Angelica keiskei extracting solution with anti-wrinkle and firming effects and preparation method of angelica keiskei extracting solution

Abstract

The invention provides an angelica keiskei extracting solution with anti-wrinkle and firming effects and a preparation method of the angelica keiskei extracting solution. The preparation method comprises the following steps: extracting angelica keiskei by adopting hot water, adding EDTA (Ethylene Diamine Tetraacetic Acid) disodium before each extraction, adding ethanol into a concentrated solution in batches, carrying out cold precipitation overnight for refining and purifying, adding p-hydroxyacetophenone, EDTA disodium, 1, 2-hexanediol and 1, 3-butanediol into a filtrate, and uniformly stirring and mixing to obtain the angelica keiskei extract. The angelica keiskei extracting solution prepared by the method improves the discoloration problem under the conditions of high temperature and illumination, and meanwhile, the prepared angelica keiskei extract has remarkable anti-wrinkle, firming and anti-oxidation effects.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics and relates to an extract of Ashitaba with anti-wrinkle and firming effects and its preparation method. Background Technology

[0002] Skin aging is the result of the combined effects of endogenous and exogenous factors. Endogenous aging includes factors such as gene programming and metabolic decline, while exogenous aging includes factors such as environmental stress and ultraviolet radiation. The core mechanism of aging is oxidative damage to cells. A series of factors caused by oxidative damage, such as inflammatory responses, loss of structural proteins, and decreased barrier function, will deepen wrinkles, cause loss of elasticity, sagging of the skin contour, dullness of skin tone, and weaken the skin's repair ability.

[0003] Modern research has confirmed that the total flavonoids, especially chalcones, contained in Ashitaba possess multi-target antioxidant, synergistic anti-inflammatory, and collagen degradation-inhibiting effects. Currently, there are reports on the application of Ashitaba extract in the food and health product fields, with efficacy studies primarily focusing on antioxidant and anti-aging aspects. However, there is still considerable room for research into the application of Ashitaba extract in the field of cosmetic anti-aging, particularly regarding its efficacy in promoting collagen and elastin regeneration in skin fibroblasts and inhibiting matrix metalloproteinase-1 (MMP-1) production.

[0004] Currently, few cosmetic products use Ashitaba extract as an anti-aging ingredient, primarily because traditionally extracted Ashitaba extract is unstable under high temperatures and light, exhibiting severe discoloration. This significantly limits its application in cosmetics. Research shows that the easy discoloration of natural plant extracts upon exposure to high temperatures or light is due to chemical reactions of unstable components, with oxidation being the primary cause. Phenolic components in the extract (such as flavonoids, phenolic acids, tannins, and anthraquinones) have reducing properties and are easily oxidized by oxygen in the air under the catalysis of light, heat, metal ions, or enzymes, producing colored substances such as quinones, which are typical examples of easily oxidized and discolored components. Since many of the active ingredients in Ashitaba are of this type, it is prone to discoloration. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an Ashitaba extract with anti-wrinkle and firming effects and its preparation method. This invention uses hot water extraction of Ashitaba, adding disodium EDTA before each extraction. The concentrated extract is purified by adding ethanol in portions and allowing it to cool and precipitate overnight. The filtrate is then mixed with p-hydroxyacetophenone, disodium EDTA, 1,2-hexanediol, and 1,3-butanediol. The Ashitaba extract prepared by this method improves the discoloration problem under high temperature and light conditions, and the prepared Ashitaba extract exhibits significant anti-wrinkle, firming, and antioxidant effects. Currently, there are no reports on the anti-wrinkle and firming effects of Ashitaba, nor are there studies on the combined use of disodium EDTA and Ashitaba extract for color protection purposes.

[0006] The technical solution of the present invention is: an Ashitaba extract with anti-wrinkle and firming effects, characterized in that its raw materials and weight parts are: 10-20 parts of Ashitaba extract (based on dry weight) after extraction, neutralization and addition of EDTA disodium, 0.3-1.2 parts of p-hydroxyacetophenone, 0.3-1.2 parts of 1,2-hexanediol, 20-30 parts of 1,3-butanediol, and 60-70 parts of purified water.

