In-vitro biochemical test method for evaluating soothing efficacy of cosmetics and raw materials

Abstract

The invention discloses an in-vitro biochemical test method for evaluating the soothing effect of cosmetics and raw materials. The in-vitro biochemical test method comprises the following steps: step 1, preparing a working solution; 2, preparing a sample solution; step 3, enzyme activation and culture; step 4, loading samples in groups; 5, adding a substrate and reacting; step 6, developing treatment; step 7, determining absorbance; 8, calculating, analyzing and processing data; and step 9, result judgment: analyzing and evaluating the soothing effect of the sample through a statistical means. According to the method, based on a hyaluronidase inhibition test, the activity of hyaluronidase is indirectly evaluated by measuring the change of a light absorption value at 520nm after beta-N-acetyl glucosamine reacts with a color developing agent, so that whether a to-be-detected sample has a soothing effect or not is judged, and the method is economical, rapid, controllable in test process, standardized in test system and high in repeatability. And large result deviation caused by individual differences of human bodies can be avoided.

Description

Technical Field

[0001] The present invention relates to a testing method, specifically, an in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials; it belongs to the technical field of cosmetics testing. Technical Background

[0002] The soothing efficacy is a common efficacy claim in the cosmetics industry, which means that after the use of cosmetics, it can reduce skin irritation, inflammation and allergic reactions, and relieve skin discomfort. Cosmetics with soothing efficacy should be evaluated through the cosmetics efficacy claim evaluation test method, and the literature data or research data analysis results can be combined at the same time for efficacy claim evaluation. According to the requirements of the cosmetics efficacy claim evaluation project, the soothing efficacy can be evaluated by any one of the three methods: human efficacy evaluation test, consumer use test and laboratory test.

[0003] At present, the cosmetics industry lacks a standardized and reliable method to evaluate the soothing efficacy of products. Traditional methods often rely on in vivo tests or complex cell experiments, which are costly and time-consuming. In addition, the existing evaluation methods often lack quantitative analysis and it is difficult to objectively evaluate the strength of the product's soothing efficacy.

[0004] In view of the above problems, it is very necessary to develop a soothing efficacy test and evaluation method with low cost and objective and reliable results. Summary of the Invention

[0005] Therefore, the purpose of the present invention is to provide an in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials. This method is based on the hyaluronidase inhibition test, and indirectly evaluates the activity of hyaluronidase by measuring the change in absorbance at 520 nm after the reaction of β-N-acetylglucosamine with the chromogenic agent, so as to judge whether the sample to be tested has soothing efficacy. This method is economical and fast, the test process is controllable, and the test system is standardized, which can avoid large result deviations caused by human individual differences.

[0006] An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials successively includes the following steps:

[0007] Step 1, Preparation of working solution

[0008] Prepare acetate buffer working solution, hyaluronidase working solution, potassium hyaluronate working solution, calcium chloride working solution, acetylacetone working solution, sodium carbonate working solution and positive control working solution respectively;

[0009] Step 2, Preparation of sample solution

[0010] Place the sample to be tested until the temperature is balanced with the room temperature, and then prepare it into a sample solution;

[0011] Step 3: Enzyme activation and cultivation

[0012] Set up six test groups, namely the sample group, the sample background group, the negative control group, the negative control background group, the positive control group, and the positive control background group. Add 1 mL of hyaluronidase solution to the sample group, the negative control group, and the positive control group, and add 1 mL of acetic acid buffer solution with pH = 5.6 to the sample background group, the negative control background group, and the positive control background group. Add 0.2 mL of calcium chloride solution to all test groups and incubate at 37°C for 20 min;

[0013] Step 4: Grouping and loading samples

[0014] Take 0.4 mL of the prepared sample solution for the sample group and the sample background group, and take 0.4 mL of the sample solvent for the negative control group, the negative control background group, the positive control group, and the positive control background group. Place each group in a 37°C water bath or incubator and incubate for 20 min;

[0015] Step 5: Adding substrate and reacting

[0016] Add 1 mL of potassium hyaluronate solution to the sample group, the negative control group, and the positive control group, and add 1 mL of acetic acid buffer solution with pH = 5.6 to the sample background group, the negative control background group, and the positive control background group. Incubate at 37°C for 40 min;

[0017] Step 6: Color development treatment

[0018] Add 0.5 mL of acetylacetone solution to all test groups, immediately place in an ice bath for 15 min after boiling water bath for 20 min, add 1.5 mL of Ehrlich solution to each test group, shake well, and place at 37°C for 40 min.

