Application of bardoxolone in whitening

Abstract

The application belongs to the technical field of cosmetics, and discloses application of bardoxolone in whitening. It is found for the first time that bardoxolone has a whitening related effect, which is specifically manifested in that the generation of melanin can be inhibited, the activity of tyrosinase can be inhibited, and the expression of a gene related to the synthesis of melanin can be inhibited, the inhibition effect is strong, the effective concentration is low, and the component has no irritation to red blood cells, which indicates that the component has a certain safety as a potential raw material component of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to the application of bardoxolone in whitening. BACKGROUND

[0002] Human melanin synthesis is a very complex biological process, which is mainly synthesized by melanocytes, transmitted to adjacent keratinocytes through the dendritic processes of melanocytes, and accompanied by the differentiation and movement of keratinocytes to the upper layer of the epidermis, thereby affecting the presentation of skin color. Once abnormal synthesis, metabolism, uneven distribution, etc. of melanin occur in the body, it is easy to cause local pigment deposition of the skin, and cause problems such as chloasma and age spots, which not only affect the appearance, but also may cause melanoma and other lesions. At present, most of the available whitening raw materials on the market are inhibitors of tyrosinase activity, such as kojic acid, arbutin, resveratrol, glabridin, benzene glycol resorcinol, and rhododendrol. Some of the ingredients can inhibit melanin transport, such as nicotinamide, or reduce melanin, such as vitamin C, thereby achieving the effect of whitening.

[0003] Bardoxolone (CAS No. 218600-44-3), also known as CDDO or RTA401, is a new type of activator of nuclear regulatory factor NRF2, belonging to oleanane type triterpenoids, which can be used for chronic kidney disease research. It has been disclosed that bardoxolone is an effective inhibitor of necroptosis, which can inhibit Z-VAD-FMK-induced necroptosis. At present, most of the existing patents around bardoxolone are mainly concentrated in the field of medicine, such as the existing patent technology which points out that it can be used for preparing N-Myc positive tumor treatment drugs (publication number: CN115252623A), its methyl derivative can be applied to preparing drugs for inhibiting respiratory syncytial virus infection (publication number: CN116473979A), and drugs for improving viral myocarditis or viral myocardial injury (publication number: CN116509874A), etc. At present, there is no application of bardoxolone in whitening. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides the application of bardoxolone in whitening. It is found for the first time that bardoxolone has a significant effect in whitening, including inhibiting tyrosinase activity, inhibiting melanin production, and inhibiting the expression of melanin synthesis related genes.

[0005] In a first aspect of the present application, the application of bardoxolone in preparing products with whitening effect is provided.

[0006] In some embodiments of the present application, the amount of bardoxolone added to the product is ≤ 10 wt%, preferably ≤ 5 wt%, further preferably ≤ 1 wt%.

[0007] In some embodiments of the present application, the amount of bardoxolone added to the product is 0.0000001 wt% (0.001 ppm) - 1 wt%, preferably 0.000005 wt% (0.05 ppm) - 1 wt%, further preferably 0.00005 wt% (0.5 ppm) - 1 wt%.

[0008] In some embodiments of the present application, the amount of bardoxolone added to the product is 0.001 wt% - 1 wt%, preferably 0.01 wt% - 1 wt%, further preferably 0.01 wt% - 0.5 wt%, more further preferably 0.01 wt% - 0.2 wt%.

[0009] In some embodiments of the present application, the whitening comprises at least one of inhibiting tyrosinase activity, inhibiting melanin content, and inhibiting expression of a melanin synthesis-related gene.

[0010] In some embodiments of the present application, the melanin synthesis-related gene comprises at least one of a TYR gene, a TYRP1 gene, a TRP2 gene, a PMEL gene, and a GPNMB gene.

[0011] In a second aspect of the present application, a composition having whitening efficacy is provided, the composition comprising bardoxolone or one or more of pharmaceutically acceptable acids, bases, salts, esters, solvates, stereoisomers, tautomers thereof.

[0012] In some embodiments of the present application, the amount of bardoxolone in the composition is ≤ 10 wt%, preferably ≤ 5 wt%, further preferably ≤ 1 wt%.

[0013] In some embodiments of the present application, the amount of bardoxolone in the composition is 0.0000001 wt% (0.001 ppm) - 1 wt%, preferably 0.000005 wt% (0.05 ppm) - 1 wt%, further preferably 0.00005 wt% (0.5 ppm) - 1 wt%.

