mesh size of about 20-50 mesh. An appropriate amount of Fucus vesiculosus powder was weighed and added to deionized water at a mass ratio of 1:15. The pH was adjusted to 3 with an appropriate amount of hydrochloric acid solution and heated in a water bath at 80℃ for 2 hours. After cooling, the filtrate was filtered through 200-mesh gauze. The filtrate was then ultrafiltered through a 0.45μm ultrafiltration membrane with a rejection rate of 10 kDa to obtain Fucus vesiculosus extract.

[0067] (3) Mix the extracts of Vanilla sphaerocephala, Fucus vesiculosus, and soluble collagen evenly according to the proportions shown in Table 1, and stir in a homogenizer at room temperature for 20 minutes to obtain the composition. The total mass of the composition is 100g.

[0068] Table 1 shows the composition ratios of the compositions in Examples 1-9 and Comparative Examples 1-3 (the composition ratios of *Erigeron breviscapus* fruit extract, soluble collagen, and *Fucus vesiculosus* extract are by mass, and the total mass of the compositions after different ratios is kept consistent). Efficacy tests were performed on the compositions obtained in the Examples and Comparative Examples.

[0069] Table 1. Composition of the formulations in the examples and comparative examples

[0070]

[0071] Example 1: Measurement of mitochondrial activity

[0072] JC-1 is a fluorescent probe used to detect mitochondrial membrane potential (MMP) in cells, tissues, or purified mitochondrial membranes. Changes in MMP are detected by the change in fluorescence color: when the MMP is high, JC-1 aggregates in the mitochondrial matrix, forming a polymer and producing red fluorescence; when mitochondria are damaged, the MMP decreases, and JC-1 cannot aggregate in the mitochondrial matrix, remaining as a monomer and producing green fluorescence.

[0073] HaCaT cells were irradiated with a certain dose of UVB to cause cell damage. Then, JC-1 staining was used to compare the changes in fluorescence intensity of mitochondrial membrane potential between the test sample group and the model control group. The ratio of red to green fluorescence intensity of mitochondrial membrane potential was calculated, i.e., mitochondrial membrane red / green fluorescence intensity ratio = S(red fluorescence intensity) / S(green fluorescence intensity) * 100%, which can determine the strength of mitochondrial activity.

[0074] The specific operating steps are as follows:

[0075] 1) Cell plating: Seed 5 x 10⁶ cells / year. 4 / well human dermal fibroblast HDF (purchased from Boxi Biotechnology) were cultured in 24-well plates at an experimental environment (37 ℃, 5% CO2) for 24 ± 2 h.

[0076] 2) Drug administration: The experimental groups were blank control group, positive control group and test sample group. Each well was inoculated with the above experimental groups and cultured in the experimental environment for 4 hours. After the experiment, D-Hanks were washed 1-2 times and UVB was used to create the model.

[0077] The blank control group was a blank control, with only an equal amount of complete culture medium added;

[0078] Positive control group: 100 ng / ml TGF-β1 solution was added; 100 ng / ml TGF-β1 solution preparation: 10 μg / mL TGF-β1 (purchased from GenScript, model Z03411) was diluted to 100 ng / mL using complete culture medium to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0079] Test sample group: The example composition or comparative composition was diluted to a concentration of 1% using a complete culture medium. The percentage refers to the mass ratio of the composition to the sample solution.

[0080] The complete culture medium was DMEM medium (purchased from Gibco, model number 10567014), which included DMEM, low sugar, GlutaMAX™ additive, and pyruvate.

[0081] 3) After modeling, D-Hanks wash 1-2 times, as in step 2), add the corresponding substances to the blank control group, positive control group and test sample group respectively, and continue to culture in the experimental environment for 24±2h; irradiate with a certain dose of UVB (intensity of 50mJ), and after the end, stain with JC-1, observe and photograph under a fluorescence microscope.

[0082] 4) Analyze the fluorescence intensity (S) of each group and calculate the rate of decrease in mitochondrial membrane potential.

[0083] The experimental results are shown in Table 2.

[0084] Table 2

[0085]

[0086] It can be seen from the above table:

[0087] 1) The mitochondrial membrane electro-red / green fluorescence intensity ratios of the compositions obtained in Examples 1-9 were all higher than those in Comparative Examples 1-3, indicating that the compositions obtained in Examples 1-9 had a better effect on enhancing mitochondrial activity than those in Comparative Examples 1-3.

