A method for combined evaluation of in vitro and in vivo redness reduction soothing efficacy of a cosmetic raw material

Abstract

The application discloses a combined evaluation method for the in-vitro and in-vivo redness-reducing soothing effect of a cosmetic raw material, which comprises in-vitro model evaluation, adoption of a human immortalized umbilical vein endothelial cell model induced by TNF-alpha, acquisition of the in-vitro model score by detecting the secretion amount of prostacyclin I2 factor, in-vivo model evaluation, adoption of a chicken embryo chorioallantoic membrane model, acquisition of the in-vivo model score by the degree of blood vessel contraction caused by the action of the sample to be tested on the chicken embryo chorioallantoic membrane, and comprehensive evaluation of the redness-reducing soothing effect of the sample to be tested based on the in-vitro model score and the in-vivo model score. The technical scheme of the application makes up for the defect of the prior art that there is no cosmetic raw material soothing effect evaluation method for the mechanism of increased vascular reactivity, the effect evaluation is more accurate, and the method has the advantages of comprehensiveness, short test cycle, simple operation, economy and convenience, and provides an effective reference for the screening and compounding scheme of redness-reducing soothing raw materials.

Description

Technical Field

[0001] This application belongs to the field of cosmetic raw material functional testing technology, specifically relating to a combined evaluation method for the in vitro and in vivo 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 (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) and blocking the production of inflammatory mediators such as prostaglandins (PG), prostacyclin (PGI2), and leukotrienes (LT).

[0006] Currently, human 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] On the other hand, the chicken embryo chorioallantoic membrane assay (HET-CAM), as a classic in vivo substitution test, is mainly used to evaluate eye and skin irritation due to its vascular structure being similar to that of mammals and its high sensitivity to stimuli. However, this model has traditionally been used primarily for safety assessment (detecting irritation) and rarely for efficacy evaluation, especially in combination with in vitro models to form a complete efficacy evaluation system, which has not yet been reported.

[0008] Therefore, it is necessary to develop an in vitro model based on human immortalized umbilical vein endothelial cells (HUVEC 40) with PGI2 as the key indicator, combined with an in vivo chicken embryo model, to evaluate the redness-reducing and soothing effects of cosmetic raw materials in a multidimensional, rapid, convenient and comprehensive manner. Summary of the Invention

[0009] To address the aforementioned technical issues, this application provides a combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic ingredients.

[0010] This application provides a combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials, including the following steps:

[0011] S1: In vitro model evaluation; The in vitro model score was obtained by detecting the secretion of prostacyclin I2 factor using a TNF-α-induced human immortalized umbilical vein endothelial cell model.

[0012] S2: In vivo model evaluation; using the chicken embryo chorioallantoic membrane model, the score of the in vivo model is obtained by measuring the degree of vasoconstriction caused by the action of the test raw material sample on the chicken embryo chorioallantoic membrane;

[0013] S3: Comprehensive evaluation; Based on the score of the in vitro model obtained by detecting the secretion of prostacyclin I2 factor in step S1 and the score of the in vivo model obtained by detecting the degree of vasoconstriction caused by the action of the test raw material sample on the chicken embryo chorioallantoic membrane in step S2, a comprehensive evaluation is conducted to determine the redness-reducing and soothing efficacy of the test raw material sample.

[0014] Furthermore, in some embodiments, the scores of the in vitro model and the in vivo model are weighted and integrated to obtain a combined evaluation score, which comprehensively assesses and determines the redness-reducing and soothing efficacy of the raw material sample to be tested; the weight distribution of the scores of the in vitro model and the in vivo model is (5.5-6.5):(3.5-4.5); preferably, the weight distribution of the scores of the in vitro model and the in vivo model is 6:4.

[0015] Furthermore, in some embodiments, the raw material sample to be tested is a cosmetic raw material to be tested; the raw material sample to be tested includes chemicals, biological products, or plant extracts.

[0016] Furthermore, in some embodiments, the in vitro model evaluation includes the following steps:

[0017] 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.

[0018] 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.

[0019] After pretreatment, except for the control group, all other groups were induced with TNF-α inducer and cultured for a longer period. Cell culture supernatant from all groups was collected, and the secretion of prostacyclin I2 factor in each group was detected. The data were then statistically analyzed.

