Whitening composition for activating autophagy pathway, application and cosmetic

Abstract

The invention belongs to the technical field of cosmetics, and discloses a whitening composition for activating an autophagy pathway, which comprises the following components by weight: 15-25 parts of 4-butylresorcinol; 25 to 40 parts of a diglucosyl gallic acid solution; 2.5 to 4 parts of trehalose; 1 to 2 parts of sodium mannose phosphate; according to the whitening composition, the 4-butylresorcinol and the diglucosyl gallic acid are used as main whitening components, and the trehalose and the sodium mannose phosphate are used as cell autophagy active components, so that the whitening effect of the whitening components can be ensured, and the purpose of remarkably improving the whitening effect is achieved. Meanwhile, the invention further provides application and cosmetics of the composition.

Description

Technical Field

[0001] The present invention relates to the field of new daily chemical materials, and in particular to a whitening composition for activating an autophagy pathway, its use, and cosmetics. Background Art

[0002] 4-Butylresorcinol and diglucosylgallic acid are common whitening ingredients in the field. 4-Butylresorcinol's whitening mechanism is primarily achieved by inhibiting the activity of tyrosinase and dihydroxyindolecarboxylic acid oxidase (TRP-1), key enzymes in melanin production. Diglucosylgallic acid's whitening mechanism is primarily achieved by targeting tyrosinase activity and interfering with melanocyte proliferation.

[0003] Among existing whitening technologies, most achieve whitening through active whitening. Defensive whitening has been a perspective that researchers have not paid much attention to. The so-called defensive whitening mainly refers to factors that delay darkening, such as cellular autophagy. Autophagy whitening is a method of processing existing melanin by clearing already generated melanosomes through the cell's own autophagy process; while defensive whitening is a method of preventing the generation, transport, or deposition of melanin through various mechanisms.

[0004] In our ongoing research, we have discovered a close relationship between autophagy and whitening ingredients. Based on this, we propose the present invention. Summary of the Invention

[0005] The purpose of the present invention is to provide a whitening composition that activates the autophagy pathway. The whitening composition uses 4-butylresorcinol and diglucosyl gallic acid as main whitening ingredients, and uses trehalose and sodium mannose phosphate as cell autophagy active ingredients, which can ensure the whitening effect of the whitening ingredients and achieve the purpose of significantly improving the whitening effect.

[0006] At the same time, the invention also provides the use and cosmetics of the composition.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A whitening composition comprising the following components in parts by weight:

[0009] 15-25 parts of 4-butylresorcinol;

[0010] 25-40 parts of diglucosyl gallic acid solution;

[0011] 2.5-4 parts of trehalose;

[0012] 1-2 parts of sodium mannose phosphate;

[0013] The concentration of diglucosyl gallic acid in the diglucosyl gallic acid solution is 2.5 wt %.

[0014] The present invention uses 4-butylresorcinol and diglucosyl gallic acid as the main whitening ingredients, and uses trehalose and sodium mannose phosphate as cell autophagy active ingredients. Whitening ingredients can inhibit and block melanin, reducing the content of melanin, but are not as effective in eliminating existing melanin (such as acne scars). The present invention uses cell autophagy active ingredients, which can promote the decomposition of melanosomes by lysosomes in keratinocytes during keratin metabolism, thereby achieving melanosome autophagy.

[0015] The present invention uses trehalose and sodium mannose phosphate as the cell autophagy active ingredients. The two have a relatively obvious synergistic effect in the whitening effect of the whitening ingredients. The synergistic mechanism is:

[0016] Sodium mannose phosphate participates in ATP synthesis, providing energy to cells, maintaining normal cell metabolism, reducing the accumulation of reactive oxygen species (ROS), and reducing the stimulation of oxidative damage on melanin production; it synergizes with trehalose to activate AMP-dependent protein kinase (AMPK), further inducing autophagy and regulating energy metabolism, indirectly affecting melanin synthesis; 4-butylresorcinol promotes the degradation of tyrosinase by the ubiquitin-proteasome system by activating the p38 MAPK signaling pathway, while enhancing cellular autophagy levels; diglucosyl gallic acid inhibits the transport of melanosomes from melanocytes to the stratum corneum.

