Gene level anti-aging composition for inhibiting DNA methylation, application and cosmetic

Abstract

The invention belongs to the field of daily chemicals, and discloses a gene-level anti-aging composition for inhibiting DNA methylation, which comprises the following components in percentage by weight: 50-70% of hydrolyzed royal jelly protein complex; 30 to 50 percent of panthenol; and 1-3% of a kelp extract. The composition takes a hydrolyzed royal jelly protein compound, panthenol and a kelp extract as main active ingredients, the composition is proved to have an excellent apparent anti-aging function through an epigenetics anti-aging test, and an excellent anti-aging effect can be achieved under the condition that the hydrolyzed royal jelly protein compound, the panthenol and the kelp extract are relatively small in dosage through related anti-aging tests. Meanwhile, the invention further discloses application of the composition and cosmetics.

Description

Technical Field

[0001] The present invention relates to the daily chemical field, and in particular to a gene-level anti-aging composition for inhibiting DNA methylation, its application and cosmetics. Background Art

[0002] Hydrolyzed Royal Jelly Protein is a small molecule peptide substance obtained by enzymatic hydrolysis of the protein extracted from royal jelly. Royal jelly itself is rich in nutrients such as protein, fatty acid, carbohydrate, hormone, and mineral, and the hydrolysis treatment makes it more easily absorbed by the skin to exert skin care effects;

[0003] Hydrolyzed Royal Jelly Protein has various functions in the skin care field:

[0004] Antioxidation: It can eliminate free radicals and prevent skin aging.

[0005] Moisturizing and repair: It promotes the production of collagen, increases skin elasticity, relieves skin irritation, and prevents wrinkles and relaxation.

[0006] Whitening and soothing: It can be used in products for whitening and lightening spots, anti-allergy, anti-irritation, etc. to improve the skin condition.

[0007] Anti-inflammatory and regeneration: It reduces skin inflammation and erythema and promotes the regeneration of epidermal cells;

[0008] Panthenol is mainly used as a skin moisturizer. Panthenol is an alcohol derivative of pantothenic acid. Panthenol has many forms, and the common ones are D-panthenol (right-handed), DL-panthenol (racemic from left to right), L-panthenol (left-handed), calcium pantothenate, etc. It is commonly used as a moisturizer and skin conditioner, can promote the metabolism of proteins, fats, and carbohydrates in the human body, protect the skin, prevent small wrinkles and inflammation. At the same time, it increases the content of hyaluronic acid. On the other hand, panthenol is a non-greasy moisturizing ingredient with strong moisturizing effects, similar to the sebum film of the skin, and is easily absorbed, penetrated, and infiltrated into the skin cutin layer.

[0009] Kelp extract is a fine powder concentrated and extracted from kelp, yellowish-brown, rich in various beneficial ingredients, and has a wide range of applications.

[0010] The applications of hydrolyzed royal jelly protein, panthenol, and kelp extract in the skin can be seen in the following literature:

[0011] The patent publication number is CN105640854A, and the subject is a caviar royal jelly firming eye gel and its preparation method. It discloses that hydrolyzed royal jelly protein is used to promote collagen production and prevent relaxation and wrinkle formation; components such as Laminaria ochotensis extract and hydrolyzed soy protein can counteract external damage (ultraviolet lamp, etc.). Due to its complex components, we cannot clarify whether there is an internal correlation between the above two;

[0012] In a large number of patent documents, panthenol is only considered to be used as a moisturizer. For example, in CN116687781A, a composition for enhancing mitochondrial energy and its application; in CN116392436A, a composition for repairing oily and sensitive skin barriers, hydrolyzed royal jelly protein is considered to be an effective active ingredient, while panthenol is used as an ordinary moisturizer.

[0013] In the research of this project, we found that there is a rather ingenious internal synergy among the above three, and based on this, this case is proposed. Summary of the Invention

[0014] The object of the present invention is to provide a gene-level anti-aging composition for inhibiting DNA methylation. The composition uses hydrolyzed royal jelly protein complex, panthenol, and kelp extract as the main active ingredients. The composition has passed epigenetic anti-aging tests, proving that it has excellent epigenetic anti-aging functions. Through relevant anti-aging tests, it is found that the above four can achieve excellent anti-aging effects with less dosage.

[0015] Meanwhile, the present invention also discloses the application of the composition and cosmetics.

[0016] To achieve the above object, the present application discloses:

[0017] A gene-level anti-aging composition for inhibiting DNA methylation, comprising the following components in weight percentage:

[0018] Hydrolyzed royal jelly protein complex 50 - 70%;

[0019] Panthenol 30 - 50%;

[0020] Kelp extract 1 - 3%.