[0020] The test raw material samples were scored based on the difference in prostacyclin I2 factor secretion between the sample group and the model group to obtain the in vitro model score;

[0021] The in vivo model evaluation includes the following steps:

[0022] Preparation of chicken embryo chorioallantoic membrane;

[0023] Hollow isolation structures were placed in areas containing blood vessels on the chorioallantoic membrane of chicken embryos as the treatment area; negative control group, positive drug group, and sample group were set up.

[0024] Physiological saline was added to the hollow isolation structure corresponding to the negative control group; cyclooxygenase inhibitor was added to the hollow isolation structure corresponding to the positive drug group; the test raw material sample was added to the hollow isolation structure corresponding to the sample group; each group was observed at least twice at different time points, and the degree of vasoconstriction was scored; the average score of multiple observations of a single chicken embryo was used as the score of a single chicken embryo; the average score of at least three independent chicken embryos was used as the final score of the in vivo model of the test raw material sample.

[0025] Based on the predetermined scoring criteria, the final score of the in vivo model is converted into a redness-reducing and soothing efficacy level score to obtain the in vivo model score.

[0026] Furthermore, in some embodiments, the test raw material samples are scored based on the difference in prostacyclin I2 factor secretion between the sample group and the model group to obtain an in vitro model score. When p < 0.05, there is a significant difference between the prostacyclin I2 factor secretion between the sample group and the model group, it is indicated by "*" and the score is 1; p < 0.01, there is a highly significant difference, it is indicated by "**" and the score is 2; p < 0.001, there is an extremely significant difference, it is indicated by "***" and the score is 3.

[0027] Furthermore, in some embodiments, when p < 0.05 between the prostacyclin I2 secretion levels of the model group and the control group in the in vitro model evaluation, there is a significant difference in the prostacyclin I2 secretion levels between the model group and the control group, and the in vitro model is successfully constructed;

[0028] When the positive drug group significantly inhibited the secretion of prostacyclin I2 compared with the model group in the in vitro model evaluation, and when p<0.05 between the prostacyclin I2 secretion levels of the positive drug group and the model group, there was a significant difference in the prostacyclin I2 secretion levels between the positive drug group and the model group, the in vitro model evaluation was effective.

[0029] Furthermore, in some embodiments, the average score of the degree of vasoconstriction observed multiple times in a single chicken embryo is used as the score for a single chicken embryo, and the scoring criteria are as follows:

[0030] 0 points, no vasoconstriction, all blood vessels in the affected area showed no changes in morphology and diameter compared with the initial state or negative control group, blood flow was smooth and uninterrupted;

[0031] 1 point, slight constriction of small blood vessels, slight narrowing of the diameter of small blood vessels in the affected area, but blood flow is not interrupted;

[0032] 2 points, small blood vessels constrict significantly, the diameter of small blood vessels in the affected area is significantly reduced, accompanied by reduced blood flow or local interruption of blood flow;

[0033] 3 points, slight constriction of the medium vessels, slight narrowing of the diameter of the medium vessels in the affected area, the constriction is clearly visible, and the blood flow in the main trunk of the medium vessels is still maintained;

[0034] 4 points, significant vasoconstriction of medium blood vessels, significant reduction in the diameter of medium blood vessels in the affected area, accompanied by significant slowing or partial interruption of blood flow;

[0035] 5 points, the large blood vessels constrict slightly, the diameter of the large blood vessels in the affected area becomes slightly thinner, and blood flow is smooth;

[0036] 6 points, large blood vessels constrict significantly, the diameter of large blood vessels in the affected area is significantly reduced, almost closed, and blood flow stops completely or almost completely.

[0037] Furthermore, in some embodiments, the average of at least three independent chicken embryo scores is used as the final in vivo model score of the test raw material sample; according to a predetermined scoring standard, the final in vivo model score is converted into a redness-reducing and soothing efficacy level score to obtain the in vivo model score; the predetermined scoring standard is:

[0038] 0 points ≤ final score of in vivo model < 1 point: no vasoconstrictive effect, score is 0;

[0039] 1 point ≤ final in vivo model score < 3 points: mild vasoconstriction effect, score 1;

[0040] 3 points ≤ final in vivo model score < 5 points: definitely has a vasoconstrictive effect, score is 2;

[0041] 5 points ≤ final score of in vivo model ≤ 6 points: significant vasoconstrictive effect, score 3.

[0042] Furthermore, in some embodiments, the evaluation of the in vivo model is valid when the final score of the in vivo model in the negative control group is 0;

[0043] The in vivo model is considered valid when the final score of the positive drug group model is not lower than 3.

