Layered anti-aging composition and application thereof

Abstract

The invention discloses a layered anti-aging composition and application thereof. The skin care product is prepared from the following components in parts by mass: 4 to 10 parts of a vanilla planifolia fruit extract, 0.01 to 0.2 part of soluble collagen and 10 to 20 parts of a fucus extract. The composition has a layered anti-aging skin care strategy (layering generally refers to different skin parts, namely cell layer-dermis layer-epidermis layer), the composition can promote expression of LOR and relative expression quantity of TGM5 (epidermis), increase the content of I-type procollagen C-terminal peptide and the content of elastin (dermis), and improve the anti-aging effect of the composition. The traditional Chinese medicine composition has the effects of enhancing mitochondrial activity and increasing ATP content (cell layer), namely progressive from epidermis-dermis-cell layer by layer, has related effects on each layer of skin, and has the effects of enhancing skin elasticity, tightening skin, resisting aging and repairing skin barrier.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics, and particularly relates to a layered anti-aging composition and its application. Background Art

[0002] With the increase of age, the accumulation of endogenous stress and exogenous stimuli will affect skin health. Intrinsic reasons: mainly formed by the passage of time, so it is also called the natural aging reason. Genes are the main cause of skin natural aging, which determines the speed and degree of aging. But at the same time, there are also some endogenous factors, including nutrition, endocrine and immunity, etc., which also affect skin aging through the whole body. Extrinsic reasons: mainly caused by the ultraviolet radiation of the sun, so it is also called the photoaging reason. Ultraviolet rays are the most important extrinsic factor causing skin aging, which will damage the elastic fibers and collagen fibers in the dermis layer, accelerate skin aging and relaxation, and cause wrinkles and spots.

[0003] The skin is composed of the epidermis and the dermis. Due to different layers of the skin facing different stimuli, different characteristics and problems will appear during the aging process. The aging of the epidermis is manifested as dryness, fine lines, dull skin, etc.; the aging of the dermis is manifested as loss of skin elasticity, decreased fullness, and the appearance of wrinkles. Skin aging is not only the changes in the epidermis and the dermis, but also the aging of the skin's component unit - cells. In 2023, "Cell" announced that the twelve hallmarks of aging are: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, inactivation of macroautophagy, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and microbiota dysbiosis.

[0004] It can be seen that the causes of skin aging are caused by many factors, and single treatment may not be able to improve the multi-level aging problems.

[0005] Vanilla planifolia fruit is famous for its charming and unique aroma.

[0006] Collagen is a structural protein, accounting for 30% of the total human protein. As the main component of the extracellular matrix, more than 80% of the dermis layer is collagen.

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

[0008] In order to comprehensively solve the problem of aging, the present invention provides a layered anti-aging composition and its application. The composition of the present invention adopts a layered anti-aging skin care strategy, that is, from the epidermis - dermis - cells in layers, enhancing mitochondrial vitality, increasing the content of ATP, increasing the content of collagen and elastin, and promoting the expression of corneodesmosin, having the effects of enhancing skin elasticity, anti-aging and repairing the skin barrier.

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

[0010] The present invention provides a layered anti-aging composition, which includes 4 - 10 parts of Vanilla planifolia fruit extract, 0.01 - 0.2 parts of soluble collagen and 10 - 20 parts of Fucus vesiculosus extract, and the above parts are all parts by mass.

[0011] In the present invention, the composition preferably consists of the 4 - 10 parts of Vanilla planifolia fruit extract, the 0.01 - 0.2 parts of soluble collagen and 10 - 20 parts of Fucus vesiculosus extract.

[0012] In the present invention, the mass ratio of the soluble collagen to the Fucus vesiculosus extract is preferably 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 the present invention, the dosage of the Vanilla planifolia fruit extract can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts, and is preferably 6 - 10 parts.

[0014] In the present invention, the Vanilla planifolia fruit extract can be prepared by conventional methods in the art, and is preferably prepared by the following method: soaking the Vanilla planifolia fruit in a solvent, and microfiltering the obtained filtrate after solid-liquid separation to obtain the Vanilla planifolia fruit extract.

[0015] Among them, the Vanilla planifolia fruit generally refers to the pod of Vanilla pompona Schiede.

[0016] Among them, before the soaking of the Vanilla planifolia fruit, it is generally first pulverized. After the pulverization, the mesh number of the Vanilla planifolia fruit powder can be about 10 meshes.

[0017] Among them, the mass ratio of the Vanilla planifolia fruit to the solvent can be 1:(5 - 10), such as 1:5 or 1:10.

[0018] Among them, the solvent is generally an alcohol solvent, and preferably ethanol.