[0017] The above-mentioned whitening composition includes the following components in parts by weight:

[0018] 18-23 parts of 4-butylresorcinol;

[0019] 30-35 parts of diglucosyl gallic acid solution;

[0020] 3-3.5 parts of trehalose;

[0021] 1.3-1.8 parts of sodium mannose phosphate.

[0022] At the same time, the present invention also discloses the use of the whitening composition for preparing cosmetics.

[0023] Finally, the present invention also discloses a cosmetic with whitening effect, comprising the composition as described above.

[0024] Preferably, the cosmetic contains 0.5 to 3 wt% of the composition.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses 4-butylresorcinol and diglucosyl gallic acid as the main whitening ingredients, and uses trehalose and sodium mannose phosphate as cell autophagy active ingredients; whitening ingredients can inhibit and block melanin and reduce the content of melanin, but they are not as effective in eliminating existing melanin (such as acne scars, etc.). The present invention uses cell autophagy active ingredients, which can promote the decomposition of melanosomes by lysosomes in keratinocytes during keratin metabolism, thereby achieving autophagy of melanosomes. By combining whitening ingredients and cell autophagy active ingredients, a more obvious whitening and lifting effect can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result of B16 cell viability test;

[0028] Figure 2 Cluster diagram, volcano diagram and scatter diagram of differentially expressed genes between the sample group and the blank control group;

[0029] Figure 3 This is a graphic of the GO function enrichment analysis results of differentially expressed genes;

[0030] Figure 4 This is a graphic display of the KEGG pathway functional enrichment analysis results of differentially expressed genes. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] 4-Butylresorcinol: The supplier is Shanghai Xuntian Biotechnology Co., Ltd., and the content of 4-butylresorcinol is 100 wt%.

[0033] Diglucosyl gallic acid solution: The supplier is Suzhou Nakang Biotechnology Co., Ltd., and the content of diglucosyl gallic acid is 2.5 wt%.

[0034] Trehalose: The supplier is Nanjing Spike Biotechnology Co., Ltd., and the trehalose content is 100 wt%.

[0035] Sodium mannose phosphate: The supplier is Beijing Yanzhishan Technology Co., Ltd., and the content of sodium mannose phosphate is 48wt%.

[0036] Part I. Examples and Comparative Examples

[0037] The formulation shown in Table 1 was used to prepare an aqueous solution. The preparation method was as follows:

[0038] Step 1: Add 4-butylresorcinol and diglucosyl gallic acid solution to water and stir evenly;

[0039] Step 2: Add trehalose and sodium mannose phosphate to the solution in step 1, stir evenly, filter, and add a preservative.

[0040] Table 5 Recipe

[0041]

[0042]

[0043] Part 2 Whitening Performance Test

[0044] 1. Tyrosinase inhibition rate test

[0045] Purpose and principle of the test

[0046] Tyrosinase is a key enzyme in the biosynthesis of melanin in the skin. It acts on dopa to form dopaquinone, which then undergoes a series of reactions to form melanin. In a phosphate solution at pH 6.8, tyrosinase catalyzes the conversion of dopa into dopaquinone, with absorbance measured at 475 nm on a spectrophotometer. Cosmetics that inhibit tyrosinase activity can reduce the conversion of dopa into dopaquinone, thereby lowering absorbance. Changes in absorbance can be used to assess the inhibitory effect of cosmetics on tyrosinase activity.

[0047] This test refers to the laboratory method (HMC-WI-032 tyrosinase inhibition rate) and compares the tyrosinase inhibition rate test results of the test sample with that of the negative control. If the inhibition rate of the test sample is higher than that of the negative control and the difference is significant (P<0.05), the test sample can be considered to have whitening effect.

