Whitening composition acting on Mitf gene target, composition, application and cosmetic

Abstract

The invention belongs to the field of cosmetics, and discloses a whitening composition acting on a Mitf gene target spot, which comprises the following components in percentage by weight: 20-30wt% of resorcinol derivative, 20-30wt% of Mitf gene, and the balance of water. 10 to 20 weight percent of gallic acid and a derivative thereof; 2-8 wt% of a barley extract; and the balance of a solvent. According to the composition, resorcinol derivatives are used as basic whitening components, the melanin transfer inhibiting function of gallic acid and derivatives of gallic acid and the antioxidant and sedative effects of barley extract are achieved, the effective acting target of the composition is Mitf genes, and the composition can be used for whitening the skin through combined use of the substances, so that the skin whitening effect of the composition is improved. The whitening effect of the resorcinol derivative can be better exerted. Meanwhile, the invention further discloses application and cosmetics of the composition.

Description

Technical Field

[0001] The present invention relates to the daily chemical field, and specifically to a whitening composition acting on the Mitf gene target, a composition, uses, and cosmetics. Background Art

[0002] A patent application with the publication number CN113576933A and the theme of a composition with whitening efficacy, its preparation method and application discloses a composition, which includes the following components: α-hydroxy acid, resorcinol derivative, ascorbic acid and its derivatives, tranexamic acid, and gallic acid and its derivatives.

[0003] It is recorded in the specification that resorcinol derivatives have extremely strong inhibitory activity on tyrosinase, thus inhibiting melanin synthesis, and are a very effective type of skin whitening agent; gallic acid is a potent tyrosinase inhibitor and an excellent antioxidant, which can effectively inhibit melanin transfer to achieve a whitening effect. At the same time, gallic acid can also prevent DNA damage and inhibit skin inflammation.

[0004] In the research of this project, it is found that 4-butylresorcinol, one of the resorcinol derivatives, has an obvious inhibitory effect on melanogenesis in B16F10 cells, and diglucosyl gallate, one of the derivatives of gallic acid, has a negative inhibitory effect on melanogenesis in B16F10 cells, that is, it may promote melanogenesis. Of course, due to the many whitening pathways, the above experimental results do not mean that the derivatives of gallic acid have no whitening effect.

[0005] This project aims to develop a composition that can exhibit excellent whitening effects.

[0006] Therefore, the technical problem to be solved in this case is: how to improve the whitening effect of a whitening formulation based on resorcinol derivatives. Summary of the Invention

[0007] The purpose of the present invention is to provide a whitening composition acting on the Mitf gene target. This composition uses resorcinol derivatives as the basic whitening component, and has the functions of inhibiting melanin transfer of gallic acid and its derivatives, the antioxidant and sedative effects of barley extract. The effective target of this composition is the Mitf gene. By using the combination of the above substances, the whitening effect of resorcinol derivatives can be better exerted.

[0008] At the same time, the present invention also discloses the application of this composition and cosmetics.

[0009] The present invention discloses a whitening composition, which includes the following components in weight percentage:

[0010] Resorcinol derivative 20-30 wt%;

[0011] Gallic acid and its derivatives: 10 - 20 wt%;

[0012] Barley extract: 2 - 8 wt%;

[0013] The balance is solvent.

[0014] In the composition of the present invention, the individual uses of each substance are as follows:

[0015] Resorcinol derivatives: inhibit melanin synthesis;

[0016] Gallic acid and its derivatives: control the expression of MITF (melanin production factor) and stop the process of melanin production;

[0017] Saturate the keratinocyte receptor and stop melanin transfer.

[0018] Barley extract: effectively inhibit all stages of signal transduction and melanogenesis pathways;

[0019] Antioxidant function, inhibit melanin deposition, and have a sedative and soothing effect.