[0019] Among them, the soaking is generally carried out at room temperature. The soaking time can be conventional in the art, preferably 2 - 5 h, such as 3 h.

[0020] Among them, the method of solid - liquid separation can be conventional in the art, such as filtration. The filtration is generally carried out with gauze. The mesh number of the gauze can be 600 - 200 mesh.

[0021] Among them, generally, the filtrate obtained by solid - liquid separation is concentrated. The concentration is generally carried out under reduced pressure.

[0022] Among them, the microfiltration generally uses a microfiltration membrane. The pore size of the microfiltration membrane is preferably 2.5 - 10 μm, such as 2.5 μm, 6 μm, 8 μm or 10 μm.

[0023] In a preferred embodiment, the extract of Vanilla planifolia fruit is prepared by the following method: crushing the Vanilla planifolia fruit, soaking it in ethanol at room temperature for 3 h (the mass ratio of Vanilla planifolia fruit to ethanol is 1:10), filtering through a 200 - mesh gauze to obtain the supernatant, concentrating the supernatant under reduced pressure to obtain the concentrated solution of Vanilla planifolia fruit, and filtering through a microfiltration membrane to obtain the extract of Vanilla planifolia fruit.

[0024] In the present invention, the dosage of the 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 the present invention, the soluble collagen can be a conventional recombinant collagen in the art, and can be one or more of recombinant I, III, V, VII and XVII, such as recombinant type III collagen.

[0026] In the present invention, the dosage 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 the present invention, the Fucus vesiculosus extract can be prepared by a conventional method in the art, preferably prepared by the following method: heating a mixed solution containing Fucus vesiculosus, and ultrafiltering the filtrate obtained by solid - liquid separation to obtain the Fucus vesiculosus extract.

[0028] Among them, the Fucus vesiculosus is generally repeatedly rinsed with water before use, washed clean of sediment and salt, and dried. The drying temperature can be 40 - 60 °C.

[0029] Among them, before the Fucus vesiculosus is used, it is generally first crushed. After crushing, the mesh number of the Fucus vesiculosus powder can be 20 - 50 mesh.

[0030] Among them, in the mixed solution containing Fucus vesiculosus, the solvent used is generally water. The water is generally deionized water.

[0031] Among them, in the mixed solution containing Fucus vesiculosus, the mass ratio of Fucus vesiculosus to the solvent can be 1:(10 - 15), preferably 1:15.

[0032] Among them, the pH value of the mixed solution containing Fucus vesiculosus can be 3 - 3.5, such as 3. Generally, hydrochloric acid is used to adjust the pH value of the system.

[0033] Among them, the heating time can be 2 - 5 h, such as 2 h.

[0034] Among them, the heating temperature can be 60 - 80 °C, such as 80 °C.

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

[0036] Among them, during ultrafiltration, the rejection rate of the ultrafiltration membrane is preferably 10 kDa.

[0037] Among them, during ultrafiltration, the pore size of the ultrafiltration membrane is preferably 0.45 - 0.8 μm, such as 0.45 μm or 0.6 μm.

[0038] In a preferred embodiment, the Fucus vesiculosus extract is prepared by the following method: Wash Fucus vesiculosus with water, dry it at 40 °C, and pulverize it to obtain Fucus vesiculosus powder with a mesh number of about 20 - 50; Adjust the pH value of the mixture of Fucus vesiculosus powder and water to 3 with hydrochloric acid solution, with the mass ratio of Fucus vesiculosus powder to water being 1:15, heat at 80 °C for 2 h; Cool, filter through a 200-mesh gauze to obtain a filtrate, and ultrafilter the filtrate through a 0.45-μm ultrafiltration membrane with a rejection rate of 10 kDa to obtain the Fucus vesiculosus extract.

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

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

[0041] The dosage of the preservative can be conventional in the art, such as 70 - 90%, and the above percentage is the mass percentage of it in the total amount of the composition.

[0042] In the present invention, the composition can have the effect of layered anti-aging, and is preferably applied to cosmetics as an ingredient for enhancing skin elasticity, firming, anti-aging, or repairing the barrier.

[0043] In the present invention, the composition preferably has the efficacy of promoting the expression of loricrin (LOR), the relative expression level of transglutaminase 5 (TGM5), increasing the content of type I procollagen C-terminal propeptide and collagen content, increasing the elastin content, enhancing mitochondrial activity or increasing the ATP content.

[0044] In the present invention, the composition can be used in cosmetics after dilution. The dilution can be carried out with distilled water. The concentration after dilution is preferably 2-10%, and the percentage is the percentage of the mass of the composition in the total mass of the diluted liquid.

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

[0046] The even mixing is generally carried out by stirring at room temperature for 20 min with a homogenizer.

