Use of a succinic acid derivative in cosmetics

Abstract

The application discloses application of a succinic acid derivative in cosmetics, in particular, application of the succinic acid derivative in preparation of cosmetics with antioxidation, soothing and / or whitening effects. The prepared cosmetics have good antioxidation, soothing and whitening effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular, application of succinic acid derivatives in cosmetics. BACKGROUND

[0002] Succinic acid, also known as butanedioic acid, is a typical symmetrical dibasic acid. Due to the two carboxyl functional groups in the molecule, the two methylene groups are activated, and have many reaction characteristics such as halogenation, dehydration, esterification, sulfonation, acylation, oxidation, reduction, etc. Therefore, succinic acid is often used as a chemical raw material and intermediate to prepare a series of active succinic acid derivatives, which are widely used in chemical, food, pharmaceutical and other industries. Succinic acid can be used to prepare surfactants, cleaning agents, additives and foaming agents. Due to the structure of polycarboxylic acid, it can also be used as an ionophore agent for corrosion prevention, such as corrosion and pitting in the electroplating industry. In the food industry, it is used as an acidifier, pH modifier, antibacterial agent, fortifier and emulsifier. In addition, succinic acid is also widely used in the medical industry, including the production of medicines, antibiotics, amino acids and vitamins.

[0003] Succinic acid derivatives have more excellent performance than conventional surfactants due to their unique structure, among which sulfosuccinic acid salts are the most widely used. Most sulfosuccinic acid monoesters are gradually unable to meet the requirements due to their single structure. The sulfosuccinic acid diester surfactant in the prior art is a symmetrical sulfosuccinic acid diester, which has improved surface activity performance, but its structure is still relatively simple and cannot meet the requirements for its surface activity. In addition, its water solubility, wettability, dispersibility and foam stability are insufficient, which limits its application range. Moreover, the structure connected to the sulfosuccinic acid ester salt in the prior art is a single long-chain hydrocarbon, which has insufficient acid and alkali resistance and stability, resulting in poor bioavailability.

[0004] At present, there are few studies on succinic acid derivatives (i.e., structural features with both succinic acid ester and succinamide structures). In the daily chemical industry, they are only used as emulsifiers, and have the limitation of low bioavailability.

[0005] Therefore, it is urgent to study a succinic acid derivative with good acid and alkali resistance and stability, and unique biological activity, i.e., a compound with good antioxidant, soothing or whitening ability, which has the potential to be used as an effective cosmetic raw material in the daily chemical industry. SUMMARY

[0006] The purpose of the present application is to provide a succinic acid derivative for use in cosmetics. The succinic acid derivative of the present application has not only good bioavailability, but also good antioxidant, anti-inflammatory and whitening effects when used in cosmetics.

[0007] The technical scheme of the present application: the application of a succinic acid derivative in cosmetics, the application of the succinic acid derivative in preparing cosmetics with antioxidant, soothing and / or whitening effects; the structural formula of the succinic acid derivative is:

[0008]

[0009] In the aforementioned application of a succinic acid derivative in cosmetics, the CAS number of the succinic acid derivative is 1449076-27-0, the molecular formula is C 15 H 27 NO3, and the molecular weight is 269.385.

[0010] In the aforementioned application of a succinic acid derivative in cosmetics, the addition amount of the succinic acid derivative in cosmetics with antioxidant effect is 0.0001%-2% in mass percentage.

[0011] In the aforementioned application of a succinic acid derivative in cosmetics, the addition amount of the succinic acid derivative in cosmetics with soothing effect is 0.0001%-2% in mass percentage.

[0012] In the aforementioned application of a succinic acid derivative in cosmetics, the addition amount of the succinic acid derivative in cosmetics with whitening effect is 0.0001%-2% in mass percentage.

[0013] Compared with the prior art, the present application applies a succinic acid derivative with a structural formula of to cosmetics, which not only has good antioxidant, soothing and whitening effects, but also has good bioavailability. Experiments show that,

[0014] 1) The experimental sample has a melanin inhibitory effect at concentrations of 62.5 μM and 125 μM, supporting the whitening effect claim of the experimental sample when applied in cosmetic formulations.