[0020] After conducting an experiment on inhibiting melanin production in B16F10 cells by combining the above three substances, it was found that gallic acid and its derivatives, and barley extract can promote the inhibitory ability of resorcinol derivatives on melanin production. We believe that the possible synergistic principle is as follows: diglucosyl gallic acid can inhibit the release of PGE2 prostaglandin, thereby down-regulating tyrosinase and inhibiting the expression of this enzyme, inhibiting and reducing melanin production from the source. Barley extract contains aloe barley polyphenols, which have high antioxidant activity. At the same time, diglucosyl gallic acid also has good antioxidant activity, improving the ability of 4 - butylresorcinol to inhibit melanin production.

[0021] The above combination was sent for external inspection to detect the effect of this composition on the transcriptome of B16F10 cells in order to explore its potential whitening mechanism and efficacy. After testing, it was found that it has the strongest inhibitory ability on the expression of MITF gene, Tyr gene, Oca2 gene, Rab27a gene, and Mc1r gene, and MITF is a key regulatory factor for the development and function of melanocytes. It can regulate the expression of multiple melanin synthesis-related genes such as TYR, TYRP1, and TYRP2. It is like a "master switch" that activates their transcription by binding to the promoter regions of these genes, thereby promoting the synthesis of melanin in melanocytes. We believe that at least one of gallic acid and its derivatives, and barley extract has an inhibitory and promoting effect on the expression with obvious inhibitory effects on the MITF gene, Tyr gene, Oca2 gene, Rab27a gene, and Mc1r gene, significantly improving the inhibitory ability of resorcinol derivatives on these two genes.

[0022] In the above-mentioned whitening composition, the resorcinol derivative is at least one of 4-butylresorcinol, hexylresorcinol, phenethylresorcinol, and dimethoxytolyl-4-propylresorcinol.

[0023] In the above-mentioned whitening composition, the gallic acid and its derivatives are at least one of diglucosyl gallic acid, gallic acid, propyl gallate, ethylhexyl gallate, and ellagic acid.

[0024] Meanwhile, the present invention also discloses the use of the whitening composition as described above in any one of the preceding paragraphs for preparing cosmetics.

[0025] Finally, the present invention also discloses a cosmetic containing the whitening composition as described above in any one of the preceding paragraphs.

[0026] In the above-mentioned cosmetic, the cosmetic contains 1-4 wt% of the whitening composition.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The present invention uses a resorcinol derivative as the basic whitening ingredient, and with the function of gallic acid and its derivatives in inhibiting melanin transfer, and the antioxidant and sedative effects of barley extract, the whitening effect of the composition can be better exerted, and at the same time, it has good skin mildness. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the skin color collection positions at multiple points on the face in the volunteer experiment. DETAILED DESCRIPTION OF THE INVENTION

[0030] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0031] Before elaborating on the method of the present invention, the sources of the reagents used in the present invention will be described first;

[0032] 4-Butylresorcinol: sourced from Shanghai Xuntian Biotechnology Co., Ltd. with a content of 100%;

[0033] Gallic Bright White: sourced from Suzhou Nakang Biotechnology Co., Ltd. containing 2.5 wt% of diglucosyl gallic acid;

[0034] HodVital: sourced from BioSpectrum, Inc. containing 2 wt% of barley (HORDEUM VULGARE) extract.

[0035] Examples and Comparative Examples

[0036] A whitening composition is prepared by dissolving resorcinol derivatives and butylene glycol, and then successively adding gallic acid and its derivatives and barley extract solution, mixing evenly, standing, and filtering to obtain the finished product.