[0048] Test indicators: If the tyrosinase inhibition rate of the sample is higher than that of the negative control and there is a significant difference, then the test sample can be considered to have whitening effect.

[0049] Experimental materials and methods

[0050] 1.1 Instruments and Equipment

[0051] BSA224S Analytical Balance

[0052] RT-6100 ELISA Analyzer

[0053] 1.2 Reagents

[0054] Polyphenol oxidase (mushroom), BR

[0055] Levodopa, BR

[0056] 1.3 Test methods

[0057] (1) Treatment of control and test samples

[0058] Sample group: The sample was diluted with pure water to a concentration of 3%;

[0059] Positive control (kojic acid, purity ≥96%): Dilute with pure water to a positive control concentration of 0.1%;

[0060] Negative control: pure water.

[0061] (2) Test operation steps

[0062] Set up sample tubes, sample background tubes, enzyme reaction tubes, and solvent background tubes. Set up three parallel tubes for each group. Add different reagent solutions to each of the four groups, shake gently, and let stand at room temperature for 5 minutes. Transfer each reaction solution into a 1 cm cuvette and measure the absorbance at 475 nm.

[0063] (3) Calculation formula

[0064] Tyrosinase inhibition rate (%) = [1-(T-T0) / (C-C0)]

[0065] Where: T—absorbance of the sample tube, i.e., absorbance of the solution after the sample reacts with tyrosinase;

[0066] T0—sample background absorbance value;

[0067] C—the average of three absorbance values of the enzyme reaction tube, i.e., the absorbance value of the reaction between tyrosinase and DOPA when no sample is added;

[0068] C0—Solvent background absorbance value.

[0069] This test method is an in vitro method suitable for evaluating cosmetics claiming to achieve whitening effects by inhibiting tyrosinase activity. The test results are shown in Table 2 below.

[0070] Table 2 Tyrosinase inhibition rate results

[0071] Test results (%) Example 1 43.42 Example 2 55.48 Example 3 61.32 Example 4 44.99 Example 5 58.34 Comparative Example 1 45.35 Comparative Example 2 51.21 Comparative Example 3 49.36 Positive control 97.28 Negative control -3.36

[0072] Result Analysis

[0073] 1. All examples and comparative examples showed significant differences compared to the negative control, indicating that all examples and comparative examples of the present invention had good whitening effects;

[0074] 2. It can be seen from Examples 1 to 3 that the whitening effect of the present invention is concentration-dependent, and the whitening effect gradually improves with increasing concentration.

[0075] 3. Comparisons between Example 2 and Comparative Example 1, Comparative Example 2 and Comparative Example 1, and Comparative Example 3 and Comparative Example 1 show that 4-butylresorcinol and diglucosylgallic acid provide the primary whitening effect. The use of either trehalose or sodium mannose phosphate can enhance the whitening effect, but the magnitude of the improvement is not particularly significant. When trehalose and sodium mannose phosphate are used simultaneously, they exhibit a certain degree of synergy.

[0076] 2. Melanin index test

[0077] The test subjects were 30 people, aged 18 to 60, including 18 males and 12 females, and the test area was the back.

[0078] First, the MED (minimal erythema dose) of the test site for each subject should be determined. Then, each test area should be selected at the test site and irradiated once a day at a dose of 0.75 times the MED at the same irradiation point using a solar simulator for 4 consecutive days to establish a melanization model.

[0079] Four areas were selected on the back of the volunteers, of which three areas were used to apply the three test products, and the other area was used as a positive control (7% ascorbic acid); the test products and the positive control were applied to the test area twice a day, with an interval of at least 4 hours between the two applications, and the application area was 6 cm 2 , the coating amount is 2.00±0.05mg / cm 2 The interval between each test area should be no less than 1.0 cm.

[0080] Apply the test substance continuously for 2 weeks. Perform visual assessment and instrumental testing of skin color 1 week and 2 weeks after application, and record the results.

