Multi-dimensional comprehensive detection system for anti-aging effect of skin care product

Abstract

The invention discloses a skin care product anti-aging effect multi-dimensional comprehensive detection system, which comprises an in-vitro cell model experiment module for detecting indexes such as cell activity, proliferation, migration, apoptosis and antioxidant enzyme activity by simulating natural aging and light aging conditions; the content change of skin structure components such as collagen, elastic fibers and laminin is quantitatively analyzed, the DNA damage repair capacity is reflected by measuring the expression quantity of gammaH2AX, epigenetics research is developed, and the methylation state change of a CpG island is monitored; and the tissue engineering and three-dimensional skin model experiment module is used for performing morphological observation, biochemical index detection and skin physiological parameter evaluation under the condition of simulating a real skin environment by utilizing an artificial skin model. The whole process from the in-vitro cell model to the clinical human body test is covered, and the actual efficiency of the skin care product for delaying or improving the skin aging process can be accurately and objectively evaluated through multi-angle and multi-level data integration analysis.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics testing, and in particular to a multi-dimensional comprehensive testing system and method for the anti-aging effect of skin care products. Background Art

[0002] In the field of cosmetics technology, especially in the development and efficacy verification of anti-aging skin care products, a variety of patent technologies have proposed evaluation strategies at different levels and angles. For example, Chinese invention patent CN114807115A discloses a method for constructing a senescent cell model and evaluating anti-aging efficacy. This method creates a corresponding relationship between the senescent cell model and the actual age by associating different generations of senescent cells cultured in vitro with the skin aging state of people of different age groups, thereby using these models to accurately simulate and test the efficacy of anti-aging products.

[0003] At the same time, China's invention patent CN114698572A elaborates on the "D-galactose-induced AL zebrafish aging model and method for evaluating anti-aging efficacy". This invention innovatively uses D-galactose to treat zebrafish to simulate the aging process of organisms, and uses this as a basis to evaluate the effects of anti-aging products. Despite this, the efficacy evaluation system of most current anti-aging skin care products still has limitations, mainly manifested in over-reliance on a single or a few physiological and biochemical indicators, which makes it difficult to fully reflect the multi-factor and multi-level effects of skin care products on the complex skin aging process.

[0004] In view of this, the present invention aims to provide a more comprehensive and scientific multi-dimensional integrated detection system and experimental method thereof, combining in vitro cell models, three-dimensional skin models, molecular biology and epigenetic research, and rigorous clinical trial design, so as to achieve a three-dimensional, multi-level, and comprehensive accurate evaluation of the anti-aging efficacy of skin care products, effectively make up for the deficiencies in the existing technology, and thus effectively promote technological innovation and progress in the field of anti-aging product research and development in the cosmetics industry. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a multi-dimensional comprehensive detection system and method for the anti-aging effect of skin care products. The invention aims to solve the problem that the evaluation system of the efficacy of anti-aging skin care products in the prior art is imperfect, lacks comprehensiveness and objectivity, and by constructing a comprehensive evaluation system including an in vitro cell model experiment module, a molecular biology and cell biochemistry index detection module, a tissue engineering three-dimensional skin model experiment module, and a clinical research verification module, a three-dimensional, multi-level, and comprehensive scientific evaluation of the actual effectiveness of skin care products in delaying or reversing skin aging can be achieved.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0007] A multi-dimensional comprehensive detection system for the anti-aging effect of skin care products, including an in vitro cell model experiment module and a tissue engineering and three-dimensional skin model experiment module.

[0008] The in vitro cell model experimental module uses human skin fibroblasts, immortalized keratinocytes or epidermal cells differentiated from induced pluripotent stem cells to simulate natural aging and photoaging conditions, and uses CCK-8 kit method, MTT method, EdU labeling method

[0009] Determine and compare the changes in cell proliferation ability among different treatment groups;

[0010] The tissue engineering and three-dimensional skin model experimental module uses artificial skin models with different age characteristics to perform morphological observations, biochemical index measurements (such as TEWL value, lactate dehydrogenase activity) and skin elasticity tests under conditions simulating real skin environments to evaluate the anti-aging properties of skin care products in three-dimensional models.

[0011] Preferably, the in vitro cell model experiment module includes a scratch healing experiment and a Transwell invasion experiment submodule, which are used to evaluate the effect of skin care products on cell migration ability.

[0012] Preferably, the in vitro cell model experiment module includes a molecular biology and cell biochemistry index detection submodule, which evaluates the effect of skin care products on cellular homeostasis by detecting changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and quantifying the autophagy-related protein LC3II / I ratio and p62 level.

[0013] Preferably, the molecular biology and cell biochemistry index detection submodule includes a DNA damage repair mechanism evaluation unit, which reflects the anti-aging effect of skin care products by measuring the change in γH2AX expression, and measuring mitochondrial membrane potential and oxygen consumption rate (OCR).

[0014] Preferably, the in vitro cell model experiment module includes an epigenetic research submodule, which selects the CpG island methylation status of the promoter region of aging-related candidate genes as a detection indicator to explore whether skin care products have the ability to regulate the expression of genes related to skin aging.

[0015] Preferably, the detection system includes a clinical research verification module, which designs and executes a double-blind, placebo-controlled, randomized clinical trial to collect skin physiological parameter data and subjective feedback from subjects after using skin care products, so as to verify the anti-aging effect of skin care products in actual human applications.

[0016] Preferably, the in vitro cell model experiment module establishes a natural aging model and a photoaging model for cells of different generations, sets different ultraviolet dose gradients, and performs detailed grouping treatment in combination with different concentration combinations of skin care products.

[0017] Preferably, the detection system achieves a comprehensive and three-dimensional evaluation of the anti-aging effects of skin care products by comparing and analyzing the differences between the skin care product treatment group and the control group in terms of cell viability, functional testing, molecular markers, three-dimensional skin model experimental results and clinical trial data.

[0018] Preferably, the detection system evaluates the effect of skin care products on inhibiting aging-related secretory phenotypes by detecting changes in the expression of inflammatory factors and growth factors such as IL6, IL8, TNFα, TGFβ1 and MMPs.

[0019] The present invention also proposes a comprehensive detection method based on the multi-dimensional comprehensive detection system for the anti-aging effect of skin care products, which specifically includes the following steps.

[0020] 1. In vitro cell model experimental process:

[0021] 1.1 Cell recovery and culture

[0022] HDFs, HaCaT or iPSC differentiated cells were extracted from the liquid nitrogen bank, thawed and washed, inoculated into basal culture medium, and cultured at 37°C and 5% CO2.

[0023] Cell generation and aging model establishment: According to the generation, the skin conditions of young (20th-30th generation), middle-aged (31st-40th generation) and old (41st-50th generation) were simulated. The photoaging model was established by using different doses of UVA / UVB (50mJ / cm 2 Up to 500mJ / cm 2 ) were continuously irradiated to construct a control group A and ultraviolet irradiation groups B1-B3 and C1-C3.

