A whitening composition

Abstract

This invention discloses a skin-whitening composition comprising a succinic acid derivative and tranexamic acid, wherein the mass ratio of the succinic acid derivative to tranexamic acid is 1:1 to 1:50. This invention has the advantages of low cost, good skin-whitening effect, and good safety.

Description

Technical Field

[0001] This invention belongs to the field of skin whitening products, and particularly relates to a whitening compound and composition. Background Technology

[0002] Most people admire "fair skin as beauty," especially Chinese women. Globally, about 7 out of 20 people use skin whitening products, making them an important category in the cosmetics market.

[0003] As is well known, Chinese people have long pursued a complexion as beautiful as peach blossoms and skin as smooth as cream. In ancient times, women would apply lead powder to their faces to achieve beautiful, fair skin. Lead-containing whitening products were famous for their rapid effects and obvious whitening results, but their use was strictly prohibited due to their high toxicity, strong irritation, instability, and tendency to accumulate in the body.

[0004] Currently, skin whitening products prioritize safety and effectiveness, with plant extracts and chemically synthesized ingredients being the preferred choices. However, plant extracts suffer from issues such as batch instability and irritation from impurities, which still require improvement. Chemically synthesized ingredients, on the other hand, are characterized by high purity, well-defined molecular structures, ease of mass production, and relatively clear safety assessment methods, making them widely used in the cosmetics industry. Ergothioneine, for example, is a commonly used chemically synthesized ingredient in the cosmetics industry.

[0005] Application No. 202311103185.4 discloses a succinic acid derivative (3,3,5-trimethylcyclohexylsuccinate dicarboxamide) with a specific structure, which is chemically synthesized and possesses antioxidant, soothing, and whitening effects, as well as good bioavailability. This succinic acid derivative is a newly developed substance, and its current preparation cost is relatively high.

[0006] In addition, experiments have shown that tranexamic acid also has a whitening effect, but the whitening effect is weak. The concentration of tranexamic acid added generally needs to reach more than 5%, but tranexamic acid with a concentration of more than 5% will cause high irritation to the skin, which may lead to other skin problems. Summary of the Invention

[0007] The purpose of this invention is to provide a whitening composition. This invention has the advantages of low cost, good whitening effect, and good safety.

[0008] The technical solution of this invention: A whitening composition comprising a succinic acid derivative and tranexamic acid, wherein the structure of the succinic acid derivative is as follows.

[0009]

[0010] In the aforementioned whitening composition, the mass ratio of the succinic acid derivative to tranexamic acid is 1:1 to 1:50.

[0011] In the aforementioned whitening composition, the mass ratio of the succinic acid derivative to tranexamic acid is 1:2 to 1:20.

[0012] In the aforementioned whitening composition, the mass ratio of the succinic acid derivative to tranexamic acid is 1:10.

[0013] In the aforementioned whitening composition, the proportion of the composition added to the whitening product is 0.1% to 4%.

[0014] Compared with existing technologies, the beneficial effects of this invention are that, through secondary development of existing succinic acid derivatives, the combination of succinic acid derivatives and tranexamic acid yields a composition that can better inhibit skin pigmentation, effectively prevent or improve skin pigmentation-related spots, and achieve skin whitening. Furthermore, the succinic acid derivatives in this invention achieve the effects of single use at higher concentrations with lower dosages, significantly reducing costs and avoiding the development of other skin problems, thus improving safety. In summary, this invention has the advantages of low cost, good whitening effect, and good safety. Attached Figure Description

[0015] Figure 1 This is a graph showing the changes in melanin content synergistically regulated by ubiquitination and autophagy after W335 and TXA are combined. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0017] Example. A skin-whitening composition comprising a succinic acid derivative and tranexamic acid, wherein the structure of the succinic acid derivative is as follows:

[0018]

[0019] The mass ratio of succinic acid derivative to tranexamic acid is 1:2, 1:5 or 1:10, with 1:10 being the optimal ratio.

[0020] The composition is added to the whitening product at a rate of 0.1% to 4%.

[0021] Experimental example.