[0021] The hydrolyzed royal jelly protein complex is rich in proteins, sugars, fatty acids, vitamin B, and various antibacterial components, which activate the proteasome to degrade aging proteins. Panthenol enhances the skin barrier function and reduces the release of inflammatory factors, and the chronic activation of inflammatory signals will accelerate aging through epigenetic mechanisms; kelp extract can activate the synthesis of nc886 to activate skin vitality. The three work together to reduce the negative impact of epigenetic aging.

[0022] In the above gene-level anti-aging composition for inhibiting DNA methylation, it comprises the following components in weight percentage:

[0023] 55 - 65% hydrolyzed royal jelly protein complex;

[0024] 35 - 45% panthenol;

[0025] 1.5 - 2.5% kelp extract.

[0026] Meanwhile, the present invention also discloses the use of the gene - level anti - aging composition for inhibiting DNA methylation as described above in preparing cosmetics.

[0027] In the above - mentioned use, the cosmetic has the function of regulating the expression of methylated genes.

[0028] In the above - mentioned use, the methylated genes are one or more combinations of methylated - related collagen expression genes, matrix metalloproteinase expression genes, autophagy expression genes, GAG - type glycosaminoglycan expression genes, anti - aging expression genes, antioxidant expression genes, and growth factor expression genes.

[0029] Finally, the present invention also discloses a cosmetic containing the composition as described above.

[0030] In regular use, we recommend that the dosage of the above composition be 0.5wt% - 5wt%.

[0031] This application has at least the following beneficial effects:

[0032] The composition of the present invention uses hydrolyzed royal jelly protein complex, panthenol, and kelp extract as the main active ingredients. This composition has passed epigenetic anti - aging tests, proving that it has excellent epigenetic anti - aging functions. Through relevant anti - aging tests, it is found that the above four components can achieve excellent anti - aging effects with relatively small dosages.

[0033] We speculate that the deep - layer synergistic mechanism is as follows:

[0034] The hydrolyzed royal jelly protein complex is rich in proteins, carbohydrates, fatty acids, vitamin B, and various antibacterial components, which activate the proteasome to degrade aging proteins. Panthenol enhances the skin barrier function and reduces the release of inflammatory factors, and the chronic activation of inflammatory signals will accelerate aging through epigenetic mechanisms; the kelp extract can activate the synthesis of nc886 to activate skin vitality. The three work together to reduce the negative impact of epigenetic aging. Description of the Drawings

[0035] Figure 1 It is a graphical display of the results of the KEGG pathway functional enrichment analysis of differential genes. Detailed Embodiments

[0036] The present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that for those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. Unless otherwise specified, the parts used in the embodiments of the present invention are all parts by weight.

[0037] Raw material source

[0038] Hydrolyzed royal jelly protein complex: The supplier is Mibelle Group Biochemistry, and the specification is: RoyalEpigen P5 content 0.014 wt%;

[0039] Panthenol: The supplier is Shanghai Xuntian Biotechnology Co., Ltd., and the specification is: D-panthenol content 100%;

[0040] Kelp extract: The supplier is BioSpectrum, Inc., and the specification is: Epseama content 1.5 wt%.

[0041] Preparation of the first part of the composition

[0042] The hydrolyzed royal jelly protein complex, panthenol, and kelp extract are mixed in the following proportions and then diluted to a preset concentration after mixing.

[0043] The formulation table can be referred to Table 1;

[0044] Table 1 Formulation table

[0045]

[0046] Second part Performance test

[0047] 2.1 Safety test

[0048] Examples 1, 3, and 5 are selected for testing;

[0049] Multiple skin irritation tests

[0050] Detection method:

[0051] Test substance: Take an appropriate amount of the sample of Example 1 and mix it well at 1:19 (sample: pure water, volume ratio) as the test substance.

[0052] Experimental animals: 4 ordinary-grade New Zealand rabbits;

[0053] Twenty-four hours before the experiment, the hair on both sides of the back of New Zealand rabbits was shaved off. The hair removal area on both the left and right sides was approximately 3 cm × 3 cm. The right side was used as the drug application area, and the left side was used as the blank control area. 0.5 mL of the sample of Example 7 was measured and evenly applied to the skin of the animal application area once a day for 14 consecutive days. Starting from the second day, the hair was cut before each application. After removing the residual test substance with pure water for 1 hour, the results were observed. The skin irritation / corrosion test Table 1 in the "Technical Specifications for Cosmetic Safety" (2015 edition) was used for scoring. The average score per animal per day was calculated according to the following formula, and the skin irritation intensity was determined according to Table 2 of the skin irritation / corrosion test in the "Technical Specifications for Cosmetic Safety" (2015 edition).

