Method and model for evaluating in-vitro red fading and relieving effects of cosmetic raw materials

Abstract

The invention relates to the technical field of function testing of cosmetic raw materials, and particularly discloses a method and a model for evaluating in-vitro red fading and relieving effects of cosmetic raw materials, and the method comprises the following steps: inducing human immortalized umbilical vein vascular endothelial cells by adopting a TNF-alpha inducer to establish a red fading and relieving in-vitro model; the method specifically comprises the following steps: after inoculating and culturing human immortalized umbilical vein vascular endothelial cells, setting a control group, a modeling group, a positive drug group and a sample group, respectively adding special culture mediums into the control group and the modeling group, adding an epoxidase inhibitor into the positive drug group, adding a raw material sample to be detected into the sample group, and pre-treating; then adding a TNF-alpha inducer into each group except the control group, and carrying out induction culture; the prostacyclin I2 factor is adopted as a detection index to evaluate the red fading and relieving effects of the raw material sample to be detected. The method is short in test period, simple to operate, economical and convenient, and provides reliable data for further research of efficacy verification in the cosmetic industry.

Description

Technical Field

[0001] This application belongs to the field of functional testing technology for cosmetic raw materials, specifically relating to a method and model for evaluating the in vitro redness-reducing and soothing effects of cosmetic raw materials. Background Technology

[0002] According to the consensus reached by the Sensitive Skin Special Interest Group of the International Forum for Research on Itch (IFSI) using the Delphi method, sensitive skin is defined as "a syndrome characterized by discomfort such as stinging, burning, pain, and itching in response to stimuli that should not normally cause abnormal sensations." Subjective symptoms often manifest as burning, stinging, itching, and tightness, with or without objective signs such as erythema, scaling, and telangiectasia. Studies have shown that sensitive skin has become one of the most prevalent skin health problems today.

[0003] Currently, the formation mechanism of sensitive skin mainly involves four aspects: impaired skin barrier function, abnormal function of the skin's sensory nervous system, increased vascular reactivity, and immune inflammatory response. In evaluating the soothing efficacy of cosmetics, commonly used methods include laboratory tests (such as biochemical methods, cell biology methods, 3D skin model substitution methods, and animal experiments) and human clinical trials. Existing in vitro cell models mostly focus on immune inflammatory mechanisms, such as LPS-induced macrophage inflammation models or UVB-induced keratinocyte models, detecting inflammatory factors such as interleukins IL-6, IL-8, IL-1β, and TNF-α, as well as inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and endothelin-1 (ET-1), and also inflammatory-related enzymes such as COX-2, 5-LOX, and iNOS, and proteins from the NF-κB and MAPK signaling pathways. In addition, there are also models based on C48 / 80 or substance P inducing mast cell degranulation and histamine release. In terms of skin barrier repair, the HaCaT cell scratch assay is commonly used to assess cell migration ability; in terms of nerve sensory abnormalities, the capsaicin receptor TRPV1 expression inhibition assay is often used.

[0004] It is worth noting that redness in sensitive skin is mainly related to increased vascular reactivity caused by inflammatory factors, which in severe cases can even lead to persistent vasodilation and the formation of spider veins. However, there is currently a lack of in vitro efficacy evaluation methods targeting the mechanisms of increased vascular reactivity, and existing studies mostly rely on human trials for verification, which limits the efficient and compliant screening of raw materials and the process of product development.

[0005] Studies have shown that tumor necrosis factor (TNF) can induce endothelial cell inflammatory responses, promote leukocyte adhesion, and increase the synthesis of prostacyclin I2 (PGI2). PGI2, as a metabolite of arachidonic acid, is an important vasodilator that can inhibit platelet aggregation and promote vasodilation. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin reduce inflammation and vascular responses by inhibiting the activity of cyclooxygenase (COX) and lipoxygenase (LOX), thereby blocking the production of inflammatory mediators such as prostaglandins (PG), prostacyclin (PGI2), and leukotrienes (LT).

[0006] Currently, human immortalized umbilical vein endothelial cells (HUVEC 40) and the PGI2 factor have been widely used in the biomedical field, such as in tissue repair, wound treatment, ischemic disease treatment, and gene therapy (see patents CN201810135019.5, CN202411717796.2, CN200980117998.0, and SGSG196247A1). However, in the cosmetics field, especially for skin redness caused by increased vascular reactivity, there are no reports on in vitro soothing efficacy evaluation methods based on the HUVEC model and the PGI2 index.

