WO2020256297A1 - Anti-aging composition comprising albatrellus confluens, and preparation method for same - Google Patents

Abstract

The present invention relates to an antioxidant composition for inhibiting aging induced by the environment, such as heat and humidity, and to a preparation method for the composition. According to the present invention: a composition comprising effective components such as grifolin and polyphenols can be obtained; antioxidant effects can be enhanced without cytotoxicity; skin aging and a decrease in skin elasticity can be prevented by inhibiting the influx of calcium ions into cells, thereby inhibiting TRPV1 and MMP-1 activation; and skin aging can be inhibited more effectively by improving an immediate skin cooling effect.

Description

Anti-aging composition containing fermented mulberry mushroom and method for producing same

The present invention relates to an antioxidant composition with improved content of an active ingredient and a method for manufacturing the same, and to an anti-aging composition for inhibiting aging due to environments such as heat and moisture, and a method for manufacturing the same.

This application claims the benefit of priority based on Korean Patent Application No. 2019-0072250 filed on June 18, 2019, and all contents disclosed in the documents of the Korean patent application are included as part of this specification.

Skin is largely divided into three layers: epidermis, dermis, and subcutaneous fat tissue in order from the outside, and functions to protect the human body from physical and chemical stimulation by the external environment. In particular, the skin carries about 65-70% of the moisture that the human body has, that is, various physiologically active substances necessary for the human body, and it is important to control the evaporation of this moisture to the outside of the body, helping to keep the skin soft and moist. Plays a role.

The condition of the skin is aging due to various causes such as changes in the external environment or changes in life patterns, artificial temperature control of cooling/heating, solar heat or sunlight, various stresses generated in social life and skin stress caused by environmental pollution. Is promoted.

Among the causes of skin aging, efforts have been made to lower the temperature of the skin in the prior art in order to prevent damage caused by thermal stimulation. For the purpose of cooling the skin surface, conventional products use a composition containing components such as mint, menthol, and erythritol.

However, it has been reported that ingredients such as mint and menthol can cause unpleasant sensations such as pain when applied near the eyes, and erythritol can cause allergies when applied to the skin, and can cause gastrointestinal disorders when taken. .

Accordingly, there is a need for a study on a composition for improving the anti-aging effect while minimizing irritation in the skin and body.

It is an object of the present invention to provide an anti-aging composition.

In addition, it is to provide a method for preparing the composition.

In order to solve the above problems, the present invention

Adding 20 times the weight of distilled water to the powder of Albatrellus confluens (Alb. & Schwein.) Kotl. & Pouz.), mixing and stirring to prepare a powder solution;

Sterilizing the powder solution at 110 to 130° C. for 10 to 20 minutes to extract hot water;

To 100 parts by weight of sterilized and hot water-extracted powder solution, 1 part by weight of ethanol extract of mulberry mushroom was added, and Saccharomyces cerevisiae cultured in a nutrient medium (YM broth, BD) for 24 hours ( Saccharomyces cerevisiae , Center for Biological Resources) , KCTC 7296) 5 parts by weight of inoculation and fermentation at 25 to 35°C for 60 to 80 hours;

Sterilizing the fermented powder liquid at 110 to 130° C. for 10 to 20 minutes; And

It provides a method for producing an anti-aging composition comprising a fermented mulberry mushroom comprising the step of centrifuging and filtering the sterilized fermentation powder to obtain a supernatant.

According to an embodiment, the composition comprises fermented or non-fermented woolly shield mushroom ( Albatrellus ovinus (Schaeff.) Kotl. & Pouz.), flower shield mushroom ( Albatrellus dispansus (Lloyd) Canf. & Gilb.), green shield mushroom. ( Albatrellus caeruleoporus (Peck) Pouz.), gray-blue shield mushroom ( Albatrellus yasudae (Lloyd) Pouzar), long legs shield mushroom ( Albatrellus pes- caprae), Albatrellus cristatus and one or more extracts selected from the group consisting of Albatrellus ellisii . can do.

According to an embodiment, the ethanol extract of mulberry mushroom

Adding 70% ethanol of 10 times the weight of the shield mushroom powder;

Stirring at 30 to 50° C. for 2 to 4 hours;

Centrifuging at 6000rpm for 20 minutes and then filtering with 0.45um; And

It can be prepared by a method comprising the step of sequentially producing a powder by concentration.

According to one embodiment, the anti-aging may include one or more uses selected from the group consisting of anti-heat aging, skin elasticity, skin barrier protection, antioxidant, moisture retention, and antibacterial.