[0037] The formula refers to Table 1 below;

[0038] Table 1 Formula Table of Whitening Composition Unit: g

[0039] 4-Butylresorcinol Diglucosyl gallic acid Barley extract Butylene glycol Example 1 20 10 2 68 Example 2 30 20 8 42 Example 3 25 15 5 55 Comparative Example 1 25 20 0 55 Comparative Example 2 25 0 20 55 Comparative Example 3 25 0 0 75 Comparative Example 4 0 15 5 85

[0040] Performance Test

[0041] 1. Melanin Production Inhibition Experiment

[0042] 1.1 Experimental Reagents

[0043] B16F10 mouse melanoma cells (B16F10) were purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for Type Culture Collection. Ordinary 96-well plates and 12-well plates were purchased from ThermoFisher. DMEM medium and fetal bovine serum (FBS) were purchased from Gibco. Arbutin was purchased from Sigma. CCK reagent was purchased from Tongren Biology (Cat. 40203ES80).

[0044] 1.2 Sample Information

[0045] A total of 7 samples were detected this time. The samples were Example 1 to Example 3 and Comparative Example 1 to Comparative Example 4;

[0046] 1.3 Experimental Method

[0047] 1.3.1 B16F10 Cell Viability Test

[0048] B16F10 cells were amplified and cultured in DMEM containing 10% FBS under the culture conditions of 5% CO2 and 37°C. When the cells grew to 80-90% confluence, they were digested with trypsin and inoculated into 96-well plates. After the cells were attached and cultured in the 96-well plates for 48 h, samples with different concentrations were prepared to treat the cells (N = 3). After 72 h, CCK-8 reagent was added according to the instructions and incubated for 1 h, and the OD value was read at 450 nm with an enzyme-labeled instrument. The relative cell activity value was obtained by calculating the ratio of the average OD values of the sample group and the control group, and the cytotoxic effect of the sample was judged.

[0049] Relative cell activity % = (OD value of sample group / OD value of control group) * 100%

[0050] 1.3.2 B16F10 Cell Melanin Content Test

[0051] Select B16 cells in the best growth state and seed them in a 12-well plate at a density of 1x10 5 / ml. After they adhere and grow for 48 h, treat the cells with samples at three safe concentrations, using arbutin (10 mM) as a positive control. After 72 h of treatment, add 1N NaOH solution containing 10% DMSO to the cells, seal at 80 °C and incubate for 1 hour, then measure the absorbance at 405 nm to evaluate the total melanin content of the cells.

[0052] 1.4 Experimental results

[0053] 1.4.1 Effect of samples on the viability of B16F10 melanocytes

[0054] After treating B16F10 melanocytes with samples at different concentrations of active ingredients, as shown in Table 2, the samples of Example 1 had no effect on reducing cell viability at a concentration of 0.312%. The samples of Example 2 had no effect on reducing cell viability at a concentration of 0.156%. The samples of Example 3 had no effect on reducing cell viability at a concentration of 0.156%. The samples of Comparative Example 1 had no effect on reducing cell viability at a concentration of 0.156%. The samples of Comparative Example 3 and Comparative Example 4 had no effect on reducing cell viability at a concentration of 2.5%. The samples of Comparative Example 2 had no effect on reducing cell viability at a concentration of 0.625%. According to the cell viability data, 0.1% was uniformly selected as the detection concentration for the subsequent melanogenesis experiments of the 7 samples.

[0055] Table 2 B16F10 melanocyte viability data

[0056]

[0057]

[0058]

[0059] 1.4.2 Effect of samples on melanogenesis in B16F10 cells

[0060] After B16F10 cells were treated with different samples, the melanin production is shown in Table 3;

[0061] Table 3 Relative melanin content in B16F10 cells

[0062]

[0063] Result analysis:

[0064] 1. From Comparative Example 3 and Comparative Example 4, it can be seen that 4 - butylresorcinol has an obvious melanogenesis inhibitory effect; barley extract and diglucosyl gallic acid have a mild melanogenesis inhibitory effect.

[0065] 2. From Examples 1 to 3, it can be seen that at the concentration of Example 3, its performance reaches a level close to that of arbutin. When the concentration is further increased, the improvement in performance is limited.