[0019] Step 7: Absorbance measurement

[0020] Take the solution after the color development reaction of each group and place it in a 96-well plate, 200 μL per well, with 3 replicates for each group, and measure the absorbance at 520 nm using an enzyme-linked immunosorbent assay reader.

[0021] Step 8: Calculating and analyzing data

[0022] Calculation formula for hyaluronidase inhibition rate:

[0023] Inhibition rate (%) = 100 - [OD520nm(Sa) - OD520nm(Sb)] / [OD520nm(NCa) - OD520nm(NCb)] × 100;

[0024] Where:

[0025] OD520nm(NCa) - The average absorbance value of the negative control group at 520 nm;

[0026] OD520nm(NCb) —— The average absorbance value of the negative control background group at 520 nm;

[0027] OD520nm(Sa) —— The average absorbance value of the test substance group at 520 nm;

[0028] OD520nm(Sb) —— The average absorbance value of the test substance background group at 520 nm;

[0029] Step 9, Result determination

[0030] Statistical and significance analysis is carried out using statistical software to calculate the P - value. P < 0.05 indicates a significant difference, otherwise there is no statistical difference. When there is a significant difference (P < 0.05) in the change value of NADPH content between the solvent control group (BC) and the negative control group (NC) after 1 - 1.5 h of reaction, the test is valid. The 5α - reductase inhibition rate of the sample group is significantly higher than that of the negative control group (NC), and there is a significant difference (P < 0.05), then it can be determined that the sample has a soothing effect.

[0031] Specifically, the statistical software is GraphPad Prism, EXCEL or others.

[0032] Further, the preparation process of the acetic acid buffer working solution in step 1 is as follows: Weigh 4.75 g of sodium acetate trihydrate and add 0.289 mL of glacial acetic acid to make 400 mL of acetic acid buffer solution (pH = 5.6), and store it at 4 °C.

[0033] Further, the preparation process of the hyaluronidase working solution in step 1 is as follows: Prepare a hyaluronidase solution with a concentration of 1995 U / mL by dissolving hyaluronidase in an acetic acid buffer solution with pH = 5.6, and dispense 4 mL into each tube and store it at - 20 °C.

[0034] Further, the preparation process of the potassium hyaluronate working solution in step 1 is as follows: Accurately weigh 50 mg of potassium hyaluronate, and make up the volume to 80 mL with acetic acid buffer solution to obtain a 0.625 mg / mL potassium hyaluronate solution, dispense 4 mL into each tube and store it at - 20 °C.

[0035] Further, the preparation process of the calcium chloride working solution in step 1 is as follows: Take 10 μL of 2.5 M calcium chloride solution and make up the volume to 10 mL with pure water to obtain a 2.5 mmol / L calcium chloride solution, and store it at 4 °C.

[0036] Further, the preparation process of the sodium carbonate working solution in step 1 is as follows: Accurately weigh 66.25 g of sodium carbonate, and make up the volume to 500 mL with ultrapure water to obtain a 1.25 mol / L sodium carbonate solution, and store it at 4 °C.

[0037] Further, the preparation process of the acetylacetone working solution in Step 1 is as follows: Accurately measure 300 μL of acetylacetone, dissolve it in 10 mL of 1.25 mol / L sodium carbonate solution, and shake well to obtain the acetylacetone solution.

[0038] Further, the preparation process of the sodium carbonate working solution in Step 1 is as follows: Weigh 10 g of solid sodium hydroxide, add pure water to dissolve it slowly and make the volume up to 50 mL.

[0039] Further, the preparation process of the positive control working solution in Step 1 is as follows: Select dipotassium glycyrrhizinate as the positive control, weigh 0.3 g of dipotassium glycyrrhizinate powder, add 9.7 mL of pure water to prepare a 3% dipotassium glycyrrhizinate solution, and store it at 4°C.