[0007] The preparation method of the above-mentioned Ashitaba extract includes the following steps: (1) Soak Ashitaba leaves in cold water overnight. After soaking, extract twice. Add 10 times the amount of water for the first extraction and 8 times the amount of water for the second extraction. Add 0.5±0.1% of disodium EDTA solvent before each extraction. The extraction temperature is 90±5℃. After extraction, filter and combine the two filtrates and concentrate under reduced pressure. (2) The concentrate was purified by adding ethanol in several batches and allowing it to settle overnight. The first addition of ethanol brought the alcohol content to 20%–30%, and the mixture was allowed to settle overnight. The supernatant was then filtered and concentrated under reduced pressure. The second addition of ethanol brought the alcohol content to 30%–40%, and the mixture was allowed to settle overnight. The supernatant was then filtered and concentrated under reduced pressure. The third addition of ethanol brought the alcohol content to 50%–70%, and the mixture was allowed to settle overnight. The supernatant was then filtered and concentrated under reduced pressure. Purified water was then added and allowed to settle overnight. The supernatant was then used for solid-liquid separation. (3) After dissolving the obtained filtrate in p-hydroxyacetophenone, slowly add EDTA disodium in small amounts several times, equivalent to 1 ± 0.1% of the amount of medicinal material, and continue stirring until the solid is completely dissolved; (4) After dissolution is complete, filter the solution and add 1,2-hexanediol and 1,3-butanediol to the filtrate and stir to mix well.

[0008] Furthermore, step (1) is concentrated to a liquid relative density of 1.15 to 1.20.

[0009] Furthermore, in step (2), after the first and second addition of ethanol, the liquid is concentrated to a relative density of 1.05 to 1.10; after the third addition of ethanol, the liquid is concentrated to a relative density of 1.15 to 1.20.

[0010] Furthermore, the overnight cold sinking temperature in step (2) is -4±1℃.

[0011] Furthermore, in step (2), water is added to the third concentrate to replenish the amount of medicinal materials.

[0012] The Ashitaba extract prepared by the method of this invention improves the discoloration problem under high temperature and light conditions. Simultaneously, the prepared Ashitaba extract exhibits significant anti-wrinkle, firming, and antioxidant effects, and can be used to prepare cosmetics with anti-wrinkle and firming effects. The anti-wrinkle effect is manifested in its ability to increase type I collagen content, and the firming effect is manifested in its ability to increase elastin content, upregulate the expression levels of CSGALNACT1 and SIRT genes, and reduce matrix metalloproteinase-1 (MMP-1) content.

[0013] The technical effects of this invention are: 1. The preparation method of the present invention is simple. The prepared Ashitaba extract improves the discoloration problem under high temperature and light conditions. At the same time, the prepared Ashitaba extract has significant anti-wrinkle, firming and antioxidant effects.

[0014] 2. Based on fibroblasts, compared with the control group, the Ashitaba extract at a concentration of 2.5% (v / v) showed a significant increase in type I collagen (Collagen I), with an increase rate of 135.71%, indicating a good anti-wrinkle effect. Compared with the control group, the Ashitaba extract at a concentration of 2.5% (v / v) showed a significant increase in elastin content, with an increase rate of 205.13%; the expression levels of CSGALNACT1 and SIRT genes were significantly upregulated, with upregulation rates of 119.05% and 66.67%, respectively; the content of matrix metalloproteinase-1 (MMP-1) was significantly decreased, with an inhibition rate of 66.15%, thus exhibiting a good firming effect. Compared with the control group, the Ashitaba extract at a concentration of 2.5% (v / v) showed a significant increase in manganese superoxide dismutase (SOD2) content, with an increase rate of 101.89%, indicating a good antioxidant effect. Attached Figure Description

[0015] Figure 1 The color comparison of Ashitaba extract under different processing conditions is shown below: 1. Example 1: High temperature for 20 days; 2. Example 1: Irradiated for 20 days; 3. Comparative Example 1: High temperature for 20 days; 4. Comparative Example 1: Irradiated for 20 days; 5. Comparative Example 2: High temperature for 20 days; 6. Comparative Example 2: Irradiated for 20 days. Figure 2 This is a cell viability curve of the sample Ashitaba extract; Figure 3 This is a cell morphology diagram of the extract of Ashitaba. Figure 4 Here is a summary chart of Collagen I immunofluorescence results; Figure 5 A bar chart of Collagen I relative integrated optical density (IOD) / cell numerical values; Figure 6 Summary chart of Elastin immunofluorescence results; Figure 7 Bar chart showing the relative integrated optical density (IOD) of Elastin / cell numerical value; Figure 8 Summary of SOD2 immunofluorescence results; Note: Images were taken using a fluorescence microscope (Olympus, BX43) at 20× magnification. Blue fluorescence represents cell nuclei, and green fluorescence represents SOD2. The more and brighter the green fluorescence, the higher the SOD2 content. Figure 9 A bar chart showing the relative integrated optical density (IOD) of SOD2 per cell. Figure 10 A bar chart showing the expression level of the CSGALNACT1 gene; Figure 11 A bar chart showing the expression levels of the SIRT gene; Figure 12 The bar chart shows the results of MMP-1 content. Detailed Implementation

[0016] The effects are illustrated below with reference to the embodiments and accompanying drawings.