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

[0048] In the present invention, the composition may have the efficacy of layered anti-aging, and is preferably used as an ingredient for enhancing skin elasticity, firming, anti-aging or repairing the barrier in cosmetics.

[0049] In the present invention, the composition preferably has the efficacy of promoting the expression of loricrin (LOR), the relative expression level of transglutaminase 5 (TGM5), increasing the content of type I procollagen C-terminal propeptide and collagen content, increasing the elastin content, enhancing mitochondrial activity or increasing the ATP content.

[0050] In the present invention, the form of the cosmetics is not limited, including but not limited to aqueous solutions, emulsions, sprays, essences, creams, masks, facial cleansers, liquid foundations or cushions, etc.

[0051] In the present invention, the composition can be used in cosmetics after dilution. The dilution can be carried out with distilled water. The concentration after dilution is preferably 2-10%, and the percentage is the percentage of the mass of the composition in the total mass of the diluted liquid.

[0052] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

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

[0055] The composition of the present invention has a layered anti-aging skincare strategy ( "layered" generally refers to different skin parts, namely "cell layer - dermis - epidermis"). This composition can promote the expression of loricrin (LOR), the relative expression level of transglutaminase 5 (TGM5) (epidermis), increase the content of C-terminal propeptide of type I procollagen and the content of elastin (dermis), enhance mitochondrial activity and increase the content of ATP (cell layer). That is, starting from the epidermis - dermis - cell layer, it has relevant effects on each layer of the skin, and has the effects of enhancing skin elasticity, firmness, anti-aging and repairing the skin barrier.

[0056] In some preferred embodiments, the anti-aging effect of the composition in the epidermis layer can be demonstrated by promoting the expression of loricrin (LOR) and the relative expression level of transglutaminase 5 (TGM5); the anti-aging effect of the composition in the dermis layer can be demonstrated by increasing the content of C-terminal propeptide of type I procollagen and the content of elastin; the anti-aging effect of the composition in the cell layer can be demonstrated by enhancing mitochondrial activity and increasing the content of ATP. The composition of the present invention can be customized layer by layer according to the different causes of skin aging in each layer, and anti-aging layer by layer, and can solve the aging problem directionally from shallow to deep. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Effects of the compositions of the blank control group, negative control group, positive control group and Example 2 on the ATP content in HDF cells. Compared with the negative control group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0058] Figure 2 Effects of the compositions of the blank control group, positive control group and Example 2 on the elastin content in HDF cells. Compared with the blank control group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0059] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0060] Unless otherwise specified, the raw materials and equipment used in the examples are common raw materials and equipment in the art and are all commercially available products.

[0061] Vanilla planifolia fruit and Fucus vesiculosus are both conventional commercially available products.

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

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

[0064] The preparation method of the composition comprises the following steps:

[0065] (1) Vanilla planifolia fruit extract: The Vanilla planifolia fruit is crushed to a particle size of about 10 mesh, added with ethanol and soaked at room temperature for 3 h (ethanol: Vanilla planifolia fruit = 5:1), and the supernatant is obtained by filtering through a 200-mesh gauze. The supernatant is concentrated under reduced pressure to obtain a concentrated solution, and the Vanilla planifolia fruit extract is obtained by filtering through an 8-μm microfiltration membrane.

[0066] (2) Fucus vesiculosus extract: The Fucus vesiculosus is repeatedly rinsed with tap water to wash away sediment and salt, dried (at 40 °C) and then crushed to obtain Fucus vesiculosus powder with a mesh number of about 20 - 50. An appropriate amount of Fucus vesiculosus powder is weighed, deionized water is added, and the mass ratio of Fucus vesiculosus powder to deionized water is 1:15. The pH value is adjusted to 3 with an appropriate amount of hydrochloric acid solution, and it is heated in a water bath at 80 °C for 2 h. After cooling, the filtrate is obtained by filtering through a 200-mesh gauze, and the filtrate is ultrafiltered through a 0.45-μm ultrafiltration membrane with a cut-off rate of 10 kDa to obtain the Fucus vesiculosus extract.

[0067] (3) According to the ratio shown in Table 1, the Vanilla planifolia fruit extract, Fucus vesiculosus extract and soluble collagen are mixed evenly and stirred at room temperature for 20 min with a homogenizer to obtain the composition. Among them, the total mass of the composition is 100 g.

[0068] Table 1 shows the composition ratios of the compositions in Examples 1 - 9 and Comparative Examples 1 - 3 (the composition ratios of Vanilla planifolia fruit extract: soluble collagen: Fucus vesiculosus extract are by mass, and the total mass of the compositions after different ratio combinations remains the same). The efficacy tests are carried out on the compositions prepared in the examples and comparative examples.