[0040] Further, the preparation process of the sample solution in Step 2 is as follows: Equilibrate the test sample at 20 - 25°C for at least 30 minutes, and then dissolve it to prepare the sample solution.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] The technical solution provided by the present invention is simple and rapid to operate. The whole test process can be completed within 4 - 5 hours, greatly shortening the evaluation period; the cost is low, and there is no need for expensive cell culture equipment, and it can be completed using conventional laboratory instruments, and the results are reliable: By measuring the production amount of β-N-acetylglucosamine, the hyaluronidase inhibitory activity can be quantitatively evaluated, and the results are objectively reliable; the standardization degree is high, and the detailed operating procedures ensure the comparability and repeatability of the results; the applicable range is wide, and it can be used to evaluate the soothing effects of various cosmetic formulations and raw materials. Through this method, the cosmetic enterprise can shorten the evaluation period of the soothing effect of new products from the traditional 1 - 2 weeks to 1 - 2 days, and the evaluation cost is reduced by about 50% - 70%, significantly improving the R & D efficiency, accelerating the new product listing speed, providing scientific and reliable data support for the product soothing effect claim, and having good application prospects. Description of the Drawings

[0043] Figure 1 It is the test result chart of the hyaluronidase inhibition test;

[0044] Figure 2 It is the comparison chart of the change in the average redness content of the skin of the subjects before and after using the sample;

[0045] Figure 3 It is the comparison chart of the change in the average water content of the stratum corneum of the skin of the subjects before and after using the sample. Detailed Embodiments

[0046] The technical solutions implemented in the present invention are described clearly and completely below. Obviously, the described embodiments are merely the main test processes of the present invention. For process parameters or conditions not specifically noted, conventional techniques can be referred to.

[0047] Example

[0048] The present invention provides an in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials.

[0049] I. Reagents and materials:

[0050] Unless otherwise specified, the reagents used in this method are all of analytical grade or above, and the water is double-distilled water, ultrapure water or above.

[0051] Reagents (all directly purchased commercially): glacial acetic acid, sodium acetate trihydrate, hyaluronidase, hyaluronic acid, sodium carbonate, calcium chloride solution (2.5 M), sodium hydroxide, potassium hyaluronate, dipotassium glycyrrhizinate, acetylacetone, Ehrlich solution, PBS buffer.

[0052] Consumables: 96-well plates; pipettes and tips; EP tubes; 15 mL and 5 mL centrifuge tubes.

[0053] II. Instruments and equipment:

[0054] Oscillation plate, microplate reader, biochemical incubator, water bath.

[0055] III. Test steps:

[0056] Reagent preparation

[0057] Acetic acid buffer solution: Weigh 4.75 g of sodium acetate trihydrate and add 0.289 mL of glacial acetic acid to make 400 mL of acetic acid buffer (pH = 5.6), and store at 4 °C.

[0058] Hyaluronidase solution: Purchase hyaluronidase (from sheep testes), and prepare a hyaluronidase solution with a concentration of 1995 U / mL using acetic acid buffer with pH = 5.6. Aliquot 4 mL per tube and store at -20 °C.

[0059] Potassium hyaluronate solution: Accurately weigh 50 mg of potassium hyaluronate, and make up the volume to 80 mL with acetic acid buffer to obtain a 0.625 mg / mL potassium hyaluronate solution. Aliquot 4 mL per tube and store at -20 °C.

[0060] Calcium chloride solution: Take 10 μL of 2.5 M calcium chloride solution and make up the volume to 10 mL with pure water to obtain a 2.5 mmol / L calcium chloride solution, and store at 4 °C.

[0061] Sodium carbonate solution: Accurately weigh 66.25 g of sodium carbonate, and make up the volume to 500 mL with ultrapure water to obtain a 1.25 mol / L sodium carbonate solution. Store at 4 °C.

[0062] Acetylacetone solution: Prepare it freshly before use. Accurately measure 300 μL of acetylacetone and dissolve it in 10 mL of 1.25 mol / L sodium carbonate solution, and shake well to obtain the acetylacetone solution.

