KR102377510B1 - Cosmetic composition for preventing skin aging showing anti-stress effect, including hydrolyzate of Tremella fuciformis) - Google Patents

Abstract

The present invention relates to a cosmetic composition which helps prevent aging that may occur by stress, and more specifically, to a cosmetic composition containing a Tremella fuciformis extract that inhibits the expression of 11β-HSD1 and promotes collagen production and cell migration. Specifically, the Tremella fuciformis extract of the present invention includes extraction conditions capable of suppressing the expression of 11β-HSD1, which plays an important role in the production of cortisol that occurs aging by stress, as much as possible. That is, the cosmetic composition according to the present invention contains an enzyme-treated Tremella fuciformis hydrolysate extract that maximally inhibits the expression of 11β-HSD1 so as to prevent aging caused by stress, to promote collagen production and cell migration, and to be effective in heating wounds.

Description

Cosmetic composition for preventing skin aging showing anti-stress effect, including hydrolyzate of white wood ear mushroom {Cosmetic composition for preventing skin aging showing anti-stress effect, including hydrolyzate of Tremella fuciformis)}

The present invention relates to a cosmetic composition for preventing skin aging having an anti-stress effect. It aims to relieve stress caused by the increase of α and θ waves emitted by nerve cells in the brain and decrease of β waves by suppressing the production of cortisol, a stress substance. According to the present invention, the hydrolyzate prepared under optimal extraction conditions inhibits the expression of 11β-HSD1, which has an important effect on cortisol production, and is effective for collagen production, cell migration, and wound healing.

Stress is a feeling of anxiety and threat that humans feel psychologically or physically when they are in a difficult situation. When we encounter a stressful situation, the sympathetic part of the autonomic nervous system is activated as a body response to the stress, and the body's resources are mobilized to respond to an emergency situation. Moderate stress maintains the health of the human body and gives vitality to quickly respond to situations that may arise in the future. It serves as the driving force of life and acts as a net function that boosts the vitality of our lives as it increases efficiency and productivity. However, continuous stress may cause insomnia, depression, etc., which may lead to physical and mental dysfunction.

EEG is a radio wave that is emitted by nerve cells in the brain, and there are α, β, γ, δ, and θ depending on the frequency. The α wave is the brain wave that comes out a lot when the mind and body are relaxed and feels comfortable, and the β wave appears when doing general work or performing conscious actions and is activated when there is mental activity. The θ wave is activated when all thoughts are gone and you are in a state of calm, allowing you to exercise your physical performance to an excellent level. In humans, as the α and θ waves increase, and the β wave decreases, it indicates that the stress was relieved.

In order to cope with these stresses, energy must be produced within the body. Among the nervous systems of the body, the sympathetic nervous system starts to work, and steroid hormones are secreted from the adrenal glands. Cortisone present in the adrenal cortex is converted into cortisol, a stress hormone, and secreted by the action of 11β-hydroxysteroid dehydrogenase (11β-HSD1) reductase when stressed. The 11β-HSD1 enzyme stimulates inactive cortisone and reduces it to activated cortisol. The expression of 11β-HSD1 is related to collagen synthesis, and when 11β-HSD1 is inhibited, the number of dermal fibroblasts increases to promote collagen metabolism. Yes (Journal of Clinical Investigation, 123(7), 3051 (2013)).

Cortisol causes the body to release a lot of blood to each organ in response to stress. As a result, the pulse and respiration increase, and the muscles tense and sharpen the sensory organs to make accurate and rapid situational judgments. However, excessive stress raises blood pressure and increases the risk of hypertension, which can also cause muscle tissue damage. In addition, the ability to protect the skin from external harmful factors decreases due to damage and weakening of the skin barrier. In addition, it affects collagen synthesis, which slows wound healing due to a decrease in the ability of skin regeneration cells due to a decrease in collagen, which adversely affects skin aging.

The present invention intends to develop a material capable of relieving stress by inhibiting the expression of 11β-HSD1 enzyme that produces the stress hormone cortisol. Therefore, it is intended to develop a material effective for proliferation of dermal fibroblasts, promotion of collagen synthesis, and wound healing.

Republic of Korea Patent No. 10-1772614 (registered on August 23, 2017) discloses a cosmetic composition containing a fermented product obtained by inoculating and fermenting a white wood ear mushroom mycelium in a medium containing an epithelial cell growth factor. Republic of Korea Patent No. 10-1834795 (registered on February 27, 2018), describes a cosmetic composition for skin improvement containing nanoliposomes containing a mixed extract of white oyster mushroom and yam extracted by an ultrasonic parallel cold pressing method.

