In-vitro biochemical test method for evaluating anti-wrinkle effects of cosmetics and raw materials

Abstract

The invention discloses an in-vitro biochemical test method for evaluating the anti-wrinkle 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, adding an enzyme working solution; 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 anti-wrinkle effect of the sample through a statistical means. According to the method, based on a collagenase inhibition test, when peptide bonds between GLY and GLY in Z-GLY-PRO-GLY-GLY-PRO-ALA-OH are specifically hydrolyzed by collagenase, GLY-PRO-ALA is released, and the activity of the collagenase is indirectly evaluated by measuring the change of a light absorption value at 565 nm after the GLY-PRO-ALA reacts with a color developing agent, so that whether a sample to be detected has an anti-wrinkle effect or not is judged. The method is economical and fast, the test process is controllable, the test system is standardized, and result deviation caused by individual difference can be avoided.

Description

Technical Field

[0001] This invention relates to a testing method, specifically an in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials; it belongs to the field of cosmetic testing technology. Technical Background

[0002] The anti-wrinkle effects of cosmetics are mainly reflected in repairing cells and activating collagen production through specific ingredients, providing long-lasting and deep-reaching effects. Cosmetics with anti-wrinkle effects should undergo efficacy claim evaluation testing, which can be combined with literature review or research data analysis results for efficacy claim evaluation. Aging is a complex biochemical process, mainly caused by oxidative stress and enhanced activation of proteolytic enzymes. Wrinkles are one of the signs of aging, appearing due to the loss of collagen and elastin in the skin, leading to skin laxity and reduced elasticity. Collagenase is a type of matrix metalloproteinase that can break down collagen and degrade the extracellular matrix. Collagen is the most abundant protein in mammals, widely present in the extracellular matrix, providing an important structural matrix for tissue development, maintenance, and regeneration, and playing a crucial role in maintaining the skin's suppleness, strength, and elasticity. Therefore, counteracting the degradation of collagen by collagenase is one of the important ways to delay skin aging.

[0003] Currently, the cosmetics industry often relies on in vivo tests or complex cell experiments to evaluate the anti-wrinkle efficacy of products, which is costly and time-consuming.

[0004] Therefore, it is essential to develop a simple, rapid, and reliable in vitro evaluation method for anti-wrinkle efficacy. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials. This method is based on a collagenase inhibition assay. When the peptide bond between GLY and GLY in Z-GLY-PRO-GLY-GLY-PRO-ALA-OH is specifically hydrolyzed by collagenase, GLY-PRO-ALA is released. The activity of collagenase is indirectly evaluated by measuring the change in absorbance at 565nm after GLY-PRO-ALA reacts with a chromogenic agent, thereby determining whether the sample has anti-wrinkle efficacy. This method is economical and fast, the testing process is controllable, and the testing system is standardized, which can avoid large result deviations caused by individual differences in human bodies.

[0006] An in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials, comprising the following steps in sequence:

[0007] Step 1: Preparation of working solution

[0008] Calcium acetate working solution, sodium acetate working solution, collagenase working solution, citric acid working solution, substrate working solution, colorimetric working solution, and positive control working solution were prepared separately.

[0009] Step 2, Sample solution preparation

[0010] The sample to be tested is placed until the temperature equilibrates with room temperature, and then a sample solution is prepared.

[0011] Step 3: Adding enzyme working solution

[0012] First, preheat all the prepared working solutions at 37°C for 15 minutes. Then, set up six test groups: sample group, sample background group, negative control group, negative control background group, positive control group, and positive control background group. Add 0.5 mL of collagenase working solution to the sample group, negative control group, and positive control group, and add 0.5 mL of calcium acetate working solution to the sample background group, negative control background group, and positive control background group.

[0013] Step 4: Grouping and loading samples

[0014] Take 0.5 mL of sample solution into the sample group and sample background group, take 0.5 mL of pure water into the negative control group and negative control background group, take 0.5 mL of positive working solution into the positive control group and positive control background group, add 0.5 mL of enzyme solution to each experimental group, and add 0.5 mL of calcium acetate solution to the background group. Place each group in a 37℃ water bath or incubator at the same time and keep warm for 15 min.