[0063] Positive control solution: Select dipotassium glycyrrhizinate for testing, with a concentration of 3%. Prepare it with pure water. Weigh 0.3 g of dipotassium glycyrrhizinate powder and add 9.7 mL of pure water to prepare a 3% dipotassium glycyrrhizinate solution. Store at 4 °C.

[0064] Sodium hydroxide solution (5 mol / L): Weigh 10 g of solid sodium hydroxide, add pure water and dissolve it slowly, and make up the volume to 50 mL.

[0065] Experimental procedures

[0066] Step 1 (Solution preparation and sample treatment):

[0067] Prepare all the working solutions according to the reagent preparation for all the experiments involved;

[0068] Weigh 0.2 g of the sample to be tested, perform an equilibration treatment at 25 °C for 30 minutes, and then add 0.8 g of PBS buffer to prepare a 20% concentration sample solution;

[0069] Step 2 (Enzyme activation and incubation):

[0070] Add 1 mL of hyaluronidase solution to the sample group, negative control group, and positive control group, add 1 mL of acetic acid buffer with pH = 5.6 to the sample background group, negative control background group, and positive control background group, and add 0.2 mL of calcium chloride solution to all the test groups. Incubate at 37 °C for 20 min.

[0071] Step 3 (Grouping and loading):

[0072] Take 0.4 mL of the prepared sample solution for the sample group and sample background group, take 0.4 mL of the sample solvent for the negative control group, negative control background group, positive control group, and positive control background group, and place each group in a 37 °C water bath or incubator and incubate for 20 min.

[0073] Step 4 (Substrate addition and reaction):

[0074] Add 1 mL of potassium hyaluronate solution to the sample group, negative control group, and positive control group, add 1 mL of acetic acid buffer with pH = 5.6 to the sample background group, negative control background group, and positive control background group, and incubate at 37 °C for 40 min.

[0075] Step 5 (Color reaction):

[0076] Add 0.5 mL of acetylacetone solution to all test groups. After boiling water bath for 20 min, immediately place in ice water bath for 15 min. Then add 1.5 mL of Ehrlich solution to each test group, shake well, and incubate at 37 °C for 40 min.

[0077] Step 6 (Absorbance measurement):

[0078] Take the solution after color reaction of each group and transfer it to a 96-well plate, 200 μL per well, with 3 replicates for each group. Measure the absorbance at 520 nm using a microplate reader.

[0079] Step 7 (Result calculation, analysis, and determination):

[0080] Calculation formula for hyaluronidase inhibition rate:

[0081] Inhibition rate (%) = 100 - [OD520nm(Sa) - OD520nm(Sb)] / [OD520nm(NCa) - OD520nm(NCb)] × 100;

[0082] Where:

[0083] OD520nm(NCa) - The average absorbance value of the negative control group at 520 nm;

[0084] OD520nm(NCb) - The average absorbance value of the negative control background group at 520 nm;

[0085] OD520nm(Sa) - The average absorbance value of the test substance group at 520 nm;

[0086] OD520nm(Sb) - The average absorbance value of the test substance background group at 520 nm

[0087] Perform statistical and significance analysis on the data, calculate the P value. P < 0.05 indicates significant difference, otherwise there is no statistical difference.

[0088] When the OD value of the negative control group is higher than that of the negative control background group and there is a significant difference (P < 0.05);

[0089] The inhibition rate of the positive control group is positive and compared with the negative control group, and there is a significant difference (P < 0.05), then the test is valid.

[0090] Perform statistical and significance analysis on the data, calculate the P value. P < 0.05 indicates significant difference, otherwise there is no statistical difference.

[0091] The hyaluronidase inhibition rate of the test substance group was positive, and there was a significant difference compared with the negative control group (P < 0.05). It can be determined that the sample has the ability to inhibit the activity of hyaluronidase. The results can be used as one of the evidence supports for the claim of soothing efficacy.