[0069] Table 1 Formulation compositions of examples and comparative examples

[0070]

[0071] Effect Example 1 Determination of mitochondrial viability

[0072] JC-1 is a fluorescent probe used to detect the mitochondrial membrane potential (MMP), and can detect the mitochondrial membrane potential of cells, tissues or purified mitochondria. The change of mitochondrial membrane potential can be detected through the change of fluorescence color: when the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers and produce red fluorescence; when the mitochondria are damaged, the mitochondrial membrane potential decreases, and JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer and produces green fluorescence.

[0073] HaCaT cells were damaged by irradiating them with a certain dose of UVB. After that, JC-1 staining was used. By comparing the changes in the fluorescence intensity of the mitochondrial membrane potential between the test sample group and the model control group, the red / green fluorescence intensity ratio of the mitochondrial membrane potential was calculated, that is, the red / green fluorescence intensity ratio of the mitochondrial membrane potential = S (red fluorescence intensity) / S (green fluorescence intensity) * 100%, and the strength of mitochondrial activity could be judged.

[0074] The specific operation steps are as follows:

[0075] 1) Cell seeding: Human dermal fibroblasts HDF (purchased from Boxi Biology) at 5x10 4 / well were seeded in a 24-well plate and cultured in an experimental environment (37 °C, 5% CO2) for 24 ±2 h.

[0076] 2) Drug administration: The experimental groups were the blank control group, the positive control group, and the test sample group. The above experimental groups were seeded in each well respectively and cultured in the experimental environment for 4 h. After that, they were washed 1-2 times with D-Hanks and subjected to UVB modeling.

[0077] Among them, the blank control group was a blank control, and only an equal amount of complete medium was added;

[0078] Positive control group: Add 100 ng / ml TGF-β1 solution; Preparation of 100 ng / ml TGF-β1 solution: Dilute 10 μg / mL TGF-β1 (purchased from GenScript, model number Z03411) to 100 ng / mL with complete medium to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0079] Test sample group: Dilute the composition of the example or the composition of the comparative example to a sample solution with a concentration of 1% with complete medium, and % refers to the mass ratio of the composition to the sample solution.

[0080] The complete medium was DMEM medium (purchased from Gibco, model number 10567014), and the DMEM medium included DMEM, low glucose, containing GlutaMAXTM additive and pyruvate.

[0081] 3) After the modeling was completed, they were washed 1-2 times with D-Hanks. As in step 2), the blank control group, the positive control group, and the test sample group were respectively added with the corresponding substances and continued to be cultured in the experimental environment for 24±2 h; irradiated with a certain dose of UVB (intensity of 50 mJ). After that, JC-1 staining was performed and observed and photographed under a fluorescence microscope.

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

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

[0084] Table 2

[0085]

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

[0087] 1) The mitochondrial membrane potential red / green fluorescence intensity ratio of the compositions obtained in Examples 1-9 is higher than that of Comparative Examples 1-3, indicating that the compositions prepared in Examples 1-9 have a better effect on improving mitochondrial activity than Comparative Examples 1-3.

[0088] In terms of the mitochondrial membrane potential red / green fluorescence intensity ratio, Examples 2, 3, 5-9 can all reach above 1.88, and the best are Example 3 and Example 8.

[0089] 2) In Examples 1-3, as the content of Vanilla planifolia fruit extract gradually increases, the mitochondrial membrane potential red / green fluorescence intensity ratio gradually increases; in Examples 4, 2, 5, 6, as the content of soluble collagen gradually increases, the mitochondrial membrane potential red / green fluorescence intensity ratio changes insignificantly and has no obvious pattern; in Examples 7, 2, 8, 9, as the content of Fucus vesiculosus extract gradually increases, the mitochondrial membrane potential red / green fluorescence intensity ratio changes insignificantly and has no obvious pattern.

[0090] Thus, it can be seen that there is an obvious dose-effect relationship between the mitochondrial membrane potential red / green fluorescence intensity ratio and the Vanilla planifolia fruit extract, but as the content of the Vanilla planifolia fruit extract increases, the growth rate of the mitochondrial membrane potential red / green fluorescence intensity ratio slows down.

[0091] Effect Example 2 Determination of ATP Content

[0092] Mitochondrial dysfunction is a marker of cell aging, and aging mitochondria will show a decrease in the ATP / ADP ratio. Therefore, an increase in ATP content can be regarded as a marker of a decrease in the degree of cell aging, and thus can improve cell viability and achieve the effect of cell anti-aging.

[0093] The test scheme 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 operation steps are as follows:

[0097] 1) Cell seeding: Seed human dermal fibroblasts HDF (purchased from Boxi Biology) into a 6-well plate at an inoculation density of 5x10 4 cells / well and incubate overnight in an incubator (37°C, 5% CO2).