[0015] Step 5: Add substrate and react.

[0016] Add 1.5 mL of substrate working solution to each group and citrate buffer to the background group in centrifuge tubes. Mix well by shaking with a shaker and incubate at 37°C for 15 min.

[0017] Step 6: Color Development Process

[0018] Add 1 mL of colorimetric solution to each group in a centrifuge tube, place the tubes in a water bath, and react at 100°C for 20 min.

[0019] Step 7: Absorbance Measurement

[0020] After cooling to room temperature, samples were added to 96-well plates, and the changes in absorbance at 565 nm were measured using an ELISA reader.

[0021] Step 8: Calculate and analyze the data.

[0022] Collagenase activity: ((A) 样品 -A 背景 )*df) / (15*3.784*0.5)

[0023] in:

[0024] df: represents the dilution factor

[0025] 15: Indicates the incubation time between the enzyme and the substrate.

[0026] D: Indicates the sample dilution factor

[0027] 3.784: Extinction coefficient of 1 micromolar GLY-PRO-ALA in 5 ml and 1 cm optical path lengths.

[0028] 0.5: Volume of enzyme added to the reaction

[0029] Formula for calculating collagenase inhibition rate:

[0030] Collagenase inhibition rate (%) = (collagenase activity) NC - Collagenase activity (s) / Collagenase activity NC *100

[0031] collagenase activity NC The collagenase activity value is for the negative control group.

[0032] collagenase activity S The collagenase activity value of the sample group;

[0033] Step 9: Result Determination

[0034] Perform statistical and significance analysis on the data, calculate the P-value, P < 0.05 indicates a significant difference, otherwise there is no statistical difference.

[0035] Each experiment included a solvent control, a negative control, and a positive control. The experiment was considered successful when there was a significant difference in collagenase activity between the solvent control group and the negative control group after the reaction time (P < 0.05).

[0036] When the collagenase inhibition rate of the sample group was significantly higher than that of the negative control group, and the difference was statistically significant (P < 0.05), it can be determined that the sample has the ability to inhibit collagenase activity.

[0037] Furthermore, the preparation process of the calcium acetate working solution in step 1 is as follows: accurately weigh 158 mg of calcium acetate, and dilute it to 100 mL with ultrapure water to obtain a 0.01 mol / L calcium acetate solution.

[0038] Further, the preparation process of sodium acetate working solution in step 1 is as follows: accurately weigh 1.64g of sodium acetate, add 30mL of ultrapure water, adjust the pH to 6.3 with acetic acid, and then make up to 50mL with ultrapure water to obtain a 4mol / L sodium acetate solution.

[0039] Furthermore, the preparation process of the collagenase working solution in step 1 is as follows: accurately weigh 6.8 mg of collagenase powder, and make up to 25 mL with calcium acetate solution to obtain a collagenase working solution of 34 U / mL.

[0040] Further, the preparation process of the citric acid working solution in step 1 is as follows: accurately weigh 9.61g of citric acid and 0.79g of calcium acetate, add 300mL of ultrapure water, adjust the pH to 6.3, and then make up the volume to 500mL with ultrapure water to obtain 0.1mol / L citric acid buffer solution.

[0041] Furthermore, the preparation process of the substrate working solution in step 1 is as follows: using citrate buffer...

[0042] Z-GLY-PRO-GLY-GLY-PRO-ALA-OH was prepared as a substrate working solution at a concentration of 0.311 mg / mL.

[0043] Furthermore, the preparation process of the colorimetric working solution in step 1 is as follows: accurately weigh 2g of ninhydrin, add 75mL of 2-methoxyethanol, then add 40mg of stannous chloride, and dilute to 100mL with sodium acetate solution.

[0044] Further, the preparation process of the positive control working solution in step 1 is as follows: 1,10-phenanthroline is selected as the positive control. First, 30 mg of the positive control is weighed, and then 1666 μL of ethanol is added to prepare a 100 mmol / L stock solution. Then, it is diluted with ultrapure water to prepare a 4 mmol / L positive working solution.