[0022] I. Experimental Principle

[0023] Skin whitening products are substances that act on a specific stage of the formation and metabolism of melanin in the skin, significantly controlling and inhibiting melanin production while meeting safety requirements. This experiment used B16 cells as the experimental subject. These melanocytes are physiologically similar to human melanocytes. A B16 cell model was established, and cytotoxicity was first measured. Then, by selecting active ingredients with low cytotoxicity, such as succinic acid derivatives (W335) or tranexamic acid (TXA), and controlling the concentration (generally requiring a cell viability rate of over 80%), the melanin content was measured. The whitening effect of the active ingredients was evaluated by the relative melanin content. Following this, in the mechanistic study, previous research on whitening mechanisms mainly focused on tyrosinase or MITF. Currently, there is considerable research on whitening mechanism targets, including the effects of autophagy and ubiquitinated proteasomes on melanin production. Based on literature reports and transcriptome data analysis of W335 in melanocytes, this experiment conducted a mechanistic study based on this.

[0024] II. Experimental Objective: To investigate whether W335 and TXA have whitening effects, and whether their combination can have a synergistic effect. To explore whether the whitening effect is achieved by reducing melanin production through ubiquitination and autophagy, to determine whether the synergistic mechanism is innovative, and to find that the complex can reduce the concentration of TXA, thus achieving a better, safer, and more effective effect, providing a basis for developing more stable and effective whitening products.

[0025] III. Experimental Consumables and Reagents

[0026] (a) Consumables:

[0027] Biosafety cabinet, water bath, centrifuge, analytical balance, 96-well plate, 24-well plate, microscope, microplate reader, 30-300μL eight-channel pipette, 1000μL pipette, 200μL pipette, 1000μL pipette tip, 200μL pipette tip, 10μL pipette tip.

[0028] (II) Reagents:

[0029] Phosphate buffer (pH≈7.4), DMEM medium, 0.25% trypsin solution, antibiotics, fetal bovine serum, CCK8 reagent, dimethyl sulfoxide (DMSO), 1M NaOH solution, active ingredient W335 and tranexamic acid (TXA), 3-methyladenine (3-MA, autophagy inhibitor), MG132 (ubiquitination-proteasome inhibitor).

[0030] (III) Reagent Preparation:

[0031] 1. Preparation of W335: Reagent grade, oil-soluble raw material, requires 0.1% DMSO as a co-solvent for dissolution. Take 1 μL of W335 into 1 mL of PBS or DMEM complete culture medium containing 0.1% DMSO in an EP tube, vortex to mix, and after complete dissolution, dilute several times to obtain the stock solution concentration. Set 5–8 concentration gradients for each sample, with 5–6 replicates for each concentration.

[0032] 2. Preparation of Tranexamic Acid (TXA): Reagent grade, water-soluble raw material. Weigh 0.1g of TXA and dissolve it in a 10mL centrifuge tube containing PBS or DMEM complete culture medium. Shake to mix well and dilute several times after complete dissolution. This concentration is used as the stock solution. Set 5-8 concentration gradients for each sample to be tested, with 5-6 replicates for each concentration.

[0033] 3.3-MA Preparation: Reagent grade, can be dissolved in water, PBS or DMSO. Weigh 0.1g of 3-MA and dissolve it in a centrifuge tube containing 6.7047mL of complete medium (water, PBS or DMEM). Shake to mix well and dilute several times after complete dissolution. This stock solution has a concentration of 10mM.

[0034] 4. Preparation of MG132: Reagent grade, can be dissolved in DMSO. Weigh 0.1g of 3-MA and dissolve it in a centrifuge tube containing 2.103mL of water, PBS or DMEM complete medium. Shake to mix well and dilute several times after complete dissolution. This stock solution has a concentration of 10mM.

[0035] IV. Experimental Procedure

[0036] (I) Cytotoxicity assay

[0037] 1. Cell seeding: Melanocytes with 80% confluence were seeded into 96-well plates with 100 μL of cell suspension per well to achieve a cell count of 6–7 × 10⁶ cells. 4 One hole / hole.

[0038] 2. Observe the cells: If the cells are in good condition and there are a large number of them, the next step of the experiment can be carried out.

[0039] 3. Washing and adding samples: Discard the culture medium, wash with PBS 1-2 times, then add 100 μL / well of high-glucose DMEM complete medium, and set up a blank group and a sample group. Then add 10 μL of sample to each well of the sample group, and then incubate in a 37℃, 5% CO2 incubator for 24 h.