[0017] Example 1: Preparation method of Ashitaba extract The raw materials and their weight parts are as follows: 15 parts of Ashitaba extract (by dry weight) after extraction and neutralization with added EDTA disodium, 0.5 parts of p-hydroxyacetophenone, 0.5 parts of 1,2-hexanediol, 25 parts of 1,3-butanediol, and 65 parts of purified water.

[0018] (1) Soak Ashitaba leaves in cold water overnight. After soaking, extract twice. The first extraction time is 3 hours and the second extraction time is 2 hours. Add 10 times the amount of water for the first extraction and 8 times the amount of water for the second extraction. Add 0.5% of disodium EDTA solvent before each extraction. The stirring speed during extraction is 200 r / min and the extraction temperature is fixed at 90℃. After extraction, filter through an 80-mesh sieve. Combine the two filtrates and concentrate under reduced pressure. Keep the temperature below 80℃ and the pressure at (-0.08±0.01) MPa. Finally concentrate to a liquid relative density of 1.15~1.20. (2) The concentrate was purified by adding ethanol in several batches and allowing it to settle overnight under cold precipitation. The first addition of ethanol brought the alcohol content to 25%, and the mixture was settled overnight at -4°C. The supernatant was then filtered, and the filtrate was concentrated under reduced pressure, maintaining a temperature below 60°C and a pressure of (-0.08±0.01) MPa, until the final liquid relative density was 1.05–1.10. The second addition of ethanol brought the alcohol content to 35%, and the mixture was settled overnight at -4°C. The supernatant was then filtered, and the filtrate was concentrated under reduced pressure, maintaining a temperature below 60°C and a pressure of (-0.08±0.01) MPa, until the final liquid relative density was 1.05–1.10. The pressure was maintained at (-0.08±0.01) MPa, and the solution was finally concentrated to a liquid relative density of 1.05–1.10. A third addition of ethanol was made to bring the alcohol content to 60%. The solution was then allowed to settle overnight at -4°C. The supernatant was collected and filtered. The filtrate was concentrated under reduced pressure, maintaining a temperature below 60°C and a pressure of (-0.08±0.01) MPa, until the liquid relative density reached 1.15–1.20. The concentrate was then made up to the volume of the medicinal material by adding purified water, and the solution was allowed to settle overnight at -4°C. The supernatant was then used for solid-liquid separation. (3) Add the above-obtained filtrate to p-hydroxyacetophenone, add purified water and keep the temperature at 60°C. Stir at 200 r / min until all the solids are dissolved. Then slowly add EDTA disodium in small amounts several times, equivalent to 1% of the amount of medicinal material, and continue stirring until all the solids are dissolved. (4) After dissolution, filter the solution using a 10 μm filter plate, add 1,2-hexanediol and 1,3-butanediol to the filtrate and stir to mix.

[0019] Comparative Example 1: Preparation method of Ashitaba extract The raw materials and weight proportions are the same as in Example 1.