[0024] Skin care product treatment preparation and application: Aseptically dilute the skin care product to a preset concentration gradient (0.1%-2.0%), and perform cell exposure tests without inducing toxic reactions. The experimental combination includes a natural aging control group Dn (n=1, 2, 3 represents each age group) and skin care product treatment groups E1-E4 combined with various ultraviolet irradiation conditions, with at least three biological replicates in each group.

[0025] Cell exposure and sample collection: Apply the culture medium containing skin care products to the corresponding cells and incubate for n hours. After the end, collect cell samples for morphological observation, functional detection and molecular level analysis.

[0026] 1.2 Cell viability and function detection

[0027] Cell proliferation assessment: CCK8, MTT or EdU labeling method was used to determine the cell proliferation rate, and the proportion of fluorescent labeled cells was used to reflect the proliferation capacity of each group (skin care product treatment group, ultraviolet irradiation group, natural aging control group), to analyze the effect of skin care products on cell proliferation and anti-aging effects.

[0028] Migration ability evaluation: The effect of skin care products on cell migration was observed by using scratch healing assay and Transwell invasion assay. Time points such as 0h, 24h, and 48h were set to record the progress of scratch healing to compare the changes in cell migration ability after skin care product treatment under different conditions (including age and ultraviolet irradiation), and the possible promotion or inhibition of skin care products and their mechanisms were analyzed by combining morphological and molecular level results.

[0029] Apoptosis analysis: Flow cytometry Annexin V / PI double staining or TUNEL staining was used to quantify the changes in cell apoptosis rate after skin care product treatment. The apoptosis status of cells treated with different concentrations of skin care products, UV irradiation and natural aging was compared to evaluate the regulation of skin care products on cell apoptosis and their potential anti-aging effects.

[0030] 1.3. Molecular biology and cell biochemical index detection

[0031] 1.3.1 Antioxidant enzyme activity assay

[0032] The activities of superoxide dismutase (SOD) and catalase (CAT) were measured to evaluate the antioxidant potential of skin care products. The experimental process includes: after completing the cell treatment, the cells of each group were collected and lysed at the set time point, washed with PBS and extracted with a buffer containing protease inhibitors. The protein concentration was determined by the BCA method to standardize the enzyme activity.

[0033] In the SOD activity test, the test kit is used to react under specific conditions, and the changes in superoxide anions are measured by a spectrophotometer to calculate the SOD activity per unit protein amount.

[0034] When detecting CAT activity, the kit is also used to react with a system containing hydrogen peroxide, and the rate of water generation is monitored to determine the CAT activity per unit protein amount.

[0035] The data were summarized and compared to compare the changes in intracellular SOD and CAT activities in different skin care product treatment groups and control groups (ultraviolet irradiation, natural aging), and the effects of skin care products on the activities of these two antioxidant enzymes were analyzed to reveal their potential antioxidant effects.

[0036] 1.3.2 β-galactosidase activity assay

[0037] The activity of intracellular senescence-related β-galactosidase was detected by quantitative SAβGal staining. The experimental process includes: after 48 hours of exposure, the cells were collected and fixed in 4% paraformaldehyde, followed by X-gal staining (prepared according to the kit instructions and incubated at 37°C for 16-20 hours). The blue precipitate of each group of cells was recorded by microscope observation the next day, and the photos of at least 10 fields of view were analyzed using ImageJ software to calculate the proportion of SA-β-Gal positive cells. All data were summarized and the average positive cell proportion and standard error of the skin care product treatment group and the control group (natural aging, ultraviolet irradiation, etc.) were compared. If the proportion of the skin care product group was significantly lower, it indicated that it had an anti-aging effect and could effectively inhibit β-galactosidase activity to delay cell senescence.

[0038] 1.3.3 Mitochondrial function assessment:

[0039] JC-1 staining method for determination of mitochondrial membrane potential

[0040] The principle is based on the change of fluorescence color of JC-1 under different membrane potentials, which is red at high membrane potential and green at low membrane potential. When applied to the study of the anti-aging effect of skin care products, 5-10μM JC-1 is added to cultured cells and incubated, followed by washing and observing the change of red-green fluorescence ratio using a fluorescence microscope or flow cytometer, in order to analyze the ability of skin care products to maintain or enhance mitochondrial membrane potential, and judge their anti-aging effect accordingly.

[0041] 1.3.4 Measuring oxygen consumption rate (OCR)

[0042] Oxygen consumption rate (OCR) was measured to evaluate the effect of skin care products on improving cellular energy metabolism in different aging models and UV irradiation conditions.

[0043] If the basal OCR value of the cell population treated with skin care products is increased, it may indicate that the skin care products help to enhance cell energy production and delay aging. In particular, when the OCR level of aged cells approaches that of the young control group, it can be considered to have anti-oxidation and anti-aging properties of improved metabolism. In addition, if the skin care products can effectively inhibit the decrease in OCR caused by ultraviolet radiation or restore it to the level of non-irradiated cells, it proves that the product has the effect of photoprotection and repairing mitochondrial function, thus showing its application potential in the field of anti-photoaging.

[0044] 1.3.5 Expression of autophagy-related proteins

[0045] The ability of skin care products to regulate cell autophagy and fight against aging damage was studied by measuring the autophagy-related protein LC3II / I ratio and p62 level. After treating each group of cells according to the experimental protocol, the cells were collected and lysed at the specified time points to extract proteins, and the BCA method was used to quantify and prepare samples for Western Blot analysis. The PVDF membrane after SDS-PAGE electrophoresis was incubated with primary and secondary antibodies and chemiluminescence detection to quantify the changes in LC3II / I ratio and p62 expression.

[0046] If skin care products can increase the LC3II / I ratio and reduce the level of p62, it indicates that they may effectively activate the cellular autophagy mechanism to cope with skin cell damage caused by natural aging and photoaging. Compared with the untreated control group, the change trend of autophagy activity after skin care product treatment was evaluated to determine its anti-aging effect.

[0047] 1.3.6 SASP factor detection

[0048] The expression changes of inflammatory factors and growth factors such as IL6, IL8, and TNFα were detected by ELISA or qRT-PCR technology to evaluate the inhibitory effect of skin care products on aging-associated secretory phenotype (SASP) factors and anti-aging properties. The experimental steps include:

[0049] According to the established protocol, cell samples from each group were collected, including the natural aging control group Dn, different ultraviolet irradiation groups, and skin care product treatment groups E1 to E4. After 48 hours, they were washed with PBS and RNA and / or protein were extracted.

[0050] RNA was quantified and reverse transcribed into cDNA for subsequent mRNA analysis.

[0051] The ELISA method was used to determine the content of IL6, IL8, TNFα and other proteins in the cell culture supernatant.

[0052] During the data analysis, the expression changes of SASP factors such as IL6, IL8, TNFα at the mRNA and / or protein levels in each group after treatment with skin care products were observed compared with the unintervention natural aging and ultraviolet irradiation control groups, in order to evaluate the actual effect of skin care products in anti-aging.