[0044] Furthermore, in some embodiments, the seeding density of human immortalized umbilical vein endothelial cells in the in vitro model assessment was (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.

[0045] Furthermore, in some embodiments, the culture conditions for human immortalized umbilical vein endothelial cells in the in vitro model evaluation were a temperature of 37±0.5°C, a CO2 concentration of (5±1)%, and a humidity of 95%.

[0046] After inoculating and culturing human immortalized umbilical vein endothelial cells, the original special culture medium in the cell culture plate was aspirated.

[0047] Furthermore, in some embodiments, the culture medium used in the in vitro model evaluation is a primary endothelial cell-specific culture medium.

[0048] Furthermore, in some embodiments, the cyclooxygenase inhibitor used in the in vitro model evaluation is aspirin; the concentration of the cyclooxygenase inhibitor is 20-40 μM.

[0049] Furthermore, in some embodiments, the concentration of TNF-α inducer in in vitro model assessments was 10-60 ng / mL.

[0050] Furthermore, in some embodiments, the detection concentration of the test raw material sample in the in vitro model evaluation 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.

[0051] The cyclooxygenase inhibitor and the test sample were prepared using a special culture medium.

[0052] Furthermore, in some embodiments, the preprocessing time in the in vitro model evaluation is 0.5-1.5 hours.

[0053] Furthermore, in some embodiments, the in vitro model evaluation involves induction with TNF-α inducer followed by 20-24 hours of culture.

[0054] Furthermore, in some embodiments, the method for detecting the secretion of prostacyclin I2 factor in the in vitro model assessment is enzyme-linked immunosorbent assay (ELISA).

[0055] Furthermore, in some embodiments, the formula for calculating the relative expression level of prostacyclin I2 factor in in vitro model evaluation is as follows:

[0056] Relative expression level = secretion of prostacyclin I2 factor in the sample group / secretion of prostacyclin I2 factor in the model group.

[0057] Furthermore, in some embodiments, the chicken embryos used in the in vivo model evaluation are chicken embryos that have been hatched to 9-11 days old, with a weight range of 50g-68g / embryo, and the chicken embryos have good activity and intact vascular structure.

[0058] Good chicken embryo activity means that a clear vascular network and embryonic activity can be seen during egg candling, and that smooth blood flow and a moist and transparent membrane can be seen after preparing the chicken embryo chorioallantoic membrane.

[0059] The term "intact vascular structure" refers to the absence of physical damage to the chorioallantoic membrane of the chicken embryo, with a clearly defined vascular network, distinct outlines, full blood supply, and no signs of bleeding, rupture, or other damage.

[0060] Furthermore, in some embodiments, the in vivo model assessment includes regions with dense vascular distribution, smooth blood flow, and distinct vascular layers, encompassing large, medium, and small blood vessels.

[0061] Large vessels refer to the main blood vessels, which are the thickest in diameter and are the primary vessels that transport blood through the chorioallantoic membrane of the chicken embryo; small vessels refer to the capillaries, which are the smallest in diameter and are the most peripheral in the chorioallantoic membrane vascular network of the chicken embryo; medium vessels refer to the secondary branch vessels, which are formed by the convergence of small vessels and have distinct walls.

[0062] Furthermore, in some embodiments, the concentration of the test sample in the in vivo model evaluation is three times that in the in vitro model evaluation; the volume is 0.25-0.35 ml.

[0063] Furthermore, in some embodiments, the hollow isolation structure was evaluated as a Teflon ring in the in vivo model.

[0064] Furthermore, in some embodiments, the in vivo model evaluation is conducted at least twice at 4-6 min and 28-32 min after the addition of the test sample.

[0065] Furthermore, in some embodiments, the preparation of the chicken embryo chorioallantoic membrane in the in vivo model evaluation includes the following steps:

[0066] Open a window in the air cell area of ​​the chicken embryo at a specific age to expose the egg membrane inside;

[0067] Moisten by applying a saline solution to the exposed egg membrane to keep it moist;

[0068] Peel off the egg membrane to expose the underlying chorioallantoic membrane (CAM), ensuring the integrity of the CAM vascular network.

[0069] The integrity and activity of the chorioallantoic membrane vascular network in chicken embryos were assessed to screen out chicken embryo chorioallantoic membranes with good activity and intact vascular structure.

[0070] Before peeling off the egg membrane, pour out the saline solution.

[0071] Furthermore, in some embodiments, the amount of physiological saline added in the in vivo model is 0.25-0.35 ml; the amount of cyclooxygenase inhibitor added is 0.25-0.35 ml; and the concentration of the cyclooxygenase inhibitor is aspirin.