[0081] The measurement indicators are: MI (Melanin Index): This parameter characterizes the melanin content in the skin by measuring the absorption of a specific wavelength spectrum by the skin surface. The smaller the MI value, the lower the skin melanin content, and vice versa. The MI value is tested using skin melanin and hemoglobin test probes;

[0082] The test results refer to Table 3;

[0083] Table 3 MI (melanin index) test results

[0084] Before use 1 week 2 weeks Example 2 260.26 252.26 241.18 Comparative Example 2 259.98 253.78 246.46 Comparative Example 3 257.13 255.43 249.78 Positive control 259.14 248.23 236.99

[0085] Result analysis:

[0086] According to the above-mentioned melanin index test results, the combined use of trehalose and sodium mannose phosphate can make the whitening effect of 4-butylresorcinol and diglucosylgallic acid solution more stable.

[0087] Part III B16 cell test experiment

[0088] The sample of Example 2 was sent to Shanghai Aiji Biotechnology Co., Ltd., and a cell model was established by culturing B16 cells in vitro. The cells were treated with the sample, and total RNA of B16 cells was extracted for transcriptome sequencing and basic data analysis. The report number is AGSE2241216-1;

[0089] Experimental materials and reagents

[0090] B16F10 mouse melanocytes (B16) were purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences; culture medium was purchased from Lifeline Company; CCK reagent was purchased from Tongren Biotechnology; Trizol reagent, 12-well and 96-well cell culture plates were purchased from Thermo Company.

[0091] Experimental methods

[0092] B16 cell activity test

[0093] B16 cells were expanded and cultured in B16 cell culture medium at 5% CO₂ and 37°C. When cells reached 80-90% confluence, they were trypsinized and seeded into 96-well plates. After 48 hours of adherent culture in the 96-well plates, the cells were treated with various concentrations of samples (N=3). After 24 hours, CCK-8 reagent was added according to the manufacturer's instructions and incubated for 1 hour. OD values were read at 450 nm using a microplate reader. Relative cell viability values were calculated by comparing the mean OD values of the sample group to the blank control group to determine the cytotoxicity of the sample.

[0094] Relative cell activity % = (OD value of sample group / OD value of blank control group) x 100%

[0095] Transcriptome sequencing and data analysis of B16 cells

[0096] When B16 cells cultured in vitro grew to 80-90% confluence, they were digested with trypsin and seeded into 12-well plates. After the cells were cultured in the 12-well plates for 48 hours, they were treated with samples at a safe concentration (N=3). After 24 hours, the supernatant was aspirated, and Trizol reagent was added to each well. The cells were thoroughly pipetted to digest and lyse the cells, and the lysate was transferred to a deep cryostat at -80°C for storage until use. After the total RNA extraction and quality inspection were passed, library construction was performed, and the samples were sequenced using the paired-end sequencing mode of the Illumina Hiseq sequencing platform for high-throughput sequencing.

[0097] Effects of samples on B16 cell viability

[0098] After treating B16 cells with samples of different concentrations for 24 hours, the cell activity was detected using a CCK kit. The results are as follows: Figure 1 It showed that the sample had no toxic effect on cells at concentrations of 0.039% and below;

[0099] RNA sample quality control

[0100] To investigate the mechanism of the whitening effect, B16 cells were treated with a 0.03% concentration of the sample. Cell lysates from the sample and control groups were collected for total RNA extraction. The RNA quality test results, shown in Table 2, showed that both the sample and control groups were Grade A, with clear main bands and no contamination from impurities such as pigments, proteins, and carbohydrates. The total RNA content was greater than 1 μg, meeting the requirements for a single standard library construction and suitable for subsequent experiments.