[0053] 1.3.7 Analysis of skin structure and composition

[0054] Western Blot or ELISA techniques were used to detect the synthesis and degradation of skin structural proteins such as collagen I, III and laminin in each experimental group (natural aging control group Dn, ultraviolet irradiation control group B1-C3, and skin care product treatment group E1-E4). The experimental process includes cell lysis, protein quantification, ELISA operation and signal detection. By comparing and analyzing the changes in the concentration of target proteins between groups, the effect of skin care products on maintaining and improving the integrity of skin structure was evaluated.

[0055] 1.3.8 Evaluation of DNA damage repair ability

[0056] Immunofluorescence was used to detect the expression of γH2AX and evaluate the effects of skin care products on DNA damage and repair in skin cells. γH2AX, as a marker of DNA double-strand breaks, forms an observable focus at the site of damage. In anti-aging studies, the changes in γH2AX expression in cells of different groups (natural aging control group Dn, ultraviolet irradiation control group B1 to C3, and skin care product treatment group E1 to E4) were compared to determine the effects of skin care products on DNA protection and repair. The experimental process includes: sample fixation, penetration treatment, followed by γH2AX antibody incubation and fluorescently labeled secondary antibody binding, and observation and image acquisition using a fluorescence microscope. The number of γH2AX spots or the average fluorescence intensity in each cell was calculated by analysis software to measure the degree of DNA damage. If the expression of γH2AX can be significantly reduced after skin care product treatment, it shows that it has potential anti-aging effects and helps maintain DNA integrity.

[0057] 1.4 Epigenetic research

[0058] DNA methylation detection

[0059] During the aging process, DNA methylation is a key mechanism for regulating gene expression. If skin care products can reduce the methylation level of CpG islands in the promoter region of aging-related genes, they may maintain skin health and delay aging by activating the normal expression of these genes. In the experiment, DNA of cell samples under different conditions was extracted and processed, methylation sites were enriched using bisulfite conversion, and high-throughput sequencing technology was used to compare and analyze the changes in the methylation status of CpG islands of specific genes before and after the use of skin care products. When skin care products reduce the methylation of anti-aging genes or increase the methylation of pro-aging genes, it indicates that they have the ability to regulate epigenetic markers and show anti-aging effects. This method is used to evaluate the effects of skin care products on skin aging and their potential mechanisms of action.

[0060] 2. Tissue Engineering and Three-Dimensional Skin Model Experiment

[0061] In order to accurately evaluate the anti-aging effect of skin care products, commercial three-dimensional reconstructed human skin models with physiological relevance (such as EpiDerm, SkinEthic, etc.) were selected, and different age stages (young and old) and basic control models were ordered. In a sterile environment, the skin care products to be tested were diluted according to the preset concentration gradient and incubated continuously for 7 to 14 days. A blank control group and a positive control group were set up in the experiment to evaluate the effect of skin care products on skin aging by observing the morphological changes of the three-dimensional skin model (epidermal thickness, dermal structural integrity and stratum corneum arrangement) and biochemical index analysis (such as lactate dehydrogenase activity, transepidermal water loss rate and expression of collagen, elastic fibers, etc.).

[0062] By comprehensively considering various parameters, we can determine the mechanism of action of skin care products in maintaining or improving skin thickness, barrier function, stabilizing dermal structure, promoting collagen synthesis, and enhancing skin elasticity and water retention capacity, as well as their actual anti-aging effectiveness.

[0063] 3. Clinical Research

[0064] A 4-24 week, double-blind, placebo-controlled, randomized clinical trial was designed to study the effect of new skin care products on improving the symptoms of skin aging in the middle-aged and elderly, using skin color Doppler ultrasound. It is planned to recruit N healthy adults aged 40-60 years with signs of natural aging, and after passing strict screening criteria, they will be allocated to the skin care product or placebo group in a 1:1 ratio.

[0065] The subjects must meet the age requirements, have no history of serious diseases and allergies, and agree to cooperate with the whole process and accept skin ultrasound examination. Special circumstances such as recent use of anti-aging products, drugs, or medical implants are excluded.

[0066] After randomization, the subjects used the tested skin care products and placebo on the left and right sides of their faces.

[0067] Data collection covers baseline data collection and follow-up observations. In the baseline phase, skin condition is comprehensively assessed through high-resolution facial photo analysis, biophysical measurements (such as TEWL value, skin conductivity test, skin elasticity measurement), and expert scoring system. During the follow-up period, the above tests are repeated, and information on product usage experience and adverse reactions is collected.

[0068] Due to the adoption of the above technical scheme, the technical progress achieved by the present invention is as follows.

[0069] The in vitro cell model experimental module of the present invention adopts human skin fibroblasts, immortalized keratinocyte cell lines or epidermal cells differentiated from induced pluripotent stem cells as research objects, simulating natural aging and photoaging conditions, specifically including but not limited to cell viability determination, cell proliferation ability assessment, cell migration and apoptosis analysis, antioxidant enzyme activity detection and mitochondrial functional status assessment, so as to reflect the impact of skin care products on the physiological functions of skin cells.

[0070] The molecular biology and cell biochemistry index detection submodule of the present invention can quantitatively analyze the changes in the content of key skin structural components such as collagen I / III, elastic fibers and laminin before and after skin care products are treated, and detect the expression of γH2AX to evaluate the effect of skin care products on the cell DNA damage repair mechanism. In addition, a detailed analysis of RNA or protein levels is performed for the key components of autophagy-related proteins inside cells and various aging-related secretory phenotypes (SASP). These key components include but are not limited to inflammatory factors and growth factors such as IL6, IL8, TNFα, TGFβ1 and MMPs. Through this series of precise tests, it is intended to reveal the specific impact mechanism of skin care products on maintaining intracellular homeostasis, so as to comprehensively evaluate its efficacy in the field of anti-aging.

[0071] The tissue engineering and three-dimensional skin model experimental module of the present invention uses artificial skin models with different age characteristics to perform morphological observations, biochemical index measurements such as TEWL value, lactate dehydrogenase activity, etc., and skin elasticity tests under conditions simulating real skin environments, thereby evaluating the anti-aging properties of skin care products in a more realistic application environment.

[0072] The above-mentioned comprehensive detection system is applied to the rigorously designed clinical research stage. Through a double-blind, placebo-controlled, randomized clinical trial, the skin physiological parameter data and subjective feedback of the subjects are collected to verify the anti-aging effect of skin care products under actual human use conditions from a clinical perspective.