[0020] (1) Soak Ashitaba leaves in cold water overnight. After soaking, extract twice. The first extraction time is 3 hours and the second extraction time is 2 hours. Add 10 times the amount of water for the first extraction and 8 times the amount of water for the second extraction. The stirring speed during extraction is 200 r / min and the extraction temperature is fixed at 90℃. After extraction, filter through an 80-mesh sieve. Combine the two filtrates and concentrate under reduced pressure. Keep the temperature below 80℃ and the pressure at (-0.08±0.01) MPa. Finally concentrate to a liquid relative density of 1.15~1.20. (2) The concentrate was purified by adding ethanol in several batches and allowing it to settle overnight under cold precipitation. The first addition of ethanol brought the alcohol content to 25%, and the mixture was settled overnight at -4°C. The supernatant was then filtered, and the filtrate was concentrated under reduced pressure, maintaining a temperature below 60°C and a pressure of (-0.08±0.01) MPa, until the final liquid relative density was 1.05–1.10. The second addition of ethanol brought the alcohol content to 35%, and the mixture was settled overnight at -4°C. The supernatant was then filtered, and the filtrate was concentrated under reduced pressure, maintaining a temperature below 60°C and a pressure of (-0.08±0.01) MPa, until the final liquid relative density was 1.05–1.10. The pressure was maintained at (-0.08±0.01) MPa, and the solution was finally concentrated to a liquid relative density of 1.05–1.10. A third addition of ethanol was made to bring the alcohol content to 60%. The solution was then allowed to settle overnight at -4°C. The supernatant was collected and filtered. The filtrate was concentrated under reduced pressure, maintaining a temperature below 60°C and a pressure of (-0.08±0.01) MPa, until the liquid relative density reached 1.15–1.20. The concentrate was then made up to the volume of the medicinal material by adding purified water, and the solution was allowed to settle overnight at -4°C. The supernatant was then used for solid-liquid separation. (3) Add the above-obtained filtrate to p-hydroxyacetophenone, add purified water and keep the temperature at 60°C. Stir at 200 r / min until all the solids are dissolved. Then slowly add EDTA disodium in small amounts several times, equivalent to 1% of the amount of medicinal material, and continue stirring until all the solids are dissolved. (4) After dissolution, filter the solution using a 10 μm filter plate, add 1,2-hexanediol and 1,3-butanediol to the filtrate and stir to mix well.

[0021] Comparative Example 2: Preparation method of Ashitaba extract The raw materials and weight proportions are the same as in Example 1.

[0022] (1) Soak Ashitaba leaves in cold water overnight. After soaking, extract twice. The first extraction time is 3 hours and the second extraction time is 2 hours. Add 10 times the amount of water for the first extraction and 8 times the amount of water for the second extraction. Add 0.5% of disodium EDTA solvent before each extraction. The stirring speed during extraction is 200 r / min and the extraction temperature is fixed at 90℃. After extraction, filter through an 80-mesh sieve. Combine the two filtrates and concentrate under reduced pressure. Keep the temperature below 80℃ and the pressure at (-0.08±0.01) MPa. Finally concentrate to a liquid relative density of 1.15~1.20. (2) The concentrate was purified by adding ethanol in several batches and allowing it to settle overnight under cold precipitation. The first addition of ethanol brought the alcohol content to 20%–30%, and the mixture was allowed to settle overnight at -4°C. The supernatant was then filtered, and the filtrate was concentrated under reduced pressure while maintaining the temperature below 60°C and the pressure at (-0.08±0.01) MPa. The concentrate was finally concentrated to a liquid relative density of 1.05–1.10. The second addition of ethanol brought the alcohol content to 30%–40%, and the mixture was allowed to settle overnight at -4°C. The supernatant was then filtered, and the filtrate was concentrated under reduced pressure while maintaining the temperature below 60°C. The pressure was maintained at (-0.08±0.01) MPa, and the solution was finally concentrated to a liquid relative density of 1.05 to 1.10. Ethanol was added for the third time to make the alcohol content 50% to 70%, and the solution was allowed to settle overnight at -4℃. The supernatant was collected and filtered, and the filtrate was concentrated under reduced pressure while maintaining the temperature below 60℃ and the pressure at (-0.08±0.01) MPa, until the liquid relative density was finally concentrated to a liquid relative density of 1.15 to 1.20. The concentrated solution was then made up to the amount of medicinal material by adding purified water, and the solution was allowed to settle overnight at -4℃. The supernatant was collected and solid-liquid separation was performed.

[0023] (3) Add the above-obtained filtrate to p-hydroxyacetophenone, add purified water and keep the temperature at 60°C, and stir at 200 r / min until all the solids are dissolved; (4) After dissolution, filter the solution using a 10 μm filter plate, add 1,2-hexanediol and 1,3-butanediol to the filtrate and stir to mix well.

[0024] The color changes of the samples from Example 1 and Comparative Examples 1-2 after 20 days of exposure to high temperature (60℃±2℃) and light (4500±500lx) are as follows. Figure 1 As shown. Figure 1 The results show that the Ashitaba extract prepared in Example 1 of this invention can significantly improve the discoloration problem of Ashitaba extract and enhance the stability of the product as a cosmetic ingredient. Comparative Examples 1 and 2, which only added disodium EDTA before and after extraction, respectively, did not show the same effect as Example 1.