[0072] Furthermore, in some embodiments, the criteria for determining the redness-reducing and soothing efficacy of the raw material sample in step S4 are as follows:

[0073] 0 points ≤ combined evaluation score ≤ 0.6 points: No redness fading or soothing effect;

[0074] 0.6 points < combined evaluation score ≤ 1.4 points: Mild redness reduction and soothing effect;

[0075] 1.4 < combined evaluation score ≤ 2.4 points: definitely has a redness-reducing and soothing effect;

[0076] 2.4 points < combined evaluation score ≤ 3 points: significant redness reduction and soothing effect.

[0077] Compared with the prior art, the technical solution of this application has the following beneficial technical effects:

[0078] This application employs a technical solution that combines in vivo and in vitro models to evaluate the redness-reducing and soothing effects of cosmetic ingredients, thus overcoming the deficiency in existing technologies that lack evaluation methods for the soothing effects of cosmetic ingredients targeting the mechanism of increased vascular reactivity.

[0079] The in vitro TNF-α-induced immortalized human umbilical vein endothelial cell model combined with the in vivo chicken embryo model provides a more accurate efficacy evaluation. It has the advantages of being comprehensive, having a short experimental cycle, being simple to operate, and being economical and convenient. It provides an effective reference for the screening of soothing raw materials and the formulation of compounding schemes, and at the same time provides effective data for further research on the development and efficacy verification of skin care products in the cosmetics industry. Attached Figure Description

[0080] 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.

[0081] Figure 1 The survival rate of human immortalized umbilical vein endothelial cells is shown for different concentrations of the test raw materials.

[0082] Figure 2 This is a graph showing the results of in vitro model evaluation at different concentrations of TNF-α inducers. Figure 2 In this context, "Control" represents the control group. Figure 2 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 2In 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.

[0083] Figure 3 This is a graph showing the concentrations of aspirin, a cyclooxygenase inhibitor, in different positive drug studies in an in vitro model. Figure 3 In 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 concentration of 40 ng / mL for TNF-α inducer and 30 μmol for aspirin in the positive drug group; TNF-α 40 ng / mL + Aspirin 40 μM represents a concentration of 40 ng / mL for TNF-α inducer and 40 μmol for aspirin in the positive drug group. Figure 3 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).

[0084] Figure 4 This is a graph showing the relative expression levels of prostacyclin I2 in the cell supernatant of different test sample materials in an in vitro model evaluation of an immortalized human umbilical vein endothelial cell model induced by TNF-α inducer. Figure 4 In the diagram, Ctrl represents the control group, M represents the model group, PC represents the positive drug group, A represents the test sample A, B represents the test sample B, C represents the test sample C, and D represents the test sample D.

[0085] Figure 5 This is a graph showing the redness-reducing and soothing activity of raw materials used in the evaluation of chicken embryo chorioallantoic membrane in an in vivo model. Figure 5 In the diagram, PC represents the positive drug group, A represents the test sample A, B represents the test sample B, C represents the test sample C, and D represents the test sample D.

[0086] Figure 6 Flowchart of a combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials. Detailed Implementation

[0087] The following is in conjunction with the appendix Figures 1 to 6 This disclosure will be explained in detail.

[0088] 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.

[0089] The experimental materials used in the embodiments of this application are shown in Table 1.

[0090] Table 1 Experimental Materials

[0091]

[0092] Solution preparation

[0093] 0.125% trypsin digestion solution: Dilute 0.25% trypsin digestion solution with PBS buffer at a ratio of 1:1.

[0094] Thiazol blue (MTT) solution: Weigh 100 mg of MTT powder, add 20 mL of PBS buffer, vortex to dissolve, filter through a 0.22 μm filter membrane for sterilization, store at -20 °C protected from light, and thaw at 4 °C before use.

[0095] Cellular MTT assay solution: The MTT solution was diluted to 0.5 mg / mL in primary endothelial cell culture medium.

[0096] 0.9% sodium chloride solution: Weigh 9g of sodium chloride powder and dissolve it in 1mL of ultrapure water.

[0097] Preparation of liquid test sample

[0098] 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.

[0099] The mother liquor of the liquid raw material sample to be tested was diluted with a special culture medium at multiples to prepare raw material samples with concentrations of 3.16%, 3%, 1.00%, 0.316%, 0.10%, 0.0316%, and 0.24%, respectively.