[0054] Average score per animal per day = (∑erythema and edema scores / number of test animals) / 14

[0055] The experimental results are referred to Table 2;

[0056] Table 2 Results of irritation experiment

[0057]

[0058]

[0059] Test conclusion

[0060] The sample of Example 1 had no irritation to the skin of New Zealand rabbits after multiple applications.

[0061] The samples of Example 3 and Example 5 were tested according to the same experimental method, and the results are shown in Table 3 and Table 4 respectively;

[0062] Table 3 Results of irritation experiment

[0063]

[0064] Table 4 Results of irritation experiment

[0065]

[0066] The samples of Example 3 and Example 5 had no irritation to the skin of New Zealand rabbits after multiple applications.

[0067] 2.2 Skin anti-aging test

[0068] Elastase inhibition test:

[0069] 1. Test principle

[0070] As a highly selective and specific protease, elastase catalyzes the hydrolysis of many amino acids such as glycine, leucine, alanine and other hydroxyl-containing polypeptide bonds, and can decompose elastin in connective tissue proteins. Inhibiting the activity of elastase can effectively slow down the degradation of elastin and reduce skin aging, wrinkles and photoaging.

[0071] 2. Test article:

[0072] Test sample original: test concentration is 1%;

[0073] Positive control: Sivelestat sodium solution tested at a concentration of 40 μg / mL

[0074] 3. Test method:

[0075] Determination of elastase inhibition rate: 90 μL buffer, 30 μL sample diluent of different concentrations and 10 μL substrate were added to a 96-well plate, mixed and incubated at 25°C for 20 minutes. Then 20 μL elastase solution was added and incubated for 10 minutes to detect the absorbance, and the statistical difference P value was calculated to obtain the effect of the sample on the activity of elastase.

[0076] Table 5 Elastase inhibition test results

[0077]

[0078]

[0079] Results and Discussion:

[0080] 1. It can be seen from Examples 1 to 5 that the above-mentioned test performances are similar, among which Example 3 has the best effect, indicating that in the formula of the present invention, the hydrolyzed royal jelly protein complex, panthenol, and kelp extract jointly exert an elastase inhibitory effect.

[0081] 2. It can be seen from Comparative Examples 1, 2 and 3 that when panthenol is dominant, its inhibitory effect on elastase is not obvious; when the hydrolyzed royal jelly protein complex is dominant, its inhibitory effect on elastase is obvious; when the hydrolyzed royal jelly protein complex and panthenol are used in combination, the function is not as good as when the hydrolyzed royal jelly protein complex is the main component, indicating that in the formula of the present invention, the hydrolyzed royal jelly protein complex is the main substance that inhibits elastase; the use of panthenol is equivalent to diluting the hydrolyzed royal jelly protein complex, which reduces the performance of the product; at the same time, when panthenol and kelp extract are used in combination, the effect of kelp extract is not obvious; referring to Example 3, it can be seen that when the three are used in combination, they have a more obvious performance improvement.

[0082] 3. After replacing panthenol with glycerol and vitamin B5, it shows a similar trend to Comparative Example 1 and Comparative Example 2, and its combined performance is also inferior to that of Example 3. Among them, the combined performance of vitamin B5 with hydrolyzed royal jelly protein complex and kelp extract is better than that of glycerol. On the one hand, the chemical structures of vitamin B5 and panthenol are relatively similar, so their synergy is better than that of glycerol. On the other hand, although vitamin B5, glycerol, and panthenol are all moisturizers, in addition to their moisturizing effects, vitamin B5 can promote skin metabolism and repair the skin barrier, and panthenol can also repair the skin barrier and promote metabolism. It can quickly penetrate into the stratum corneum and be converted into pantothenic acid and coenzyme A, which further affects the elastase reaction pathway to achieve a better effect of inhibiting elastase. At the same time, like vitamin B5, it also has the functions of inhibiting inflammatory factors and antioxidant free radicals, and these auxiliary external factors can further inhibit the activity of elastase.

[0083] The third part: Epigenetic anti-aging test

[0084] Send the product of Example 3 to a third-party testing agency for testing;

[0085] 1. Project introduction

[0086] DNA methylation is a typical epigenetic mechanism that plays an important role in gene expression regulation and has an important mechanism in normal cell development and maintaining tissue stability. Many human diseases (including cancer) are caused by abnormal methylation.

[0087] The test sample of this project is an active ingredient raw material, and the goal is to investigate its skin care efficacy and mechanism. A cell model is established using normal human dermal fibroblasts cultured in vitro, the cells are treated with the sample, and Illumina DNA methylation chips are used for sequencing and basic data analysis to explore the deep mechanism of the skin care efficacy of the sample.