[0092] Application example:

[0093] 1. A test method for a cosmetic A with soothing efficacy:

[0094] 1.1 Sample name: Soothing body lotion

[0095] 1.2 Method:

[0096] 1.2.1 In vitro biochemical test method: The soothing efficacy of the sample was tested according to the method described in the present invention. The sample was diluted with PBS to a concentration of 20% for the hyaluronidase inhibition test, and the hyaluronidase inhibition rate was calculated to evaluate the soothing effect of the test sample.

[0097] 1.2.2 Human test method: More than 30 Chinese subjects were screened. By establishing a skin barrier damage model, the mean redness value and the water content of the skin stratum corneum before and after using the product were tested, and compared with the blank group to verify the soothing effect of the sample.

[0098] 1.2.3 Data statistical analysis: For the data of the biochemical test method, a two-tailed t-test was used. When P < 0.05, it indicated a significant difference. For the human test data, the Shapiro-Wilk Test was first used with SPSS software to perform a significance test on the normality of the data improvement value. If Sig. (two-sided) > 0.01, it was normally distributed, and a paired t-test was performed; if Sig. (two-sided) < 0.01, it was non-normally distributed, and a Wilcoxon test was performed. When P < 0.05, it indicated a significant difference.

[0099] If P > 0.05, it indicates no significant difference, denoted as "n.s.";

[0100] If P < 0.05 indicates a significant difference, and 0.01 ≤ P < 0.05, it is denoted as "*"; 0.001 ≤ P < 0.01, it is denoted as "**"; 0.0001 ≤ P < 0.001, it is denoted as "***"; P < 0.0001, it is denoted as "****".

[0101] 1.3 Results:

[0102] 1.3.1 Biochemical test results

[0103] The sample was diluted to a concentration of 20% for the hyaluronidase inhibition test. The results showed that the inhibition rate of the sample against hyaluronidase at a concentration of 20% was 27.66%. Compared with the negative control group, there was a significant difference (P < 0.05). The results are shown in Table 1. Figure 1 .

[0104] Table 1. Detection Results of Hyaluronidase Inhibition Test

[0105]

[0106] 1.3.2 Results of Human Trials

[0107] After 32 Chinese subjects used the test sample for 7 days, the following results could be obtained through instrument testing:

[0108] After continuously using the test sample for 7 days: the mean value of flushing improved by 26.89%, showing a significant difference compared with the blank group; the water content of the skin stratum corneum improved by 33.44%, showing a significant difference compared with the blank group.

[0109] In summary, it shows that the test sample has a soothing effect for 7 days. The results are shown in Table 2, Table 3 and Figure 2 、 Figure 3 .

[0110] Table 2. Descriptive Statistical Results of Each Test Index

[0111]

[0112] Table 3. Statistical Analysis Results of Each Test Index

[0113]

[0114]

[0115] As can be seen from the above, through practical application, this test method has established a criterion for judging the soothing efficacy, which is economical and fast, with a high degree of controllability in the test process, a standardized test system, and can avoid large result deviations caused by individual differences in the human body, and has good application prospects.