[0101] Sequencing data quality statistics can be found in Table 4 ;

[0102] Table 4 Sequencing and data quality control statistics

[0103] Sequencing data quality statistics (QC.xls)

[0104]

[0105] As shown in Table 3 , sequencing libraries were successfully constructed and the data quality was high, with all samples meeting Q30 > 85%;

[0106] Differentially expressed gene screening

[0107] Based on the experimental design, DESeq2 software was used to screen differentially expressed genes between different sample groups. Genes with mean differences and P-value < 0.05 were screened as differentially expressed genes between the two groups. A total of 4,800 differentially expressed genes were found between the sample group and the control group. Compared with the blank control group, the expression levels of 2,204 genes in the sample group were significantly downregulated and the expression levels of 2,596 genes were significantly upregulated. Figure 2 Cluster diagram, volcano diagram and scatter plot of differentially expressed genes between the sample group and the blank control group.

[0108] Table 5 lists some differentially expressed genes known to be associated with skin whitening and cell autophagy. The expression levels of multiple genes in the sample group showed significant positive effects compared with the control group (NT).

[0109] Table 5 Differentially expressed genes related to skin

[0110]

[0111] From the data in Table 5, it can be seen that the combination of the whitening component of the present invention and the autophagy-related component to enhance the whitening effect can be supported at the genetic level.

[0112] GO functional annotation enrichment analysis of differentially expressed genes

[0113] GO (Gene Ontology) is a widely used ontology in bioinformatics, providing a standardized platform for describing gene terms or explaining their meanings. We used TopGO software to perform GO functional enrichment analysis on the differentially expressed gene list obtained above. This analysis categorized the differentially expressed gene set according to its function, thereby achieving the goal of annotating and classifying these genes.

[0114] Figure 3 This is a graphic of the GO functional enrichment analysis results of differential genes, showing the top 20 GO entries ranked by Enrichment Factor. The size of the network node indicates the total number of candidate genes belonging to GO.

[0115] KEGG pathway enrichment analysis of differentially expressed genes

[0116] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database that systematically analyzes gene function and genomic information. It facilitates the study of genes and their expression as a holistic network. KEGG pathway enrichment analysis, similar to GO functional enrichment analysis, enriches differentially expressed genes for pathways, helping to identify biological regulatory pathways that exhibit significant differences under experimental conditions.

[0117] Figure 4 This is a graphical display of the KEGG pathway functional enrichment analysis results of differentially expressed genes. The vertical axis represents the top 20 terms / pathways ranked in descending order of enrichment factor from top to bottom. The size of the scatter points in the figure indicates the number of enriched genes falling in the pathway.

[0118] in conclusion

[0119] Transcriptomics results showed that at a concentration of 0.03%, the sample had a certain inhibitory trend on the expression of 6 melanin synthesis and transport-related genes, and a certain promoting trend on the expression of 9 cell autophagy-related genes, and had the potential for whitening effects.

[0120] Application formula

[0121] Examples of the application formulation and preparation method of the composition of the present invention can be found in Table 6;

[0122] Table 6 Application formula

[0123]

[0124]

Claims

1. A whitening composition for activating the autophagy pathway, characterized in that: It comprises the following components in parts by weight: 15-25 parts of 4-butylresorcinol; 25-40 parts of diglucosyl gallic acid solution; 2.5-4 parts of trehalose; 1-2 parts of sodium mannose phosphate; The concentration of diglucosyl gallic acid in the diglucosyl gallic acid solution is 2.5 wt %.

2. The whitening composition according to claim 1, characterized in that It comprises the following components in parts by weight: 18-23 parts of 4-butylresorcinol; 30-35 parts of diglucosyl gallic acid solution; 3-3.5 parts of trehalose; 1.3-1.8 parts of sodium mannose phosphate.

3. Use of the whitening composition according to claim 1 or 2 in preparing cosmetics.

4. A cosmetic with whitening effect, characterized in that: Comprising the composition as claimed in claim 1 or 2.

5. The cosmetic according to claim 4, characterized in that The composition contains 0.5 to 3 wt%.

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