[0100] Preparation of solid test sample

[0101] To prepare a stock solution of solid test sample with a concentration of 10 mg / mL: Weigh 100 mg of solid sample powder and place it in a 15 mL sterile centrifuge tube. Add 10 mL of special 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.

[0102] The mother liquor of the solid test raw material sample was diluted with a special culture medium at multiples to prepare test raw material samples with concentrations of 3.16 mg / ml, 1.5 mg / ml, 1.00 mg / ml, 0.316 mg / ml, 0.10 mg / ml, 0.0316 mg / ml and 0.03 mg / ml respectively.

[0103] The instruments used in the embodiments of this application are shown in Table 2.

[0104] Table 2 Instruments

[0105]

[0106] The test raw material samples (test substances) used in the embodiments of this application are shown in Table 3.

[0107] Table 3 Samples of raw materials to be tested

[0108]

[0109] The specific implementation of this application is as follows:

[0110] 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. 4Cells 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.

[0111] 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 1 As shown in Table 4.

[0112] Table 4. Dosage concentrations and efficacy concentrations of the test raw materials at CV90 in the human immortalized umbilical vein endothelial cell model.

[0113]

[0114] 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.

[0115] Depend on Figure 1As shown in Table 4, 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.

[0116] Example 1: In vitro model evaluation

[0117] This embodiment provides an in vitro model evaluation method.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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 and continued to be cultured at 37℃, 5% CO2 and saturated humidity for 24 hours.

[0122] 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.

[0123] The relative expression level of prostacyclin I2 in the model group was calculated as 100%, significance analysis was performed, and scores were obtained to obtain the in vitro model score.

[0124] Based on Example 1, this application investigated the concentrations of TNF-α inducer and the concentration of the positive control drug cyclooxygenase inhibitor aspirin. The specific parameters are shown in Tables 5 and 6.

[0125] Table 5. Concentration parameters of TNF-α inducers.

[0126]

[0127] Table 6. Concentration parameters of the positive drug cyclooxygenase inhibitor aspirin

[0128]

[0129] 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 "*", score 1; p<0.01 is considered highly significant and is indicated by "**", score 2; p<0.001 is considered extremely significant and is indicated by "***", score 3. 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.

[0130] like Figure 2 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.

[0131] 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 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 blank group. "#" represents p < 0.05, "##" represents p < 0.01, and "###" (3 points) represents p < 0.001. The results are as follows... Figure 3 As shown, Figure 3As shown, Ctrl represents the control group, and the TNF-α inducer 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 showed a significant difference compared to 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, exhibiting a highly significant difference.

[0132] 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.

[0133] Table 7 shows the evaluation parameters for the in vitro redness-reducing and soothing effects of raw material samples A, B, C, and D.

[0134]

[0135] 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.

[0136] Table 8. Evaluation Results of the TNF-α Induced Human Immortalized Umbilical Vein Endothelial Cell Model

[0137]

[0138] 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 4As 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.

[0139] Example 10 In vivo model evaluation

[0140] This embodiment provides an in vivo model evaluation method.

[0141] Chicken embryo incubation

[0142] Purchase eggs that are less than 7 days old and incubate them in an incubator until they are 9-11 days old. The incubation conditions are: room temperature 20℃-25℃, relative humidity 45%-70%; incubation temperature 37.5℃±0.5℃, relative humidity 55%-70%, and a turntable frequency of 3-6 times / h.

[0143] To prepare the chorioallantoic membrane (CAM) of chicken embryos, candling is performed on 9-day-old chicken embryos, and the location of the air cell is marked on the eggshell surface. Using dental serrated forceps, the marked portion of the eggshell is peeled away to expose the white egg membrane, handling it carefully to avoid damaging its integrity. The egg membrane is moistened with 0.9% sodium chloride (NaCl) solution using a pipette, and the next step is performed immediately, or the chicken embryo is placed in an incubator or under a light (to prevent the embryo temperature from dropping) for no more than 20 minutes, after which the 0.9% sodium chloride solution is poured out. The inner membrane is carefully removed with forceps, ensuring the vascular membrane is not damaged. The structure of the vascular system is then observed again, and its integrity and suitability for experimental use are assessed.

[0144] Teflon rings were placed in areas containing large, medium, and small blood vessels to serve as the sample application area, and the condition of the raw material sample before application was recorded by photograph. 0.9% sodium chloride was set as a negative control before each test of the raw material sample to ensure that the test conditions would not cause an irritating reaction.