[0007] As consumers increasingly demand higher standards for the safety and efficacy of cosmetics, developing scientific, reliable, and ethical in vitro evaluation methods is of great significance. Animal experiments, due to their long duration, ethical controversies, and limitations imposed by the "3R principle" (reduce, replace, optimize), have been gradually replaced by in vitro methods.

[0008] Therefore, there is a need to develop an efficient, convenient, safe, and economical in vitro method based on the human immortalized umbilical vein endothelial cell (HUVEC 40) model, using PGI2 as the key indicator, to evaluate the redness-reducing and soothing effects of cosmetic ingredients. Summary of the Invention

[0009] To address the aforementioned technical problems, this application provides a method and model for evaluating the in vitro redness-reducing and soothing effects of cosmetic raw materials.

[0010] In a first aspect, this application provides a method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic ingredients, comprising the following steps: S1: Model building; An in vitro model for redness reduction and relief was established by inducing immortalized human umbilical vein endothelial cells with TNF-α inducer, including the following steps: Immortalized human umbilical vein endothelial cells were seeded into cell culture plates and cultured. Control group, model group, positive drug group and sample group were set up. Culture medium was added to the control group and the model group; cyclooxygenase inhibitor was added to the positive drug group; and the raw material sample to be tested was added to the sample group for pretreatment. After pretreatment, except for the control group, all other groups were induced with TNF-α inducer and cultured to establish an in vitro model of redness reduction and soothing. S2: Efficacy evaluation; The redness-reducing and soothing efficacy of the raw material sample was evaluated using prostacyclin I2 as a detection index, including the following steps: Collect cell culture supernatant from all groups in step S1, detect the secretion of prostacyclin I2 factor in each group, and statistically analyze the data. Using the secretion level of prostacyclin I2 in the model group as a benchmark, the secretion inhibition rate of prostacyclin I2 in the positive drug group and the sample group was calculated. The in vitro redness-reducing and soothing efficacy of the raw material sample was evaluated based on the secretion inhibition rate of prostacyclin I2 in the sample group.

[0011] Furthermore, in some embodiments, human immortalized umbilical vein endothelial cells are seeded into cell culture plates for culture, wherein the human immortalized umbilical vein endothelial cells are passaged human immortalized umbilical vein endothelial cells in the logarithmic growth phase.

[0012] Furthermore, in some embodiments, the seeding density of human immortalized umbilical vein endothelial cells is (3.0-5.0) × 10⁻⁶. 4 Cells / well; the cell culture plate used for seeding human immortalized umbilical vein endothelial cells is a 24-well plate; the culture conditions are temperature 37±0.5℃, CO2 concentration 5±1%, and saturated humidity.

[0013] After inoculating and culturing human immortalized umbilical vein endothelial cells, the original culture medium in the cell culture plate was aspirated.

[0014] Furthermore, in some embodiments, the concentration of the cyclooxygenase inhibitor is 20-40 μM.

[0015] Furthermore, in some embodiments, the detection concentration of the test sample is determined by selecting human immortalized umbilical vein endothelial cells in the logarithmic growth phase, at a concentration of (0.5-2.0) × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in cell culture plates and cultured for 20-24 hours. Samples containing different concentrations of the test material were added. Complete culture medium without the sample was used as the control group. Cell viability was detected by the MTT assay, and cell survival rate was calculated. The concentration of the test material sample with a cell survival rate of 90% or higher was used as the detection concentration.

[0016] Furthermore, in some embodiments, step S1 involves adding culture medium to the control group and the model group, wherein the culture medium is a primary endothelial cell-specific culture medium.

[0017] The cyclooxygenase inhibitor and the test sample were prepared using a special culture medium for primary endothelial cells.

[0018] Furthermore, in some embodiments, the concentration of the TNF-α inducer is 10-60 ng / mL.

[0019] Furthermore, in some embodiments, the preprocessing time is 0.5-1.5 hours.

[0020] Furthermore, in some embodiments, the culture time after induction with TNF-α inducer is 20-24 hours.

[0021] Furthermore, in some embodiments, the method for detecting the secretion of prostacyclin I2 factor is enzyme-linked immunosorbent assay (ELISA).

[0022] Furthermore, in some embodiments, the formula for calculating the secretion inhibition rate of prostacyclin I2 factor is as follows: Relative expression level = Prostacyclin I2 secretion level in the sample group / Prostacyclin I2 secretion level in the model group; The secretion inhibition rate of prostacyclin I2 factor = (1 - relative expression level) × 100%.