[0073] The present invention can not only accurately and objectively evaluate the anti-aging effects of skin care products, but also help cosmetics companies optimize product formulas, improve product quality and market competitiveness. It is also beneficial for consumers to choose anti-aging skin care products that are more suitable for them, thereby promoting the healthy development and technological progress of the entire cosmetics industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 It is a structural framework diagram of the present invention;

[0075] Figure 2 This is a fluorescence staining comparison diagram of the effect of skin care products on the proliferation of human skin fibroblasts observed by EdU labeling in a specific embodiment of the present invention;

[0076] Figure 3 This is an experimental comparison diagram of the effect of skin care products on the migration ability of human skin fibroblasts observed by a scratch healing experiment in a specific embodiment of the present invention;

[0077] Figure 4This is an experimental comparison diagram of the effect of skin care products on apoptosis of human fibroblasts using flow cytometry Annexin V / PI double staining method in a specific embodiment of the present invention;

[0078] Figure 5 This is an experimental comparison diagram of the effect of skin care products on the activity of the antioxidant enzyme SOD in human fibroblasts using the nitroblue tetrazolium (NBT) photoreduction method in a specific embodiment of the present invention;

[0079] Figure 6 This is an experimental comparison diagram of the effect of skin care products on the β-galactosidase content in human fibroblasts using the MTT colorimetric method in a specific embodiment of the present invention;

[0080] Figure 7 This is an experimental comparison diagram of the effect of skin care products on mitochondrial membrane potential in human fibroblasts determined by JC-1 staining method in a specific embodiment of the present invention;

[0081] Figure 8 This is an experimental comparison curve diagram for analyzing the effect of skin care products on the oxygen consumption rate (OCR) of human fibroblasts in a specific embodiment of the present invention;

[0082] Fig. 9 This is a comparison diagram of mitochondria under an optical microscope during the analysis of the effect of skin care products on the oxygen consumption rate (OCR) of human fibroblasts in a specific embodiment of the present invention;

[0083] Fig.10 This is a comparison diagram of mitochondria under an electron microscope during the analysis of the effect of skin care products on the oxygen consumption rate (OCR) of human fibroblasts in a specific embodiment of the present invention;

[0084] Fig.11 This is an experimental comparison diagram of the effect of skin care products on the LC3II / I ratio and p62 level of human fibroblast phagocytosis-related protein in a specific embodiment of the present invention;

[0085] Fig.12 This is a comparative bar graph of the results of the analysis of the effects of skin care products on the LC3II / I ratio and p62 level of human fibroblast phagocytosis-related protein in a specific embodiment of the present invention;

[0086] Fig.13 This is an experimental comparison diagram of the effect of skin care products on the expression of inflammatory factors and growth factors such as IL6, IL8, and TNFα in human fibroblast SASP factors in a specific embodiment of the present invention;

[0087] Fig.14This is a comparative bar graph of the results of the analysis of the effects of skin care products on the expression of inflammatory factors and growth factors such as IL6, IL8, and TNFα in human fibroblast SASP factors in a specific embodiment of the present invention;

[0088] Fig.15 This is an experimental comparison diagram of the effect of skin care products on type I collagen in human fibroblasts in a specific embodiment of the present invention;

[0089] Fig.16 This is an experimental comparison diagram of the effect of skin care products on the laminin component of human fibroblasts in a specific embodiment of the present invention;

[0090] Fig.17 This is an experimental comparison diagram of immunofluorescence (IF) analysis of the effect of skin care products on the expression of γH2AX in human fibroblasts in a specific embodiment of the present invention;

[0091] Fig.18 This is an experimental comparison diagram of the effect of skin care products on DNA methylation of human fibroblasts in a specific embodiment of the present invention;

[0092] Fig.19 A three-dimensional reconstructed human skin model diagram in a specific embodiment of the present invention;

[0093] Fig. 20 This is an experimental comparison diagram of measuring the effect of skin care products on the lactate dehydrogenase (LDH) level of a three-dimensional reconstructed human skin model in a specific embodiment of the present invention;

[0094] Fig.21 This is an experimental comparison diagram of measuring the effect of skin care products on the transepidermal water loss rate (TEWL) of a three-dimensional reconstructed human skin model in a specific embodiment of the present invention;

[0095] Fig. 22 This is an experimental comparison diagram of measuring the effects of skin care products on the expression of collagen type I and type III and collagen fibers in a three-dimensional reconstructed human skin model in a specific embodiment of the present invention;

[0096] Fig.23 This is an experimental comparison diagram of measuring the skin color ultrasound of three typical subjects in a specific embodiment of the present invention to determine the efficacy of skin care products; DETAILED DESCRIPTION

[0097] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0098] A multi-dimensional comprehensive detection system for the anti-aging effect of skin care products, the structure of which is as follows Figure 1 As shown, it includes in vitro cell model experiment module, tissue engineering and three-dimensional skin model experiment module and clinical research verification module.

[0099] The in vitro cell model experimental module utilizes epidermal cells differentiated from human skin fibroblasts, immortalized keratinocyte cell lines or induced pluripotent stem cells to simulate natural aging and photoaging conditions, and measures and compares changes in cell proliferation ability between different treatment groups through CCK-8 kit method, MTT method, EdU labeling method, etc.

[0100] The in vitro cell model experiment module establishes a natural aging model and a photoaging model for cells of different generations, sets different ultraviolet dose gradients, and performs detailed grouping treatments in combination with different concentration combinations of skin care products.

[0101] The in vitro cell model experiment module includes a scratch healing experiment and a Transwell invasion experiment submodule, which are used to evaluate the effect of skin care products on cell migration ability.

[0102] The in vitro cell model experiment module includes a molecular biology and cell biochemistry index detection submodule, which evaluates the effect of skin care products on cellular homeostasis by detecting changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and quantifying the autophagy-related protein LC3II / I ratio and p62 level. The molecular biology and cell biochemistry index detection submodule includes a DNA damage repair mechanism evaluation unit, which determines the changes in γH2AX expression by flow cytometry AnnexinV / PI double staining or TUNEL staining, and measures mitochondrial membrane potential and oxygen consumption rate (OCR) to reflect the anti-aging effect of skin care products.

[0103] The in vitro cell model experiment module includes an epigenetic research submodule, which selects the CpG island methylation status of the promoter region of aging-related candidate genes as a detection indicator to explore whether skin care products have the ability to regulate the expression of genes related to skin aging.

[0104] The tissue engineering and three-dimensional skin model experimental module uses artificial skin models with different age characteristics to perform morphological observations, biochemical index measurements (such as TEWL value, lactate dehydrogenase activity) and skin elasticity tests under conditions simulating real skin environments to evaluate the anti-aging properties of skin care products in three-dimensional models.

[0105] The clinical research verification module designs and implements a double-blind, placebo-controlled, randomized clinical trial to collect skin physiological parameter data and subjective feedback from subjects after using skin care products, so as to verify the anti-aging effect of skin care products in actual human applications.

[0106] The present invention also proposes a comprehensive detection method based on the above-mentioned multi-dimensional comprehensive detection system for the anti-aging effect of skin care products, which is used to test the Control Series anti-aging skin care product set developed by DJI (Tianjin) Biopharmaceutical Co., Ltd., including essence water, facial essence, eye essence and cream set.

[0107] In actual operation, according to the implementation order of the product manual, the four products were quantitatively measured according to the use of one person, and aseptically diluted to the preset concentration of 1.0% for standby use. The specific detection method is as follows.

[0108] 1. Implementation of in vitro cell model experiments

[0109] 1.1 Cell culture and processing steps

[0110] (1) Cell recovery and inoculation: Human dermal fibroblasts (HDFs) were taken out from liquid nitrogen stocks and routinely cultured according to the standard operating procedures provided by each supplier.