The present invention provides a cosmetic composition for preventing skin aging that includes a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme and exhibits anti-stress and anti-wrinkle effects by suppressing the expression of 11β-HSD1 involved in cortisol production. would like to provide

The present invention provides a cosmetic composition for preventing skin aging comprising a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme.

In the cosmetic composition for preventing skin aging of the present invention, the prevention of skin aging may be preferably characterized by stress relief.

In the cosmetic composition for preventing skin aging of the present invention, the stress relief may be preferably characterized by reducing the content of cortisol.

In the cosmetic composition for preventing skin aging of the present invention, the skin aging prevention may preferably be characterized by improving skin wrinkles.

The present invention is a cosmetic for preventing skin aging that contains a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme and suppresses the expression of 11β-HSD1, thereby exhibiting an excellent effect on skin regeneration through stress relief, collagen synthesis, and cell proliferation promotion. A composition is provided.

1 is a view confirming the protein size of the enzyme-treated hydrolyzed extract of white wood ear mushroom according to an embodiment of the present invention.
2 is a diagram showing the effect of inhibiting the expression of 11β-HSD1 of an enzyme-treated hydrolyzed extract of white wood ear mushroom according to an embodiment of the present invention.
3 is a view showing the collagen production promoting effect of the enzyme-treated hydrolyzed extract of white wood ear mushroom according to an embodiment of the present invention.
4 is an enzyme-treated hydrolyzed extract of white wood ear mushroom according to an embodiment of the present invention; It is a diagram showing the effect of cell migration.
Figure 5 is a diagram showing the cell regeneration effect of the enzyme-treated hydrolyzed extract of white wood ear mushroom according to an embodiment of the present invention.

The present invention provides a cosmetic composition for preventing skin aging comprising a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme.

Tremella fuciformis ( Tremella fuciformis ) is a plant belonging to the family Hibiscus, and its origin is China, Japan, Korea, etc. It occurs in the rotten trees of broad-leaved trees in hilly areas and mountainous areas. The diameter of the fruiting body is 3-8 cm. It is pure white, similar to translucent gelatin, and is called silver ear because of its shape similar to a human ear.

White wood ear mushroom contains a lot of minerals and dietary fiber, so it can be helpful for diet as a low-calorie food when consumed. is known to be

On the other hand, in the cosmetic composition for preventing skin aging of the present invention, the hydrolyzate of white wood ear mushroom is trypsin, pepsin, papain, elastase, enzyme treatment using protease It is preferable to extract by a hydrolysis method. More preferably, it is good to extract using alcalase. In this case, the enzyme treatment is preferably performed under the conditions for optimal enzyme activity, and more preferably at a pH of 6.0 to 9.0 and a temperature of 40 to 60°C.

Alternatively, the hydrolyzate of the white wood ear mushroom may be extracted using an acid-treated hydrolysis method, and may be variously applied depending on the concentration, temperature, and time of HCl used in the acid-treated hydrolysis method. At this time, preferably, hydrolysis extraction can be performed at a concentration of HCl of 0.1 to 5.0 M, pH 2.0 to pH 4.5, a temperature of 90 to 130° C., and 6 to 48 hours.

In addition, the hydrolyzate of the white wood ear mushroom may have a low molecular weight of 10 to 100 KDa, and also contain a large amount of glycoprotein and collagen.

On the other hand, in the cosmetic composition for preventing skin aging of the present invention, the prevention of skin aging may be preferably characterized by stress relief. More preferably, stress relief can be achieved by increasing the α wave and the θ wave and decreasing the β wave.

On the other hand, in the cosmetic composition for preventing skin aging of the present invention, the stress relief may be preferably characterized by reducing the content of cortisol. More specifically, according to the following experiment, the stress relief may be achieved by reducing the cortisol content by inhibiting the expression of 11β-HSD1, which has an important effect on the production of cortisol, a stress hormone.

On the other hand, in the cosmetic composition for preventing skin aging of the present invention, the prevention of skin aging may preferably be characterized in that the skin wrinkle improvement. More specifically, according to the following experiment, the hydrolyzate of white wood ear mushroom of the present invention promotes collagen production and cell migration in the skin, and promotes cell proliferation to show skin regeneration effect, thereby improving skin wrinkles and preventing skin aging. could show

On the other hand, in the cosmetic composition of the present invention, the cosmetic composition is not necessarily limited to a specific formulation, for example, a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O/W) type cosmetic, water-in-oil It may be in any one formulation selected from the group consisting of (W/O) type cosmetics, ointments, foundations, skin covers, lipsticks, and cosmetics for the scalp.