[0045] Furthermore, the sample solution preparation process in step 2 is as follows: the sample to be tested is equilibrated at 20-25℃ for at least 30 minutes, and then dissolved to prepare a sample solution.

[0046] Furthermore, in the sample solution preparation process of step 2, for samples dissolved in DMSO or ethanol, when used for testing, the final concentration of DMSO or ethanol in the solvent control group or all test concentration groups shall not exceed 1% by volume.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The technical solution provided by this invention is simple and quick to operate, and the entire experimental process can be completed within 2 hours, greatly shortening the evaluation cycle. It is also low-cost, requiring no expensive cell culture equipment and can be completed using conventional laboratory instruments. By measuring the change in absorbance at 565nm after the synthetic peptide cleavage product reacts with the chromogenic solution, collagenase inhibitory activity can be quantitatively assessed. The results are objective, reliable, and highly standardized, and detailed operating procedures ensure the comparability and reproducibility of the results. Furthermore, it has a wide range of applications, suitable for evaluating the anti-wrinkle efficacy of various cosmetic formulations and raw materials. Using this method, cosmetic companies can shorten the evaluation cycle for the anti-wrinkle efficacy of new products from the traditional 2 weeks to 1 month to 1-2 days, reducing evaluation costs by approximately 50%-70%, significantly improving R&D efficiency, accelerating the launch of new products, and providing scientific and reliable data support for product anti-wrinkle efficacy claims, demonstrating promising application prospects. Attached Figure Description

[0049] Figure 1 This is a graph showing the results of the collagenase inhibition test.

[0050] Figure 2 This is a comparison chart showing the changes in the length of crow's feet wrinkles in the subjects before and after using the sample. Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only the main test process of the present invention. For process parameters or conditions not specifically noted, conventional techniques can be referred to.

[0052] Example

[0053] This invention provides an in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials.

[0054] I. Reagents and Materials:

[0055] Unless otherwise specified, all reagents used in this method are of analytical grade or higher, and the water is redistilled water, ultrapure water or higher grade.

[0056] Reagents (all commercially available): collagenase; Z-GLY-PRO-GLY-GLY-PRO-ALA-OH; 1,10-phenanthroline; citrate buffer; calcium acetate; sodium acetate; 2-methoxyethanol; stannous chloride; ninhydrin.

[0057] Consumables: 96-well plate; pipettes and tips; EP tubing.

[0058] II. Instruments and Equipment:

[0059] Shaking plate, microplate reader, biochemical incubator, water bath.

[0060] III. Experimental Procedure:

[0061] Reagent preparation

[0062] Calcium acetate solution: Accurately weigh 158 mg of calcium acetate and dilute to 100 mL with ultrapure water to obtain a 0.01 mol / L calcium acetate solution.

[0063] Sodium acetate solution: Accurately weigh 1.64 g of sodium acetate, add 30 mL of ultrapure water, adjust the pH to 6.3 with acetic acid, and then make up to 50 mL with ultrapure water to obtain a 4 mol / L sodium acetate solution.

[0064] Collagenase working solution: Accurately weigh 6.8 mg of collagenase powder and dilute to 25 mL with calcium acetate solution to obtain a 34 U / mL collagenase working solution.

[0065] Citrate buffer: Accurately weigh 9.61 g of citric acid and 0.79 g of calcium acetate, add 300 mL of ultrapure water, adjust the pH to 6.3, and then bring the volume to 500 mL with ultrapure water to obtain 0.1 mol / L citrate buffer.

[0066] Working substrate solution: Prepare a 0.311 mg / mL working substrate solution of Z-GLY-PRO-GLY-GLY-PRO-ALA-OH using citrate buffer.

[0067] Positive control: 1,10-phenanthroline was selected as the positive control. 30 mg of the positive control was weighed and then 1666 μL of ethanol was added to prepare a 100 mmol / L stock solution. The stock solution was then diluted with ultrapure water to prepare a 4 mmol / L positive working solution.

