CN117297999A - Composition for improving skin state and application thereof - Google Patents

Abstract

The invention discloses a composition for improving skin condition and application thereof, wherein the addition mass of ergothioneine is 0.01-20%, and the addition mass of retinol is 0.01-20%. The composition for improving skin condition of the present invention can be applied to the preparation of medical and cosmetic products. Ergothioneine can improve stability of retinol, and effectively prevent damage of light and air to retinol. Ergothioneine reduces phototoxic effect of retinol on cells and inflammatory reaction caused by retinol, and compared with retinol alone, the combination of retinol and the ergothioneine promotes proliferation and migration of keratinocytes and basal layer cells better, and shortens cell renewal cycle. Ergothioneine can improve skin irritation side effect caused by retinol, and the combination of the ergothioneine and the retinol has synergistic effect on skin health, and has effects of reducing wrinkles, refining pores, whitening skin, reducing wrinkle, reducing pigmentation, brightening skin, enhancing skin elasticity, promoting collagen and elastin production, resisting inflammation, relieving and enhancing skin barrier.

Description

Composition for improving skin condition and application thereof

Technical field

The present invention relates to the technical field of composition preparation, and in particular to a composition for improving skin condition and its application.

Background technique

Retinol (VA) is a fat-soluble vitamin A and the precursor of retinoic acid (retinoic acid). Retinol or other forms of vitamin A are necessary for vision, cell development, maintenance of skin and mucous membranes, immune function, and reproductive development and are used as an ingredient in skin care products to reduce wrinkles and other effects of skin aging. , including wrinkle reduction, pore refinement, whitening and anti-wrinkle, anti-inflammatory and soothing.

Studies have shown that retinol exerts these effects by speeding up skin cell turnover, shedding old cells and promoting the production of new cells. As an all-purpose skin care active ingredient, retinol is deeply loved by consumers, but because of the side effects of skin irritation, consumers love and hate it. Usually consumers will experience varying degrees of skin irritation when they first use it, including dryness, inflammation, burning, pain and even redness, swelling and peeling. The reason for these side effects is also due to its ability to speed up cell renewal.

Some researchers believe that retinol can cause skin cells to renew faster, causing more dead skin cells to be shed, but may cause a lag time before new, healthy cells reach the surface of the skin. The new skin is exposed to outside influences before it is ready, such as light, air pollutants, etc. The result is redness, discoloration, and irritation. In addition, retinol itself has poor stability. It can easily denature and oxidize when exposed to air and light, and may even produce substances harmful to the body. In order to avoid this problem, many products on the market have It appears as modified retinol, such as retinyl palmitate, retinyl propionate, etc.

Ergothioneine is a water-soluble small molecule amino acid that is widely found in fungi in nature and also in the livers of animals. It cannot be synthesized by the human body, but almost every tissue and organ in the human body has its transporter OCTN-1. Expression, this transporter is also expressed in skin cells. Ergothioneine is a highly efficient antioxidant that effectively scavenges various free radicals, protects mitochondrial health, delays cell aging, and promotes apoptosis of senescent cells. It has been widely used in health products and cosmetics as an antioxidant and antioxidant. Functional components of aging.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method to improve the skin condition in view of the different kinds of skin irritations caused by existing retinol when used for the first time, including dryness, inflammation, burning sensation, pain, even redness, swelling and peeling. Compositions and their applications.

In order to achieve the above-mentioned object of the invention, the present invention provides a composition for improving skin condition, wherein the added mass of ergothioneine is 0.01%-20%, and the added mass of retinol is 0.01%-20%.

In a preferred embodiment of the present invention, the ergothioneine and the retinol can be added in the same proportion or in different proportions.

In a preferred embodiment of the present invention, the retinol has a cell viability concentration of 70-80%.

The application of a composition for improving skin condition of the present invention is in the preparation of medical beauty products.

In a preferred embodiment of the present invention, the medical beauty product is one of an emulsion, an essence, a cream, a facial mask, a spray, an ampoule, and an oral agent.