[0023] Furthermore, in some embodiments, the evaluation index for the in vitro redness-reducing and soothing efficacy of the test raw material sample is: Inhibition rate <10%: No redness-reducing or soothing effect; 10% ≤ Inhibition rate < 25%: Mild redness-reducing and soothing effect; 25% ≤ Inhibition rate < 35%: Moderate redness-reducing and soothing effect; 35% ≤ Inhibition rate < 50%: Good redness-reducing and soothing effects; Inhibition rate ≥50%: Significant redness-reducing and soothing effects.

[0024] Furthermore, in some embodiments, the raw material sample to be tested is a cosmetic raw material, including chemicals, biological products, or plant extracts.

[0025] Secondly, this application provides an in vitro redness-reducing and soothing model for cosmetic ingredients, which is constructed using the model establishment method in the evaluation method for the in vitro redness-reducing and soothing efficacy of cosmetic ingredients described in the first aspect.

[0026] Furthermore, in some embodiments, the in vitro redness-reducing and soothing model of the cosmetic ingredient includes: TNF-α inducers; Human immortalized umbilical vein endothelial cells; Cyclooxygenase inhibitors; The TNF-α inducer is used to induce prostacyclin I2 secretion in human immortalized umbilical vein endothelial cells; The cyclooxygenase inhibitor is used to inhibit the secretion of prostacyclin I2 factor after TNF-α induces human immortalized umbilical vein endothelial cells.

[0027] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: The technical solution of this application for the evaluation method of redness-reducing and soothing efficacy of cosmetic raw materials uses TNF-α-induced immortalized human umbilical vein endothelial cells as a model and prostacyclin I2 factor as a detection index. It has the advantages of short experimental cycle, simple operation, and economic convenience, providing an effective reference for the screening of redness-reducing and soothing raw materials, making up for the deficiency of existing technologies in the lack of evaluation methods for the soothing efficacy of cosmetic raw materials targeting the mechanism of increased vascular reactivity; and providing effective data for further research on the development and efficacy verification of skin care products in the cosmetic industry. Attached Figure Description

[0028] The present application will be described in further detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will understand that these drawings are drawn only for the purpose of explaining the preferred embodiments and therefore should not be construed as limiting the scope of this disclosure. Furthermore, unless specifically indicated, the drawings are only schematic representations of the composition or structure of the described objects and may contain exaggerated depictions, and the drawings are not necessarily drawn to scale.

[0029] Figure 1 The graph shows the results of studies with different concentrations of TNF-α inducers. Figure 1 In this context, "Control" represents the control group. Figure 1 In the middle, PGI2 represents prostacyclin I2 factor, HUVEC-40 represents human immortalized umbilical vein endothelial cells; TNF-α represents tumor necrosis factor α. Figure 1 In the diagram, "*" indicates that compared with the control group, p<0.05 is considered to be a significant difference, "**" indicates that p<0.01 is considered to be a highly significant difference, and "***" indicates that p<0.001 is considered to be an extremely significant difference.

[0030] Figure 2 This is a graph showing the concentration of aspirin, a cyclooxygenase inhibitor, under different positive drug assays. Figure 2In the diagram, Ctrl represents the control group; Aspirin represents aspirin; TNF-α 40 ng / ml represents the concentration of TNF-α inducer in the model group (40 ng / ml); TNF-α 40 ng / ml + Aspirin 1 μM represents the concentration of TNF-α inducer in the positive control group (40 ng / ml + 1 μm of aspirin); TNF-α 40 ng / ml + Aspirin 10 μM represents the concentration of TNF-α inducer in the positive control group (40 ng / ml + 10 μm of aspirin); TNF-α 40 ng / ml + Aspirin 20 μM represents the concentration of TNF-α inducer in the positive control group (40 ng / ml + 20 μm of aspirin); TNF-α 40 ng / ml + Aspirin 30 μM represents a TNF-α inducer concentration of 40 ng / mL and an aspirin concentration of 30 μmol in the positive control group; TNF-α 40 ng / mL + Aspirin 40 μM represents a TNF-α inducer concentration of 40 ng / mL and an aspirin concentration of 40 μmol in the positive control group. Figure 2 In the diagram, "*" indicates a significant difference between the positive drug group and the model group (p < 0.05); "**" indicates a highly significant difference (p < 0.01); and "***" indicates an extremely significant difference (p < 0.001). Similarly, "#" indicates a significant difference between the model group and the control group (p < 0.05); a highly significant difference (p < 0.01); and an extremely significant difference (p < 0.001).