[0068] Colorimetric solution: Accurately weigh 2g of ninhydrin, add 75mL of 2-methoxyethanol, then add 40mg of stannous chloride, and dilute to 100mL with sodium acetate solution.

[0069] Experimental steps

[0070] Step 1: Preparation of working solution

[0071] Calcium acetate working solution, sodium acetate working solution, collagenase working solution, citric acid working solution, substrate working solution, colorimetric working solution, and positive control working solution were prepared separately.

[0072] Step 2, Sample solution preparation

[0073] The sample to be tested is placed until the temperature equilibrates with room temperature, and then a sample solution is prepared.

[0074] Step 3: Adding enzyme working solution

[0075] First, preheat all the prepared working solutions at 37°C for 15 minutes. Then, set up six test groups: sample group, sample background group, negative control group, negative control background group, positive control group, and positive control background group. Add 0.5 mL of collagenase working solution to the sample group, negative control group, and positive control group, and add 0.5 mL of calcium acetate working solution to the sample background group, negative control background group, and positive control background group.

[0076] Step 4: Grouping and loading samples

[0077] Take 0.5 mL of sample solution into the sample group and sample background group, take 0.5 mL of pure water into the negative control group and negative control background group, take 0.5 mL of positive working solution into the positive control group and positive control background group, add 0.5 mL of enzyme solution to each experimental group, and add 0.5 mL of calcium acetate solution to the background group. Place each group in a 37℃ water bath or incubator at the same time and keep warm for 15 min.

[0078] Step 5: Add substrate and react.

[0079] Add 1.5 mL of substrate working solution to each group and citrate buffer to the background group in centrifuge tubes. Mix well by shaking with a shaker and incubate at 37°C for 15 min.

[0080] Step 6: Color Development Process

[0081] Add 1 mL of colorimetric solution to each group in a centrifuge tube, place the tubes in a water bath, and react at 100°C for 20 min.

[0082] Step 7: Absorbance Measurement

[0083] After cooling to room temperature, samples were added to 96-well plates, and the changes in absorbance at 565 nm were measured using an ELISA reader.

[0084] Step 8: Calculate and analyze the data.

[0085] Collagenase activity: ((A) 样品 -A 背景 )*df) / (15*3.784*0.5)

[0086] in:

[0087] df: represents the dilution factor

[0088] 15: Indicates the incubation time between the enzyme and the substrate.

[0089] D: Indicates the sample dilution factor

[0090] 3.784: Extinction coefficient of 1 micromolar GLY-PRO-ALA in 5 ml and 1 cm optical path lengths.

[0091] 0.5: Volume of enzyme added to the reaction

[0092] Formula for calculating collagenase inhibition rate:

[0093] Collagenase inhibition rate (%) = (collagenase activity) NC - Collagenase activity S Collagenase activity NC *100

[0094] collagenase activity NC The collagenase activity value is for the negative control group.

[0095] collagenase activity S The collagenase activity value of the sample group;

[0096] Step 9: Result Determination

[0097] Perform statistical and significance analysis on the data, calculate the P-value, P < 0.05 indicates a significant difference, otherwise there is no statistical difference.

[0098] Each experiment included a solvent control, a negative control, and a positive control. The experiment was considered successful when there was a significant difference in collagenase activity between the solvent control group and the negative control group after the reaction time (P < 0.05).

[0099] When the collagenase inhibition rate of the sample group was significantly higher than that of the negative control group, and the difference was statistically significant (P < 0.05), it can be determined that the sample has the ability to inhibit collagenase activity.

[0100] Application example:

[0101] 1. A test method for a cosmetic product with anti-wrinkle effects:

[0102] 1.1 Sample Name: Serum

[0103] 1.2 Method:

[0104] 1.2.1 In vitro biochemical test method: The anti-wrinkle efficacy of the sample was tested according to the method described in this invention. The sample was diluted to a concentration of 10% for collagenase inhibition test. The collagenase inhibition rate was calculated to evaluate the anti-wrinkle effect of the test sample.