Studies have found that ergothioneine can improve the stability of retinol and effectively prevent the damage of retinol by light and air. Through experiments, it was found that ergothioneine reduced the toxic effect of retinol on cells and the inflammatory response caused by retinol. Compared with the use of retinol alone, the combination of the two better promoted the growth of stratum corneum cells and cells. The proliferation and migration of underlying cells shorten the cell renewal cycle. Therefore, we believe that ergothioneine can improve the side effects of skin irritation caused by retinol, and the combination of the two has a synergistic effect on skin health. Skin health includes reducing wrinkles, minimizing pores, whitening and anti-wrinkle, reducing pigmentation, brightening skin tone, enhancing skin elasticity, promoting the production of collagen and elastin, anti-inflammatory and soothing, and strengthening the skin barrier.

Description of drawings

Figure 1 is a bar graph showing the viability of HaCaT cells according to different concentrations of retinol.

Figure 2 is a bar graph showing the effect of ergothioneine on improving retinol toxicity.

Figures 3a to 3c are relative expression diagrams of inflammation-related genes induced by retinol according to different concentrations of ergothioneine.

Figure 4 is a schematic diagram of cell scratch images at different time points in Example 4.

Figure 5 is a schematic diagram of cell scratch images at different time points in Example 5.

Figure 6 is a schematic diagram of the growth cycle curve of HaCaT cells in the presence of retinol according to Example 6.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments.

Example 1. Retinol cytotoxicity experiment

Select human immortalized keratinocytes (HaCaT) in the logarithmic growth phase, inoculate 1.0×10 ⁴ per well into a 96-well cell culture plate (100 μL per well), and place them in a CO ₂ incubator (37°C, 5%) Culture for 24h. Remove the culture medium in the 96-well plate and add 200 μL of fresh culture medium containing different concentrations of retinol as the administration group. Add 200 μL of fresh culture medium to the blank control and continue culturing in the incubator for 24 hours before replacing it with 100 μL of 10% CCK-8 working solution, incubated at 37°C for 1 hour, and then measured the OD value of each well at a wavelength of 450 nm. Cell viability = (sample group OD group/blank group OD value) × 100%.

According to the histogram of different concentrations of retinol on the viability of HaCaT cells (see Figure 1), it can be seen that retinol in the administration group reduces the viability of HaCaT cells as the concentration increases, that is, it exhibits toxicity when it exceeds a certain concentration. It has a stimulating effect. Therefore, we chose to use a concentration around 75% of cell viability as the concentration for subsequent retinol stimulation to complete the next test to explore the improving effect of ergothioneine on retinol in the present invention.

Example 2. Ergothioneine improves the toxic effects of retinol

The conditions and methods were the same as in Example 1. First, the toxic effects of ergothioneine containing only different concentrations on HaCaT cells were determined. The difference is that the retinol concentration with cell activity of 70-80% in Example 1 was selected as the positive control group, and three safe concentrations of ergothioneine without significant toxicity were selected as the experimental group. Ergothioneine and retinol were combined. After preparation, it was used as fresh administration medium. After incubation for 24 hours, it was replaced with 100 μL of 10% CCK-8 working solution. After 1 hour of incubation, the OD value of each well was measured at a wavelength of 450 nm to calculate cell viability. Determination of the ameliorating effect of ergothioneine on retinol toxicity.

According to the results in the bar graph (see Figure 2), the presence of ergothioneine alone at a concentration of 0.1mM-10mM has no toxic effect on HaCaT cells. Correspondingly, ergothioneine at this concentration was compounded with retinol, and it was found that ergothioneine significantly reduced the toxic effects stimulated by retinol as the concentration increased, effectively improving the side effects of retinol.

Example 3. Ergothioneine improves the inflammatory effect of retinol

Select HaCaT cells in the logarithmic growth phase and inoculate them in a 6-well plate at 6.0 × 10 ⁵ per well. The volume of each well is 1 mL. Incubate at 37°C and 5% CO ₂ for 24 hours. The normal control group is replaced with fresh culture medium. , the model control group was replaced with fresh medium containing retinol determined in Example 1, and the sample group was replaced with fresh medium containing three safe concentrations of ergothioneine and retinol compound, at 37°C, 5% Incubate with _CO2 for 24h. Collect cells: extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and target genes. β-actin was used as the internal reference for gene expression to calculate the relative RNA expression of TRPV1, IL-1β, and NF-κB genes.