In addition, in the cosmetic composition of the present invention, the hydrolyzate of the white wood ear mushroom is preferably added in an amount of 0.001 to 30% by weight based on the total weight of the cosmetic composition. When included in an amount of less than 0.001% by weight, the skin wrinkle improvement effect confirmed in the present invention is insignificant, and when it exceeds 30.0% by weight, the efficiency is lowered compared to the amount of material input, which is not economical.

In addition, the cosmetic composition of the present invention may contain auxiliary agents commonly used in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants, dyes, etc. may contain. The amount of these various adjuvants is an amount conventionally used in the art, for example, 0.001 to 30% by weight relative to the total weight of the composition. However, in any case, the auxiliary agent and its ratio will be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.

Meanwhile, all technical terms used in the present invention, unless otherwise defined, have the following definitions and correspond to the meanings commonly understood by those of ordinary skill in the art related to the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention.

The term "about" means 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 4, 3, 2 or 1%.

Throughout this specification, unless the context requires otherwise, the terms "comprises" and "comprising" include, but are not limited to, a given step or element, or group of steps or elements, but any other step or element, or It is to be understood that a step or group of elements is not excluded.

Hereinafter, the present invention will be described in more detail in the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of technical ideas equivalent thereto.

[Comparative Example 1: Preparation of hot water extract of white oyster mushroom]

After adding 5 kg of purified water to 500 g of white wood ear mushroom, the temperature was raised and extraction was performed at 100° C. for 3 hours. This was filtered through a 400 mesh filter, and then freeze-dried white wood ear mushroom powder was used.

[Example 1: Preparation of enzyme-treated hydrolyzed extract of white wood ear mushroom]

After adding 5 kg of purified water to 500 g of white wood ear mushroom, a 50% sodium hydroxide solution was added so that the pH became 9.0. After putting 5 g of alcalase here, the temperature was raised and extraction was performed with stirring at 50 °C for 3 hours. In order to remove insoluble substances in the extract, only the supernatant was used after centrifugation. This was freeze-dried to use white wood ear mushroom powder.

[Experimental Example 1: Quantitation of protein content according to extraction conditions of white wood ear mushroom]

In this experiment, it was attempted to quantify the protein content according to the extraction conditions of the white wood ear mushroom.

The protein content of Comparative Example 1 and Example 1 was quantified using the BCA (Bicinchoninic acid) method. Each BCA sample was mixed in a ratio of 50:1, 175 μl each was dispensed into each well, 25 μl of the sample was added, and reacted in an incubator at 37° C. for 30 minutes. After the reaction was completed, absorbance was measured at 540 nm. After measuring the absorbance using BSA (Bovine serum albumin) as a standard material, the protein content was calculated and shown in Table 1.

% A540 Protein (ug/ml) Mean (ug/ml) Comparative Example 1 0.1 0.1946 0.199 206.80 216.76 211.78 Example 1 0.1 0.275 0.2771 389.13 393.66 391.39

As can be seen from the results of Table 1, it was confirmed that the protein content of Example 1 of the present invention was about 2 times higher than that of Comparative Example 1.

[Experimental Example 2: Analysis using SDS-PAGE of white oyster mushroom extract]

Protein expression was confirmed through the analysis using SDS-PAGE of Comparative Example 1 and Example 1. After making a 10% acrylamide gel (Acrylamide Gel), it was hardened at room temperature for 30 minutes. Comparative Example 1 and Example 1 were loaded on a gel by 20 μl, and electrophoresis was performed until the loaded sample reached the very end on the page. For band detection, the gel was stained with coomassie brilliant blue for 30 minutes, washed with washing buffer 3 to 4 times, and photographed.

1 is a result of SDS-PAGE analysis of Comparative Example 1 and Example 1, respectively. In Comparative Example 1, it was confirmed between 24-75 kDa, but in Example 1, the protein was reduced to a low molecular weight through enzyme treatment and SDS-PAGE was not confirmed on the

[Experimental Example 3: Inhibition of 11β-HSD1 expression of enzyme-treated hydrolyzed extract of white wood ear mushroom]

In order to confirm the effect of Comparative Example 1 and Example 1 on cortisol production, the inhibitory effect of 11β-HSD1 expression was confirmed. Carbenoxolone was used as a positive control, and the experiment was performed as follows.