[0014] In some embodiments of the present application, the amount of bardoxolone in the composition is 0.001 wt% - 1 wt%, preferably 0.01 wt% - 1 wt%, further preferably 0.01 wt% - 0.5 wt%, more further preferably 0.01 wt% - 0.2 wt%.

[0015] In a third aspect of the present application, a cosmetic product with whitening efficacy is provided, which comprises bardoxolone or one or more of pharmaceutically acceptable acids, bases, salts, esters, solvates, stereoisomers, tautomers thereof.

[0016] In some embodiments of the present application, the content of bardoxolone in the cosmetic product is ≤10 wt%, preferably ≤5 wt%, and further preferably ≤1 wt%.

[0017] In some embodiments of the present application, the content of bardoxolone in the cosmetic product is 0.0000001 wt% (0.001 ppm) to 1 wt%, preferably 0.000005 wt% (0.05 ppm) to 1 wt%, and further preferably 0.00005 wt% (0.5 ppm) to 1 wt%.

[0018] In some embodiments of the present application, the content of bardoxolone in the cosmetic product is 0.001 wt% to 1 wt%, preferably 0.01 wt% to 1 wt%, further preferably 0.01 wt% to 0.5 wt%, and more further preferably 0.01 wt% to 0.2 wt%.

[0019] In some embodiments of the present application, the cosmetic product with whitening efficacy further comprises an auxiliary material, which comprises at least one of emulsifiers, humectants, thickening agents, chelating agents, and preservatives.

[0020] In some embodiments of the present application, the dosage form of the cosmetic product comprises any one of water, emulsion, spray, cream, essence, or mask.

[0021] Compared with the prior art, the present application has the following advantages:

[0022] The present application first discovers that bardoxolone has whitening-related efficacy, which is manifested in that it can inhibit melanin production, inhibit tyrosinase activity, and inhibit the expression of melanin synthesis-related genes, and has strong inhibitory effect and low effective concentration. At the same time, the ingredient has no irritation to red blood cells, indicating that it has certain safety as a potential raw material ingredient of cosmetic products. DETAILED DESCRIPTION

[0023] In order to make those skilled in the art more clearly understand the technical solutions of the present application, the following examples are given for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0024] The raw materials, reagents, and devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels or can be obtained by known methods.

[0025] Example 1

[0026] The present example provides an oleanane type triterpenoid compound, bavachromone (CAS: 218600-44-3).

[0027] Comparative Example 1

[0028] The present comparative example provides a phenolic compound, resveratrol (CAS: 501-36-0).

[0029] Comparative Example 2

[0030] The present comparative example provides a phenolic compound, hydroxytyrosol (CAS: 520-45-4).

[0031] Comparative Example 3

[0032] The present comparative example provides a flavonoid compound, glabridin (CAS: 59870-68-7).

[0033] Comparative Example 4

[0034] The present comparative example provides a flavonoid compound, phloretin (CAS: 60-82-2).

[0035] Comparative Example 5

[0036] The present comparative example provides a pentacyclic triterpenoid compound, quercetin A (CAS: 108524-94-3).

[0037] Comparative Example 6

[0038] The present comparative example provides an oleanolic acid 3-acetate (CAS: 4339-72-4).

[0039] Test Example 1 Melanin synthesis and tyrosinase activity inhibition test

[0040] 1.1 Preparation of sample solution

[0041] The substances provided in Example 1 and Comparative Examples 1-6 were dissolved in dimethyl sulfoxide (DMSO) as a solvent, respectively, to prepare sample solutions at concentrations of 0.01 μM, 0.1 μM, and 1 μM for testing.

[0042] 1.2 Experimental principle

[0043] Melanin is a biological pigment produced by melanocytes through synthesis and secretion. In melanocytes, tyrosine is catalyzed by tyrosinase and undergoes a series of reactions to ultimately generate melanin.

[0044] B16-F10 mouse melanoma cells are widely used as test cells for efficacy determination of whitening chemicals. α-MSH is an α-melanocyte-stimulating hormone that can promote B16-F10 to secrete melanin and increase intracellular tyrosinase activity. B16-F10 is induced by α-MSH to determine the inhibitory effect of the test substance on melanin synthesis and the inhibitory effect on tyrosinase activity.

[0045] 1.3 Experimental method

[0046] (1) Cell culture and treatment

[0047] B16-F10 cells were cultured in 1640 complete medium (1640 medium + 10% FBS by volume + double antibody) at 37°C in a carbon dioxide incubator with 5% CO2, and subcultured every 2-3 days.