[0015] 2) Based on fibroblasts, the reactive oxygen species (ROS) content significantly decreases after the experimental sample is treated at a concentration of 0.0063% (v / v), indicating that the sample achieves an antioxidant effect by reducing the content of reactive oxygen species (ROS) at this concentration, supporting the antioxidant effect claim of the experimental sample when applied in cosmetic formulations.

[0016] 3) Based on macrophages, the inflammatory mediator (NO) content significantly decreases after the experimental sample is treated at a concentration of 0.0016% (v / v), indicating that the sample achieves the effect of inhibiting the generation of inflammatory mediator (NO) at this concentration. The experimental sample can significantly inhibit the aggregation of neutrophils in zebrafish embryos at formulation addition concentrations of 0.1%, 0.05% and 0.025%, and has a soothing effect. This supports the soothing effect claim of the experimental sample when applied in cosmetic formulations. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a statistical chart of the effect of the experimental sample on B16F10 cell toxicity in the whitening efficacy test;

[0018] Figure 2 is a melanin production chart (100x) under a microscope of each group in the whitening efficacy test;

[0019] Figure 3 is a statistical chart of the effect of the experiment on the release of cell melanin in the whitening efficacy test;

[0020] Figure 4 is a result chart of the model control group in the neutrophil aggregation inhibition test of zebrafish embryos;

[0021] Figure 5 is a result chart of the positive control group in the neutrophil aggregation inhibition test of zebrafish embryos;

[0022] Figure 6 is a result chart of the experimental sample group (0.1% formula addition concentration) in the neutrophil aggregation inhibition test of zebrafish embryos;

[0023] Figure 7 is a result chart of the experimental sample group (0.05% formula addition concentration) in the neutrophil aggregation inhibition test of zebrafish embryos;

[0024] Figure 8 is a result chart of the test sample group in the neutrophil aggregation inhibition test of zebrafish embryos. DETAILED DESCRIPTION

[0025] The present application will be further described below in conjunction with the accompanying drawings and examples, but not as a basis for limiting the present application.

[0026] Example. Application of a succinic acid derivative in cosmetics, application of a succinic acid derivative in the preparation of cosmetics with antioxidant, soothing and / or whitening efficacy.

[0027] The structural formula of the succinic acid derivative is:

[0028] The CAS number of the succinic acid derivative is 1449076-27-0, the molecular formula is C 15 H 27 NO3, and the molecular weight is 269.385.

[0029] The addition amount of the succinic acid derivative in cosmetics with antioxidant efficacy is 0.0001%-2% by mass percentage. Further preferably, the mass percentage is 0.005%-0.007%.

[0030] The addition amount of the succinic acid derivative in the cosmetic with soothing effect is 0.0001%-2% by mass. Further preferably, 0.02%-0.01% by mass.

[0031] The addition amount of the succinic acid derivative in the cosmetic with whitening effect is 0.0001%-2% by mass. Further preferably, the addition concentration is 50-125 μM.

[0032] The succinic acid derivative was subjected to whitening, soothing and antioxidant experiments, respectively, and the specific experiments are as follows.

[0033] A. Whitening effect test

[0034] Experiment 1:

[0035] Experimental method: Effect of experimental sample on B16F10 cell release of melanin

[0036] 1.1. CCK-8 detection of sample product on B16F10 cell toxicity (screening whitening experiment concentration)

[0037] The sample was prepared with 1640 complete culture medium, and the experimental sample concentration was set to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μM, and a blank control group (i.e. normally cultured cells without sample intervention) was also set. In this experiment, the blank control group is marked as NC.

[0038] Logarithmic growth phase B16 cells were inoculated in a 96-well plate and incubated in a CO2 incubator. When the cell confluence rate reached 80%, the above prepared sample and control solution were added to each well, and incubated in a CO2 incubator for 24 h.

[0039] CCK-8 detection of sample cell proliferation activity: After incubation for 24 h, 100 μl of CCK-8 working solution was added to each well, and incubated in a CO2 incubator for 1 h. The absorbance was measured at 450 nm.