[0040] 4. Add CCK-8 reagent: After observing the cells, add 10 μL of CCK-8 reagent per well and incubate for 1-4 hours.

[0041] 5. Microplate reader detection: After incubation, the OD450 value was measured using a microplate reader. The measured data were then processed and plotted into a bar chart using Graphpad Prism software.

[0042] 6. Data analysis: If the cell survival rate reaches 80% or more compared with the control group, it indicates that the sample is non-toxic to cells.

[0043] 7. Concentration selection: Based on the toxicity test results, the ratio of W335 to TXA was selected as 1:10 for subsequent experiments.

[0044] (II) Melanin content determination—synergistic effect of W335 and TXA combination

[0045] 1. Cell seeding: Melanocytes with 80% confluence were seeded into 24-well plates with 100 μL of cell suspension per well to achieve a cell count of 9–10 × 10⁶ cells. 4 One hole / hole.

[0046] 2. Observe the cells: If the cells are in good condition and in appropriate numbers, the next step of the experiment can be carried out.

[0047] 3. Set up groups: Based on the toxicity test results and well plate conditions, set up the following groups: blank group, W335 group, TXA group and W335+TXA combination group (combination ratio 1:10).

[0048] 4. Washing and sample loading: Discard the culture medium, wash 1-2 times with PBS, then add 100 μL / well of high-glucose DMEM complete medium. Add the appropriate amount of W335 to the wells where W335 needs to be added; add the appropriate amount of TXA to the wells where TXA needs to be added, keeping the final total volume consistent. Then incubate the plate at 37°C in a 5% CO2 incubator for 48 hours.

[0049] 5. Cell observation: When observing cells under a microscope, a test can be performed when a significant color change is observed.

[0050] 6. Cell Collection: Prepare the water bath by setting it to 80°C and pre-cooling with PBS buffer. Then, digest the cells in the wells with trypsin, centrifuge, discard the supernatant, and collect the cells into new EP tubes. Wash twice with pre-cooled PBS to terminate the drug reaction.

[0051] 7. Cell lysis: After washing, add 100 μL of 1M NaOH to each tube, then place the EP tube on a foam float and heat in a water bath for about 5-10 minutes until the melanin is completely dissolved.

[0052] 8. Melanin absorbance measurement: Take 70 μL of lysis buffer from each tube into a 96-well plate and measure the absorbance value using an ELISA reader at OD405.

[0053] 9. Calculation of results: Melanin content (%) = (Sample melanin content value OD / Blank melanin content value OD).

[0054] 10. Draw a bar chart: Based on the calculated results, draw a bar chart using Graphpad Prism software.

[0055] (III) Melanin Content Determination—Mechanism Study of W335 and TXA Combination

[0056] 1. Cell seeding: Melanocytes with 80% confluence were seeded into 24-well plates with 100 μL of cell suspension per well to achieve a cell count of 9–10 × 10⁶ cells. 4 One hole / hole.

[0057] 2. Observe the cells: If the cells are in good condition and in appropriate numbers, the next step of the experiment can be carried out.

[0058] 3. Set the groups: Set the corresponding groups according to the orifice plate: blank group, W335 group, W335+MG132 group, W335+MG132+3-MA group, TXA group, TXA+MG132 group, TXA+MG132+3-MA group, W335+TXA compound group (compound ratio is 1:10), W335+TXA+MG132 group and W335+TXA+MG132+3-MA group.

[0059] 4. Washing and sample loading: Discard the culture medium, wash 1-2 times with PBS, then add 100 μL / well of high-glucose DMEM complete medium. Add the appropriate amount of W335 to the wells where W335 needs to be added; add the appropriate amount of TXA to the wells where TXA needs to be added; then add the appropriate concentration of MG132 or 3-MA to the wells where inhibitors need to be added, ensuring a consistent total volume. Incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0060] 5. Cell observation: When observing cells under a microscope, a test can be performed when a significant color change is observed.

[0061] 6. Cell collection: Prepare the constant temperature water bath to 80℃ and pre-cool the PBS buffer in advance.

[0062] Then, trypsin was used to digest the cells in the well plate, the supernatant was discarded by centrifugation, and the cells were collected into a new EP tube.