Human fibroblasts were aliquoted in the number of 1 × 10 ⁵ in a 6-well plate, and then cultured in an incubator at 37° C. and 5% CO ₂ condition for 24 hours. After removing the medium and adding DPBS, the remaining cell groups except for the UVB non-irradiated group were irradiated with UVB at 15 mJ/cm ² , and white oyster mushroom extract was added at 3, 10, and 30 μg/ml, respectively, and added for 24 hours. incubated with Thereafter, the cells of each sample were recovered and RNA was isolated using 1 mL of Trizol (RNA iso, DAKARA, Japan). After RNA was quantified at 260 nm using a UV detector, cDNA was synthesized in an amplifier (Reverse Transcriptase Mix, ELPIS biotech, Korea). The relative expression level of 11β-HSD1 was finally analyzed in comparison with beta-actin.

2 is a result of evaluation of the expression inhibitory effect of 11β-HSD1 for Example 1 and Comparative Example 1. As shown in FIG. 2, Example 1 confirmed that the expression level of 11β-HSD1 decreased from 3 μg/ml. In addition, it was confirmed that the inhibition rate was up to 52% higher than that of Comparative Example 1 at the same concentration.

[Experimental Example 4: Collagen-promoting effect of enzyme-treated hydrolyzed extract of white wood ear mushroom]

To confirm the wrinkle improvement effect of the enzyme-treated hydrolyzed extract of white wood ear mushroom, the collagen production promoting effect was confirmed.

Human skin fibroblasts (HDFn) were aliquoted in a 24-well plate at a concentration of 1 × 10 ⁵ cells/well and then stabilized at 37° C. in a 5% carbon dioxide incubator (CO _2- incubator). At the same time as exchange with FBS-free medium, 500 μM of cortisone and samples were treated, and TGF-β at a concentration of 10 ng/ml was used as a positive control. After 24 hours, the supernatant was collected, and the remaining cells were treated with MTT solution to confirm cytotoxicity. A procollagen type I peptide EIA kit was used to check the collagen expression level using the collected supernatant. After the collagen ELISA assay, absorbance was measured at a wavelength of 450 nm to calculate the amount of collagen produced, and then the collagen expression increase rate (%) was calculated compared to the control group.

3 is a result of a collagen production promotion evaluation experiment for Example 1 and Comparative Example 1. As shown in FIG. 3, Example 1 promoted collagen production from 3 μg/ml. In addition, at the same concentration, it was confirmed that the collagen content was up to 16% higher than that of Comparative Example 1.

[Experimental Example 5: Effect of Cell Migration of Enzyme-treated Hydrolyzed Extract of White Wood Ear Mushroom]

In order to confirm the wrinkle improvement effect of the enzyme-treated hydrolyzed extract of white wood ear mushroom, the cell migration effect was confirmed.

500 μl of human-derived fibroblasts (HDFn) were dispensed in a 24-well plate at 1 × 10 ⁵ cells/ml, and the cells were stabilized in a 37 ^o C, 5% CO ₂ cell incubator for one day. After making a scratch by scraping the bottom of the plate using a blue tip, the cells were washed twice with 1 × PBS to remove the detached cells. It was exchanged with F12/DMEM medium containing 1% FBS, and 500 μM of cortisone and samples were diluted by concentration and reacted for 48 hours in a cell incubator. After making the scratch, the same location was taken using a microscope at 0 and 48 hours.

4 is a cell migration evaluation experiment result for Comparative Example 1 and Example 1 after treatment with cortisone. As shown in FIG. 4, Example 1 promotes cell migration in the skin, and it was confirmed that cells migrated similarly to the case where cortisone was not treated.

[Experimental Example 6: Cell Proliferation Effect of Enzyme-treated Hydrolyzed Extract of White Wood Ear Mushroom]

In order to confirm the wrinkle improvement effect of the hydrolyzed extract treated with the enzymatic process of white wood ear mushroom, the cell proliferation effect was confirmed.

500 μl of human-derived fibroblasts (HDFn) were dispensed in a 24-well plate at 1 × 10⁴cells/ml, and the cells were stabilized in a 37 ^o C, 5% CO ₂ cell incubator for one day. After making a scratch by scraping the bottom of the plate using a blue tip, the cells were washed twice with 1 × PBS to remove the detached cells. It was exchanged with F12/DMEM medium containing 1% FBS, and samples of 500 μM were diluted according to concentration and reacted for 48 hours in a cell incubator. After treatment with 50 μg of MTT solution, the reaction was carried out for 2 hours, and after removing the supernatant, 200 μl of DMSO was added to dissolve the generated formazan. Absorbance was measured at 540 nm with a microplate reader.

5 is a cell proliferation effect test results for Example 1. As shown in FIG. 5 , Example 1 promotes cell proliferation in the skin and is determined to exhibit a skin regeneration effect.