[0040] Cell survival rate calculation formula: Survival rate = (OD sample group / OD blank control group) %

[0041] 1.2. Alkaline lysis method for detecting the effect of experimental sample on B16F10 cell release of melanin

[0042] The experimental groups were set according to the following method

[0043]

[0044] Note: Except for the BC group, all other groups added IBMX (100 uM) to intervene the cells.

[0045] 1) Take the logarithmic growth phase B16 cells to inoculate in 24-well plates, incubate in an incubator;

[0046] 2) After 24h of incubation, prepare the above experimental samples with DMEM medium and add to the corresponding wells, and place in a CO2 incubator for 24h of intervention;

[0047] 3) After 24h, repeat the operation in 2) above;

[0048] 4) After the intervention, collect each group of cells respectively, add 10% DMSO-NaOH to dissolve and boil to lyse the cells, collect melanin, and detect the absorbance at 405nm wavelength;

[0049] 5) Melanin release rate calculation formula: (OD sample group-OD blank control group) / (OD model control group-OD blank control group) x 100%

[0050] Experimental results

[0051] CCK-8 results show that experimental samples at a concentration of 125μM and below have no toxic effect on B16F10 cells, and the cell survival rate in this concentration range is higher than 90%, therefore the selected concentration for this whitening efficacy detection is 125, 62.5 and 31.25μM (see Figure 1 , Table 1).

[0052] Table 1 Experimental sample toxicity data table on B16F10 cells

[0053]

[0054] In the melanin inhibition experiment results, the IBMX induction group (i.e. model control group NC) has a significantly increased melanin release compared with the blank control group, and the arbutin group (i.e. positive control group PC) has a significantly reduced melanin release rate compared with the NC group, indicating that the experimental results are effective.

[0055] The experimental results show that the experimental samples have a melanin inhibition effect at a certain concentration (62.5μM and 125μM), and the inhibition effect is significantly different from the NC group (see Figure 2 , Figure 3 , Table 2).

[0056] Table 2 Experimental sample data table on cell melanin release

[0057]

[0058] Note: significance is indicated by *, *** in the figure indicates P≤0.005 compared with the NC group, and ** indicates P≤0.01 compared with the NC group.

[0059] II. Summary

[0060] The experimental sample had melanin inhibiting effect at 62.5 μM and 125 μM concentrations, and the inhibiting effect was significant compared with the NC group.

[0061] B. Antioxidant efficacy test

[0062] Experiment 1: Detection of reactive oxygen species (ROS) content based on UVA irradiation of fibroblasts

[0063] Experimental method: fibroblast-based test method

[0064] (1) Inoculation: inoculate cells into a 6-well plate at a seeding density of 1.6 x 10 5

[0065] (2) Liquid preparation: prepare the experimental sample working solution according to the test groups (Table 3).

[0066] (3) Dosing: according to the test groups, when the plating rate of the cells in the 6-well plate reaches 30% to 50%, dosing is performed, and 3 replicate wells are set up for each group. The sample group is added with 2 mL of culture solution containing the experimental sample at the corresponding concentration per well; the negative control group is added with 2 mL of culture solution per well; the positive control group is added with 2 mL of positive control culture solution per well; and the blank control group is added with 2 mL of culture solution per well. After the sample is added, the 6-well plate is placed in an incubator (37°C, 5% CO2) for 24 h.

[0067] (4) ROS content test: after 24 h of incubation, the cells are washed with PBS, 1 mL of probe working solution (the probe is diluted 1:1000 in serum-free culture medium) is added per well, and the cells are incubated at 37°C for 20 min. The probe working solution is aspirated, the cells are washed with PBS 3 times, the positive control group is added with positive standard working solution prepared with PBS, and the other wells are added with 1 mL of PBS; UVA irradiation is performed on the remaining groups except the blank control group, and the irradiation dose is 30 J / cm2. After irradiation, the culture medium is directly replaced with 2 mL per well, and the cells are incubated in the incubator for 30 min. After incubation, the cells are digested with trypsin (0.25%), washed with PBS, and subjected to machine detection.