[0111] (2) Cell passage and aging model establishment:

[0112] Table 1

[0113]

[0114]

[0115] (3) Preparation and application of skin care products to be tested:

[0116] Dilution and aseptic processing of skin care products: Aseptically dilute the skin care products to be tested to a preset concentration of 1.0% to ensure that the test is carried out without inducing cytotoxic reactions.

[0117] 1.2 Cell viability and function detection

[0118] 1.2.1 Assessment of cell proliferation ability:

[0119] The cells were grouped according to the cell passage and aging model described in 1.1. The EdU labeling method was used for more intuitive data results. See the attached data analysis. Figure 2 .

[0120] Attached Figure 2 Description: Cell type: human skin fibroblasts HDFs; Experimental method: EdU labeling method; Duration: 72h.

[0121] The cell proliferation rate of the young control group increased by 185%; the cell proliferation rate of the middle-aged control group increased by 196%; the cell proliferation rate of the elderly control group increased by 177%; and the cell proliferation rate of the UVA irradiation group increased by 161%.

[0122] Conclusion: The results of cell image analysis showed that UVA irradiation significantly reduced the number of cells, reflecting its negative impact on cells. In contrast, the skin care product treatment group still maintained a high number of cells after UVA irradiation, indicating that skin care products have the effect of protecting cells from damage. At the same time, in the control groups of different ages, skin care product treatment led to an increase in the number of cells, confirming that skin care products have a positive effect on promoting vitality or proliferation of cells of all ages.

[0123] 1.2.2 Evaluation of cell migration ability:

[0124] The scratch healing experiment was used to observe the effect of skin care products on the migration ability of human skin fibroblasts.

[0125] Cell grouping and treatment:

[0126] D-Control group: untreated skin fibroblasts. B-Control group: D-group cells were irradiated with UVA without subsequent treatment. D-Product group: 1% skin care products were added to D-group cells for scratch healing experiment. B-Product group: 1% skin care products were added to B-group cells that had been irradiated with UVA for experiment.

[0127] Data collection and comparative analysis are attached. Figure 3 .

[0128] Conclusion: Both the D-Product group and the B-Product group treated with 1% skin care product showed enhanced cell scratch healing effects, indicating that the skin care product can promote the migration and repair of normal aging and photoaging damaged skin cells.

[0129] 1.2.3 Cell apoptosis analysis:

[0130] In this study, flow cytometry AnnexinV / PI double staining was used to evaluate the effect of skin care products on apoptosis of middle-aged HDFs. Four groups of experiments were designed: untreated control group D2, UVA irradiation group B2, 1.0% skin care product alone treatment group E2, and UVA irradiation followed by 1.0% skin care product E4 group. Each group was cultured for 3 days. The specific operation process includes collecting and preparing cells from each group, labeling with AnnexinV and PI dyes, setting up blank, single staining and experimental tube controls, and analyzing the proportion of live cells and cells at different apoptotic stages by flow cytometry, so as to compare and analyze the regulatory effects of skin care products alone and their combined effects with UVA on cell apoptosis.

[0131] Data collection and comparative analysis are attached. Figure 4 .

[0132] Conclusion: Skin care products significantly reduced the apoptosis rate of HDFs cells under simulated photoaging conditions, from 9.77% in the untreated UVA irradiation group to 3.33%, indicating that it has a certain photoprotective effect on skin cells. Compared with the natural aging group without any intervention (cell apoptosis rate of 3.02%), skin care products alone can reduce the apoptosis rate of middle-aged cells to 1.52%, reflecting the obvious protective effect of the skin care products in resisting the natural aging process.

[0133] 1.3. Molecular biology and cell biochemical index detection

[0134] The test plan approach includes the following key steps:

[0135] Cell recovery and culture:

[0136] Brief description of cell recovery and culture process: Thaw and culture human skin fibroblasts according to standard procedures, adjust cell density and culture routinely. Construct 7 experimental groups D1-E4 according to the cell passage and aging model described in 1.1:

[0137] 1.3.1 Determination of antioxidant enzyme activity:

[0138] The cells of each group were collected, washed with PBS and gently lysed after 72 hours to release enzymes, and the total protein concentration was determined by BCA and other methods. SOD activity was detected by NBT photoreduction method, etc., based on its catalytic O2 - The principle of clearance rate is to set up controls, add reaction system and measure the change in absorbance at a specific wavelength to calculate activity.

[0139] Conclusion: Based on the data obtained, see the attached Figure 5 .

[0140] Attached Figure 5 Description: Nitroblue tetrazolium (NBT) photoreduction method; Cell type: Human dermal fibroblasts HDFs.

[0141] After treatment with skin care products, the SOD activity of each age control group increased: the young group increased from 0.49 to 0.68, the middle-aged group increased from 0.41 to 0.56, and the elderly group increased from 0.35 to 0.51, indicating that skin care products can effectively enhance the activity of cellular SOD. UVA irradiation reduced the SOD activity of the middle-aged group from 0.46 to 0.36, but after adding skin care products, it recovered to 0.52, confirming that skin care products have the effect of restoring and improving the SOD activity of cells after UVA irradiation.

[0142] 1.3.2SA-β-Gal: β-galactosidase activity assay

[0143] The test plan approach includes the following key steps:

[0144] Human skin fibroblasts were thawed and cultured according to standard procedures, and the cell density was adjusted and cultured routinely. Four experimental groups were constructed: untreated middle-aged control group D2, UVA irradiation group B2, middle-aged control group E2 treated with 1.0% skin care products, and group E4 treated with UVA first and then with skin care products. The treatment time of each group was 3 days.

[0145] Detection of cell viability and β-galactosidase activity: The MTT method was used to determine the cell survival rate and metabolic capacity of each group, and β-galactosidase activity was evaluated by staining or biochemical testing to determine the degree of skin aging and the effect of skin care products on photoaging. Relevant data were collected and statistically analyzed, and the differences between different treatment groups were compared to further study the effects of skin care products in anti-UVA-induced photoaging and anti-aging across age groups.

[0146] Data analysis is attached Figure 6 .

[0147] Attached Figure 6 Description: MTT colorimetric assay; Cell type: Human skin fibroblasts HDFs

[0148] UVA radiation increases the level of β-galactosidase in the skin, accelerating the photoaging process; while skin care products can effectively reduce the level of this enzyme in cells irradiated by UVA, demonstrating its anti-photoaging efficacy. At the same time, skin care products can also reduce the level of β-galactosidase in the middle-aged control group, reflecting a positive improvement effect on naturally aged skin.

[0149] 1.3.3 Mitochondrial function assessment:

[0150] JC-1 staining method for determination of mitochondrial membrane potential

[0151] Human skin fibroblasts were thawed and cultured according to standard procedures, and the cell density was adjusted and cultured routinely. Four experimental groups were constructed: untreated middle-aged control group D2, UVA irradiation group B2, middle-aged control group E2 treated with 1.0% skin care products, and group E4 treated with UVA first and then with skin care products. The treatment time of each group was 7 days.