According to another embodiment of the present invention, there is provided a cosmetic composition containing the anti-aging composition as described above.

According to another embodiment of the present invention, it provides a health functional food composition containing the anti-aging composition as described above.

Other specifics of the embodiments of the present invention are included in the detailed description below.

The present invention can solve the disadvantages of the conventional anti-aging composition by providing a composition having an increased content of an active ingredient for anti-aging. By providing a composition that minimizes irritation in the skin and the body and improves the anti-aging effect, it can be usefully used as an external preparation for skin, a cosmetic composition, a functional food composition, and the like.

1 is a graph showing the polyphenol content.

2 is a graph showing the DPPH analysis result.

3 is a graph showing the cytotoxicity results.

4 is a graph showing the result of calcium inflow.

5 is a graph showing the degree of MMP-1 gene expression.

6 is a graph showing the level of expression of the TRPV1 gene.

7 is a graph showing the temperature change of the stratum corneum.

8 is a photograph of the thermal imaging camera.

9 and 10 are graphs showing the results of HPLC analysis of grifolin.

Since the present invention can apply various transformations and have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

Hereinafter, an anti-aging composition and a method for preparing the same according to an embodiment of the present invention will be described in more detail.

The composition according to the invention is for suppressing skin aging, in particular skin aging due to heat. In the skin, there is a protein TRPV1 (The transient receptor potential cation channel subfamily V member 1) that acts as a temperature sensor.According to the composition of the present invention, the composition of the present invention regulates the activity of these proteins to prevent skin damage caused by thermal stimulation and evaporate moisture Can be suppressed.

The composition according to the present invention may include a mushroom fermented product. Mushrooms used in the mushroom fermentation can be used in the genus Shield Mushroom, and specific examples include Albatrellus confluens (Alb. & Schwein.) Kotl. & Pouz.), Wool Shield Mushroom ( Albatrellus ovinus (Schaeff.) Kotl.& Pouz.), flower shield mushroom ( Albatrellus dispansus (Lloyd) Canf. & Gilb.), green shield mushroom ( Albatrellus caeruleoporus (Peck) Pouz.), gray-blue shield mushroom ( Albatrellus yasudae (Lloyd) Pouzar), long legs Shield mushrooms ( Albatrellus pes- caprae ), Albatrellus cristatus and may contain one or more selected from the group consisting of Albatrellus ellisii , fermented or non-fermented ones may be used. For example, it may include a bundle of shield mushrooms, a wool shield mushroom, or a combination thereof. Multiple shield mushrooms or woolly shield mushrooms can play a role in antioxidant, anti-aging, and inhibition of TRPV1 activity.

According to one embodiment, the mushroom fermentation product may be fermented by enzymes or yeast, for example, germinating yeast, that is, mushrooms fermented by Saccharomyces cerevisiae (Budding yeast; Saccharomyces cerevisiae ) It may be a fermented product.

According to one embodiment, the mushroom fermentation product,

Adding 20 times the weight of distilled water to the shield mushroom powder, mixing and stirring to prepare a powder solution;

Sterilizing the powder solution at 110 to 130° C. for 10 to 20 minutes to extract hot water;

To 100 parts by weight of the sterilized and hot-water-extracted powder solution, 1 part by weight of the shield mushroom ethanol extract was added, and then Saccharomyces cerevisiae cultured in a nutrient medium (YM broth, BD) for 24 hours was added to 5 Inoculating parts by weight and fermenting at 25 to 35° C. for 60 to 80 hours;

Sterilizing the fermented powder liquid at 110 to 130° C. for 10 to 20 minutes; And

It can be prepared by a method comprising the step of obtaining a supernatant by centrifuging the sterilized fermented powder liquid at 6000 rpm for 20 minutes and then filtering 0.2um.

According to the present invention, compared to non-fermented mushroom extract, fatty acids such as oleic acid and linoleic acid, magnesium (Mg), zinc (Zn), minerals such as copper (Cu), and By including fermented mushrooms with an increased content of active ingredients such as grifolin, polyphenol, and tannic acid, it lowers the temperature of the skin, replenishes moisture, and protects and strengthens damaged skin barriers. . Among them, gripholine, in particular, regulates a temperature receptor called TRPV1 (transient receptor potential cation channel subfamily V member 1) to inhibit aging due to an increase in the temperature of the skin, plays an anti-inflammatory and anti-cancer role, and is thus produced by the method according to the present invention. Mushroom fermentations containing foline can be prepared.