[0063] The drug reaction was then terminated by washing twice with pre-cooled PBS.

[0064] 7. Cell lysis: After washing, add 100 μL of 1M NaOH to each tube, then place the EP tube on a foam float and heat in a water bath for about 5-10 minutes until the melanin is completely dissolved.

[0065] 8. Melanin absorbance measurement: Take 70 μL of lysis buffer from each tube into a 96-well plate and measure the absorbance value at OD 405 nm using a microplate reader.

[0066] 9. Calculation of results: Melanin content (%) = (Sample melanin content value OD / Blank melanin content value OD)

[0067] 10. Draw a bar chart: Based on the calculated results, draw a bar chart using Graphpad Prism software.

[0068] (V) Results Analysis

[0069] 1. The toxic effects of W335 or TXA on melanocytes.

[0070] Based on the toxicity test results, as shown in Table 1, we found that when melanocytes were treated with W335 active ingredient, the cell viability was above 80% when the W335 concentration was ≤0.01%, indicating that it was non-toxic to the cells. As shown in Table 2, we found that when melanocytes were treated with TXA active ingredient, the cell viability was above 80% when the TXA concentration was ≤0.1%, indicating that it was non-toxic to the cells.

[0071]

[0072] Table 1 (Cytotoxic effects of W335 on melanocytes)

[0073]

[0074] Table 2 (Toxic effects of TXA on melanocytes)

[0075] Note: Mean±SD represents the mean ± standard deviation.

[0076] 2. The synergistic effect of W335 and TXA in reducing melanin content in melanocytes.

[0077] Based on the toxicity test results, we found that W335 or TXA was non-toxic to cells at concentrations ≤0.01% or ≤0.1%. Combining this with previous experiments, we decided to use a 1:10 ratio of W335 to TXA for subsequent melanin content determination in this experiment. The results are shown in Table 3. We found that compared to W335 or TXA alone, the melanin content in melanocytes was significantly reduced at a medium dose with a 1:10 ratio of W335 to TXA.

[0078]

[0079] Table 3 (Synergistic effect of W335 combined with tranexamic acid on changes in melanin content)

[0080] Note: Statistical analysis was performed between groups. "SD" represents standard deviation, "*" indicates significance, ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.

[0081] 3. The combination of W335 and TXA reduces melanin production through the synergistic regulation of ubiquitination-proteasome and autophagy.

[0082] Based on the above results, we found that compared with the single-component groups (335 or TXA), the melanin content was significantly reduced when the W335 to TXA ratio was 1:10. Therefore, in this experiment, we still used the W335 to TXA ratio of 1:10 to explore the mechanism. According to literature reports, some melanin can regulate its content by controlling autophagy or ubiquitination-proteasome action. In this experiment, we found a similar phenomenon, as shown in the results below. Figure 1 As shown, the experimental results revealed that compared with the control group, except for the W335+TXA+MG132+3-MA group, the melanin content of all other groups decreased significantly, and the melanin content of the compound group was significantly lower than that of the single component group; compared with the W335 group, the melanin content of the group with the corresponding MG132 or 3-MA was inhibited to a certain extent; compared with the TXA group, the melanin content of the group with the corresponding MG132 or 3-MA was inhibited to a certain extent.

[0083] 3,3,5-trimethylcyclohexylsuccinate dimethylamide (W335), along with tranexamic acid, both possess whitening effects. When combined, they exhibit synergistic effects. Mechanism analysis revealed that their whitening effect involves melanin regulation induced by ubiquitination and autophagy, demonstrating an innovative synergistic mechanism. Furthermore, the complex can reduce the concentration of tranexamic acid, resulting in a better, safer, and more effective effect, thus providing possibilities for developing more stable and effective whitening agent compositions.

Claims

1. A whitening composition, characterized in that: This includes succinic acid derivatives and tranexamic acid. The structure of the succinic acid derivatives is as follows. ； The mass ratio of the succinic acid derivative to tranexamic acid is 1:1 to 1:

50.

2. The whitening composition according to claim 1, characterized in that: The mass ratio of the succinic acid derivative to tranexamic acid is 1:2 to 1:

20.

3. The whitening composition according to claim 2, characterized in that: The mass ratio of the succinic acid derivative to tranexamic acid is 1:10.

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