[0098] 2) Preparation of the liquid: Prepare the test substance working solution according to Table 3 of the test protocol.

[0099] Among them, the blank control group is a blank control, only adding an equal amount of complete medium;

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

[0101] Test sample group: Dilute the composition of the example or the composition of the comparative example with complete medium to a sample solution with a concentration of 1%, where % refers to the mass ratio of the composition to the sample solution.

[0102] The complete medium is DMEM medium (purchased from Gibco, model number 10567014), and the DMEM medium includes DMEM, low glucose, containing GlutaMAXTM additive and pyruvate.

[0103] 3) Stimulation and administration: According to the test protocol, when the cell seeding rate in the 6-well plate reaches 40% - 60%, perform grouped UVA stimulation. After stimulation, the dosage per well is 2 mL, and each group is set with 3 replicate wells, and incubate in an incubator (37 °C, 5% CO2) for 24 h.

[0104] 4) Sample collection: After culturing for 24 h, digest and collect the cells into an EP tube, and store them frozen in a -80 °C refrigerator.

[0105] 5) Detection by chemiluminescence method: Collect the cell culture supernatant and detect it according to the ATP kit instructions.

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

[0107] Table 4

[0108]

[0109] Among them: MM: millimole, representing the amount of ATP. MG: milligram, representing the amount of the sample.

[0110] ATP content (MM / MG): Represents the ATP content per milligram of the sample, reflecting the relative level of ATP in the cells.

[0111] As 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 prepared in Examples 1-9 had a better effect on promoting mitochondrial activity than those in Comparative Examples 1-3.

[0113] In terms of the ATP content (MM / MG), Examples 2, 3, 5-9 could all reach above 62, and the best ones were Example 3 and Example 9.

[0114] 2) In Examples 1-3, as the content of Vanilla planifolia fruit extract gradually increased, the ATP content (MM / MG) gradually increased; in Examples 4, 2, 5, 6, as the content of soluble collagen gradually increased, the ATP content (MM / MG) changed insignificantly without an obvious pattern; in Examples 7, 2, 8, 9, as the content of Fucus vesiculosus extract gradually increased, the ATP content (MM / MG) increased slowly.

[0115] Thus, there was an obvious dose-effect relationship between the ATP content (MM / MG) and the Vanilla planifolia fruit extract, but as the content of the Vanilla planifolia fruit extract increased, the growth rate of the ATP content (MM / MG) slowed down.

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

[0117] Effect Example 3 Determination of elastin content

[0118] Fibroblasts are the main cell components of the skin, distributed in the dermis of the skin. 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 content of elastin determines the firmness of the skin and thus affects the skin condition. A decrease in the content of elastin will lead to skin aging. Therefore, the effect of the sample on the content of elastin can be tested by the enzyme-linked immunosorbent assay (ELISA) method to evaluate the firming effect of the sample.

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

[0121] Table 5

[0122]

[0123] Preparation of the working solution:

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

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

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

[0127] The complete medium was DMEM medium (purchased from Gibco, model number 10567014), and the DMEM medium included DMEM, low glucose, containing GlutaMAXTM additive and pyruvate.

[0128] The specific operation steps are as follows:

[0129] 1) Cell seeding: Human dermal fibroblasts HDF (purchased from Boxi Biology) were seeded into a 96-well plate at a density of 8×10 4 / mL, 100 μl per well. The seeded cell culture plate was placed in an incubator and cultured for another 24 h (5% CO2, 37 °C).

[0130] 2) Administration of drugs: After 24 h of cell culture, 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, mixed well, and placed in the incubator for 24 h ± 1 h.

[0131] 3) Cell viability detection: After 24 h, the supernatant was collected (for ELISA detection), 100 μl of CCK-8 working solution was added to each well, and it was placed in the incubator for 1 h to 4 h. The absorbance value was measured at 450 nm, and the experimental data was saved in electronic format for subsequent analysis.

[0132] 4) ELISA detection: ELISA detection was performed after collecting the cell culture supernatant.

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

[0134] Table 6

[0135]

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

[0137] 1) The elastin content of the compositions obtained in Examples 1-9 was higher than that in Comparative Examples 1-3, indicating that the compositions prepared in Examples 1-9 had a better firming effect than 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 flat - leaf vanilla fruit extract gradually increases, the increase in elastin content is slow and not significant; in Examples 4, 2, 5, and 6, as the content of soluble collagen gradually increases, the elastin content gradually increases significantly; in Examples 7, 2, 8, and 9, as the content of Fucus extract gradually increases, the increase in elastin content is slow and not significant.

[0140] Thus, it can be seen that there is an obvious dose - effect relationship between elastin content and soluble collagen.