[0088] 2. Experimental materials and reagents

[0089] Human primary dermal fibroblasts and culture medium are purchased from Lifeline Company; CCK reagent is purchased from Tongren Biotech (Cat#: 40203ES80); Trizol reagent (Cat#: 15596026), 6-well, 12-well, and 96-well cell culture plates are purchased from Thermo Company.

[0090] Main equipment

[0091] Cell culture incubator, inverted fluorescence microscope, biosafety cabinet, Illumina sequencing platform.

[0092] Sample preparation

[0093] A total of 1 sample was detected this time, and its specific information is shown in Table 1:

[0094] Table 1. Formulation information of test samples

[0095] Physical and chemical properties Storage conditions Sample preparation Emulsion Store at 4°C Filter through a 0.22 μm filter after diluting the medium

[0096] Experimental method

[0097] Activity test of human primary dermal fibroblasts

[0098] Human primary dermal fibroblasts were amplified and cultured in fibroblast medium under the culture conditions of 5% CO2 and 37°C. When the cells grew to 80-90% confluence, they were digested with trypsin and seeded into a 96-well plate. After the cells were adherently cultured in the 96-well plate for 48 h, samples with different concentrations were prepared to treat the cells (N = 3). After 24 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 blank control group, and the cytotoxic effect of the sample was judged.

[0099] Relative cell activity % = (OD value of sample group / OD value of blank control group) x 100%

[0100] Transcriptome sequencing and data analysis of human primary dermal fibroblasts

[0101] When the fibroblasts cultured in vitro grew to 80-90% confluence, they were digested with trypsin and seeded into a 12-well plate. After the cells were adherently cultured in the 12-well plate for 48 h, the cells were treated with the sample 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 digested and lysed, and the lysate was transferred to -80°C for deep cryopreservation for later use. After the total DNA extraction and quality inspection were qualified, library construction was carried out, and high-throughput sequencing of the samples was performed using the DNA methylation chip Illumina InfiniumMethylationEPIC BeadChip.

[0102] Test results:

[0103] Figure 1 It is a graphical display of the results of KEGG pathway functional enrichment analysis of differential genes. The vertical axis from top to bottom is the top 20 terms / pathways ranked in descending order according to Enrichment Factor. The size of the scatter points in the figure indicates the number of enriched genes falling in that pathway.

[0104] The methylation results showed that the sample at a concentration of 0.5% could significantly regulate the expression of methylation-related genes such as collagen, matrix metalloproteinase, autophagy, GAG glycosaminoglycan, anti-aging, antioxidant, and growth factor. Therefore, the sample has the potential for certain skin care and anti-aging effects.

[0105] Part IV Application Formulation

[0106] The following shows the application of the composition of the present invention in skin care products with a specific formulation as shown in Table 8 below:

[0107] Table 7 Skin Care Product Formulation

[0108]

[0109]

[0110] In the present invention, the preparation process of the skin care product is as follows:

[0111] Add the raw materials of Phase A to the water pot in sequence, stir at 40 rpm until evenly dispersed, and heat to 80 °C; transfer to the main pot, add the raw materials of Phase B to the oil pot, stir at 700 rpm, heat to 80 °C, pump the raw materials of the oil pot to the main pot, stir at 40 rpm, and the homogenization speed is 1000 - 3000 r / min; the homogenization time is 10 min, emulsify until evenly fine and without particles, cool to 45 °C, add the raw materials of Phase C, and stir evenly to obtain the target product.

[0112] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claims involved.

Claims

1. A gene-level anti-aging composition for inhibiting DNA methylation, characterized in that, Comprising the following components in weight percentages: Hydrolyzed royal jelly protein complex 50 - 70%; Panthenol 30 - 50%; Kelp extract 1 - 3%.

2. The gene-level anti-aging composition for inhibiting DNA methylation according to claim 1, wherein, Comprising the following components in weight percentages: Hydrolyzed royal jelly protein complex 55 - 65%; Panthenol 35 - 45%; Kelp extract 1.5 - 2.5%.

3. Use of the gene-level anti-aging composition for inhibiting DNA methylation as described in claim 1 or 2 in the preparation of cosmetics.

4. The use according to claim 3, characterized in that, The cosmetics have the function of regulating the expression of methylated genes.

5. The use according to claim 4, wherein, The methylated genes are one or a combination of methylated collagen-related expression genes, matrix metalloproteinase expression genes, autophagy expression genes, GAG glycosaminoglycan expression genes, anti-aging expression genes, antioxidant expression genes, growth factor expression genes.

6. A cosmetic, characterized in that, Containing the composition as described in claim 1 or 2.

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