[0105] 1.2.2 Human Testing Method: The test sample was used continuously by more than 30 subjects. The length of crow's feet wrinkles before and after use was compared to verify the anti-wrinkle efficacy of the sample. Change rate (percentage) = (mean value after use - mean value before use) / mean value before use.

[0106] 1.2.3 Statistical Analysis: For biochemical test data, a two-tailed t-test was used, with P < 0.05 indicating a significant difference. For human test data, SPSS software was used to perform a Shapiro-Wilk Test to test the significance of the improved values' normal distribution. If Sig. (two-tailed) > 0.05, the data was normally distributed, and a paired t-test was performed; if Sig. (two-tailed) < 0.05, the data was not normally distributed, and a Wilcoxon test was performed. For comparisons of ordinal data before and after the data change, a two-sample rank-sum test was used.

[0107] If P > 0.05, it indicates no significant difference, denoted as "ns";

[0108] If P < 0.05 indicates a significant difference, and 0.01 ≤ P < 0.05 is marked with "*"; 0.001 ≤ P < 0.01 is marked with "**"; 0.0001 ≤ P < 0.001 is marked with "***"; and P < 0.0001 is marked with "****".

[0109] If the result is improved and P < 0.05, it is considered effective and marked with "√"; if the result is improved but P > 0.05 or P < 0.05, but the result worsens, it is considered invalid and marked with "×".

[0110] 1.3 Results:

[0111] 1.3.1 Biochemical Test Results

[0112] The sample was diluted to a 10% concentration for collagenase inhibition assay. The results showed that the sample inhibited collagenase by 68.33% at the 10% concentration, which was significantly different from the negative control group (P < 0.05). See Table 1 for details. Figure 1 .

[0113] Table 1. Results of Collagenase Inhibition Test

[0114]

[0115] 1.3.2 Results of Human Trials

[0116] In a study of 32 healthy Chinese subjects who used the test sample for 28 days, the length of their crow's feet wrinkles improved by 34.19%, which was statistically significant compared to before use (P < 0.05), indicating that the test sample has an anti-wrinkle effect. Detailed results are shown in Tables 2 and 3. Figure 2 .

[0117] Table 2. Descriptive Statistical Results of Crow's Feet Wrinkle Length

[0118]

[0119] Table 3. Statistical Analysis Results of Crow's Feet Wrinkle Length

[0120]

[0121] As can be seen from the above, this experimental method has established a standard for judging anti-wrinkle efficacy through practical application. It is economical and quick, the experimental process is highly controllable, the experimental system is standardized, and it avoids large result deviations caused by individual differences in the human body, thus showing good application prospects.