[Preparation Examples 1 and 2: Preparation of a cosmetic composition comprising a white wood ear mushroom extract]

A skin wrinkle improvement cosmetic composition containing 0.5% by weight of Comparative Examples 1 and 1 was prepared according to the composition of Table 2 below, and was referred to as Preparation Example 1 and Preparation Example 2. The Preparation Examples 1 and 2 can be applied in various formulations, which are intended to only be described in detail, not to limit the present invention.

The cosmetic composition of this preparation example was prepared according to the contents of Table 2 below.

division ingredient Preparation Example 1
(weight%) Preparation 2
(weight%) A Comparative Example 1 0.5 - Example 1 - 0.5 EDTA-2Na 0.02 0.02 Purified water to 100 to 100 B cetostearyl alcohol 2.0 2.0 Glyceryl Stearate 1.5 1.5 microcrystallin 0.7 0.7 squalane 5.0 5.0 liquid paraffin 3.0 3.0 trioctanoin 5.0 5.0 polysorbate 1.2 1.2 Sorbitan Stearate 0.5 0.5 tocopheryl acetate 0.2 0.2 cyclomethicone 3.0 3.0 BHT 0.05 0.05 C fragrance, preservative appropriate amount appropriate amount Sum 100 100

[Experimental Example 7: EEG measurement result by white oyster mushroom cosmetic composition]

An EEG measurement experiment was conducted to confirm the stress relief effect of the enzyme-treated hydrolyzed extract of white wood ear mushroom.

Excessive exercise or excessive drinking was refrained from prior to EEG measurement, and smoking, beverages, drugs, and chewing gum were prohibited on the day of the experiment. The experiment was conducted on 5 males and females in their 20s and 30s who were currently healthy and did not take drugs. For EEG, electrodes for EEG measurement were attached to the frontal, parietal, and temporal lobes according to the International Electrode Arrangement Act, and GRASS equipment, MP 100 system, and Acknowledgment program were used. Each cosmetic composition was applied to the face and measured after a certain period of time had elapsed. After the measured data was analyzed as a power spectrum by the FFT method, α/(α+β+θ) and β/(α+β+θ) were obtained according to the frequency band. Preparation Examples 1 and 2 were measured for improvement, and the experimental results are shown in Table 3 below.

Preparation Example 1 Preparation 2 frontal lobe 23% 53% parietal lobe 21% 55% temporal lobe 17% 50%

In Preparation Example 2, the alpha wave was measured higher than in Preparation Example 1, which means that the test subject felt relaxation and comfortable emotions of the human body. Based on the measurement results of alpha waves, it can be confirmed that the stress relief effect is excellent when the composition of the present invention is used.

[Experimental Example 8: Human application test according to the white wood ear mushroom cosmetic composition]

In this experiment, a human application test was carried out on the skin wrinkle improvement effect of a cosmetic containing white oyster mushroom extract. The test was evaluated through the actual use test of each cosmetic.

That is, the human application test confirmed and compared and evaluated the skin wrinkle improvement effect due to the use of the cosmetics containing Preparation Example 1 and Preparation Example 2.

The experimenter (20 - 35 years old female) divided 10 women into groups A and B for 20 women, and the A group applied Preparation Example 1 and the B group applied Preparation Example 2 to the face to conduct the experiment. The skin wrinkle improvement effect was evaluated through visual observation and instrument measurement (cutometer SEM 575, C+K Electronic Co., Germany) of an experienced doctor for each subject's wrinkle improvement effect before and after using the product for 10 weeks. It is shown in Table 4 below.

Wrinkle improvement effect Improvement (%) Great a little does not exist group A 2 4 4 40.0 group B 6 2 2 70.0

As can be seen from the results of Table 4, the cosmetic composition of Preparation Example 2 of the present invention showed a high improvement rate compared to Preparation Example 1. In particular, the cosmetic composition containing Preparation Example 2 showed an excellent anti-wrinkle effect.

Claims (4)

Contains a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme,
The hydrolyzate contains a low molecular weight protein,
The low molecular weight protein is a cosmetic composition for anti-stress, characterized in that the molecular weight is less than 24 kDa.
The method of claim 1,
A cosmetic composition for anti-stress, characterized in that the stress relief is achieved by reducing the cortisol content.
Contains a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme,
The hydrolyzate contains a low molecular weight protein,
The low molecular weight protein is a cosmetic composition for skin regeneration, characterized in that the molecular weight is less than 24 kDa.
Contains a hydrolyzate obtained by treating white wood ear mushroom with a proteolytic enzyme,
The hydrolyzate contains a low molecular weight protein,
The low molecular weight protein is a cosmetic composition for improving skin wrinkles, characterized in that the molecular weight is less than 24 kDa.

Images