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

[0142] Effect Example 4 Determination of the content of type I procollagen C - terminal peptide to judge the expression of type I collagen

[0143] Type I collagen is one of the main components of the dermal extracellular matrix. Type I procollagen synthesized intracellularly is secreted extracellularly. Under the action of peptidase, the propeptides attached to its amino - terminal and carboxyl - terminal are cut off to form tropocollagen, and tropocollagen molecules polymerize into collagen fibers to form the extracellular matrix.

[0144] By measuring the up - regulation rate of the content of type I procollagen C - terminal peptide after administration in the blank control group, the positive control group, and the test sample group, evaluate whether the test substance has efficacy in promoting collagen synthesis. The determination of the content of type I procollagen C - terminal peptide uses the enzyme - linked immunosorbent assay (ELISA). The specific principle is as follows: After type I procollagen C - terminal peptide specifically binds to the antibody coated on the enzyme - linked immunosorbent assay plate, it binds to the anti - type I procollagen C - terminal peptide antibody with a substrate label. After the substrate is catalyzed by the enzyme, a colored product is generated, and the content of type I procollagen C - terminal peptide is positively correlated with the depth of the color of the colored product. Measure the optical density (OD value) at a wavelength of 450 nm with an enzyme - linked immunosorbent assay reader, and calculate the content of type I procollagen C - terminal peptide.

[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 the working solution:

[0149] The blank control group was a blank control, and only an equal amount of complete medium was added;

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

[0151] Preparation of the sample solution: The compositions of the examples and comparative examples were diluted to a 1% sample solution, where % refers to the mass ratio of the composition to the sample solution.

[0152] The complete medium was DMEM medium (purchased from Gibco, product number 10567014), and the DMEM medium included DMEM, low glucose, GlutaMAXTM additive, and pyruvate.

[0153] The specific operation steps are as follows:

[0154] 1) Cell seeding: Human dermal fibroblasts HDF (purchased from Boxi Biotech) were seeded into a 96-well plate at a density of 8×10 4 / mL, 100 μl per well. The seeded cell culture plate was placed in an incubator and cultured for another 24 h (5% CO2, 37 °C).

[0155] 2) Administration of drugs: After 24 h of cell culture, the supernatant was aspirated, and 100 μl of the sample to be tested and 100 μL of the 100 ng / ml TGF-β1 solution were added to each well, mixed, and placed in an incubator for 24 h ± 1 h.

[0156] 3) Cell viability detection: After 24 h, the supernatant was collected (for ELISA detection), and 100 μl of CCK-8 working solution was added to each well and placed in an incubator for 1 h to 4 h. The absorbance was measured at 450 nm.

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

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

[0159] Table 8

[0160]

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

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

[0163] For the content of C-terminal propeptide of type I procollagen, the optimal ones are Example 3, Example 5, and Example 6.

[0164] 2) In Examples 1 - 3, as the content of Vanilla planifolia fruit extract gradually increased, the content of C-terminal propeptide of type I procollagen increased slowly and insignificantly; in Examples 4, 2, 5, and 6, as the content of soluble collagen gradually increased, the content of C-terminal propeptide of type I procollagen gradually increased significantly; in Examples 7, 2, 8, and 9, as the content of Fucus vesiculosus extract gradually increased, the content of C-terminal propeptide of type I procollagen increased slowly and insignificantly.

[0165] Thus, it can be seen that there is an obvious dose - effect relationship between the content of C-terminal propeptide of type I procollagen and soluble collagen.

[0166] Effect Example 5 Determination of relative expression level of loricrin

[0167] Loricrin (LOR) is the most abundant component in the epidermal cornified envelope, accounting for 70 - 85% of the cornified envelope. It is a very hydrophobic and insoluble protein that easily polymerizes in ambient air through disulfide cross - linking, making it suitable as a structural strengthening protein. LOR is expressed in the granular layer and cross - links with filaggrin (FLG), involucrin (IVL), envoplakin, and periplakin scaffolds through 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 selected to determine the promoting effect of the composition on the expression of the skin barrier - related gene LOR in cells, and to explore its repair efficacy.

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

[0169] Table 9

[0170]

[0171] Preparation of working solution:

[0172] The blank control group was a blank control, only adding an equal amount of complete medium;

[0173] 1 μmoL / ml retinoic acid solution: Retinoic acid (purchased from Siga) was diluted to 1 μmoL / ml with complete medium to obtain a retinoic acid solution with a concentration of 1 μmoL / ml.

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

[0175] The complete medium is Gibco's cell culture medium DMEM, High Glucose, GlutaMAX™, Pyruvate (Cat. No. 10569-010).