[0145] 0.3 ml of 3% Teflon ring sample A was directly added to the treatment area of ​​the sample. The contraction changes of the chorioallantoic membrane blood vessels of chicken embryos were observed and photographed at 0 min, 5 min and 30 min. The degree of vascular contraction was scored. The average score at 5 min and 30 min was used as the score of the degree of chorioallantoic membrane blood vessel contraction of one chicken embryo. The experiment was repeated three times, and the average score of the degree of chorioallantoic membrane blood vessel contraction of three chicken embryos was finally obtained as the final score for the in vivo model evaluation of sample A.

[0146] Based on predetermined judgment criteria, the final score of the in vivo model evaluation of raw material sample A is converted into a redness-reducing and soothing efficacy level score.

[0147] Based on Example 10, in vivo model evaluation was performed on the positive drug aspirin and test raw material samples B, C, and D. Specific parameters are shown in Table 9, and the scores for the in vivo model evaluation are shown in Tables 10 and 11. Figure 5 As shown (PC represents the positive drug aspirin).

[0148] Table 9. Evaluation parameters of the in vivo chicken embryo chorioallantoic membrane vasoconstriction model for positive drugs and test raw material samples A, B, C, and D.

[0149]

[0150] As shown in Table 9, the detection concentration was three times the concentration used to evaluate cell efficacy in the in vitro model.

[0151] Table 10 Evaluation Results of the Chicken Embryo Villi Allantoic Membrane Model

[0152]

[0153] Depend on Figure 5 It can be seen that the positive drug group and sample group A exhibited slight vasoconstriction, sample group B showed significant vasoconstriction in small vessels, while samples C and D showed no vasoconstriction effect. Referring to Table 10, in the in vivo model assessment, the positive drug group and sample group A scored 3 points, indicating slight vasoconstriction; sample group B scored 2 points, indicating significant vasoconstriction in small vessels; and samples C and D scored 0 points, indicating no vasoconstriction effect.

[0154] The scores from the in vitro model evaluation and the in vivo model evaluation of the raw material samples A, B, C, and D were weighted and integrated according to a weight ratio of 6:4 to obtain a comprehensive score, and the redness-reducing and soothing effects were determined, as shown in Table 11.

[0155] Table 11 Combined Evaluation Results

[0156]

[0157] This application investigated the weighting of in vitro and in vivo model evaluation scores. The evaluation results of the TNF-α-induced HUVEC-SV4O cell models in Examples 1 and 7-9 were summed with the evaluation results of the chicken embryo chorioallantoic membrane model in Examples 10-14, respectively, at weights of 5:5 and 7:3, to obtain the combined evaluation results. The results are shown in Table 12.

[0158] Table 12. Weighting of in vitro model assessment scores versus in vivo model assessment scores.

[0159]

[0160] Table 12 shows that when the weight ratio of the in vitro model assessment score to the in vivo model assessment score is 5:5, it cannot effectively distinguish between samples with mild, certain, and significant redness-reducing and soothing effects. If the weight ratio of the in vitro model assessment score to the in vivo model assessment score is 7:3, the score cannot effectively screen samples. Therefore, a weight ratio of 6:4 is chosen for the summation.

[0161] Efficacy verification

[0162] To better verify the feasibility and reliability of the combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of the cosmetic raw materials in this application, samples A, B, C, and D of the raw materials to be tested were simultaneously applied to skin tests for comparison. The specific procedures are as follows:

[0163] At least 30 lactate-positive subjects were recruited. On the day of the experiment, filter paper (0.8±0.1 cm in diameter) soaked in 50 μL of 10% lactate solution was applied to one cheek of each subject. The filter paper was removed when the subject felt stinging or itching. Antera 3D was used to take pictures and the average initial value of skin redness was analyzed. Samples A, B, C, and D were treated with the concentration added according to the chicken embryo chorioallantoic membrane vasoconstriction model for 15 min, and pictures were taken again to analyze the average value of skin redness. GraphPadPrism was used to statistically analyze the significance of the difference from the initial value. The statistical results are shown in Table 13.

[0164] Table 13 Analysis results of average skin redness

[0165]

[0166] As shown in Tables 11 and 13, the skin test results are consistent with the efficacy test results of this application, indicating that the technical solution of this application is feasible and reliable.