[0088] Regarding the ratio of red to green fluorescence intensity of mitochondrial membrane, Examples 2, 3, and 5-9 all achieved a value of 1.88 or higher, with Examples 3 and 8 being the most optimal.

[0089] 2) In Examples 1-3, as the content of the extract of *Erigeron breviscapus* gradually increased, the ratio of red to green fluorescence intensity of the mitochondrial membrane gradually increased; in Examples 4, 2, 5, and 6, as the content of soluble collagen gradually increased, the ratio of red to green fluorescence intensity of the mitochondrial membrane did not change significantly and there was no obvious pattern; in Examples 7, 2, 8, and 9, as the content of *Fucus vesiculosus* extract gradually increased, the ratio of red to green fluorescence intensity of the mitochondrial membrane did not change significantly and there was no obvious pattern.

[0090] This shows that there is a significant dose-response relationship between the mitochondrial membrane red / green fluorescence intensity ratio and the extract of *Vanilla sylvestris*, but the increase in the mitochondrial membrane red / green fluorescence intensity ratio slows down as the content of *Vanilla sylvestris* extract increases.

[0091] Example 2: Measurement of ATP content

[0092] Mitochondrial dysfunction is a hallmark of cellular aging, and aging mitochondria exhibit a decreased ATP / ADP ratio. Therefore, increased ATP levels can be seen as a sign of reduced cellular aging, which can enhance cellular vitality and achieve anti-aging effects.

[0093] The test plan is shown in Table 3. The experiment is divided into a blank control group, a negative control group, a positive control group, and a test sample group.

[0094] Table 3

[0095]

[0096] The specific operating steps are as follows:

[0097] 1) Cell plating: at 5 x 10 4 Human dermal fibroblasts (HDF) (purchased from Boyi Biotechnology) were seeded into 6-well plates at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0098] 2) Solution preparation: Prepare the working solution of the test substance according to Table 3 of the test plan.

[0099] The blank control group was a blank control, with only an equal amount of complete culture medium added;

[0100] Positive control group: 100 ng / ml TGF-β1 solution was added; Preparation method of 100 ng / ml TGF-β1 solution: 10 μg / mL TGF-β1 (purchased from GenScript, model Z03411) was diluted to 100 ng / mL using complete culture medium to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0101] Test sample group: The example composition or comparative composition was diluted to a concentration of 1% using a complete culture medium. The percentage refers to the mass ratio of the composition to the sample solution.

[0102] The complete culture medium was DMEM medium (purchased from Gibco, model number 10567014), which included DMEM, low sugar, GlutaMAX™ additive, and pyruvate.

[0103] 3) Stimulation and drug administration: According to the test protocol, when the cell seeding rate in the 6-well plate reaches 40%~60%, group UVA stimulation is performed. After stimulation, the drug administration volume is 2mL per well, and each group is set with 3 replicates. The cells are incubated in an incubator (37℃, 5% CO2) for 24 h.

[0104] 4) Sample collection: After culturing for 24 hours, digest and collect the cells in EP tubes and freeze them at -80°C.

[0105] 5) Chemiluminescence detection: Collect cell culture supernatant and perform detection according to the ATP kit instructions.

[0106] The test results are shown in the table below.

[0107] Table 4

[0108]

[0109] Where: MM: millimoles, representing the amount of ATP. MG: milligrams, representing the amount of sample.

[0110] ATP content (MM / MG): This represents the amount of ATP in each milligram of sample, reflecting the relative level of ATP in cells.

[0111] It can be seen from the above table:

[0112] 1) The ATP content of the compositions obtained in Examples 1-9 was higher than that in Comparative Examples 1-3, indicating that the compositions obtained in Examples 1-9 had a better effect on promoting mitochondrial activity than those in Comparative Examples 1-3.

[0113] Regarding the ATP content (MM / MG), Examples 2, 3, and 5-9 all achieved a value of 62 or higher, with Examples 3 and 9 being the most optimal.

[0114] 2) In Examples 1-3, the ATP content (MM / MG) gradually increased as the content of the flat-leaved fragrant orchid fruit extract gradually increased; in Examples 4, 2, 5, and 6, the ATP content (MM / MG) did not change significantly and showed no obvious pattern as the content of soluble collagen gradually increased; in Examples 7, 2, 8, and 9, the ATP content (MM / MG) increased slowly as the content of the fucus vesiculosus extract gradually increased.