According to one embodiment, the shield mushroom ethanol extract,

Adding 70% ethanol of 10 times the weight of the shield mushroom powder;

Stirring at 30 to 50° C., eg 45° C. for 2 to 4 hours, eg 3 hours; And

Centrifuging at 6000rpm for 20 minutes and then filtering with 0.45um; And

It can be prepared by a method comprising the step of producing a powder by concentration in sequence.

The shield mushroom extract may be added at any stage of the manufacturing step of the shield mushroom fermentation product, but, for example, when added at the stage before strain inoculation, which is the initial stage of fermentation, the polyphenol content and efficacy may be increased through fermentation.

The composition of the present invention prepared by the method as described above can improve the anti-aging effect, the anti-aging effect is one selected from the group consisting of anti-heat aging, skin elasticity, skin barrier protection, antioxidant, moisture retention and antibacterial It may include more than one.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Example: Mushroom fermentation production

In order to manufacture the fermented mushrooms, Albatrellus confluens ( Albatrellus confluens , Finnish water supply, powder) was used. Distilled water in an amount corresponding to 20 times the weight of the mulberry powder was added, mixed, and stirred to prepare a powder solution. The powder was sterilized at 121° C. for 15 minutes to pre-ferment. After adding 1 part by weight of ethanol extract of mulberry mushroom to 100 parts by weight of sterilized and hot water-extracted powder, Saccharomyces cerevisiae cultured for 24 hours in a nutrient medium (YM broth, BD), https:/ /kctc.kribb.re.kr/ KCTC, Center for Biological Resources, KCTC 7296) 5 parts by weight of the culture solution was inoculated and fermented at 30°C for 72 hours.

The mulberry ethanol extract was added with 70% ethanol of 10 times the weight of the mulberry powder, stirred at 45° C. for 3 hours, centrifuged at 6000 rpm for 20 minutes, filtered with 0.45 um, filtered mulberry ethanol The extract was concentrated to prepare a powder.

The fermented powder was sterilized at 121° C. for 15 minutes, centrifuged at 6000 rpm for 20 minutes, and then filtered with 0.2 μm to obtain a supernatant as a fermented mushroom.

The properties of the obtained fermented mushroom are as follows.

Appearance: Liquid

Color: light brown or brown

Fragrance: A characteristic smell

Specific gravity (d20/20): 1.000~1.040

pH (25℃): 5.64±1

Refractive index (n20/D): 1.3000-1.4000

Total microorganism count: <100 CFU/g

Comparative Example: Preparation of mushroom extract

In order to prepare a mushroom extract, distilled water in an amount corresponding to 20 times the weight thereof was added to Albatrellus confluens (Finnish water supply, powder), and mixed and stirred to prepare a powder solution. The powder solution was centrifuged at 6000 rpm for 20 minutes and filtered with 0.2 μm after centrifugation at 6000 rpm for 20 minutes by adding 1% of 70% ethanol extract prepared in the same manner to the hot water extract sterilized at 121°C for 15 minutes. The extract was prepared.

Experimental Example 1: Polyphenol content

Quantification using Folin&Ciocalteu's phenol reagent to compare the total polyphenol content of each of the Albatrellus confluens extract according to the comparative example and the Albatrellus confluens ferment extract according to the example. I did. As a standard material, gallic acid was used. The sample and the standard were mixed with 50% Folin&Ciocalteu's phenol in a 4:1 ratio and reacted at room temperature for 5 minutes, and then an equal amount of 2% Na ₂ CO ₃ solution was added and reacted at room temperature for 30 minutes. Standard substances were used at concentrations of 100, 80, 60, 40, and 20ug/ml, and each absorbance was used to draw a standard curve later. After the reaction, the absorbance was measured at 750 nm using the absorbance measuring instrument Multiskan GO (Thermo Fisher Scientific), and the amount of total polyphenol before and after fermentation was compared with the absorbance value of the standard material. The results are shown in Fig. 1, and as shown in Fig. 1, it can be seen that the mushroom fermented product included in the present invention has a total polyphenol content increased by about 25% compared to the non-fermented fermented extract.