[0176] The specific operation steps are as follows:

[0177] 1) Cell seeding: Seed HaCat cells (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) into a 96-well plate at a density of 3×10 5 / mL, 100 μl per well. Place the seeded cell culture plate in an incubator and continue culturing for 24 h (5% CO2, 37 °C).

[0178] 2) Drug administration: After culturing the 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: Extract RNA using the Cell to RT One Step Reagent.

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

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

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

[0183] Table 10

[0184]

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

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

[0187] In terms of the relative expression level of LOR, Examples 3, 5-6, 8-9 are all above 400, and the best are Examples 8-9.

[0188] 2) In Examples 1-3, as the content of Vanilla planifolia fruit extract gradually increased, the relative expression level of LOR increased slowly and insignificantly; in Examples 4, 2, 5, and 6, as the content of soluble collagen gradually increased, the relative expression level of LOR gradually increased, with a significant change; in Examples 7, 2, 8, and 9, as the content of Fucus vesiculosus extract gradually increased, the relative expression level of LOR gradually increased, with the most significant change. That is, the up-regulation rate of the relative expression level of LOR in Examples 1-3, Examples 4, 2, 5-6 was lower than that in Examples 7, 2, 8-9.

[0189] It can be seen that there is an obvious dose-effect relationship between the relative expression level of LOR and the Fucus vesiculosus extract.

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

[0191] TGM is a calcium-dependent protease, with a total of 9 types, which can catalyze the isomerization synthesis of γ-glutamyl lysine between proteins. TGM plays a role in the ester bond formed between proteins and Ⅲ-hydroxyceramide, and this cross-action is very necessary for the assembly of the cornified cell envelope. TGM5 is a subtype of transglutaminase, which exists in the upper layer of the epidermis and promotes the cross-linking of FLG, LOR, keratin, etc. to form KIF. In this experimental protocol, the human keratinocyte cell line HaCaT was selected 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 scheme is shown in Table 11. The experiment was divided into a blank control group, a positive control group, and a test sample group.

[0193] Table 11

[0194]

[0195] Preparation of the working solution:

[0196] The blank control group was a blank control, and only an equal amount of complete medium was added;

[0197] 1 μmoL / ml retinoic acid solution: Retinoic acid (purchased from Siga) was diluted to 1 μmoL / ml with complete medium to obtain a retinoic acid solution with a concentration of 1 μmoL / ml.

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

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

[0200] The specific operation steps are as follows:

[0201] 1) Cell seeding: Seed HaCat cells (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) into a 96-well plate at a density of 3×10 5 / mL, 100 μl per well. Place the seeded cell culture plate in an incubator and continue culturing for 24 h (5% CO2, 37 °C).

[0202] 2) Drug administration: After culturing the 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 the incubator for 24 h ± 1 h for treatment.

[0203] 3) RNA extraction: Extract RNA using a cell-to-RT one-step reagent.

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

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

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

[0207] Table 12

[0208]

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

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

[0211] Regarding the relative expression level of TGM5, Examples 2-3, 5-6, 8-9 are all above 300, and the best are Examples 8-9.

[0212] 2) In Examples 1-3, as the content of Vanilla planifolia fruit extract gradually increases, the relative expression level of TGM5 increases slowly and insignificantly; in Examples 4, 2, 5, 6, as the content of soluble collagen gradually increases, the relative expression level of TGM5 increases slowly and insignificantly; in Examples 7, 2, 8, 9, as the content of Fucus vesiculosus extract gradually increases, the relative expression level of TGM5 gradually increases significantly. That is, the upregulation rates of the relative expression levels of TGM5 in Examples 1-3, Examples 4, 2, 5-6 are all lower than those in Examples 7, 2, 8-9.

[0213] It can be seen that there is an obvious dose-effect relationship between the relative expression level of TGM5 and the extract of Fucus vesiculosus.

Claims

1. A layered anti-aging composition, characterized in that: The invention comprises 4-10 parts of vanilla flat-leaf fruit extract, 0.01-0.2 parts of soluble collagen and 10-20 parts of fucus vesiculosus extract, and the above parts are all parts by mass.

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 Vanilla planifolia fruit extract is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts, preferably 6-10 parts; (2) the amount of the soluble collagen 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, preferably 0.01-0.1 parts; and, (3) The amount of the Fucus vesiculosus extract is 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.

3. The layered anti-aging composition according to claim 1, characterized in that The layered anti-aging composition satisfies the following conditions (1) and / or (2): (1) The mass ratio of the Vanilla lanceolata fruit extract, 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, preferably (6-10):(0.01-0.08):(10-15); (2) The composition further contains a preservative; The type of the preservative is preferably one or more of 1,3-butylene glycol, 1,2-propylene glycol, dipropylene glycol and glycerol; The amount of the preservative is preferably 70-90%, where the percentage is the mass percentage of the total amount of the composition.