[0025] Test Example: Sample Efficacy Test Results 1. Cytotoxicity test 1.1 Test Method 1.1.1 Cell viability testing methods 1) Cell seeding: After reviving fibroblasts (batch number: Fb230131, provided by Guangdong Boxi Biotechnology Co., Ltd.), when the plate-laying rate reaches about 60%, the cells are seeded into 96-well plates and incubated overnight in a CO2 incubator (37℃, 5% CO2).

[0026] 2) Experimental Groups: The experiment included a zeroing group, a solvent control group, a positive control group, and a sample group. In the sample group, each sample was set with 8 concentrations, and 3 replicate wells were set for each concentration.

[0027] 3) Solution preparation: Prepare working solutions of different concentrations according to the test concentration gradient table, as shown in Table 1.

[0028] Table 1 Test Concentration Setting Table

[0029] 4) Drug administration: Drug administration was performed when the cell seeding rate in the 96-well plates reached 50%–60%. For the solvent control group, 200 μL of DMEM cell culture medium was added to each well; for the positive control group, 200 μL of DMEM cell culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of DMEM cell culture medium containing the corresponding concentration of the sample was added to each well; for the zeroing group, no cells were seeded, only 200 μL of DMEM cell culture medium was added. After drug administration, the 96-well plates were placed in a CO2 incubator (37℃, 5% CO2) and cultured for 24 h.

[0030] 5) Detection: After culturing cells for 24 hours, discard the supernatant, add 100 μL of MTT working solution (0.5 mg / mL) to each well, and incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant, add 150 μL of DMSO to each well, and read the OD value at 490 nm.

[0031] 6) Calculation of relative cell viability: According to the formula, relative cell viability (%) = (OD of sample well - OD of zeroing well) / (OD of solvent control well - OD of zeroing well) × 100%.

[0032] 1.1.2 Cell morphology testing methods 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 24-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2); 2) Experimental Groups: The experiment included a solvent control group and a sample group. Within the sample group, each sample was configured with five concentration gradients (10%, 5%, 2.5%, 1.25%, 0.625%). 3) Drug administration: Drug administration was performed when the cell deposition rate in the 24-well plate reached 50%–60%. For the solvent control group, 1 mL of culture medium was added to each well; for the sample group, 1 mL of culture medium containing the corresponding concentration of the test sample was added to each well. After drug administration, the 24-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 hours. 4) Take photos: After incubation, discard the supernatant and take photos under an inverted microscope.

[0033] 1.2 Test Results The MTT assay results of the Ashitaba extract are shown in Table 2.

[0034] Table 2. MTT assay results of Ashitaba extract samples

[0035] Cell viability curve of Ashitaba extract sample as shown in the figure Figure 2 As shown, the cell morphology image of the extract of *Ashitsubo* is as follows. Figure 3 As shown. Based on MTT and morphological results, the Ashitaba extract was considered to be fibroblast-based and did not exhibit significant cytotoxicity at a concentration range of 2.5%.

[0036] 2. Elastin and Collagen I Efficacy Testing 2.1 Test Method 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 6-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0037] 2) Solution preparation: Prepare working solutions of the test substances according to the test groups, as shown in Table 3.

[0038] Table 3 Test Groups

[0039] 3) Drug administration: According to the test groups in Table 3, when the cell deposition rate in the 6-well plate reached 30%~50%, drug administration was performed in groups, with 3 replicates per group. 2 mL of DMEM cell culture medium was added to each well of the blank control group and negative control group; 2 mL of DMEM cell culture medium containing vitamin E was added to each well of the positive control group; and 2 mL of DMEM cell culture medium containing the corresponding concentration of the sample was added to each well of the sample group. After drug administration, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) and cultured for 24 h. 4) UVA irradiation: According to the test groups in Table 3, except for the blank control group, the other groups were subjected to UVA irradiation with an irradiation dose of 30 J / cm². 2 The irradiation time was 20 minutes. After irradiation, the samples were placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 hours. 5) Immunofluorescence detection: Cells were fixed with 4% paraformaldehyde for 30 min, and then immunofluorescence detection was performed. The staining results were observed and photographed under a fluorescence microscope, and the images were collected and analyzed using Image-Pro® Plus software.

[0040] 6) Calculation of improvement rate: Improvement rate (%) = (sample group - negative control group) / negative control group × 100%.