[0031] Figure 3 The survival rate of human immortalized umbilical vein endothelial cells is shown for different concentrations of the test raw materials.

[0032] Figure 4 This is a graph showing the relative expression levels of prostacyclin I2 in the cell supernatant of different test sample materials in a TNF-α-induced model of immortalized human umbilical vein endothelial cells. Figure 4 In the diagram, Ctrl represents the control group; M represents the model group; and PC represents the positive drug group.

[0033] Figure 5 The graph shows the relative expression levels of prostacyclin I2 in the cell supernatant of the raw material samples used in TNF-α-induced HDFa, HaCaT, and RAW264.7 cell models. Ctrl represents the control group, TNF-α 40 ng / ml represents the concentration of TNF-α inducer in the model group (40 ng / ml), and Aspirin 40 μM represents the concentration of aspirin in the positive control group (40 μmol). Detailed Implementation

[0034] The following is in conjunction with the appendix Figures 1 to 5 This disclosure will be explained in detail.

[0035] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0036] The experimental materials used in the embodiments of this application are shown in Table 1.

[0037] Table 1: Experimental Materials

[0038] Solution preparation 0.125% trypsin digestion solution: Dilute 0.25% trypsin digestion solution with PBS buffer at a ratio of 1:1.

[0039] MTT (thiazolyl blue) solution: Weigh 100 mg of MTT powder, add 20 mL of PBS buffer, vortex to dissolve, and filter through a 0.22 μm filter membrane for sterilization. Store at 20℃ away from light. Thaw at 4℃ before use.

[0040] Cellular MTT assay solution: The MTT solution was diluted to 0.5 mg / mL in primary endothelial cell culture medium.

[0041] Preparation of liquid test sample To prepare a 10% concentration liquid stock solution of the raw material sample to be tested: Take a sterile centrifuge tube, add 900 μL of special culture medium, add 100 μL of liquid raw material sample to be tested, vortex or repeatedly blow and mix it thoroughly to obtain a 10% concentration liquid stock solution of the raw material sample to be tested.

[0042] The mother liquor of the liquid raw material sample to be tested was diluted with primary endothelial cell culture medium at multiple ratios to prepare raw material samples with concentrations of 3.16%, 1.00%, 0.316%, 0.10%, and 0.0316%, respectively.

[0043] Preparation of solid test sample To prepare a stock solution of solid test sample with a concentration of 10 mg / mL: Weigh 10 mg of solid sample powder and place it in a 15 mL sterile centrifuge tube. Add 10 mL of primary endothelial cell culture medium, vortex and sonicate gently until the powder is completely dissolved and the solution is clear, thus obtaining a stock solution of solid test sample with a concentration of 10 mg / mL.

[0044] The mother liquor of the solid test raw material sample was diluted multiple times with primary endothelial cell culture medium to prepare test raw material samples with concentrations of 3.16 mg / mL, 1.00 mg / mL, 0.316 mg / mL, 0.10 mg / mL, and 0.0316 mg / mL, respectively.

[0045] The instruments used in the embodiments of this application are shown in Table 2.

[0046] Table 2: Instruments

[0047] The test raw material samples (test substances) used in the embodiments of this application are shown in Table 3.

[0048] Table 3: Samples of raw materials to be tested

[0049] Example 1 This embodiment provides a method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials, including the following steps: S1: Model establishment: A redness-reducing in vitro model was established by inducing human immortalized umbilical vein endothelial cells with TNF-α inducer.

[0050] HUVEC-SV4O cells were cultured in primary endothelial cell culture medium at 37°C, 5% CO2, and saturated humidity in a CO2 incubator. When the cell confluence reached approximately 80%, the cells were passaged at a 1:2 ratio. Before passage, the original culture medium was discarded, and the cells were washed 1-2 times with PBS buffer. Then, 0.125% trypsin was added, and the cells were digested in an incubator for 1 min. An equal volume of culture medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min, and then collected for passage.