[0115] This shows that there is a clear dose-response relationship between ATP content (MM / MG) and the extract of Vanilla sylvestris var. sylvestris, but the increase in ATP content (MM / MG) slows down as the content of Vanilla sylvestris var. ...

[0116] Figure 1 The effects of the blank control group, negative control group, positive control group, and the composition of Example 2 on ATP content in HDF cells were compared with the negative control group. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0117] Example 3: Determination of elastin content

[0118] Fibroblasts are the main cellular components of the skin, distributed in the dermis. They are the most common cells in loose connective tissue and can produce a large amount of collagen and elastic fibers, playing an important role in maintaining the structural stability and elasticity of the skin.

[0119] The amount of elastin determines the firmness of the skin and thus affects its condition; a decrease in elastin content leads to skin aging. Therefore, the firming effect of a sample can be evaluated by testing its elastin content using an enzyme-linked immunosorbent assay (ELISA).

[0120] The test plan is shown in Table 5. The experiment is divided into a blank control group, a positive control group, and a test sample group.

[0121] Table 5

[0122]

[0123] Preparation of working fluid:

[0124] The blank control group was a blank control, with only an equal volume of complete culture medium added;

[0125] TGF-β1 solution: 10 μg / mL TGF-β1 (purchased from GenScript, model Z03411) was diluted to 100 ng / mL using complete culture medium to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0126] Preparation of sample solutions: The compositions of the examples and comparative examples were diluted with complete culture medium to a concentration of 1%, where % refers to the mass ratio of the composition to the sample solution.

[0127] The complete culture medium was DMEM medium (purchased from Gibco, model number 10567014), which included DMEM, low sugar, GlutaMAX™ additive, and pyruvate.

[0128] The specific operating steps are as follows:

[0129] 1) Cell plating: with a cell concentration of 8 × 10⁻⁶ 4 Human dermal fibroblasts (HDF) (purchased from Boxi Biotechnology) were seeded into 96-well plates at a density of 100 μl per well. The seeded cell culture plates were then placed in an incubator and cultured for 24 h (5% CO2, 37℃).

[0130] 2) Drug administration: After culturing cells for 24 h, the supernatant was aspirated, and 200 μL of 1% test sample and 200 μL of 100 ng / ml TGF-β1 solution were added to each well. The mixture was then placed in an incubator for 24 h ± 1 h.

[0131] 3) Cell viability assay: After 24 h, collect the supernatant (for ELISA detection), add 100 μl of CCK-8 working solution to each well, and incubate for 1 h to 4 h. Measure the absorbance at 450 nm. Save the experimental data electronically for subsequent analysis.

[0132] 4) ELISA detection: After collecting the cell culture supernatant, perform ELISA detection.

[0133] The test results are shown in Table 6.

[0134] Table 6

[0135]

[0136] It can be seen from the above table:

[0137] 1) The elastin content of the compositions obtained in Examples 1-9 is higher than that in Comparative Examples 1-3, indicating that the compositions obtained in Examples 1-9 have a better firming effect than those in Comparative Examples 1-3.

[0138] In terms of elastin content, Examples 5 and 6 are optimal.

[0139] 2) In Examples 1-3, as the content of the extract of *Erigeron breviscapus* gradually increased, the elastin content increased slowly and not significantly; in Examples 4, 2, 5, and 6, as the content of soluble collagen gradually increased, the elastin content gradually increased, and the change was significant; in Examples 7, 2, 8, and 9, as the content of *Fucus vesiculosus* extract gradually increased, the elastin content increased slowly and not significantly.

[0140] This shows that there is a clear dose-response relationship between elastin content and soluble collagen.

[0141] Figure 2 The effect of the composition of Example 2 on the elastin content in HDF cells was compared with the blank control group and the positive control group. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0142] Example 4: The content of C-terminal peptide of type I procollagen was measured to determine the expression of type I collagen.

[0143] Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen synthesized in cells is secreted into the extracellular space. Under the action of endopeptidase, the propeptides attached to its amino and carboxyl terms are cleaved to form procollagen. Procollagen molecules polymerize into collagen fibers to form the extracellular matrix.