Experimental Example 2: Antioxidant Analysis

DPPH (2,2-diphenyl-1-picryl hydrezyl) to confirm the antioxidant and effect of the Albatrellus confluens extract according to the comparative example and the Albatrellus confluens ferment extract according to the example Radical scavenging ability analysis was performed. 1 mM DPPH solution was dissolved in methanol and used immediately. The sample solution and the DPPH solution were mixed at a ratio of 1:1, and left in the dark at room temperature for 20 minutes. At this time, 0.1 mg/ml of ascorbic acid was used as a positive control. After 20 minutes of prevention, absorbance was measured at 540 nm using an absorbance measuring instrument Multiskan GO (Thermo Fisher Scientific). The radical ratio (%) compared to the control group (control) was calculated. The results are shown in Fig. 2, and as shown in the graph, it can be seen that the antioxidant efficacy of Albatrellus confluens ferment extract is higher than that of the sample before fermentation, especially at a concentration of 1000 ug/ml of the fermented product. It was confirmed that 80% of the free radical scavenging ability was shown.

Experimental Example 3: Cytotoxicity

To confirm the cytotoxicity of the Albatrellus confluens extract according to the comparative example and the Albatrellus confluens ferment extract according to the example, the effect on the proliferation of HaCaT keratinocytes was tested, and the cultured HaCaT keratinocytes were treated with Albatrellus confluens ferment extract and Albatrellus confluens extract at respective concentrations, and then cell viability was measured. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was measured using a reagent, and MTT was absorbed into cells to form formazan by succinate dehydratase in mitochondria. Accumulation of substances in cells means mitochondrial activity, broadly, cellular activity, so the toxicity of cells can be evaluated.

Each cell was aliquoted into a 96 well plate at 2 X 10 ⁴ cells/well and cultured in a cell incubator for 24 hours. After cultivation, the medium was replaced with a medium containing 1% FBS, resulting in starvation and cultured for 24 hours. Albatrellus confluens extract and Albatrellus confluens ferment extract were treated at respective concentrations and cultured for 24 hours, followed by treatment with MTT solution and cultured for 4 hours. After cultivation, the medium was removed and the cells were completely dissolved with dimethyl sulfoxide (DMSO), and absorbance was measured at 450 nm with an absorbance measuring instrument Multiskan GO (Thermo Fisher Scientific). Based on the results, the cell viability was expressed as% by comparing the cell viability with the group not treated with the sample through the formula for calculating the cell viability below.

Cell viability (%) = [absorbance of the sample added group (450 nm) / absorbance of the sample-free group (450 nm)] × 100

The results are shown in FIG. 3. HaCaT were identified which does not exhibit the toxic concentration of the bundle shield mushroom extract on keratinocytes (Albatrellus confluens extract), the bundle shield mushroom fermentation (Albatrellus confluens ferment extract). In a later experiment, the concentration of the sample was determined based on the toxicity result. As shown in the graph of FIG. 3, it can be seen that the fermented product does not exhibit cytotoxicity at a concentration of 250 ug/ml or less for HaCaT cells.

Experimental Example 4: Calcium inflow

TRPV1 is activated by the detection of thermal or chemical stimulation to induce the influx of calcium (Ca ²⁺ ) into the cell, which induces cell senescence. Therefore, the degree of cell aging was confirmed by checking the influx of calcium.

To confirm whether the Albatrellus confluens ferment extract and Albatrellus confluens extract inhibit the influx of calcium ions into the cells, calcium ions were stained with fluorescence to visually check the change in the amount. When TRPV1 (The transient receptor potential cation channel, subfamily V, member 1) is activated by stimulation such as temperature above 42℃, capsaicin, and acidic conditions, calcium ions from the outside of the cell move inside the cell. The degree of activity of TRPV1 can be confirmed by checking the change in calcium ion in the cell.

Each cell was aliquoted into a 96 well plate at 1 X 10 ⁴ cells/well and incubated in a cell incubator for 24 hours. After cultivation, the medium was replaced with a medium containing 1% FBS, resulting in starvation and cultured for 24 hours. Albatrellus confluens ferment extract and Albatrellus confluens extract were treated according to the concentration and incubated for 30 minutes. At this time, 5 μM capsazepine was used as a control. Calcium Kit; The loading solution was treated with Fluo-8 No wash Calcium Assay Kit (abcam), and incubated for 30 minutes at 37°C and 30 minutes at room temperature in dark conditions. After treatment with 5 μM capsaicin, cells were immediately photographed with a Cytell device, and the amount of calcium ion change in the cells was checked. The results are shown in Fig. 4, and it can be seen that the influx of calcium ions into the cells due to capsaicin stimulation was suppressed by the fermented mulberry mushroom.