[0116] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials, characterized in that, The steps are as follows: Step 1: Preparation of working solution Prepare acetate buffer working solution, hyaluronidase working solution, potassium hyaluronate working solution, calcium chloride working solution, acetylacetone working solution, sodium carbonate working solution and positive control working solution respectively; Step 2: Sample solution preparation The sample to be tested is placed until its temperature is balanced with room temperature, and then prepared into a sample solution; Step 3: Enzyme activation and incubation Six test groups were set up, namely, sample group, sample background group, negative control group, negative control background group, positive control group, and positive control background group. 1 mL of hyaluronidase solution was added to the sample group, negative control group, and positive control group, and 1 mL of pH = 5.6 acetate buffer was added to the sample background group, negative control background group, and positive control background group. 0.2 mL of calcium chloride solution was added to all test groups, and the mixture was kept at 37°C for 20 min. Step 4: Grouping and loading Take 0.4 mL of the prepared sample solution to the sample group and sample background group, and take 0.4 mL of the sample solvent to the negative control group, negative control background group, positive control group, and positive control background group. Place each group in a 37°C water bath or incubator at the same time and keep warm for 20 minutes. Step 5: Add substrate and react Add 1 mL of potassium hyaluronate solution to the sample group, negative control group, and positive control group, and 1 mL of acetate buffer (pH 5.6) to the sample background group, negative control background group, and positive control background group, and keep at 37°C for 40 min; Step 6: Color development Add 0.5 mL of acetylacetone solution to all test groups, boil in water for 20 min, then immediately place in ice water for 15 min, add 1.5 mL of Ehrlich solution to all test groups, shake thoroughly, and place at 37°C for 40 min. Step 7: Absorbance measurement The solution after color development reaction in each group was taken into a 96-well plate, 200 μL per well, 3 replicate wells per group, and the absorbance at 520 nm was measured using an enzyme-labeled instrument. Step 8: Calculate and analyze the data The calculation formula for hyaluronidase inhibition rate is: Inhibition rate (%) = 100 - [OD520nm (Sa) - OD520nm (Sb)] / [OD520nm (NCa) - OD520nm (NCb)] × 100; in: OD520nm(NCa)——the average absorbance value of the negative control group at 520nm; OD520nm(NCb)——the average absorbance value of the negative control background group at 520nm; OD520nm (Sa)——the average absorbance value of the test substance group at 520nm; OD520nm (Sb)——the average absorbance value of the background group of the test substance at 520nm; Step 9: Result determination Statistical analysis and significance analysis were performed using statistical software to calculate the P value. A P value < 0.05 indicates significant difference, otherwise there is no statistical difference. When there is a significant difference (P < 0.05) in the change value of NADPH content between the solvent control group (BC) and the negative control group (NC) after 1 - 1.5 h of reaction, the test is valid. The 5α - reductase inhibition rate of the sample group is significantly higher than that of the negative control group (NC), and there is a significant difference (P < 0.05), indicating that the sample has a soothing effect.

2. The in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that The preparation process of the acetic acid buffer working solution in step 1 is as follows: Weigh 4.75 g of sodium acetate trihydrate, add 0.289 mL of glacial acetic acid, and make up to 400 mL of acetic acid buffer solution (pH = 5.6), and store at 4 °C.

3. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the hyaluronidase working solution in step 1 is as follows: Dissolve hyaluronidase in acetic acid buffer solution with pH = 5.6 to prepare a hyaluronidase solution with a concentration of 1995 U / mL, and dispense 4 mL per tube and store at -20 °C.

4. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the potassium hyaluronate working solution in step 1 is as follows: Accurately weigh 50 mg of potassium hyaluronate, and make up the volume to 80 mL with acetic acid buffer solution to obtain a 0.625 mg / mL potassium hyaluronate solution, and dispense 4 mL per tube and store at -20 °C.

5. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the calcium chloride working solution in step 1 is as follows: Take 10 μL of 2.5 M calcium chloride solution, and make up the volume to 10 mL with pure water to obtain a 2.5 mmol / L calcium chloride solution, and store at 4 °C.

6. The in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that The preparation process of the sodium carbonate working solution in step 1 is as follows: Accurately weigh 66.25 g of sodium carbonate, and make up the volume to 500 mL with ultrapure water to obtain a 1.25 mol / L sodium carbonate solution, and store at 4 °C.

7. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the acetylacetone working solution in step 1 is as follows: Accurately measure 300 μL of acetylacetone, dissolve it in 10 mL of 1.25 mol / L sodium carbonate solution, and shake well to obtain an acetylacetone solution.

8. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the sodium carbonate working solution in step 1 is as follows: Weigh 10 g of solid sodium hydroxide, add pure water and slowly dissolve it, and make up the volume to 50 mL.

9. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the positive control substance working solution in step 1 is as follows: Select dipotassium glycyrrhizinate as the positive control substance, weigh 0.3 g of dipotassium glycyrrhizinate powder, add 9.7 mL of pure water to prepare a 3% dipotassium glycyrrhizinate solution, and store at 4 °C.

10. An in vitro biochemical test method for evaluating the soothing efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the sample solution in step 2 is as follows: Equilibrate the sample to be tested at 20 - 25 °C for at least 30 minutes, and then dissolve it to prepare a sample solution.

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