[0144] The efficacy of the test substance in promoting collagen synthesis was evaluated by measuring the upregulation rate of type I procollagen C-terminal peptide (CTP) content in the blank control group, positive control group, and test sample group after drug administration. The CTP content of type I procollagen was determined using an enzyme-linked immunosorbent assay (ELISA). The principle is as follows: after the type I procollagen CTP specifically binds to the antibody coated on the ELISA plate, it binds to the substrate-labeled anti-type I procollagen CTP antibody. The substrate is catalyzed by the enzyme to generate a colored product. The CTP content of type I procollagen is positively correlated with the intensity of the colored product. The optical density (OD value) was measured at 450 nm using an ELISA reader to calculate the type I procollagen CTP content.

[0145] The test plan is shown in Table 7. The experiment is divided into a blank control group, a positive control group, and a test sample group.

[0146] Table 7

[0147]

[0148] Preparation of working fluid:

[0149] The blank control group was a blank control, with only an equal volume of complete culture medium added;

[0150] TGF-β1 solution: 10 μg / mL TGF-β1 (purchased from GenScript, model Z03411) was diluted to 100 ng / mL using complete culture medium to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0151] Preparation of sample solution: The sample solution was prepared by diluting the compositions of the examples and comparative examples to a concentration of 1%, where % refers to the mass ratio of the composition to the sample solution.

[0152] The complete culture medium was DMEM medium (purchased from Gibco, model number 10567014), which included DMEM, low sugar, GlutaMAX™ additive, and pyruvate.

[0153] The specific operating steps are as follows:

[0154] 1) Cell plating: with a cell concentration of 8 × 10⁻⁶ 4 Human dermal fibroblasts (HDF) (purchased from Boxi Biotechnology) were seeded into 96-well plates at a density of 100 μl per well. The seeded cell culture plates were then placed in an incubator and cultured for 24 h (5% CO2, 37℃).

[0155] 2) Drug administration: After culturing cells for 24 h, the supernatant was aspirated, and 100 μl of the test sample and 100 μL of 100 ng / ml TGF-β1 solution were added to each well. The mixture was then placed in an incubator for 24 h ± 1 h.

[0156] 3) Cell viability assay: After 24 h, collect the supernatant (for ELISA assay), add 100 μl of CCK-8 working solution to each well, and incubate for 1 h to 4 h. Measure the absorbance at 450 nm.

[0157] 4) ELISA detection: After collecting the cell culture supernatant, ELISA was performed to detect the content of type I procollagen C-terminal peptide to determine the expression of type I collagen.

[0158] The experimental results are shown in Table 8.

[0159] Table 8

[0160]

[0161] It can be seen from the above table:

[0162] 1) The collagen content of the compositions obtained in Examples 1-9 is higher than that in Comparative Examples 1-3, indicating that the compositions obtained in Examples 1-9 have better anti-aging effects than those in Comparative Examples 1-3.

[0163] Regarding the content of type I procollagen C-terminal peptide, Examples 3, 5, and 6 are optimal.

[0164] 2) In Examples 1-3, as the content of *Erigeron breviscapus* fruit extract gradually increased, the content of type I procollagen C-terminal peptide increased slowly and not significantly; in Examples 4, 2, 5, and 6, as the content of soluble collagen gradually increased, the content of type I procollagen C-terminal peptide increased gradually and significantly; in Examples 7, 2, 8, and 9, as the content of *Fucus vesiculosus* extract gradually increased, the content of type I procollagen C-terminal peptide increased slowly and not significantly.

[0165] This shows that there is a clear dose-response relationship between the content of type I procollagen C-terminal peptide and soluble collagen.

[0166] Example 5: Determination of the relative expression level of LOR protein.

[0167] Loricrin (LOR) is the most abundant component of the keratinized capsule of the epidermis, accounting for 70-85% of its composition. It is a highly hydrophobic, insoluble protein that readily polymerizes in ambient air via disulfide cross-linking, making it suitable as a structural reinforcement protein. LOR is expressed in the granular layer and cross-links with filaggrin (FLG), inner laminarin (IVL), desmosome proteins envoplakin and periplakin scaffolds via transglutaminase 1, tightly connecting intercellular lipids and keratinocytes, maintaining the orderly arrangement of keratinocytes in the stratum corneum, and increasing the stability of the epidermal permeability barrier. The human keratinocyte cell line HaCaT was used to determine the promoting effect of the composition on the expression of the skin barrier-related gene LOR within the cells, exploring its repair efficacy.

[0168] The test plan is shown in Table 9. The experiment is divided into a blank control group, a positive control group, and a test sample group.