[0122] 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials, characterized in that, The steps are as follows: Step 1: Preparation of working solution Calcium acetate working solution, sodium acetate working solution, collagenase working solution, citric acid working solution, substrate working solution, colorimetric working solution, and positive control working solution were prepared separately. Step 2, Sample solution preparation The sample to be tested is placed until the temperature equilibrates with room temperature, and then a sample solution is prepared. Step 3: Adding enzyme working solution First, preheat all the prepared working solutions at 37°C for 15 minutes. Then, set up six test groups: sample group, sample background group, negative control group, negative control background group, positive control group, and positive control background group. Add 0.5 mL of collagenase working solution to the sample group, negative control group, and positive control group, and add 0.5 mL of calcium acetate working solution to the sample background group, negative control background group, and positive control background group. Step 4: Grouping and loading samples Take 0.5 mL of sample solution into the sample group and sample background group, take 0.5 mL of pure water into the negative control group and negative control background group, take 0.5 mL of positive working solution into the positive control group and positive control background group, add 0.5 mL of enzyme solution to each experimental group, and add 0.5 mL of calcium acetate solution to the background group. Place each group in a 37℃ water bath or incubator at the same time and keep warm for 15 min. Step 5: Add substrate and react. Add 1.5 mL of substrate working solution to each group and citrate buffer to the background group in centrifuge tubes. Mix well by shaking with a shaker and incubate at 37°C for 15 min. Step 6: Color Development Process Add 1 mL of colorimetric solution to each group in a centrifuge tube, place the tubes in a water bath, and react at 100°C for 20 min. Step 7: Absorbance Measurement After cooling to room temperature, samples were added to 96-well plates, and the changes in absorbance at 565 nm were measured using an ELISA reader. Step 8: Calculate and analyze the data. Collagenase activity: ((A) 样品 -A 背景 )*df) / (15*3.784*0.5) in: df: represents the dilution factor 15: Indicates the incubation time between the enzyme and the substrate. D: Indicates the sample dilution factor 3.784: Extinction coefficient of 1 micromolar GLY-PRO-ALA in 5 ml and 1 cm optical path lengths. 0.5: Volume of enzyme added to the reaction Formula for calculating collagenase inhibition rate: Collagenase inhibition rate (%) = (collagenase activity NC - collagenase activity s) / collagenase activity NC *100 Collagenase activity NC represents the collagenase activity value of the negative control group. Collagenase activity S represents the collagenase activity value of the sample group; Step 9: Result Determination Statistical and significance analysis was performed on the data, and the p-value was calculated. P < 0.05 indicated a statistically significant difference; otherwise, there was no statistically significant difference. Each experiment included a solvent control, a negative control, and a positive control. The experiment was considered successful when there was a significant difference in collagenase activity between the solvent control group and the negative control group after the reaction time (P < 0.05). When the collagenase inhibition rate of the sample group was significantly higher than that of the negative control group, and the difference was statistically significant (P < 0.05), it can be determined that the sample has the ability to inhibit collagenase activity.

2. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the calcium acetate working solution in step 1 is as follows: accurately weigh 158 mg of calcium acetate, and dilute it to 100 mL with ultrapure water to obtain a 0.01 mol / L calcium acetate solution.

3. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of sodium acetate working solution in step 1 is as follows: accurately weigh 1.64g of sodium acetate, add 30mL of ultrapure water, adjust the pH to 6.3 with acetic acid, and then make up to 50mL with ultrapure water to obtain a 4mol / L sodium acetate solution.

4. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the collagenase working solution in step 1 is as follows: accurately weigh 6.8 mg of collagenase powder, and make up to 25 mL with calcium acetate solution to obtain a collagenase working solution of 34 U / mL.

5. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the citric acid working solution in step 1 is as follows: accurately weigh 9.61g of citric acid and 0.79g of calcium acetate, add 300mL of ultrapure water, adjust the pH to 6.3, and then make up the volume to 500mL with ultrapure water to obtain 0.1mol / L citric acid buffer solution.

6. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the substrate working solution in step 1 is as follows: Z-GLY-PRO-GLY-GLY-PRO-ALA-OH is prepared into a substrate working solution of 0.311 mg / mL using citrate buffer.

7. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the colorimetric working solution in step 1 is as follows: accurately weigh 2g of ninhydrin, add 75mL of 2-methoxyethanol, then add 40mg of stannous chloride, and dilute to 100mL with sodium acetate solution.

8. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The preparation process of the positive control working solution in step 1 is as follows: 1,10-phenanthroline is selected as the positive control. First, 30 mg of the positive control is weighed, and then 1666 μL of ethanol is added to prepare a 100 mmol / L stock solution. Then, it is diluted with ultrapure water to prepare a 4 mmol / L positive working solution.

9. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 1, characterized in that, The sample solution preparation process in step 2 is as follows: the sample to be tested is equilibrated at 20-25℃ for at least 30 minutes, and then dissolved to prepare a sample solution.

10. The in vitro biochemical test method for evaluating the anti-wrinkle efficacy of cosmetics and raw materials according to claim 9, characterized in that, In the preparation of the sample solution in step 2, for samples dissolved in DMSO or ethanol, when used for testing, the final concentration of DMSO or ethanol in the solvent control group or all test concentration groups shall not exceed 1% by volume.

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