[0041] 7) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical results are two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0042] 2.2 Test Results 2.2.1 Results of Type I Collagen Test The summary figure of Collagen I immunofluorescence results is shown below. Figure 4 As shown in Table 4, the summary table of Collagen I immunofluorescence results is presented. The bar chart of Collagen I relative integrated optical density (IOD) / cell count is shown below. Figure 5 As shown.

[0043] Table 4 Summary of Collagen I Immunoassay Fluorescence Results

[0044] Note: Integrated optical density (IOD) / cell number reflects the content of Collagen I. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0045] The test results show that: compared with BC, the Collagen I content in the NC group was significantly lower, indicating that the stimulation conditions in this test were effective. Compared with NC, the Collagen I content in the PC group was significantly higher, indicating that the positive control in this test was effective. Compared with NC, the Collagen I content in the Ashitaba extract at a concentration of 2.5% was significantly higher, with an increase rate of 135.71%.

[0046] 2.2.2 Elastin Test Results A summary table of Elastin immunofluorescence results is shown below. Figure 6 As shown in Table 5, the summary table of Elastin immunofluorescence analysis results is presented. The bar chart of Elastin relative integrated optical density (IOD) / cell count is shown below. Figure 7 As shown.

[0047] Table 5 Summary of Elastin Immunofluorescence Analysis Results

[0048] Note: Integrated optical density (IOD) / cell number reflects the elastin content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0049] The results showed that the elastin content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. The elastin content in the PC group was significantly higher than that in the NC group, indicating that the positive control in this test was effective. Compared with the NC group, the elastin content in the *Ashitsubaki* extract at a concentration of 2.5% was significantly higher, with an increase rate of 205.13%.

[0050] 3. Manganese superoxide dismutase (SOD2) assay method 3.1 Test Method 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 24-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0051] 2) Solution preparation: Prepare the working solution of the test substance according to the test group, as shown in Table 6.

[0052] Table 6 Test Groups

[0053] 3) Drug administration: According to the test groups, when the cell deposition rate in the 24-well plate reached 30%~50%, drug administration was performed in groups, with 3 replicates per group. 1 mL of DMEM cell culture medium was added to each well of the blank control group and negative control group, 1 mL of DMEM cell culture medium containing vitamin E was added to each well of the positive control group, and 1 mL of DMEM cell culture medium containing the corresponding test sample was added to each well of the sample group. After drug administration, the 24-well plate was incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours.

[0054] 4) UVA irradiation: Except for the blank control group, all other groups underwent UVA irradiation at a dose of 30 J / cm². 2 The irradiation time was 20 minutes. After the irradiation was completed, the samples were placed in a CO2 incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0055] 5) Immunofluorescence detection: Cells were fixed with 4% paraformaldehyde for 30 minutes, and then immunofluorescence detection was performed. The cells were photographed and observed under a microscope, and the images were collected and analyzed.

[0056] 6) Calculation of improvement rate: Improvement rate (%) = (sample group - negative control group) / negative control group × 100%.

[0057] 7) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical results are two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0058] 3.2 Test Results The summary figure of SOD2 immunofluorescence results is shown below. Figure 8 As shown in Table 7, the summary table of SOD2 immunofluorescence analysis results is presented. The bar chart of SOD2 relative integrated optical density (IOD) / cell count is shown in Table 7. Figure 9 As shown.

[0059] Table 7 Summary of SOD2 Immunofluorescence Analysis Results

[0060] Note: Integrated optical density (IOD) / cell number reflects the SOD2 content. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0061] The test results showed that the SOD2 content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. The SOD2 content in the PC group was significantly higher than that in the NC group, indicating that the positive control in this test was effective. Compared with the NC group, the SOD2 content of the Ashitaba extract at a concentration of 2.5% was significantly higher, with an increase rate of 101.89%.

[0062] 4. Detection of CSGALNACT1 and SIRT gene expression levels and matrix metalloproteinase 1 (MMP-1) function. 4.1 Test Method 1) Cell seeding: After cell resuscitation, when the cell plating rate reaches about 60%, seed the cells into 6-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0063] 2) Solution preparation: Prepare the working solution of the test substance according to the test group, as shown in Table 8.