[0066] 3. From Example 3, Comparative Example 1 and Comparative Example 2, it can be seen that when either barley extract or diglucosyl gallic acid is used, the improvement effect on the melanogenesis inhibition of 4 - butylresorcinol is limited.

[0067] From the above cases, it can be seen that the present invention uses a combination of barley extract, diglucosyl gallic acid and 4 - butylresorcinol, which can significantly improve the ability of 4 - butylresorcinol to inhibit melanogenesis.

[0068] According to the existing literature records, we believe that the mechanism of the synergistic combination of the above three is as follows:

[0069] Diglucosyl gallic acid can inhibit the release of PGE2 prostaglandin, thereby down - regulating tyrosinase and inhibiting the expression of this enzyme, inhibiting and reducing melanogenesis from the source. The barley extract contains aloe - barley polyphenols, which have high antioxidant activity. At the same time, diglucosyl gallic acid also has good antioxidant activity, improving the ability of 4 - butylresorcinol to inhibit melanogenesis.

[0070] In order to further verify the possible action pathways of barley extract and diglucosyl gallic acid, the present invention verifies the efficacy of the above - mentioned composition through the DPPH free - radical scavenging experiment.

[0071] 2. DPPH free - radical scavenging rate

[0072] The DPPH free - radical scavenging rate is an index to measure the antioxidant ability of a substance, which is determined by the DPPH (1,1 - diphenyl - 2 - picrylhydrazyl) free - radical scavenging experiment. The basic principle of this experiment is to utilize the strong absorption characteristics of DPPH free - radicals at 517 nm. When a sample is added to the DPPH solution, if the sample can scavenge DPPH free - radicals, the absorbance will decrease, and thus the scavenging rate can be calculated.

[0073] Experimental steps

[0074] Prepare DPPH solution: Prepare a 0.1 mmol / L DPPH solution using anhydrous ethanol and store it in the dark.

[0075] Sample treatment: Add 2 mL of the test sample solution (diluted at a concentration of 0.1 wt%) and 2 mL of the DPPH solution into the same test tube, shake well, and let it stand in the dark at room temperature for 30 minutes.

[0076] Absorbance measurement: Use a spectrophotometer to measure the absorbance at 517 nm, and record the absorbance (A0) without adding the sample and the absorbance (A 样品 ) after adding the sample respectively.

[0077] Calculation formula

[0078] The calculation formula for the DPPH free radical scavenging rate is:

[0079] DPPH free radical scavenging rate (%) = (A0 - A 样品 ) / A0 × 100;

[0080] where A0 is the absorbance of DPPH at 517 nm without adding the sample, and A 样品 is the absorbance of DPPH at 517 nm after adding the sample.

[0081] The test formulation refers to Table 4;

[0082] Table 4 Test Formulation Unit: g

[0083] Diglucosyl gallic acid Barley extract Butylene glycol Sample 1 15 5 80 Sample 2 20 0 80 Sample 3 0 20 80

[0084] The test results refer to Table 5;

[0085] Table 5 Test Results

[0086] DPPH free radical scavenging rate % Sample 1 63.5 Sample 2 42.7 Sample 3 35.2

[0087] From the test results of the above DPPH free radical scavenging rate, it can be seen that at least part of the factor for improving the melanin production inhibition effect of the composition of the present invention comes from the strong antioxidant synergy between diglucosyl gallic acid and barley extract.

[0088] 3. Research on the whitening mechanism

[0089] Send the sample of Example 3 to Shanghai Aiji Biotechnology Co., Ltd. for testing, and the test report number is AGSE20240912 - 2;

[0090] The detection method is:

[0091] When the in vitro cultured B16F10 cells grew to 80 - 90% confluence, they were digested with trypsin and seeded into 12-well plates. After the cells were adherently cultured in the 12-well plates for 48 h, the cells were treated with the samples at a safe concentration (N = 3). After 24 h, the supernatant was aspirated, and Trizol reagent was added to each well. The cells were thoroughly pipetted and lysed, and the lysate was transferred to -80 °C for deep cryopreservation for later use. After the total RNA extraction passed the quality inspection, library construction was carried out, and high-throughput sequencing of the samples was performed using the paired-end sequencing mode of the Illumina Hiseq sequencing platform.