[0051] Human immortalized umbilical vein endothelial cells in logarithmic growth phase and in good condition were selected at a concentration of 4.0 × 10⁻⁶. 4 500 μL of cells per well were seeded into 24-well plates and cultured at 37°C, 5% CO2, and saturated humidity for 24 h. The original culture medium was then removed, and control, model, positive control, and sample groups were established. The control group and the model group were given a special culture medium for primary endothelial cells. The positive drug group was given a cyclooxygenase inhibitor aspirin at a concentration of 40 μM prepared in the culture medium. The sample group was given a test sample A at a concentration of 1.00% prepared in the culture medium for pretreatment. After 1 hour of pretreatment, except for the control group, all other groups were induced by adding TNF-α inducer at a concentration of 40 ng / mL prepared in the culture medium. The groups were then cultured at 37℃, 5% CO2 and saturated humidity for 24 hours to establish an in vitro model of redness reduction and soothing.

[0052] S2: Efficacy evaluation, using prostacyclin I2 factor as the detection index to evaluate the redness-reducing and soothing efficacy of the raw material sample to be tested.

[0053] The culture supernatant of each group of cells was collected and centrifuged at 1000×g (1000 times the acceleration of gravity, equivalent to applying a force 1000 times its own weight to the supernatant sample) at 4℃ for 20 min. The supernatant was then collected, and the prostacyclin I2 secretion level in the supernatant was detected using an ELISA kit according to the instructions. A standard curve was calculated using data analysis software, and the OD value of the test sample A was substituted into the standard curve to calculate the prostacyclin I2 content. The relative expression level of prostacyclin I2 in the model group (with 100% as the threshold) and the secretion inhibition rate of prostacyclin I2 were calculated to evaluate the efficacy of the sample in relieving redness.

[0054] The calculation formula is: relative expression level = prostacyclin I2 factor content in the sample group / prostacyclin I2 factor content in the model group, and prostacyclin I2 factor secretion inhibition rate = (1 - relative expression level) × 100%.

[0055] Based on Example 1, this application investigated the concentrations of TNF-α inducer and the concentration of the positive drug cyclooxygenase inhibitor aspirin. The specific parameters are shown in Tables 4 and 5.

[0056] Table 4: Concentration parameters of TNF-α inducers

[0057] Table 5: Concentration parameters of the positive control drug cyclooxygenase inhibitor aspirin

[0058] Examples 1-4 and Comparative Examples 1-3 of this application studied the induction of human immortalized umbilical vein endothelial cells with different concentrations of TNF-α inducer to find the optimal induction concentration. The concentration of TNF-α inducer was plotted on the x-axis, and the relative expression level of prostacyclin I2 was plotted on the y-axis using GraphPad Prism statistical analysis and plotting software. The results are expressed as Mean ± SD, where the height of each data point represents the mean of that group, and an error bar representing the standard deviation is added. One-way comparisons were used between the groups of each example and comparative example. ANOVA (one-way ANOVA) statistical analysis is used to compare whether there are significant differences between the means of three or more independent groups. All statistical analyses are two-tailed tests, designed to detect differences in any direction (increase or decrease) to evaluate whether there are differences between multiple groups. "*" represents the TNF-α inducer model group compared with the control group. p<0.05 is considered significant and is indicated by "*"; p<0.01 is considered highly significant and is indicated by "**"; p<0.001 is considered extremely significant and is indicated by "***". Based on the specific p-value obtained from the test, the groups with significant differences are marked on the graph with asterisks (*, *, **). The more asterisks, the more significant the difference and the more reliable the result.

[0059] like Figure 1 As shown, Control represents the control group; in the control group, the relative value of prostacyclin I2 factor was 1, representing the basal prostacyclin I2 factor secretion level of cells under normal conditions; as the concentration of TNF-α inducer increased, the inflammatory stimulation on vascular endothelial cells became stronger, and the relative value of prostacyclin I2 factor showed a significant, dose-dependent increase, reaching a maximum value at a concentration of 40 ng / mL; after 40 ng / mL, the relative value of prostacyclin I2 factor began to decrease again. This is attributed to the fact that when the concentration of TNF-α inducer is too high, it begins to have a significant toxic effect on human immortalized umbilical vein vascular endothelial cells, leading to a significant decrease in cell activity, thereby resulting in the inability to effectively synthesize and secrete prostacyclin I2 factor.