[0152] JC-1 staining to detect mitochondrial membrane potential:

[0153] After washing the cells, incubate the cells with 2.5-5μM JC-1 under appropriate conditions, so that normal mitochondria show red fluorescence and damaged mitochondria show green fluorescence. The changes in membrane potential of each group were compared by observing and taking fluorescence images under a microscope, and then the signal intensity of FL1 (green) and FL2 (red) channels was analyzed by flow cytometry, and the JMR ratio was calculated to quantitatively evaluate the mitochondrial membrane potential level. Data analysis and conclusion:

[0154] Firstly, the difference in JMR between the D2 group without UVA irradiation and the B2 group with UVA irradiation was compared to verify the validity of the photoaging model.

[0155] The changes in mitochondrial membrane potential between the E2 group using 1.0% skin care products and the untreated D2 group were analyzed to determine the anti-aging properties of the skin care products under normal physiological conditions.

[0156] Finally, a comparison was made between the E4 group that applied skin care products after UVA irradiation and the B2 group that was simply exposed to UVA irradiation, revealing the protective and repairing effects of skin care products under photoaging conditions.

[0157] Data analysis is attached Figure 7 .

[0158] Attached Figure 7 Description: JC-1 staining method; Cell type: Human skin fibroblasts HDFs

[0159] Conclusion: UVA irradiation caused a significant increase in the JMR values ​​of the B2 and D2 groups, revealing its accelerated aging effect; while the E2 group showed a lower mitochondrial membrane potential than the D2 group, proving that skin care products have anti-aging effects under normal conditions. In addition, the comparison between the E4 group and the B2 group showed that skin care products can effectively protect and repair cells under photoaging conditions.

[0160] 1.3.4 Measuring oxygen consumption rate (OCR)

[0161] Human skin fibroblasts were thawed and cultured according to standard procedures, and the cell density was adjusted and cultured routinely. Four experimental groups were constructed: untreated middle-aged control group D2, UVA irradiated group B2, middle-aged control group E2 treated with 1.0% skin care products, and group E4 treated with skin care products after UVA irradiation. The treatment time of each group was 7 days. According to the experimental protocol, OCR detection was performed on cells under different conditions. In the Seahorse XF system, the cells were first prepared and incubated, and then the skin care products or control group culture medium were added, and further incubated to allow the skin care products to take effect. Finally, the real-time oxygen consumption rate (OCR) of each treatment group was measured on the analyzer, and mitochondrial function parameters such as basal OCR, ATP coupling efficiency, and maximum respiratory capacity were explored using drug inhibitors.

[0162] Data analysis is attached Figures 8 to 10 .

[0163] Conclusion: Skin care products can enhance cell energy metabolism: By increasing the basal OCR value, it shows that skin care products can enhance the cell energy production capacity to delay aging. Skin care products show photoprotection and repair functions: Under ultraviolet irradiation, skin care products can effectively inhibit the decline of cell OCR and restore it to a near normal level, proving that it has significant photoprotection and damaged mitochondrial repair effects, thus reflecting the anti-photoaging effect.

[0164] 1.3.5 Expression of autophagy-related proteins

[0165] By measuring the LC3II / I ratio and p62 level, the effects of skin care products on regulating cell autophagy and fighting natural aging and photoaging were explored. In the experiment, human skin fibroblasts (HDFs) were used to simulate the middle-aged state (D2 group) and the photoaging model (B2 group). A control group D2, a UVA irradiation group B2, a 1.0% skin care product treatment group E2, and an E4 group treated with skin care products after UVA irradiation were set up, and each group was treated for 7 days. Western Blot analysis was used to detect protein expression in cell lysates, including changes in LC3B and p62, to evaluate the ability of skin care products to activate cell autophagy and resist aging damage. If the LC3II / I ratio increases and the p62 level decreases after skin care product treatment, it indicates that the skin care product has a good anti-aging effect.

[0166] See attached for analysis of the results Fig.11 , 12 .

[0167] After skin care product treatment, the LC3II / I ratio increased significantly, reflecting the enhanced cell autophagy activity, especially in the E2 group and the E4 group treated with UVA irradiation, indicating that the skin care product can effectively promote the autophagy process of normal and photoaged skin cells. At the same time, p62 protein was significantly reduced under the action of skin care products, further proving that skin care products can clear metabolic waste and abnormal protein aggregation in damaged or aged cells by enhancing autophagy. Therefore, skin care products have strongly confirmed their important role in activating skin cell autophagy, repairing photoaging damage, and maintaining skin health by upregulating the LC3II / I ratio and downregulating p62 expression.

[0168] 1.3.6 SASP factor detection

[0169] By detecting the expression changes of inflammatory factors and growth factors such as IL6, IL8, TNFα in cells, we evaluate whether skin care products can inhibit or reduce the level of SASP (Senescence-Associated Secretory Phenotype) factors related to aging, thereby verifying their anti-aging effects.

[0170] The steps are as follows:

[0171] Human skin fibroblasts were thawed and cultured according to standard procedures, and the cell density was adjusted and cultured routinely. Four experimental groups were constructed: untreated middle-aged control group D2, UVA irradiation group B2, middle-aged control group E2 treated with 1.0% skin care products, and group E4 treated with skin care products after UVA irradiation. The treatment time of each group was 7 days. After washing the cells with PBS buffer, total RNA and protein were obtained using RNA extraction kit or lysis buffer. RNA was quantified and reverse transcribed into cDNA; at the same time, ELISA detection technology was used to determine the content of proteins such as IL6, IL8 and TNFα in the cell culture supernatant according to the instructions of the kit.

[0172] Conclusion analysis is attached Fig.13 , 14 : Cells treated with skin care products showed a significant reduction in the expression of SASP factors, especially in the natural aging and photoaging models, revealing that the skin care product has the potential to inhibit inflammation, delay aging and resist photoaging, and exhibits certain anti-aging effects.

[0173] 1.3.7 Analysis of skin structure and composition

[0174] Quantify the synthesis and degradation of key skin structural components such as collagen I, collagen III, and laminin.

[0175] Experimental operation steps:

[0176] Human skin fibroblasts were thawed and cultured according to standard procedures, and the cell density was adjusted and cultured routinely. Four experimental groups were constructed: untreated middle-aged control group D2, UVA irradiation group B2, middle-aged control group E2 treated with 1.0% skin care products, and group E4 treated with UVA first and then with skin care products. The treatment time of each group was 7 days.

[0177] Lyse the cells to release collagen I / III, laminin and other structural components, collect the supernatant after centrifugation for total protein concentration determination, and dilute the sample according to the requirements of the ELISA kit. Perform ELISA experiments according to the supplier's instructions, including preparing 96-well plates, adding diluted samples and standards, and color development after enzyme reactions after blocking, antibody incubation, washing and other steps. Finally, read the absorbance value of each well at a specific wavelength using an ELISA reader, and calculate the target protein concentration based on the standard curve. Comparing the skin care product treatment group (1%) with the control groups D2 and B2, the changes in collagen I, III and laminin content were recorded and analyzed in the E2 and E4 groups.