[0064] Table 8 Test Groups

[0065] 3) Drug administration: According to the test groups, when the cell deposition rate in the 6-well plates reached 30%~50%, drug administration was performed in groups, with 3 replicates per group. 2 mL of DMEM cell culture medium was added to each well of the blank control group and negative control group; 2 mL of DMEM cell culture medium containing TGF-β1 was added to each well of the positive control group; and 2 mL of DMEM cell culture medium containing the corresponding test sample was added to each well of the sample group. After drug administration, the 6-well plates were placed in a CO2 incubator (37℃, 5% CO2) for 24 h.

[0066] 4) UVA irradiation: Except for the blank control group, all other groups underwent UVA irradiation at a dose of 30 J / cm². 2 The irradiation time was 20 minutes. After the irradiation was completed, the samples were placed in a CO2 incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0067] 5) Gene expression detection: After incubation, wash twice with 1 mL / well of PBS, add 1 mL of AG RNAex ProReagent to each well, lyse the cells by pipetting, and collect the samples. Extract RNA, reverse transcribe it into cDNA, and then perform real-time PCR detection. -△△CT The method is used to calculate the results.

[0068] 6) ELISA detection: Collect cell culture supernatant and perform ELISA detection according to the ELISA kit instructions.

[0069] 7) Calculation of upregulation rate and inhibition rate: Upregulation rate (%) = (Sample group - Negative control group) / Negative control group × 100%; Inhibition rate (%) = (Negative control group - Sample group) / Negative control group × 100%.

[0070] 8) Statistical Analysis of Results: GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical results are two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0071] 4.2 Test Results 4.2.1 CSGALNACT1 and SIRT Test Results Table 9 summarizes the CSGALNACT1 gene detection results, and the bar chart for CSGALNACT1 gene expression level is shown below. Figure 10 As shown.

[0072] Table 9 Summary of CSGALNACT1 gene detection results

[0073] Note: Results were calculated using the 2-ΔΔCT method. When performing statistical analysis using the t-test method, the fold increase of mRNA in the BC group was normalized. Compared with the BC group, significance is indicated by #, with P-value < 0.05 indicated by # and P-value < 0.01 indicated by ##. Compared with the NC group, significance is indicated by *, with P-value < 0.05 indicated by * and P-value < 0.01 indicated by **.

[0074] Compared with the BC group, the expression level of CSGALNACT1 gene was significantly downregulated in the NC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the expression level of CSGALNACT1 gene was significantly upregulated in the PC group, indicating that the positive control in this test was effective. Compared with the NC group, the expression level of CSGALNACT1 gene in the extract of Ashitaba at a concentration of 2.5% was significantly upregulated, with an upregulation rate of 119.05%.

[0075] Table 10 summarizes the SIRT gene detection results. A bar chart of SIRT gene expression levels is shown below. Figure 11 As shown.

[0076] Table 10 Summary of SIRT Gene Detection Results

[0077] Note: Results were calculated using the 2-ΔΔCT method. When performing statistical analysis using the t-test method, the fold increase of mRNA in the BC group was normalized. Compared with the BC group, significance is indicated by #, with P-value < 0.05 indicated by # and P-value < 0.01 indicated by ##. Compared with the NC group, significance is indicated by *, with P-value < 0.05 indicated by * and P-value < 0.01 indicated by **.

[0078] Compared with the BC group, the SIRT gene expression level in the NC group was significantly downregulated, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the SIRT gene expression level in the PC group was significantly upregulated, indicating that the positive control in this test was effective. Compared with the NC group, the SIRT gene expression level in the Ashitaba extract at a concentration of 2.5% was significantly upregulated, with an upregulation rate of 66.67%.

[0079] 4.2.2 Matrix metalloproteinase 1 (MMP-1) test results The summary table of MMP-1 content results is shown in Table 11, and the bar chart of MMP-1 content results is shown in Table 11. Figure 12 As shown.

[0080] Table 11 Summary of MMP-1 Content Results

[0081] Note: When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by #, with P-value < 0.05 indicated by # and P-value < 0.01 indicated by ##; significance compared to the NC group is indicated by *, with P-value < 0.05 indicated by * and P-value < 0.01 indicated by **.

[0082] Compared with the BC group, the MMP-1 content in the NC group was significantly increased, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the MMP-1 content in the PC group was significantly decreased, indicating that the positive control in this test was effective. Compared with the NC group, the MMP-1 content in the Ashitaba extract at a concentration of 2.5% was significantly decreased, with an inhibition rate of 66.15%.