[0092] The test results are shown in Table 6 below;

[0093] Table 6 Gene Sequencing Results Table

[0094]

[0095] The above test results show that the combination of the present invention has an obvious inhibitory effect on the gene pathways: MITF gene, Tyr gene, Oca2 gene, Rab27a gene, and Mc1r gene; the combination of the present invention has a relatively obvious whitening effect.

[0096] 4. Volunteer Experiment

[0097] The samples of Example 3 of the present invention and Comparative Example 1 and Comparative Example 2 were subjected to volunteer experiments.

[0098] Experimental instrument: Colormeter CL400 skin color tester (Courage + Khazaka, Germany)

[0099] Experimental principle: The Colorimeter CL400 skin color tester mainly emits white LED light through the probe, measures the light reflected by the skin and displays the corresponding values. It is corrected by a special color matrix. The color of the measured skin is represented by XYZ-values and is calculated into relevant L*a*b* values.

[0100] ITA° = arctan[(L* - 50) / b*] × 180 ÷ 3.1416

[0101] Thirty subjects aged between 20 and 30 years old (excluding pregnant or lactating women) were selected and divided into three groups of 10 people each; those without serious systemic diseases, immunodeficiency or autoimmune diseases; the test sites had not received skin treatment, beauty treatment, or other tests that might affect the results; those without active allergic diseases; those without highly sensitive constitutions; those who had not used hormonal drugs and immunosuppressants in the recent month; those who had not participated in other clinical trials at the test sites in the recent month;

[0102] Test steps:

[0103] 1) After the subject cleans the face, sit quietly in a laboratory at a temperature of 21±1°C and 50±10% RH for 30 minutes, and then the tester operates the relevant instrument to collect skin color data of the subject's face.

[0104] 2) The collection positions of skin color at multiple points on the face are shown in Figure 1 ;

[0105] The test results of ITA° can be seen in Table 7;

[0106] Table 7 Test Results Table

[0107] Before testing After testing Example 3 50.57 62.71 Comparative Example 1 50.61 58.20 Comparative Example 2 50.59 55.65

[0108] From the above test results, it can be seen that the combination of the present invention can significantly and rapidly improve skin color brightness and has a more excellent whitening effect.

[0109] The above composition can be used in commonly used cosmetics in this field to achieve the whitening effect. The following takes lotion and essence water as examples to list its specific applications, and the specific formulations refer to Table 8 and Table 9;

[0110] Table 8 Lotion Formulation Table

[0111]

[0112]

[0113] Table 9 Essence Water Formulation Table

[0114]

[0115]

Claims

1. A whitening composition acting on the Mitf gene target, characterized in that, Comprising the following components by weight percentage: Resorcinol derivative 20 - 30 wt%; Gallic acid and its derivatives 10 - 20 wt%; Barley extract 2 - 8 wt%; The balance of solvent.

2. The whitening composition according to claim 1, characterized in that, The resorcinol derivative is at least one of 4 - butylresorcinol, hexylresorcinol, phenethylresorcinol, dimethoxytolyl - 4 - propylresorcinol.

3. The whitening composition according to claim 1, wherein The gallic acid and its derivatives are at least one of diglucosyl gallic acid, gallic acid, propyl gallate, ethylhexyl gallate, ellagic acid.

4. Use of the whitening composition as described in any one of claims 1 - 3 for preparing cosmetics.

5. A cosmetic, characterized in that, Containing the whitening composition as described in any one of claims 1 - 3.

6. The cosmetic according to claim 5, characterized in that, The cosmetics contain 1 - 4 wt% of the whitening composition.

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