[0060] Examples 1, 5-6, and Comparative Examples 4-6 of this application studied different concentrations of the positive control drug, the cyclooxygenase inhibitor aspirin, to determine the optimal effective concentration. The plotting and analysis methods were similar to those used in the study of TNF-α inducer concentrations. "*" represents the positive control group compared to the TNF-α inducer model group; "#" represents the TNF-α inducer model group compared to the control group. This indicates a significant difference between the TNF-α inducer model group and the control group. "#" represents p < 0.05, "##" represents p < 0.01, and "###" represents p < 0.001. The results are as follows: Figure 2 As shown, Figure 2As shown, Ctrl represents the control group, and the TNF-α modeling group is marked with "###", indicating successful modeling. The stimulation of TNF-α inducer significantly (p<0.001) elicited a cellular response, resulting in a large release of prostacyclin I2, which was significantly different from the untreated control group, proving successful modeling. For the aspirin concentration groups (Aspirin 1μM-40μM), the inhibitory effect of aspirin on prostacyclin I2 release was strongest with the addition of 40μM aspirin, showing a highly significant difference.

[0061] This application investigated the selection of detection concentrations for the test sample using the cytotoxic MTT assay. Human immortalized umbilical vein endothelial cells in logarithmic growth phase and in good condition were selected, and a concentration of 1.0 × 10⁻⁶ was used. 4 Cells were seeded per well in 96-well plates at 100 μL per well. 100 μL of PBS was added to the outermost ring of each well. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 h. The original culture medium was removed, and control and sample groups were set up. The sample groups were prepared with a concentration gradient of the test sample (substance) using the dedicated culture medium. Six concentrations were set for each test sample (substance): liquid test sample concentrations (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, and 0.0316%, and solid test sample concentrations were 10 mg / mL, 3.16 mg / mL, 1.0 mg / mL, 0.316 mg / mL, 0.10 mg / mL, and 0.0316 mg / mL, 100 μL per well. Each experimental well was divided into three replicates. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 18–24 h.

[0062] Cell viability was assessed using the MTT assay. The sample concentration that resulted in 90% cell viability (CV90) was calculated using dose-response curves. All subsequent redness-reducing and soothing efficacy experiments were conducted at concentrations not exceeding this CV90 value to ensure that the observed efficacy was not caused by cytotoxicity. Results are as follows: Figure 3 As shown in Table 6.

[0063] Table 6: Drug concentration and efficacy concentration of the test raw material sample at CV90 in the human immortalized umbilical vein endothelial cell model

[0064] The CV90 concentration refers to the safe concentration, which is the highest permissible concentration determined by toxicity tests such as the MTT assay, that maintains cell viability at 90%. It is used to assess safety and ensure that the concentration used in subsequent efficacy studies will not cause toxicity to cells, thus avoiding false positives. The efficacy concentration refers to the effective concentration, which, within the safe concentration range, is the lowest concentration determined by efficacy experiments (prostacyclin I2 factor inhibition assay) that produces a significant expected effect; it identifies the lowest dose at which the active ingredient takes effect.

[0065] Depend on Figure 3 As shown in Table 6, the raw material concentration of sample A at a cell viability of 90% (CV90) in the human immortalized umbilical vein endothelial cell model was 1.15%, with an efficacy concentration of 1.00%; the raw material concentration of sample B at a cell viability of 90% (CV90) in the human immortalized umbilical vein endothelial cell model was 32.64 μg / mL, with an efficacy concentration of 10.00 μg / mL; the raw material concentration of sample C at a cell viability of 90% (CV90) in the human immortalized umbilical vein endothelial cell model was 10.00 mg / mL, with an efficacy concentration of 0.50 mg / mL; and the raw material concentration of sample D at a cell viability of 90% (CV90) in the human immortalized umbilical vein endothelial cell model was 1.27%, with an efficacy concentration of 0.08%. Subsequent administration was carried out at the concentration of each raw material sample corresponding to a cell viability of 90% (CV90) in the human immortalized umbilical vein vascular endothelial cell model, or at a lower concentration. Specifically, incubation was carried out according to the efficacy concentration of each raw material sample to be tested.

[0066] Based on Example 1, this application evaluated the in vitro redness-reducing and soothing effects of the test raw material samples B, C, and D, and the specific parameters are shown in Table 7.

[0067] Table 7: Evaluation parameters of the in vitro redness-reducing and soothing efficacy of raw material samples A, B, C, and D.

[0068] This application collected cell supernatants from the sample groups and positive drug groups of Examples 1, 7-9, and 9. Following the instructions of the prostacyclin I2 factor ELISA kit, the secretion of prostacyclin I2 factor in the cell supernatants of each example's sample group and positive drug group was detected and calculated, and the relative secretion amount was calculated. The plotting and analysis methods were similar to those used in the study of TNF-α inducer concentrations. The results are as follows: Figure 4 As shown in Table 8.