[0048] (2) Determination of intracellular melanin content

[0049] Logarithmic growth phase B16-F10 cells were selected, digested with 0.25% trypsin, inoculated in 6-well culture plates, 2mL medium per well, and placed in a 37°C, 5% CO2 incubator. The next day after inoculation, different concentrations of test substances and α-MSH (1 μM) were added, and a control group (containing only 1 μM α-MSH) was set up, 2mL per well, and 3 parallel samples were prepared for each concentration. After incubation at 37°C, 5% CO2 for 48h, the supernatant was discarded, 0.5mL 0.25% trypsin digestion solution was added to each well and digested at room temperature for 1min. Stop digestion by adding 1mL medium, and blow into a single cell suspension. Take 20μL for cell counting, take 0.7mL cell suspension and centrifuge at 2000r / min for 5min, discard the supernatant, add 1mL 1mol / L NaOH solution (containing 10% DMSO by mass fraction), and incubate in a 80°C water bath for 30min, then measure the absorbance at 490nm on a microplate reader.

[0050] (3) Determination of intracellular tyrosinase activity

[0051] After trypsin digestion and counting of the cells treated with the above drugs (the specific steps are the same as 1.3-(2)), 0.7mL of cell suspension was centrifuged at 2000r / min for 5min, the supernatant was discarded, 1mL of 0.5% sodium deoxycholate solution was added, and the cells were lysed by ice bath for 15min to prepare the tyrosinase-containing extract. After preheating at 37°C, 0.5mL of 0.3% dopa solution was added, and the absorbance was measured at 475nm on a microplate reader after 10min of reaction at 37°C.

[0052] (4) Results statistics

[0053] The OD value is calibrated with the number of cells.

[0054] Melanin production inhibition rate (%) = [1 - (As / Ds) / (Ac / Dc)] x 100;

[0055] Tyrosinase activity inhibition rate (%) = [1 - (As / Ds) / (Ac / Dc)] x 100;

[0056] In the formula, As is the absorbance of the sample group, Ac is the absorbance of the control group, Ds is the cell concentration of the sample group, and Dc is the cell concentration of the control group.

[0057] 1.4 Experimental results

[0058] The results are shown in Tables 1-2.

[0059] Table 1

[0060]

[0061] Table 2

[0062]

[0063]

[0064] It can be understood that melanin is mainly produced by melanocytes, and the content of melanin in the skin is closely related to the depth, dullness, and uneven color of human skin. Tyrosinase is a key enzyme for catalyzing melanin synthesis, so the melanocytes can be used to evaluate the whitening effect of the test substance by evaluating the content of melanin and the activity of tyrosinase.

[0065] Currently, in the whitening market, resveratrol (Comparative Example 1) and phenylethyl resorcinol (Comparative Example 2) with phenolic structure, glabridin (Comparative Example 3) and phloretin (Comparative Example 4) with flavonoid structure are known cosmetic raw material ingredients with good whitening effect. As can be seen from the results of Tables 1 and 2, Example 1 (Bardoxolone) showed a significant effect of inhibiting melanin production and tyrosinase activity, and at the same concentration (0.01-1 μM), the inhibitory effect of Example 1 was significantly better than that of Comparative Examples 1-4. Comparative Examples 5 (Quercetin A) and 6 (Oleuropein 3-acetate), which are also triterpenoid compounds, are not in the current cosmetic raw material directory, and they did not show a significant inhibitory effect at a concentration of 0.01-1 μM, further indicating that Example 1 has a certain unique structure-activity.

[0066] The results of Table 1 and Table 2 above show that Example 1 (Bardoxolone) has a strong effect of inhibiting melanin production and tyrosinase activity, and it has a wider effective concentration range compared to Comparative Examples 1-4, which suggests that Example 1 (Bardoxolone) can achieve a whitening effect even better than existing whitening active ingredients in practical applications by adding a small amount.

[0067] Test Example 2: Inhibition of melanin synthesis-related gene expression

[0068] 1. Preparation of sample solution

[0069] The substances provided in Example 1 and Comparative Example 3 were dissolved in DMSO as a solvent to prepare sample solutions with a molar concentration of 1 μM, and the test was performed.

[0070] 1.2 Experimental principle

[0071] An α-MSH-induced B16-F10 mouse melanoma cell model was used, which increases the ability to secrete melanin and upregulates the expression of melanin synthesis-related genes under the action of α-MSH. The effect of the test substance on the expression of melanin synthesis-related genes in B16-F10 cells was determined by qPCR.