[0169] Table 9

[0170]

[0171] Preparation of working fluid:

[0172] The blank control group was a blank control, with only an equal volume of complete culture medium added;

[0173] 1 μmol / ml retinoic acid solution: Retinoic acid (purchased from Siga) was diluted to 1 μmol / ml using complete culture medium to obtain a 1 μmol / ml retinoic acid solution.

[0174] Preparation of sample solutions: The compositions of the examples and comparative examples were diluted with complete culture medium to a concentration of 1%, where % refers to the mass ratio of the composition to the sample solution.

[0175] The complete culture medium was Gibco's DMEM cell culture medium, High Glucose, GlutaMAX™, Pyruvate (model 10569-010).

[0176] The specific operating steps are as follows:

[0177] 1) Cell plating: with a cell concentration of 3×10⁻⁶ 5 HaCat cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were seeded into 96-well plates at a density of 100 μl / mL. The seeded cell culture plates were then placed in an incubator and cultured for 24 h (5% CO2, 37℃).

[0178] 2) Drug administration: After culturing cells for 24 h, add 100 μl of 1% test sample and 100 μl of 1 μmol / ml retinoic acid solution to each well, mix well, and place in an incubator for 24 h ± 1 h.

[0179] 3) RNA extraction: RNA was extracted using a cell-to-reverse transcription one-step reagent.

[0180] 4) cDNA synthesis: cDNA was synthesized using a cDNA synthesis kit.

[0181] 5) PCR detection: RT-PCR detection was performed using BeyoFast™ SYBR Green qPCR Mix (2X, Low ROX).

[0182] The experimental results are shown in Table 10.

[0183] Table 10

[0184]

[0185] It can be seen from the above table:

[0186] 1) The relative expression levels of LOR in the compositions obtained in Examples 1-9 were all higher than those in Comparative Examples 1-3, indicating that the sage extract prepared in Examples 1-9 had a higher barrier repair effect than that in Comparative Examples 1-3.

[0187] Regarding the relative expression level of LOR, Examples 3, 5-6, and 8-9 all exceeded 400, with Examples 8-9 being the optimal one.

[0188] 2) In Examples 1-3, the relative expression level of LOR increased slowly and not significantly with the gradual increase of the content of *Erigeron breviscapus* fruit extract; in Examples 4, 2, 5, and 6, the relative expression level of LOR increased gradually and significantly with the gradual increase of the content of soluble collagen; in Examples 7, 2, 8, and 9, the relative expression level of LOR increased gradually and significantly with the gradual increase of the content of *Fucus vesiculosus* extract. That is, the upregulation rate of relative LOR expression in Examples 1-3 and Examples 4, 2, and 5-6 was lower than that in Examples 7, 2, and 8-9.

[0189] This shows that there is a clear dose-response relationship between the relative expression level of LOR and the extract of Fucus vesiculosus.

[0190] Example 6: Determination of the relative expression level of TGM5

[0191] TGM is a calcium-dependent protease, of which there are nine types, that catalyzes the isomerization of γ-glutamine lysine between proteins. TGM plays a role in the ester bond formation between proteins and III-hydroxyceramides; this cross-linking is essential for the assembly of the keratinocyte membrane. TGM5 is a subtype of glutamine transaminase, located in the upper epidermis, promoting the cross-linking of FLG, LOR, keratin, etc., to form KIF. This experimental protocol used the human keratinocyte cell line HaCaT to determine the promoting effect of cosmetic raw materials on the expression of the skin barrier-related gene TGM5 in cells, and to explore the repair efficacy of the raw materials.

[0192] The test plan is shown in Table 11. The experiment is divided into a blank control group, a positive control group, and a test sample group.

[0193] Table 11

[0194]

[0195] Preparation of working fluid:

[0196] The blank control group was a blank control, with only an equal volume of complete culture medium added;

[0197] 1 μmol / ml retinoic acid solution: Retinoic acid (purchased from Siga) was diluted to 1 μmol / ml using complete culture medium to obtain a 1 μmol / ml retinoic acid solution.

[0198] Preparation of sample solutions: The compositions of the examples and comparative examples were diluted with complete culture medium to a concentration of 1%, where % refers to the mass ratio of the composition to the sample solution.

[0199] The complete culture medium was Gibco's DMEM cell culture medium, High Glucose, GlutaMAX™, Pyruvate (model 10569-010).