The relative expression diagram of ergothioneine on retinol-induced inflammation-related genes according to different concentrations is shown in Figure 3. It can be seen from Figure 3 that ergothioneine significantly reduces TRPV1 and IL produced by retinol as the concentration increases. -1β and NF-κB gene expression, which shows that ergothioneine can effectively alleviate the inflammatory effect on cells in the presence of retinol and repair the skin barrier.

Example 4. Ergothioneine cooperates with retinol to promote the proliferation and migration of stratum corneum cells

Select HaCaT in the logarithmic growth phase, inoculate 5.0 × 10 ⁵ per well into a 12-well cell culture plate (1 mL per well), and culture in a CO ₂ incubator (37°C, 5%) for 24 hours. After marking the positioning line at the bottom of the culture plate, use a 1mL pipette to make straight scratches on the cells in each well. Remove the culture medium in the 6-well plate, wash 3 times with pre-cooled PBS to wash away cell residues, and add 1mL containing Safe concentrations of ergothioneine alone, retinol alone, and fresh serum-free medium mixed with ergothioneine and retinol were used as the administration group. 1 mL of fresh culture medium was added to the blank control. EGF growth factor was used in the control group. Continue After culturing in the incubator for 12, 24, and 48 hours, the original scratches were photographed and compared with the photos taken at 0 h. The cell scratch area was calculated using Image J image analysis software to analyze the migration ability of HaCaT cells. After taking pictures at 48 hours, add CCK-8 working solution to measure the cell activity of each group, and calculate the proliferation of HaCaT cells.

Referring to Figure 4, according to the cell scratch images at different time points, it can be seen that the migration ability of HaCaT cells is significantly improved in the presence of ergothioneine and retinol, and the scratch area gradually decreases over time, and finally almost returns to Before scratching. At the same time, the migration ability of the compound group of ergothioneine and retinol was weaker than that of the retinol group, indicating that the compound compound of ergothioneine and retinol can regulate the migration of skin keratinocytes. In addition, according to the cell viability after 48 hours, both ergothioneine and retinol have a certain proliferation effect on HaCaT cells. These results indicate that ergothioneine, retinol and their compounds may have the ability to quickly repair skin. Efficacy of barriers and postoperative trauma recovery.

Example 5. Ergothioneine cooperates with retinol to promote the proliferation and migration of basal layer cells

Select human skin fibroblasts (HSF) in the logarithmic growth phase, inoculate 1.0×10 ⁵ per well into a 12-well cell culture plate (1 mL per well), and culture in a CO ₂ incubator (37°C, 5%). 24h. After marking the positioning line at the bottom of the culture plate, use a 1mL tip to make straight scratches on the cells in each well. Remove the culture medium in the 6-well plate, wash 3 times with pre-cooled PBS to wash away cell residues, and add 1mL containing Safe concentrations of ergothioneine alone, retinol alone, and fresh serum-free medium mixed with ergothioneine and retinol were used as the administration group. 1 mL of fresh culture medium was added to the blank control. EGF growth factor was used in the control group. Continue After culturing in the incubator for 12, 24, and 48 hours, the original scratches were photographed and compared with the photos taken at 0 h. Image J image analysis software was used to calculate the cell scratch area and analyze the migration ability of HSF cells. After taking pictures at 48 hours, CCK-8 working solution was added to measure the cell activity of each group, and the proliferation of fibroblasts was calculated.

Referring to Figure 5, according to the cell scratch images at different time points, it can be seen that the migration ability of HSF cells is significantly improved in the presence of ergothioneine and retinol, and the scratch area gradually decreases with time, and finally almost returns to Before scratching. At the same time, the migration ability of the compound group of ergothioneine and retinol is higher than that of the retinol group, indicating that the compound compound of ergothioneine and retinol can regulate the migration of dermal fibroblasts. In addition, according to the cell viability after 48 hours, both ergothioneine and retinol have a certain proliferation effect on HSF cells. These results indicate that ergothioneine can significantly promote the proliferation and migration of dermal fibroblasts, which This effect is also shown when compounded with retinol, which is higher than that of the retinol alone group. This shows that ergothioneine can improve the proliferation and differentiation rate of dermis cells in the presence of retinol, which is lower than the proliferation and differentiation rate of stratum corneum cells. For skin sensitivity problems, it promotes the metabolism of full-thickness skin cells and improves the skin toxic effects of retinol.