[0072] 1.3 Experimental method

[0073] (1) Cell culture and treatment

[0074] The same as Step 1.3(1) in Test Example 1.

[0075] (2) Extraction of cell RNA

[0076] RNA: Add cell lysis solution to each well in a six-well plate to lyse the cells; add chloroform (1:5 by volume ratio to the lysis solution) to the lysis solution, shake well, and then stand at room temperature for 5 min before placing in a 4°C centrifuge at 12000 rpm for 15 min; aspirate the upper colorless liquid into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, stand at room temperature for 10 min, and then place in a 4°C centrifuge at 12000 rpm for 10 min to remove the supernatant; add 500 μL of 75% ethanol, and then place in a 4°C centrifuge at 7500 rpm for 5 min, discard the supernatant, and stand at room temperature to dry for 1-2 min; then add an appropriate amount of DEPC water to dissolve the RNA precipitate.

[0077] (3) cDNA synthesis and RT-PCR

[0078] The reverse transcription kit and the fluorescent quantitative PCR kit used in the experiment were purchased from Nanjing Nuo Weizan Biological Technology Co., Ltd. According to the kit instructions, the steps were performed in a relative quantitative manner to calculate the expression of related genes.

[0079] 1.4 Experimental results

[0080] The results are shown in Table 3.

[0081] Table 3

[0082]

[0083] It can be understood that tyrosinase (TYR) is a key enzyme in the melanin synthesis reaction, which participates in two melanin production processes, and tyrosinase-related protein 1 (TYRP1) and tyrosinase-related protein 2 (TRP2) play an important role in the synthesis of eumelanin. TYRP1 has 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidase activity, and TRP2 can quickly convert dopachrome to DHICA. Premelanosome protein (PMEL) is the structural basis of the fibrils in the melanosome protein, and glycoprotein non-metastatic melanoma protein b (GPNMB) plays an important role in the formation of late-stage melanosome. Its role may be related to the transport or transfer of melanosome to keratinocytes. Therefore, by detecting the expression levels of TYR, TYRP1, TRP2, PMEL, and GPNMB genes, it can be determined whether the test substance has whitening activity.

[0084] Comparative Example 3 (glabridin) is a common whitening ingredient on the market, which has been reported to inhibit the expression of related genes in the melanin synthesis pathway. As can be seen from the results of Table 3, at the same concentration (1 μM), the effect of Example 1 (badosilone) on inhibiting the expression of TYR, TYRP1, TRP2, PMEL, and GPNMB genes is obviously better than that of Comparative Example 3. The results of Table 3 show that Example 1 (badosilone) not only can inhibit the activity of tyrosinase, but also can reduce the level of melanin synthesis-related genes, thereby reducing the content of tyrosinase.

[0085] Test Example 3 Red blood cell hemolysis test

[0086] 1.1 Experimental principle

[0087] Ocular irritants can denature proteins or dissolve cell membranes. The test substance is applied to red blood cells to detect whether hemolysis occurs and whether denaturation occurs to hemoglobin released from the cells. By measuring the amount of leaked hemoglobin and the degree of denaturation, the damage to the cell membrane caused by the test substance can be evaluated, and it can be inferred whether it may have ocular irritation. The red blood cell hemolysis test simulates the damage to the cornea.

[0088] 1.2 Experimental method (Refer to EURL ECVAM DB-ALM Protocol No. 99 and EURL ECVAM DB-ALM Protocol No. 37)

[0089] (1) The test substance, Example 1 and Comparative Examples 1-4, was prepared into a suspension with a mass fraction of 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, etc. in a series of gradients using physiological saline as the solvent, and mixed with an equal volume of red blood cell suspension. After incubation at 37°C for 3 h, the supernatant was separated by centrifugation, and the absorbance was measured at 410 nm, 540 nm, and 575 nm on a microplate reader. The results were statistically analyzed. Physiological saline was used as the negative control (i.e. complete hemolysis), and 0.1% SDS solution was used as the positive control (i.e. complete hemolysis).

[0090] (2) Results statistics

[0091] Hemolysis rate (%) = (A 410nm -C 410nm ) / (B 410nm -C 410nm ) x 100;

[0092] Protein denaturation index DI (%) = (A 575nm / A 540nm ) / (B 575nm / B 540nm ) x 100;

[0093] In the formula, A is the average absorbance of the test sample, B is the average absorbance of the positive control, and C is the average absorbance of the negative control. In the calculation of DI%, the average absorbance of the sample at a concentration of 1% is used.