[0069] Table 8: Evaluation Results of the TNF-α Induced Human Immortalized Umbilical Vein Endothelial Cell Model

[0070] Figure 4 In the diagram, Ctrl represents the control group, M represents the model group, and PC represents the positive drug group. "*" indicates a comparison between the sample group (positive drug group, sample group) and the model group. Figure 4 It can be seen that the in vitro redness-reducing and soothing efficacy of the tested raw material samples A, B, C, and D showed extremely significant differences between the model group and the control group (###), which proves that the in vitro redness-reducing and soothing model was successfully established; Figure 4 As shown in Table 8, among the in vitro redness-reducing and soothing effects of test raw material samples A, B, C, and D, the positive drug group scored **, meaning that compared with the model group, the positive drug could significantly (p<0.01) inhibit the secretion of prostacyclin I2, proving the effectiveness and reliability of the experimental system. The score of test raw material sample A was higher than that of the positive drug group, indicating that at the experimental concentration, test raw material sample A had an extremely significant inhibitory effect on the secretion of prostacyclin I2 (p<0.001), making it a very efficient potential soothing raw material. Test raw material samples B, C, and D could all significantly (p<0.05) inhibit the secretion of prostacyclin I2, but compared with the positive drug group and test raw material sample A, the effect was at the same effective but lower level.

[0071] This application calculated the inhibition rate of prostacyclin I2 secretion in the cell supernatant of the sample groups in Examples 1 and 7-9. When the inhibition rate was <10%, it indicated that the test sample had no redness-reducing and soothing effect; when 10% ≤ inhibition rate <25%, it indicated that the test sample had a slight redness-reducing and soothing effect; when 25% ≤ inhibition rate <35%, it indicated that the test sample had a moderate redness-reducing and soothing effect; when 35% ≤ inhibition rate <50%, it indicated that the test sample had a good redness-reducing and soothing effect; and when 50% ≤ inhibition rate, it indicated that the test sample had a significant redness-reducing and soothing effect. The results are shown in Table 9.

[0072] Table 9: Inhibition Rate and Evaluation Results

[0073] As shown in Table 9, the efficacy intensity is: Sample A > Positive Control Group (PC) > Sample D ≈ Sample C ≈ Sample B. Sample A achieved a prostacyclin I2 inhibition rate of 37.57%, demonstrating an ability to inhibit prostacyclin I2 production and potentially reduce vasodilation and skin redness even surpassing that of the classic anti-inflammatory drug aspirin. Sample A is a highly effective candidate ingredient. The positive control group (PC) showed an inhibition rate of 31.04%, achieving the expected effect and proving the effectiveness and reliability of the experimental model. The positive control group serves as a qualified baseline, providing a reference for evaluating the efficacy of the sample ingredients. Samples B, C, and D showed inhibition rates of approximately 24%, significantly weaker than Sample A and the positive control group. Therefore, the experimental protocol of this application successfully screened out the star ingredient—Sample A—from multiple candidate ingredients. Sample A is not only effective but also superior to commonly used positive control drugs, exhibiting excellent redness-reducing and soothing potential in the in vitro model.

[0074] Based on Example 1, this application employs the same in vitro redness-reducing and soothing efficacy evaluation method. An in vitro redness-reducing and soothing model was established using HDFa, HaCaT, and RAW264.7 cells, and the test raw material samples were evaluated. Specific parameters are shown in Table 10, and the evaluation results of the test raw material samples are shown below. Figure 5 .

[0075] Table 10: Parameters of in vitro models of redness reduction and soothing effects in different cells

[0076] Depend on Figure 5 It can be seen that, for the HDFa cell model, the relative value of prostacyclin I2 in the model group was only slightly higher than that in the control group. In the HDFa cell model, the induction effect of TNF-α inducer was extremely weak, and the model was not successfully established. Compared with the model group, the relative value of prostacyclin I2 in the positive drug group was almost unchanged, and aspirin did not show the expected inhibitory effect at all. Therefore, the HDFa cell model failed and could not be used to evaluate the samples.

[0077] In the HaCaT cell model, the relative value of prostacyclin I2 in the model group was about 25% higher than that in the control group. TNF-α inducers showed a certain induction trend in the HaCaT cell model, but the effect was still weak. Compared with the model group, the relative value of prostacyclin I2 in the positive drug group was only slightly lower, with negligible inhibitory effect, indicating that the positive drug group was ineffective. Therefore, the HaCaT cell model was not ideal, the positive drug was ineffective, and the model was unreliable.