[0178] Conclusion analysis is attached Fig.15 , 16: After skin care product treatment, collagen I, III and laminin increased significantly, suggesting that the active ingredients such as peptides and vitamin C in the products can effectively stimulate fibroblasts to synthesize more of these key structural proteins. This change is reflected in the skin repair and anti-aging effects, that is, enhancing skin elasticity and firmness, improving epidermal-dermal connection, and thus improving the overall health of the skin and its anti-aging ability.

[0179] 1.3.8 Evaluation of DNA damage repair ability

[0180] The expression of γH2AX is detected by immunofluorescence (IF) to evaluate the effect of skin care products on cell DNA damage and repair. γH2AX is a biomarker for DNA double-strand breaks. It will be phosphorylated and form aggregation focuses when damage occurs. In anti-aging research, detecting the changes in γH2AX expression in cells before and after treatment with skin care products can indirectly reflect its effect on improving DNA protection and repair capabilities. If skin care products can effectively reduce the expression level of γH2AX, it means that the product helps reduce DNA damage and promote skin cell repair.

[0181] The detection method is as follows:

[0182] Human skin fibroblasts were thawed and cultured according to standard procedures, and the cell density was adjusted and cultured routinely. Four experimental groups were constructed: untreated middle-aged control group D2, UVA irradiation group B2, middle-aged control group E2 treated with 1.0% skin care products, and group E4 treated with UVA first and then with skin care products. The treatment time of each group was 7 days.

[0183] Cell fixation and permeabilization: Fix cells with 4% paraformaldehyde and permeabilize with 0.5% Triton X-100 to expose the target protein. γH2AX staining process: Block nonspecific binding sites. Add primary antibody (anti-human γH2AX antibody) for incubation, then wash and add fluorescently labeled secondary antibody. Optional nuclear dye DAPI staining can be added, and unbound secondary antibody can be removed by washing.

[0184] Microscope observation and image acquisition: Observe the cell slides under a fluorescence microscope and capture γH2AX and nuclear signals through filters. Appropriately adjust the exposure time and gain to avoid overexposure and ensure quantitative accuracy.

[0185] The conclusion of data analysis is attached. Fig.17 The experimental results showed that the cell nucleus (blue) was observed by DAPI staining, and the DNA damage sites (green) were revealed by γH2AX fluorescence staining. DNA damage was obvious in the natural aging control group D2; the damage was reduced after 1% skin care product treatment (E2 group). Ultraviolet irradiation caused the damage of the control group B2 to increase, while the damage of the E4 group treated with the same treatment was reduced. Based on this, it was concluded that skin care products can effectively reduce DNA damage and promote repair, showing a good anti-aging effect.

[0186] 1.4 Epigenetic research

[0187] DNA methylation detection

[0188] During aging, changes in CpG island methylation in the promoter region affect gene expression, and high methylation may lead to decreased activity of skin repair and renewal genes. Skin care products may improve skin condition, promote regeneration, and enhance elasticity by regulating the methylation of CpG islands of specific aging genes to restore their normal transcription and function, thereby achieving anti-aging effects. Therefore, detecting CpG island methylation in the promoter of related genes is an important biomarker for evaluating the anti-aging properties of skin care products.

[0189] Experimental groups and treatments:

[0190] Four experimental groups were set up: a middle-aged control group D2 without any treatment, a group B2 after UVA irradiation, a middle-aged control group E2 treated with 1.0% skin care products, and a group E4 treated with 1.0% skin care products after UVA irradiation. The treatment time was 7 days. Cell samples were collected and high-quality DNA was extracted for epigenetic analysis. CpG sites related to aging were accurately selected for methylation research, and the methylation status was distinguished by bisulfite conversion. High-throughput sequencing technology (such as WGBS, etc.) was used to compare the changes in DNA methylation levels before and after skin care product treatment, focusing on the CpG islands of key genes for skin aging. If the methylation of the promoter region of the anti-aging gene is reduced after skin care product treatment, its expression may be activated; if the methylation of the adverse aging-related gene is increased, its activity is inhibited. These changes all show that skin care products delay the skin aging process by regulating gene methylation and have potential anti-aging effects.

[0191] See attached data Fig.18 , the conclusions are shown in Table 2.

[0192] Table 2

[0193]

[0194] Skin care product verification: It demonstrates anti-aging effects at the cellular level by activating gene expression related to skin health and anti-aging. Specific pathways include: promoting repair and regeneration, regulating the cell cycle, enhancing DNA repair and antioxidant and anti-inflammatory capabilities, supporting autophagy and inflammation inhibition, maintaining protein homeostasis, and enhancing stem cell vitality and tissue repair function. The coordinated upregulation of these key genes indicates that the skin care product has the ability to comprehensively improve skin condition and delay the aging process, which is reflected in improving metabolism, enhancing repair ability, resisting oxidative stress and inflammation, and stabilizing skin structure and function.

[0195] 2. Tissue Engineering and Three-Dimensional Skin Model Experiment

[0196] In order to accurately simulate real skin and evaluate the anti-aging performance of skin care products, this study selected the EpiDerm 3D reconstructed human skin model from MatTek Corporation as the experimental object, see Appendix Fig.19 .

[0197] Order 3D skin models of different ages to simulate the natural aging stages and the effects of photoaging, including young controls (representing early aging) and aged models (reflecting advanced or photoaged skin states).

[0198] Experimental preparation: All steps were performed aseptically in a clean bench to ensure that the skin care products were not contaminated.

[0199] The skin care product to be tested was diluted aseptically at a concentration of 1.0%, and the culture medium was changed every morning and evening, and the model was treated continuously for 14 days.

[0200] Model treatment and control settings: The three-dimensional skin model was immersed in a solution containing skin care products and incubated at 37°C and 5% CO2. Four control groups were set up: untreated natural aging control B1, UVA irradiation simulated photoaging model B2 (medium dose 250mJ / cm 2 , once a day, 30 minutes each time), and two skin care product treatment groups E1 and E2 (E2 was treated with UVA first and then skin care products).

[0201] Morphological assessment: An inverted microscope was used to observe and record the changes in aging indicators such as epidermal thickness, dermal integrity, and stratum corneum arrangement of the model every week.

[0202] Table 3

[0203]

[0204]

[0205] Biochemical index analysis:

[0206] Evaluation of skin barrier function: The supernatant of the three-dimensional skin model was collected during the culture process, and the lactate dehydrogenase (LDH) level was measured to evaluate the barrier integrity, and the transepidermal water loss (TEWL) value was measured to reflect the changes in barrier function.

[0207] Protein expression analysis: The proteins in the three-dimensional skin model tissue are extracted by chemical methods, and the changes in the content of structural components closely related to skin aging (such as type I and type III collagen, elastic fibers, etc.) are quantitatively analyzed using Western Blot or ELISA technology.

[0208] The tested skin care products showed positive effects in anti-aging, which are reflected in: increasing epidermal thickness and combating skin thinning, see Table 3; optimizing dermal structural integrity and collagen fiber arrangement, maintaining skin stability and delaying the aging process, see Table 3; improving stratum corneum structure and strengthening skin barrier function, see Table 3; significantly reducing lactate dehydrogenase (LDH) activity and enhancing skin barrier protection, see Appendix Fig. 20 ; Reduce transepidermal water loss (TEWL) and improve skin's water retention and moisturizing properties, see attached Fig.21 ; Promote the increase of collagen type I and type III expression, improve skin elasticity and firmness, see attached Fig. 22 .