[0167] Based on the in vitro model evaluation results, in vivo model evaluation results, and in vitro-in vivo combination evaluation results in Tables 8, 10, and 11, the order of efficacy of test ingredients A, B, C, D, and aspirin (PC) for the in vitro model evaluation results is: test ingredient A > aspirin (PC) > B = C = D = 1. In the in vitro model evaluation, test ingredient A has the highest efficacy, followed by aspirin. Test ingredients B, C, and D all have efficacy, and their efficacies are consistent.

[0168] Based on the in vivo model evaluation results, the efficacy order of each ingredient is as follows: ingredient A = aspirin (PC) > B > C = D = 0. In the in vivo model evaluation, ingredient A and aspirin have the highest efficacy and their efficacy is consistent. Ingredient B has the next highest efficacy, while ingredients C and D have no efficacy.

[0169] In the in vitro and in vivo combination evaluation, the efficacy order of each raw material was: test raw material A > aspirin > B > C = D = 0.6. In the in vitro and in vivo combination evaluation, test raw material A had the highest efficacy, followed by aspirin, test raw material B had some efficacy, and test raw materials C and D had no efficacy.

[0170] In the efficacy verification of skin tests, the efficacy order of each raw material was A>B>C=D, with raw material A having the highest efficacy, raw material B having some efficacy, and raw materials C and D having no efficacy.

[0171] Based on the above analysis, the evaluation results of test ingredients C and D in the in vitro model are inconsistent with the efficacy verification results of the skin experiment; the evaluation results of test ingredients A and aspirin in the in vivo model are inconsistent with the efficacy verification results of the skin experiment; however, in the in vitro and in vivo combined evaluation, the evaluation results of test ingredients A, B, C, and D are consistent with the efficacy verification results of the skin experiment. Therefore, the combined evaluation method for the redness-reducing and soothing effects of cosmetic ingredients provided in this application is more accurate and reliable than the evaluation of a single in vitro or in vivo model.

[0172] Based on the specific implementation of this application described above, this application provides a combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials, such as... Figure 6 As shown.

[0173] 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 combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials, characterized in that, Includes the following steps: S1: In vitro model evaluation; a TNF-α-induced human immortalized umbilical vein endothelial cell model was used, and the score of the in vitro model was obtained by detecting the secretion of prostacyclin I2 factor; the human immortalized umbilical vein endothelial cells were HUVEC-SV40 cells; S2: In vivo model evaluation; using the chicken embryo chorioallantoic membrane model, the score of the in vivo model is obtained by measuring the degree of vasoconstriction caused by the action of the test raw material sample on the chicken embryo chorioallantoic membrane; The in vivo model evaluation includes the following steps: Preparation of chicken embryo chorioallantoic membrane; Hollow isolation structures were placed in areas containing blood vessels on the chorioallantoic membrane of chicken embryos as the treatment area; negative control group, positive drug group, and sample group were set up. Physiological saline, cyclooxygenase inhibitor, and test raw material sample were added to the hollow isolation structures corresponding to the negative control group, positive drug group, and sample group, respectively; each group was observed at least twice at different time points, and the degree of vasoconstriction was scored. S3: Comprehensive evaluation; Based on the score of the in vitro model obtained by detecting the secretion of prostacyclin I2 factor in step S1 and the score of the in vivo model obtained by the degree of vasoconstriction caused by the action of the raw material sample on the chorioallantoic membrane of chicken embryo in step S2, the scores of the in vitro model and the in vivo model are weighted and integrated to obtain a combined evaluation score, and the redness-reducing and soothing efficacy of the raw material sample is comprehensively evaluated and determined. The weighting of the in vitro model score and the in vivo model score was (5.5-6.5):(3.5-4.5); The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic ingredients is a method for evaluating the soothing effects of cosmetic ingredients based on the mechanism of increased vascular reactivity.

2. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 1, characterized in that, The in vitro model evaluation includes 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 for a longer period. Cell culture supernatant from all groups was collected, and the secretion of prostacyclin I2 factor in each group was detected. The data were then statistically analyzed. The test raw material samples were scored based on the difference in prostacyclin I2 factor secretion between the sample group and the model group to obtain the in vitro model score; The in vivo model evaluation uses the average score of multiple observations of a single chicken embryo as the score of a single chicken embryo; the average score of at least three independent chicken embryos is used as the final in vivo model score of the raw material sample to be tested; according to the predetermined scoring criteria, the final in vivo model score is converted into a redness-reducing and soothing efficacy level score to obtain the in vivo model score.

3. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 2, characterized in that, The culture conditions for human immortalized umbilical vein endothelial cells in the in vitro model evaluation were: temperature 37±0.5℃, CO2 concentration (5±1)%, and humidity 95%. The culture medium used in the in vitro model evaluation was a special culture medium for primary endothelial cells; The cyclooxygenase inhibitor used in the in vitro model evaluation was aspirin; the concentration of the cyclooxygenase inhibitor was 20-40 μM. In the in vitro model evaluation, the concentration of TNF-α inducer was 10-60 ng / mL; In the in vitro model evaluation, the detection concentration of the test 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 more than 90% was used as the detection concentration. The concentration of the test sample in the in vivo model evaluation was three times that in the in vitro model evaluation. In vivo model evaluation indicated that the hollow isolation structure was a Teflon ring; In the in vivo model evaluation, at least two observation times should be 4-6 min and 28-32 min after the addition of the test raw material sample.

4. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 2, characterized in that, The test raw material samples were scored based on the difference in prostacyclin I2 factor secretion between the sample group and the model group to obtain the in vitro model score. When p < 0.05, the score was 1; when p < 0.01, the score was 2; and when p < 0.001, the score was 3.

5. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 4, characterized in that, When p < 0.05 between the prostacyclin I2 secretion levels of the model group and the control group in the in vitro model evaluation, there is a significant difference in the prostacyclin I2 secretion levels between the model group and the control group, and the in vitro model is successfully constructed. When the positive drug group significantly inhibited the secretion of prostacyclin I2 compared with the model group in the in vitro model evaluation, and when p<0.05 between the prostacyclin I2 secretion levels of the positive drug group and the model group, there was a significant difference in the prostacyclin I2 secretion levels between the positive drug group and the model group, the in vitro model evaluation was effective.

6. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 2, characterized in that, The average score of vasomotor contraction observed multiple times in a single chicken embryo was used as the score for that individual chicken embryo. The scoring criteria were as follows: 0 points, no vasoconstriction, all blood vessels in the affected area showed no changes in morphology and diameter compared with the initial state or negative control group, blood flow was smooth and uninterrupted; 1 point, slight constriction of small blood vessels, slight narrowing of the diameter of small blood vessels in the affected area, but blood flow is not interrupted; 2 points, small blood vessels constrict significantly, the diameter of small blood vessels in the affected area is significantly reduced, accompanied by reduced blood flow or local interruption of blood flow; 3 points, slight constriction of the medium vessels, slight narrowing of the diameter of the medium vessels in the affected area, the constriction is clearly visible, and the blood flow in the main trunk of the medium vessels is still maintained; 4 points, significant vasoconstriction of medium blood vessels, significant reduction in the diameter of medium blood vessels in the affected area, accompanied by significant slowing or partial interruption of blood flow; 5 points, the large blood vessels constrict slightly, the diameter of the large blood vessels in the affected area becomes slightly thinner, and blood flow is smooth; 6 points, large blood vessels constrict significantly, the diameter of large blood vessels in the affected area is significantly reduced, almost closed, and blood flow stops completely or almost completely.

7. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 6, characterized in that, The average score of at least three independent chicken embryo scores is used as the final in vivo model score for the test raw material sample; according to a predetermined scoring standard, the final in vivo model score is converted into a redness-reducing and soothing efficacy level score to obtain the in vivo model score; the predetermined scoring standard is: If the score is 0 or less than the final score of the in vivo model and less than 1, the score is 0. If a score of 1 or less is less than 3 points in the final in vivo model score, the score is 1. A score of 3 or less (final in vivo model score < 5) is considered a score of 2. A score of 5 or less is considered a final score of 6 or less for the in vivo model, and the score is 3.

8. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 7, characterized in that, The evaluation of the in vivo model is valid when the final score of the in vivo model in the negative control group is 0. The in vivo model is considered valid when the final score of the positive drug group in vivo model is not lower than 3.

9. The combined evaluation method for the in vitro and in vivo redness-reducing and soothing effects of cosmetic raw materials as described in claim 1, characterized in that, The criteria for determining the redness-reducing and soothing effects of the tested raw material samples are as follows: 0 points ≤ combined evaluation score ≤ 0.6 points: No redness fading or soothing effect; 0.6 points < combined evaluation score ≤ 1.4 points: Mild redness reduction and soothing effect; 1.4 < combined evaluation score ≤ 2.4 points: definitely has a redness-reducing and soothing effect; 2.4 points < combined evaluation score ≤ 3 points: significant redness reduction and soothing effect.

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