[0068] (5) Result statistical analysis: GraphPad Prism is used for plotting, and the results are expressed as Mean ± SD. t-test statistical analysis is used for comparison between groups. P < 0.05 is considered to have significant difference, and P < 0.01 is considered to have extremely significant difference.

[0069] Table 3

[0070]

[0071] Experimental results:​

[0072] Table 4. ROS content result summary table

[0073]

[0074] Note: MFI represents the mean fluorescence intensity. The significance compared with the BC group is indicated by #, P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##; the significance compared with the NC group is indicated by *, P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **.

[0075] Inhibition rate = (NC-sample) / NC x 100%.

[0076] The ROS content of the NC group increased significantly compared with the BC group, indicating that the stimulation conditions of this test were effective.

[0077] The ROS content of the PC group decreased significantly compared with the NC group, indicating that the positive control of this test was effective.

[0078] The ROS content of the experimental sample at a concentration of 0.0063% decreased significantly compared with the NC group, with an inhibition rate of 10.06%.

[0079] Conclusion:

[0080] Based on fibroblasts, the ROS content of the experimental sample at a concentration of 0.0063% (v / v) decreased significantly compared with the control group, with an inhibition rate of 10.06%, indicating that the sample at this concentration achieved antioxidant effects by reducing the ROS content.

[0081] C. Soothing efficacy test

[0082] Experiment 1: Based on the ROS content detection of fibroblasts after UVA irradiation

[0083] Table 5

[0084]

[0085] Positive control group (dexamethasone) working solution preparation: 2 μL of 10% mother liquor was dissolved in 2 mL of culture medium to prepare 0.01% dexamethasone

[0086] (1) Cell inoculation: 2.2 x 10 5The cells were inoculated into 6-well plates at a seeding density of 1 cell / well, 2 mL of cell suspension was added to each well, and the inoculated cell culture plates were placed in an incubator for further culture for 24 h (37°C, 5% CO2).

[0087] (2) The old cell culture solution in the hole was sucked and discarded. 1.8 mL of culture solution containing the corresponding concentration of experimental sample was added to each hole of the sample group; 1.8 mL of solvent control culture solution was added to each hole of the negative control group; 1.8 mL of positive control culture solution was added to each hole of the positive control group; 1.8 mL of normal culture solution was added to each hole of the blank control group.

[0088] (3) After the administration was completed, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) for 2 h.

[0089] (4) LPS stimulation: After 2 h of administration, 200 μL of normal culture solution was added to the blank control group, and 200 μL of prepared LPS-containing working solution was added to each hole of the remaining groups, which were placed in a CO2 incubator (37°C, 5% CO2) for further culture for 22 h.

[0090] (5) NO detection: The cell culture supernatant was collected, and NO detection was performed according to the NO kit instructions.

[0091] Experimental results

[0092] According to the specific test scheme, the NO content was detected, and the detection results are shown in the following chart 6

[0093] Table 6. NO content result summary table

[0094]

[0095] Note: When statistical analysis is performed by t-test method, compared with BC group, the significance is represented by #, P-value <0.05 is represented by #, and P-value <0.01 is represented by ##; compared with NC group, the significance is represented by *, P-value <0.05 is represented by *, and P-value <0.01 is represented by **.

[0096] Inhibition rate = (NC-sample) / NC x 100%.

[0097] Compared with the BC group, the NO content of the NC group increased significantly, indicating that the stimulation conditions of this test were effective.

[0098] Compared with the NC group, the NO content of the PC group decreased significantly, indicating that the positive control of this test was effective.

[0099] Compared with the NC group, the NO content of the experimental sample group W335 at a concentration of 0.0016% decreased significantly, with an inhibition rate of 49.64%.

[0100] IV. Conclusion

[0101] Based on macrophages, compared with the control group, the experimental sample at a concentration of 0.0016% (v / v) significantly reduced the content of inflammatory mediators (NO), with an inhibition rate of 49.64%, indicating that the sample at this concentration achieved soothing effect by inhibiting the secretion of inflammatory mediators (NO).