[0200] The specific operating steps are as follows:

[0201] 1) Cell plating: with a cell concentration of 3×10⁻⁶ 5 HaCat cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were seeded into 96-well plates at a density of 100 μl / mL. The seeded cell culture plates were then placed in an incubator and cultured for 24 h (5% CO2, 37℃).

[0202] 2) Drug administration: After culturing cells for 24 h, add 100 μl of 1% test sample and 100 μl of 1 μmol / ml retinoic acid solution to each well, mix well, and place in an incubator for 24 h ± 1 h.

[0203] 3) RNA extraction: RNA was extracted using a cell-to-reverse transcription one-step reagent.

[0204] 4) cDNA synthesis: cDNA was synthesized using a cDNA synthesis kit.

[0205] 5) PCR detection: RT-PCR detection was performed using BeyoFast™ SYBR Green qPCR Mix (2X, Low ROX).

[0206] The test results are shown in Table 12.

[0207] Table 12

[0208]

[0209] It can be seen from the above table:

[0210] 1) The relative expression levels of TGM5 in the compositions obtained in Examples 1-9 were all higher than those in Comparative Examples 1-3, indicating that the compositions obtained in Examples 1-9 had better barrier repair effects than those in Comparative Examples 1-3.

[0211] Regarding the relative expression level of TGM5, Examples 2-3, 5-6, and 8-9 all exceeded 300, with Example 8-9 being the optimal one.

[0212] 2) In Examples 1-3, the relative expression level of TGM5 increased slowly and not significantly with the gradual increase of the content of *Erigeron breviscapus* fruit extract; in Examples 4, 2, 5, and 6, the relative expression level of TGM5 increased slowly and not significantly with the gradual increase of the content of soluble collagen; in Examples 7, 2, 8, and 9, the relative expression level of TGM5 increased gradually and significantly with the gradual increase of the content of *Fucus vesiculosus* extract. That is, the upregulation rate of the relative expression level of TGM5 in Examples 1-3 and Examples 4, 2, and 5-6 was lower than that in Examples 7, 2, and 8-9.

[0213] This shows that there is a clear dose-response relationship between the relative expression level of TGM5 and the extract of Fucus vesiculosus.

Claims

1. A layered anti-aging composition characterized in that, It includes 4-10 parts of flat-leaved vanilla fruit extract, 0.01-0.2 parts of soluble collagen and 10-20 parts of fucus vesiculosus extract, all of which are parts by weight; The flat-leaved vanilla fruit extract is prepared by the following method: the flat-leaved vanilla fruit is soaked in a solvent, and the filtrate obtained by solid-liquid separation is microfiltered to obtain the flat-leaved vanilla fruit extract; the solvent is ethanol. The *Fucus vesiculosus* extract is prepared by the following method: heating a mixture containing *Fucus vesiculosus*, ultrafiltration of the filtrate obtained from solid-liquid separation, and obtaining the *Fucus vesiculosus* extract; the solvent used in the mixture containing *Fucus vesiculosus* is water; the pH value of the mixture containing *Fucus vesiculosus* is 3-3.

5. The soluble collagen is recombinant type III collagen.

2. The layered anti-aging composition according to claim 1, characterized in that, The layered anti-aging composition satisfies one or more of the following conditions: (1) The amount of the flat-leaf vanilla fruit extract is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts; (2) The amount of the soluble collagen used is 0.02 parts, 0.03 parts, 0.04 parts, 0.05 parts, 0.06 parts, 0.08 parts, 0.09 parts, 0.1 parts, 0.12 parts, or 0.15 parts; and, (3) The amount of the extract of the seaweed is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 parts.

3. The layered anti-aging composition according to claim 1, characterized in that, The layered anti-aging composition satisfies one or more of the following conditions: (1) The amount of the flat-leaved vanilla fruit extract is 6-10 parts; (2) The amount of the soluble collagen used is 0.01-0.1 parts; and, (3) The amount of the Fucus vesiculosus extract is 10-15 parts.

4. The layered anti-aging composition according to claim 1, characterized in that, The layered anti-aging composition satisfies (1) and / or (2) of the following conditions: (1) The mass ratio of the soluble collagen to the *Fucus veitchii* extract is 6:0.05:12, 8:0.05:12, 10:0.05:12, 8:0.01:12, 8:0.08:12, 8:0.05:10, 8:0.05:13 or 8:0.05:15; (2) The composition also contains a preservative.