[0083] 5. Conclusion 1) Anti-wrinkle effect: Based on fibroblasts, compared with the control group, the sample Ashitaba extract showed a significant increase in type I collagen content at a concentration of 2.5% (v / v), with an increase rate of 135.71%, indicating that the sample can increase type I collagen content at this concentration and has anti-wrinkle effects.

[0084] 2) Firming effect: Based on fibroblasts, compared with the control group, the elastin content of the Ashitaba extract at a concentration of 2.5% (v / v) was significantly increased, with an increase rate of 205.13%; the expression levels of CSGALNACT1 and SIRT genes were significantly upregulated, with upregulation rates of 119.05% and 66.67%, respectively; and the content of matrix metalloproteinase 1 (MMP-1) was significantly decreased, with an inhibition rate of 66.15%. This indicates that the sample at this concentration can increase elastin content, upregulate the expression levels of CSGALNACT1 and SIRT genes, and decrease the content of matrix metalloproteinase 1 (MMP-1), thus exhibiting a firming effect.

[0085] 3) Antioxidant effects: Based on fibroblasts, compared with the control group, the extract of *Ashitsubaki* showed a significant increase in manganese superoxide dismutase (SOD2) content at a concentration of 2.5% (v / v), with an increase rate of 101.89%. This indicates that the sample can increase the content of manganese superoxide dismutase (SOD2) at this concentration and has antioxidant effects.

Claims

1. An extract of Ashitaba (Fragrance of Ashitaba) with anti-wrinkle and firming effects, characterized in that, The raw materials and their weight parts are as follows: 10-20 parts of Ashitaba extract after extraction and neutralization with added EDTA disodium, 0.3-1.2 parts of p-hydroxyacetophenone, 0.3-1.2 parts of 1,2-hexanediol, 20-30 parts of 1,3-butanediol, and 60-70 parts of purified water.

2. The method for preparing Ashitaba extract as described in claim 1, characterized in that, Includes the following steps: (1) Soak Ashitaba leaves in cold water overnight. After soaking, extract twice. Before each extraction, add 0.5±0.1% of disodium EDTA solvent. The extraction temperature is 90±5℃. After extraction, filter, combine the two filtrates and concentrate under reduced pressure. (2) The concentrate was purified by adding ethanol in several batches and allowing it to settle overnight. The first addition of ethanol brought the alcohol content to 20%–30%, and the mixture was allowed to settle overnight. The supernatant was then filtered and the filtrate was concentrated under reduced pressure. The second addition of ethanol brought the alcohol content to 30%–40%, and the mixture was allowed to settle overnight. The supernatant was then filtered and the filtrate was concentrated under reduced pressure. The third addition of ethanol brought the alcohol content to 50%–70%, and the mixture was allowed to settle overnight. The supernatant was then filtered and the filtrate was concentrated under reduced pressure. Water was then added and allowed to settle overnight. The supernatant was then used for solid-liquid separation. (3) After dissolving the obtained filtrate in p-hydroxyacetophenone, slowly add EDTA disodium in small amounts several times, equivalent to 1 ± 0.1% of the amount of medicinal material, and continue stirring until the solid is completely dissolved; (4) After dissolution, filter the solution and add 1,2-hexanediol and 1,3-butanediol to the filtrate and stir to mix.

3. The method for preparing Ashitaba extract as described in claim 2, characterized in that, Step (1) involves concentrating the liquid until the relative density is 1.15 to 1.

20.

4. The method for preparing Ashitaba extract as described in claim 2, characterized in that, In step (2), after the first and second additions of ethanol, the liquid is concentrated to a relative density of 1.05 to 1.10; after the third addition of ethanol, the liquid is concentrated to a relative density of 1.15 to 1.

20.

5. The method for preparing Ashitaba extract as described in claim 2, characterized in that, The overnight cold settling temperature in step (2) is -4±1℃.

6. The method for preparing Ashitaba extract as described in claim 2, characterized in that, In step (2), water is added to the third concentrate to replenish the amount of medicinal materials.

7. The use of the Ashitaba extract according to claim 1 in the preparation of cosmetics with anti-wrinkle and firming effects.

8. The application as described in claim 7, characterized in that, The anti-wrinkle effect is reflected in its ability to increase the content of type I collagen.

9. The application as described in claim 7, characterized in that, The firming effect is manifested in increasing elastin content, upregulating the expression levels of CSGALNACT1 and SIRT genes, and reducing matrix metalloproteinase 1 content.

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