[0102] Experiment 2: Zebrafish embryo neutrophil aggregation inhibition test

[0103] Experimental method:

[0104] (1) Select healthy zebrafish embryos 3 days after fertilization. Set up model control group (copper sulfate working solution), positive control group (copper sulfate working solution + indomethacin working solution) and experimental sample group (copper sulfate working solution + experimental sample) for testing.

[0105] (2) Model control group setting

[0106] Randomly select 24 fish embryos into a 3 cm culture dish, and add 5 mL of fish embryo culture solution containing 0.16 mg / L copper sulfate pentahydrate.

[0107] (3) Positive control group setting

[0108] Randomly select 24 fish embryos into a 3 cm culture dish, and add 5 mL of fish embryo culture solution containing 0.16 mg / L copper sulfate pentahydrate and 0.0036 mg / L indomethacin.

[0109] (4) Experimental sample treatment

[0110] Randomly select 24 fish embryos into a 3 cm culture dish, and add 5 mL of fish embryo culture solution containing 0.16 mg / L copper sulfate pentahydrate and experimental sample. Place in a 28°C±1°C incubator for 40-45 min.

[0111] (5) Fish embryo fixation and staining

[0112] Fix each test group of fish embryos in paraformaldehyde for at least 1 h, then treat the fish embryos with PBST for 3 times, 5 min each time, then treat with 50% ethanol for 3 min. Stain the fish embryos with Sudan black staining solution at room temperature for 1 h, then immerse in 70% ethanol for 4 times, 5 min each time, then treat with PBST for 2 times, 5 min each time.

[0113] Treat the fish embryos with bleach solution for 10 min, if treated in a test tube, keep the lid open. Then treat the fish embryos with 70% ethanol solution

[0114] Treat for 5 min, PBST for 1 min, Clearing Solution 1 for 15 min, Clearing Solution 2 for 10 min, and PBST for 3 min.

[0115] (6) Microscopic analysis of samples

[0116] Place the fish embryo on its side and then take a picture of the tail under a stereomicroscope.

[0117] (7) Data and result calculation

[0118] Count the number of neutrophils in the lateral line region of the fish embryo three quarters of the way from the anus.

[0119] (8) Calculation of neutrophil aggregation inhibition rate:

[0120] Inhibition rate = (C - S) / C x 100%; where:

[0121] S - average number of neutrophils in the fish embryo in the test group;

[0122] C - average number of neutrophils in the fish embryo in the model control group;

[0123] Results of the experiment

[0124] Table 7

[0125]

[0126] Representative graph of the soothing efficacy test results (* dark dots are stained neutrophils).

[0127] Model control group: no significant soothing effect (0% neutrophil aggregation inhibition rate), as shown in Figure 4 .

[0128] Example of positive control group: 0.0036 mg / L indomethacin; significant soothing effect (30% neutrophil aggregation inhibition rate), as shown in Figure 5 .

[0129] Experimental sample group: 0.1% formulation addition concentration (36% neutrophil aggregation inhibition rate); significant soothing effect, as shown in Figure 6 .

[0130] Experimental sample group:

[0131] Significant soothing effect: 0.05% formulation addition concentration (37% neutrophil aggregation inhibition rate), as shown in Figure 7 .

[0132] Test sample group:

[0133] Significant soothing effect: 0.025% formulation addition concentration (30% neutrophil aggregation inhibition rate), as shown in Figure 8

[0134] IV. Conclusion

[0135] The sample at 0.1%, 0.05% and 0.025% formulation addition concentration had a neutrophil aggregation inhibition rate of 36% (p=0.0021), 37% (p=0.00080) and 30% (p=0.012) in zebrafish embryos, respectively.

[0136] The experimental sample can significantly inhibit the aggregation of neutrophils in zebrafish embryos, has a soothing effect, and supports the soothing efficacy claim.​

Claims

1. The application of a succinic acid derivative in cosmetics, characterized in that: Application of succinic acid derivatives in the preparation of cosmetics with soothing effects; the structural formula of the succinic acid derivatives is: ； The CAS number of the succinic acid derivative is 1449076-27-0, and its molecular formula is C2. 15 H 27 NO3 has a molecular weight of 269.

385. The succinic acid derivative is added in cosmetics with soothing effects at a rate of 0.0001%-2% by mass.

Images