5. The layered anti-aging composition according to claim 1, characterized in that, The mass ratio of the flat-leaved vanilla fruit extract, the soluble collagen, and the fucus vesiculosus extract is (6-10):(0.01-0.08):(10-15).

6. The layered anti-aging composition according to claim 4, characterized in that, The preservatives are 1,3-butanediol and / or 1,2-propanediol.

7. The layered anti-aging composition according to claim 4, characterized in that, The amount of the preservative is 70-90%, and the percentage is its mass percentage of the total composition.

8. The layered anti-aging composition according to claim 1, characterized in that, The layered anti-aging composition comprises 4-10 parts of the flat-leaved vanilla fruit extract, 0.01-0.2 parts of soluble collagen, and 10-20 parts of fucus vesiculosus extract.

9. The layered anti-aging composition according to claim 1, characterized in that, In the preparation method of the flat-leaved vanilla fruit, one or more of the following conditions are met: (1) The flat-leaved fragrant orchid fruit is crushed before soaking; (2) The soaking is performed at room temperature; (3) The soaking time is 2-5 hours; (4) The mass ratio of the flat-leaved fragrant fruit to the solvent is 1:(5-10); (5) The solid-liquid separation method is gauze filtration; (6) The microfiltration uses a microfiltration membrane with a pore size of 2.5~10μm.

10. The layered anti-aging composition according to claim 1, characterized in that, In the preparation method of the flat-leaved vanilla fruit, one or more of the following conditions are met: (1) Before soaking, the flat-leaf vanilla fruit is first crushed; after crushing, the flat-leaf vanilla fruit powder has a mesh size of 10 mesh; (2) The soaking time is 3 hours; (3) The mass ratio of the flat-leaved fragrant fruit to the solvent is 1:5 or 1:10; (4) The solid-liquid separation method is gauze filtration; the mesh size of the gauze is 200-600 mesh; (5) The microfiltration uses a microfiltration membrane with a pore size of 2.5 μm, 6 μm, 8 μm or 10 μm.

11. The layered anti-aging composition according to claim 9, characterized in that, The preparation method of the flat-leaved vanilla fruit extract includes the following steps: crushing the flat-leaved vanilla fruit, soaking it in ethanol at room temperature for 3 hours, with a mass ratio of flat-leaved vanilla fruit to ethanol of 1:10, filtering it through 200-mesh gauze to obtain the supernatant, concentrating the supernatant under reduced pressure to obtain flat-leaved vanilla fruit concentrate, and filtering it through a microfiltration membrane to obtain the flat-leaved vanilla fruit extract.

12. The layered anti-aging composition according to claim 1, characterized in that, The preparation method of the Fucus vesiculosus extract satisfies one or more of the following conditions: (1) The Fucus vesiculosus should be pulverized before use; (2) In the mixture containing Fucus vesiculosus, the mass ratio of Fucus vesiculosus to solvent is 1:(10-15). (3) The pH value of the mixture containing Fucus vesiculosus is 3; (4) The heating time is 2-5 hours; (5) The heating temperature is 60-80℃; (6) The solid-liquid separation method is gauze filtration; (7) When performing ultrafiltration, an ultrafiltration membrane with a molecular weight cutoff of 10 kDa is selected.

13. The layered anti-aging composition according to claim 1, characterized in that, The preparation method of the Fucus vesiculosus extract satisfies one or more of the following conditions: (1) The Fucus vesiculosus is pulverized before use; after pulverization, the Fucus vesiculosus powder has a mesh size of 20-50 mesh. (2) In the mixture containing Fucus vesiculosus, the mass ratio of Fucus vesiculosus to solvent is 1:15; (3) The heating time is 2 hours; (4) The heating temperature is 80℃; (5) The solid-liquid separation method is gauze filtration; the mesh size of the gauze is 200-600 mesh.

14. The use of a layered anti-aging composition as described in any one of claims 1-13 in the cosmetic field.

15. The application as described in claim 14, characterized in that, (1) The composition is used in cosmetics as an ingredient that enhances skin elasticity, firmness, anti-aging, or repairs the skin barrier; and / or, (2) The composition is used in cosmetics after being diluted.

16. The application as described in claim 15, characterized in that, The concentration after dilution is 2-10%, and the percentage is the percentage of the composition mass to the total mass of the diluted liquid.

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