[0094] A curve was plotted with the sample concentration as the abscissa and the hemolysis rate as the ordinate. According to the regression equation, the concentration of the test substance that caused 50% hemolysis of red blood cells, HC50 (mg / L), was determined.

[0095] (3) Results determination

[0096] The L / D value of the sample was calculated according to the HC50 value and the DI value: L / D = HC50 / DI;

[0097] According to the RBC experiment grading standard of the European Center for Validation of Alternative Methods (ECVAM), the degree of irritation of the cosmetic was graded, and the grading standard is shown in Table 4.

[0098] Table 4

[0099] L / D Classification ＞100 Nonirritating 10 < L / D < 100 Mildly irritating 1 < L / D < 10 Slightly irritating 0.1 < L / D < 1 Moderately irritating L / D < 0.1 Severely irritating

[0100] 1.3 Experimental results

[0101] The results are shown in Table 5.

[0102] Table 5

[0103]

[0104] Comparative Examples 1-4 are all currently popular cosmetic raw material ingredients with good effects in the field of whitening. As can be seen from the results in Table 5, by comparing the irritation of different samples to red blood cells, it is found that Example 1 (bardoxolone) has no irritation and is relatively mild, which is similar to Comparative Examples 1-4. Therefore, from the perspective of safety, Example 1 (bardoxolone) of the present application has the potential to be applied in the field of cosmetics.

[0105] Although bardoxolone is not currently in the list of cosmetic raw materials, it has low irritation and good whitening effect, and the above characteristics provide good data support for its application in the field of cosmetics, especially in whitening effect products.

[0106] Application Example 1

[0107] A composition with whitening effect, consisting of 10 wt% bardoxolone and the rest of physiological saline solvent.

[0108] Application Example 2

[0109] A composition with whitening effect, consisting of 1 wt% bardoxolone and the rest of physiological saline solvent.

[0110] Application Example 3

[0111] A composition with whitening effect, consisting of 0.01 wt% bardoxolone and the rest of physiological saline solvent.

[0112] Application Example 4

[0113] A composition with whitening effect, consisting of 0.5 ppm bardoxolone and the rest of physiological saline solvent.

[0114] Application Example 5

[0115] A cosmetic water with whitening effect, consisting of the following components in mass percentage:

[0116] Glycerol 6%, sodium hyaluronate 0.08%, xanthan gum 0.05%, p-hydroxyacetophenone 0.4%, 1,2-hexanediol 1%, bardoxolone 10%, and water in the rest.

[0117] Application Example 6

[0118] A mask with whitening effect, consisting of the following components in mass percentage:

[0119] Glycerin 10%, EDTA disodium 0.01%, acryloyldimethyltaurine ammonium / VP copolymer 0.1%, sodium hyaluronate 0.05%, p-hydroxyacetophenone 0.4%, 1,2 hexanediol 1%, squalane 1.5%, badosol 0.01% and water balance.

[0120] Application Example 7

[0121] An emulsion with whitening effect, consisting of the following components in mass percentage:

[0122] Glycerin 10%, EDTA disodium 0.01%, acryloyldimethyltaurine ammonium / VP copolymer 0.1%, sodium hyaluronate 0.05%, p-hydroxyacetophenone 0.4%, 1,2 hexanediol 1%, squalane 1.5%, badosol 0.01% and water balance.

[0123] Application Example 8

[0124] An essence with whitening effect, consisting of the following components in mass percentage:

[0125] Carbomer 0.3%, aminomethylpropanol 0.1%, glycyrrhizic acid dipotassium 0.3%, trehalose 0.6%, 1,2 hexanediol 0.5%, p-hydroxyacetophenone 0.4%, ethanol 8%, badosol 0.5ppm and water balance.

[0126] It is easily conceivable that the compositions of application examples 1-4 and the cosmetics of application examples 5-8 of the present application, due to containing badosol of the present application respectively, have good whitening effect.

[0127] The above has specifically described the preferred embodiments of the present application, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without departing from the spirit of the present application, and these equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. Application of bardoxolone in preparation of a product with whitening effect.

2. Use according to claim 1, characterized in that, The addition amount of the bardoxolone in the product is ≤10 wt%.

3. Use according to claim 1, characterized in that, The whitening includes at least one of inhibiting tyrosinase activity, inhibiting melanin content, and inhibiting expression of a melanin synthesis related gene.

4. Use according to claim 3, characterized in that, The melanin synthesis related gene includes at least one of a TYR gene, a TYRP1 gene, a TRP2 gene, a PMEL gene, and a GPNMB gene.

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