[0209] 3. Clinical Research

[0210] A double-blind, placebo-controlled, randomized clinical trial for skin aging in the middle-aged and elderly was designed for 4 weeks, involving 200 healthy subjects aged 40-60 years. The study evaluated the anti-aging effects of new skin care products by skin color Doppler ultrasound. Recruited subjects must meet the criteria of age, skin aging characteristics and health status, and exclude specific populations such as those who have recently received cosmetic treatments, drug users and pregnant women.

[0211] After randomization, subjects will use the test skin care products and placebo on both sides of their faces at the same time. The data collection phase includes baseline and follow-up observations, covering indicators such as skin image analysis (wrinkle depth, texture roughness, pigmentation), biophysical measurements (TEWL value, skin conductivity, skin elasticity) and expert scoring. A comprehensive test is conducted once a week to track changes in skin condition and collect feedback information. At the same time, the subjects' product usage experience is collected to understand their subjective experience in terms of skin feel, absorbency and intuitive effects. Adverse reaction monitoring: Pay close attention to any adverse reactions that may occur during the use of skin care products by subjects, including redness, itching, allergies, etc., and keep records to timely evaluate the safety and adaptability of the product.

[0212] Table 4

[0213]

[0214] Conclusion: Table 4 shows that after four weeks of testing, the results show that skin care products have shown significant effects in anti-aging. 73% of the subjects' skin water retention capacity increased by more than 20%, and the TEWL value decreased, confirming its effectiveness in improving barrier function. More than 78% of the subjects' skin conductivity increased by more than 20%, reflecting the product's strong moisturizing ability and ability to improve the state of the stratum corneum. 89% of the subjects' skin elasticity increased by more than 20%, showing the outstanding role of skin care products in tightening the skin. In addition, 83% of the subjects confirmed that the degree of skin aging was significantly improved by more than 20 points through the expert scoring method, proving that skin care products have a strong practical effect on anti-aging phenomena such as wrinkles and sagging.

[0215] The skin ultrasound of the subjects is attached. Fig.23 A skin study on three typical subjects found that after using the skin care product for 8 weeks, the collagen content in the dermis of all subjects was significantly increased through skin color ultrasound. This evidence strongly proves that the skin care product has an effective effect in promoting collagen production.

[0216] Conclusion: In the four-week trial, the skin care product showed excellent skin feel improvement, with subjects reporting a more refreshed and less greasy skin. The results of the absorption test showed that the product was widely recognized to have good penetration and absorption properties. In terms of wrinkle removal, on the 28th day, up to 96% of the subjects reported significant anti-wrinkle effects, confirming the powerful anti-aging effect of the skin care product, see Appendix 5.

[0217] Table 5

[0218]

[0219] In addition, the safety assessment showed that none of the subjects experienced adverse reactions such as redness, swelling, itching or allergies during the entire trial period, indicating that the product is safe and well tolerated for short-term use (see Appendix 6).

[0220] Table 6

[0221] time Number of people with redness and swelling Number of people itching Number of people with allergic reactions Other adverse reactions Day 0 - - - - Day 7 0 0 0 0 Day 14 0 0 0 0 Day 21 0 0 0 0 Day 28 0 0 0 0

[0222] In summary, the test subjects generally gave positive comments on the skin feel, absorption and anti-wrinkle effects of the skin care product, and no obvious adverse reactions were observed, further confirming the effectiveness and safety of the skin care product in the field of anti-aging skin care.

[0223] The specification has described the technical solution of the present invention in detail, including the specific implementation methods of each experimental module, detection indicators, analysis methods and application fields, and has been explained in detail with the accompanying drawings. The scope of protection requested by the present invention is defined by the attached claims, in which each independent claim and its dependent claims respectively define different protection levels and scopes.

[0224] In addition, it should be understood that although this specification describes the present invention in specific embodiments, these embodiments are not restrictive, and any non-substantial improvements or equivalent substitutions made based on the concepts of the present invention should fall within the scope of protection of the present invention.

Claims

1. A multi-dimensional comprehensive detection system for the anti-aging effect of skin care products, characterized by: Including in vitro cell model experiment module and tissue engineering and three-dimensional skin model experiment module, The in vitro cell model experimental module uses epidermal cells differentiated from human skin fibroblasts, immortalized keratinocytes or induced pluripotent stem cells to simulate natural aging and photoaging conditions, and measures and compares changes in cell proliferation ability between different treatment groups using CCK-8 kit method, MTT method and EdU labeling method; The tissue engineering and three-dimensional skin model experimental module uses artificial skin models with different age characteristics to perform morphological observations, biochemical index measurements (such as TEWL value, lactate dehydrogenase activity) and skin elasticity tests under conditions simulating real skin environments to evaluate the anti-aging properties of skin care products in three-dimensional models.

2. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 1, characterized in that: The in vitro cell model experiment module includes a scratch healing experiment and a Transwell invasion experiment submodule, which are used to evaluate the effect of skin care products on cell migration ability.

3. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 1, characterized in that: The in vitro cell model experiment module includes a molecular biology and cell biochemistry index detection submodule, which evaluates the effect of skin care products on cellular homeostasis by detecting changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and quantifying the autophagy-related protein LC3II / I ratio and p62 level.

4. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 3, characterized in that: The molecular biology and cell biochemistry index detection submodule includes a DNA damage repair mechanism evaluation unit, which reflects the anti-aging effect of skin care products by measuring the changes in γH2AX expression, as well as mitochondrial membrane potential and oxygen consumption rate (OCR).

5. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 3, characterized in that: The in vitro cell model experiment module includes an epigenetic research submodule, which selects the CpG island methylation status of the promoter region of aging-related candidate genes as a detection indicator to explore whether skin care products have the ability to regulate the expression of genes related to skin aging.

6. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 1, characterized in that: The detection system includes a clinical research verification module, which designs and executes a double-blind, placebo-controlled, randomized clinical trial to collect skin physiological parameter data and subjective feedback from subjects after using skin care products, so as to verify the anti-aging effect of skin care products in actual human applications.

7. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 1, characterized in that: The in vitro cell model experiment module establishes a natural aging model and a photoaging model for cells of different generations, sets different ultraviolet dose gradients, and performs detailed grouping treatments in combination with different concentration combinations of skin care products.

8. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 1, characterized in that: The detection system achieves a comprehensive and three-dimensional evaluation of the anti-aging effects of skin care products by comparing and analyzing the differences between the skin care product treatment group and the control group in terms of cell viability, functional testing, molecular markers, three-dimensional skin model experimental results and clinical trial data.

9. A multi-dimensional comprehensive detection system for anti-aging effects of skin care products according to claim 1, characterized in that: The detection system evaluates the effect of skin care products on inhibiting aging-related secretory phenotypes by detecting the expression changes of IL6, IL8, TNFα, TGFβ1 and MMPs inflammatory factors and growth factors.

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