Example 6. Ergothioneine cooperates with retinol to regulate keratinocyte growth cycle

Select HaCaT cells in the logarithmic growth phase, inoculate 6.0×10 ⁵ per well into a 6-well cell culture plate (1 mL per well), and culture in a CO ₂ incubator (37°C, 5%) for 24 hours. Remove the culture medium in the 6-well plate, wash 3 times with pre-cooled PBS to wash away cell residues, and add 1 mL of fresh culture medium containing safe concentrations of ergothioneine and retinol as the administration group, according to Example 1 The safe retinol concentration in the sample was used as a positive control group. 1 mL of fresh culture medium was added to the blank control. After continuing to culture in the incubator for 24 hours, the cells in each well were digested with trypsin. Changes in the cell growth cycle were detected by a cell viability analyzer and analyzed. Changes in the growth and proliferation cycle of HaCaT cells by ergothioneine and retinol.

Referring to Figure 6, according to the growth cycle curve of HaCaT cells in the presence of retinol, it can be seen that when ergothioneine is combined with retinol, it effectively controls the excessive cell production cycle caused by retinol, indicating that ergothioneine It regulates cell metabolism caused by retinol and prevents skin problems caused by excessive skin renewal.

Example 7. Ergothioneine improves retinol stability

Monitor changes in the content of ergothioneine and retinol using high-performance liquid chromatography (HPLC) as follows:

HPLC method for detecting ergothioneine content:

Liquid phase: Thermo Fisher

Column: C18 reverse chromatography column

Flow rate: 1.0mL/min

Column temperature: 35℃

Mobile phase: 95% methanol-water

Detection: 325nm/full wavelength

Injection volume: 10μL

By compounding ergothioneine and retinol at different concentrations, 30% ethanol was added, and the content changes of ergothioneine and retinol were detected by HPLC at different time points: 1 day, 3 day, and 7 day. Analyze the effect of ergothioneine on the breakdown of retinol.

The results showed that the content of retinol decreased with time, indicating that retinol is easily decomposed in 30% ethanol aqueous solution. When ergothioneine is combined with retinol, this decomposition effect is significantly reduced, indicating that ergothioneine can significantly inhibit the degradation rate of retinol and improve the stability of retinol.

Example 8. Ergothioneine improves skin discomfort caused by retinol

20 women aged 25-35 who had not used retinol or EGT-related skin care products within a year. Use 1% Retinol Essence and 1% Retinol & 1% EGT Essence on the left and right sides respectively, once a day for 7 days. On Day 0 and Day 30, subjects will receive a questionnaire to fill in their facial skin feelings, and researchers will record the subject's skin status through observation and questioning. The result is as follows:

8.1 Analysis and personnel statistics of facial application of 1% retinol essence

Day 0-Number of people Day 7-number of people Percentage of people with skin discomfort Redness and swelling 0 4 20% burning sensation 0 8 40% tingling sensation 0 6 30% Peeling 0 5 25% dry 0 13 65%

8.2 Analysis and personnel statistics of facial application of 1% retinol & 1% EGT essence

Day 0-Number of people Day 7-number of people Percentage of people with skin discomfort Redness and swelling 0 1 5% burning sensation 0 3 15% tingling sensation 0 5 25% Peeling 0 3 15% dry 0 5 25%

From the analysis of the results, it can be seen that the retinol essence added with EGT can significantly alleviate the redness and swelling and other skin irritations.

Claims (4)

1. A composition for improving skin condition, characterized in that the added mass of ergothioneine is 0.01%-20%, and the added mass of retinol is 0.01%-20%. 2. A composition for improving skin condition according to claim 1, characterized in that the ergothioneine and the retinol can be added in the same proportion or in different proportions. 3. An application of the composition for improving skin condition according to any one of claims 1 to 3, which is characterized in that it is used in the preparation of medical beauty products. 4. The application according to claim 4, characterized in that the medical beauty product is one of emulsion, essence, cream, facial mask, spray, ampoule, and oral agent.