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

5. 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 vanilla oleracea fruit extract is prepared by the following method: soaking the vanilla oleracea fruit in a solvent, and microfiltrating the filtrate obtained by solid-liquid separation to obtain the vanilla oleracea fruit extract; (2) The Fucus vesiculosus extract is prepared by the following method: heating a mixed solution containing Fucus vesiculosus, and ultrafiltrating the filtrate obtained by solid-liquid separation to obtain the Fucus vesiculosus extract; and, (3) The soluble collagen is one or more of recombinant type I, III, V, VII and XVII collagen, such as recombinant type III collagen.

6. The layered anti-aging composition according to claim 5, characterized in that In the preparation method of the flat-leaf vanilla fruit, one or more of the following conditions are met: (1) The flat-leaf vanilla fruit is first crushed before the soaking; after the crushing, the mesh size of the flat-leaf vanilla fruit powder is preferably 10 meshes; (2) The immersion is performed at room temperature; (3) The soaking time is 2-5 hours, for example 3 hours; (4) The mass ratio of the flat-leaved vanilla fruit to the solvent is 1:(5-10), for example 1:5 or 1:10; (5) The solvent is an alcohol solvent, preferably ethanol; (6) The solid-liquid separation method is gauze filtration; the mesh size of the gauze is preferably 600-200 mesh; (7) The microfiltration uses a microfiltration membrane, and the pore size of the microfiltration membrane is 2.5 to 10 μm, such as 2.5 μm, 6 μm, 8 μm or 10 μm; Preferably, the preparation method of the flat-leaf vanilla fruit extract comprises the following steps: crushing the flat-leaf vanilla fruit, soaking it in ethanol at room temperature for 3 hours, the mass ratio of the flat-leaf vanilla fruit to ethanol is 1:10, filtering through 200-mesh gauze to obtain a supernatant, concentrating the supernatant under reduced pressure to obtain a flat-leaf vanilla fruit concentrate, and filtering with a microfiltration membrane to obtain the flat-leaf vanilla fruit extract.

7. The layered anti-aging composition according to claim 5, characterized in that In the preparation method of the Fucus vesiculosus extract, one or more of the following conditions are met: (1) The Fucus vesiculosus is crushed before use; after the crushing, the mesh size of the Fucus vesiculosus powder is preferably 20-50 meshes; (2) The solvent used in the mixed solution containing Fucus vesiculosus is water; (3) In the mixed solution containing Fucus vesiculosus, the mass ratio of Fucus vesiculosus to solvent is 1:(10-15), preferably 1:15; (3) The pH value of the mixed solution containing Fucus vesiculosus is 3-3.5, for example 3; (4) The heating time is 2-5 hours, for example 2 hours; (5) The heating temperature is 60-80°C, for example 80°C; (6) The solid-liquid separation method is gauze filtration; the mesh size of the gauze is preferably 600-200 mesh; (7) During the ultrafiltration, the ultrafiltration membrane has a retention rate of 10 kDa; and, (8) During the ultrafiltration, the pore size of the ultrafiltration membrane is 0.45-0.8 μm, for example, 0.45 μm or 0.6 μm; Preferably, the preparation method of the Fucus vesiculosus extract comprises the following steps: washing the Fucus vesiculosus with water, drying at 40°C, and crushing to obtain Fucus vesiculosus powder with a mesh size of about 20-50 meshes; adjusting the pH value of the mixture of the Fucus vesiculosus powder and water to 3 with a hydrochloric acid solution, the mass ratio of the Fucus vesiculosus powder to water to 1:15, and heating at 80°C for 2h; cooling, filtering through 200-mesh gauze to obtain a filtrate, and ultrafiltering the filtrate through a 0.45μm ultrafiltration membrane with a retention rate of 10 kDa.

8. The layered anti-aging composition according to claim 1, characterized in that The composition satisfies one or more of the following conditions: (1) The composition is used in cosmetics as an ingredient for enhancing skin elasticity, firming, anti-aging or barrier repair; and, (2) The composition has the effects of promoting the expression of loricrin LOR, the relative expression 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.

9. Use of the layered anti-aging composition according to any one of claims 1 to 8 in the field of cosmetics.

10. The use according to claim 9, characterized in that The application meets one or more of the following conditions: (1) The composition is used in cosmetics as an ingredient for enhancing skin elasticity, firming, anti-aging or barrier repair; (2) The composition has the effects of promoting the expression of loricrin LOR, the relative expression 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; (3) The composition is used in cosmetics after being diluted; the concentration after dilution is preferably 2-10%, where the percentage is the percentage of the mass of the composition to the total mass of the liquid after dilution.

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