[0078] For the RAW264.7 cell model, the relative value of prostacyclin I2 in the model group was significantly lower than that in the control group; compared with the model group, the relative value of prostacyclin I2 in the positive drug group was higher than that in the model group, but significantly lower than that in the control group; therefore, the RAW264.7 cell model is not usable.

[0079] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present disclosure and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A method for evaluating the in vitro redness-reducing and soothing efficacy of a cosmetic ingredient, characterized in that, Includes the following steps: S1: Model building; An in vitro model for redness reduction and relief was established by inducing immortalized human umbilical vein endothelial cells with TNF-α inducer, including the following steps: Immortalized human umbilical vein endothelial cells were seeded into cell culture plates and cultured. Control group, model group, positive drug group and sample group were set up. Culture medium was added to the control group and the model group; cyclooxygenase inhibitor was added to the positive drug group; and the raw material sample to be tested was added to the sample group for pretreatment. After pretreatment, except for the control group, all other groups were induced with TNF-α inducer and cultured to establish an in vitro model of redness reduction and soothing. S2: Efficacy evaluation; The redness-reducing and soothing efficacy of the raw material sample was evaluated using prostacyclin I2 as a detection index, including the following steps: Collect cell culture supernatant from all groups in step S1, detect the secretion of prostacyclin I2 factor in each group, and statistically analyze the data. Using the amount of prostacyclin I2 secreted in the model group as a benchmark, the secretion inhibition rate of prostacyclin I2 in the positive drug group and the sample group was calculated. The in vitro redness-reducing and soothing efficacy of the test raw material samples was evaluated based on the secretion inhibition rate of prostacyclin I2 factor in the sample group.

2. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The seeding density of immortalized human umbilical vein endothelial cells was (3.0-5.0) × 10⁻⁶. 4 Cells / well; human immortalized umbilical vein endothelial cells were seeded into cell culture plates and cultured under the following conditions: temperature 37±0.5℃, CO2 concentration 5±1%, and saturated humidity.

3. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The concentration of the cyclooxygenase inhibitor is 20-40 μM.

4. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The concentration of the TNF-α inducer is 10-60 ng / mL.

5. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The detection concentration of the raw material sample was determined by selecting immortalized human umbilical vein endothelial cells in the logarithmic growth phase, at a concentration of (0.5-2.0)×10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in cell culture plates and cultured for 20-24 hours. Samples containing different concentrations of the test material were added. Complete culture medium without the sample was used as the control group. Cell viability was detected by the MTT assay, and cell survival rate was calculated. The concentration of the test material sample with a cell survival rate of 90% or higher was used as the detection concentration.

6. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The formula for calculating the secretion inhibition rate of prostacyclin I2 is as follows: Relative expression level = Prostacyclin I2 secretion level in the sample group / Prostacyclin I2 secretion level in the model group; The secretion inhibition rate of prostacyclin I2 factor = (1 - relative expression level) × 100%.

7. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The evaluation indicators for the in vitro redness-reducing and soothing efficacy of the raw material samples to be tested are: Inhibition rate <10%: No redness-reducing or soothing effect; 10% ≤ Inhibition rate < 25%: Mild redness-reducing and soothing effect; 25% ≤ Inhibition rate < 35%: Moderate redness-reducing and soothing effect; 35% ≤ Inhibition rate < 50%: Good redness-reducing and soothing effects; Inhibition rate ≥50%: Significant redness-reducing and soothing effects.

8. The method for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials as described in claim 1, characterized in that, The raw material samples to be tested are cosmetic raw materials, including chemicals, biological products, or plant extracts.

9. An in vitro redness-reducing and soothing model for cosmetic raw materials, characterized in that, The model was constructed using the method described in claim 1 for evaluating the in vitro redness-reducing and soothing efficacy of cosmetic raw materials.

10. The in vitro redness-reducing and soothing model of cosmetic raw materials as described in claim 9, characterized in that, The in vitro redness-reducing and soothing model of the cosmetic raw materials includes: TNF-α inducers; Human immortalized umbilical vein endothelial cells; Cyclooxygenase inhibitors; The TNF-α inducer is used to induce prostacyclin I2 secretion in human immortalized umbilical vein endothelial cells; The cyclooxygenase inhibitor is used to inhibit the secretion of prostacyclin I2 factor after TNF-α induces human immortalized umbilical vein endothelial cells.

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