Experimental Example 5: TRPV1 and MMP-1 expression

Albatrellus confluens ferment extract and Albatrellus confluens extract express mRNA of TRPV1 (The transient receptor potential cation channel, subfamily V, member 1) and MMP-1 (Matrix metalloproteinase-1). An experiment was conducted to check whether the amount was controlled. After dispensing 1 X 10 ⁶ cells/well into a 6 well plate, it was cultured in a cell incubator for 24 hours. After cultivation, the medium was replaced with a medium containing 1% FBS, resulting in starvation and cultured for 24 hours. Albatrellus confluens ferment extract and Albatrellus confluens extract were treated according to the concentration and incubated for 30 minutes. At this time, 5 μM capsazepine was used as a control. 10 μM capsaicin was treated, and cultured for 24 hours. The medium was removed, RNA was extracted using an RNA extract kit (TaKaRa MiniBest RNA Extraction Kit), and RNA was quantified using a Qubit Fluorometer (Qubit 2.0 Fluorometer, life technologies). CDNA was synthesized with the same amount of RNA using a High-Capacity RNA-to-cDNA kit (applied biosystems). Taqman primers TRPV1, MMP-1, and GAPDH were purchased and used from life technologies. Using the Taqman master mix, a desired gene was amplified using Real-time PCR equipment Step One Plus Real-Time PCR (Applied Biosystem), and the change in gene expression level was confirmed. The results of confirming the expression level of the MMP-1 gene are shown in FIG. 5, and the results of confirming the expression level of the TRPV1 gene are shown in FIG. As shown in Figures 5 and 6, it can be seen that the multi-shield mushroom fermentation product ( Albatrellus confluens ferment extract) inhibits the mRNA expression of TRPV1 and MMP-1 activated by capsaicin.

Experimental Example 6: Lowering the skin temperature

Twenty-one subjects were selected, and the tester applied a cream containing Albatrellus confluens ferment extract once to the facial area, and then analyzed the immediate change in skin temperature using a thermal imaging camera. When a cream containing Albatrellus confluens ferment extract was used, it was confirmed that the skin temperature decreased to a statistically significant level compared to when a cream containing no cream was used. Specifically, FIG. 7 shows the amount of change in the temperature of the stratum corneum before and after the application of the cream containing the fermented mulberry mushroom according to the embodiment, and as can be seen from the graph, an average 2.63°C skin temperature reduction effect at the test site is shown. Can be seen. In addition, a measurement photograph of the thermal imaging camera is shown in FIG. 8, and a temperature change of the left cheek, which is a test area to which the fermentation product according to the embodiment is applied, can be confirmed.

From the above results, it was confirmed that the Albatrellus confluens ferment extract had an immediate skin temperature reduction (cooling) effect.

Experimental Example 7: Grifolin

Examples of high-performance liquid chromatography so morpholine (grifolin) analysis (high-performance liquid chromatography, HPLC ) with respect to the bundle shield mushroom fermentation (Albatrellus confluens ferment extract) was confirmed in accordance with the.

Analysis conditions are as follows.

HPLC analysis conditions of Girfolin

Instrument: Waters 2695 & 996

Detection: UV 213nm

Column: Agilent Eclipse XDB-C18, 5μm, 4.6 x 150 mm

Column Temp.: 25℃

Mobile phase: Acetonitrile 75%:0.01% phosphoric acid in DW 25%

Flow rate: 1.0 mL/min

Injection: 20 μL

The standard analysis peak of gripholine is shown in Fig. 9, and the results of the multi-leaf mushroom ferment (Albatrellus ovinus ferment extract) are shown in Fig. 10.

As can be seen from FIGS. 9 and 10, it was confirmed that the mushroom fermented product according to the present invention contained gripholine.

As can be seen from the above results, according to the present invention, a composition containing active ingredients such as griffoline and polyphenol can be obtained, antioxidant efficacy can be improved without cytotoxicity, and calcium ions can enter cells By inhibiting the activity of TRPV1 and MMP-1, it is possible to prevent skin aging and loss of elasticity, and by improving the immediate skin temperature reduction effect, skin aging can be more effectively suppressed.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the technical field to which the present invention pertains can make various modifications and variations without departing from the essential characteristics of the present invention. In addition, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain the technical idea, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.

Claims (7)

1. Adding 20 times the weight of distilled water to the mulberry powder, mixing and stirring to prepare a powder solution;

   Sterilizing the powder solution at 110 to 130° C. for 10 to 20 minutes to extract hot water;

   To 100 parts by weight of sterilized and hot water-extracted powder solution, 1 part by weight of ethanol extract of mulberry mushroom was added, and Saccharomyces cerevisiae cultured in a nutrient medium (YM broth, BD) for 24 hours ( Saccharomyces cerevisiae , Center for Biological Resources) , KCTC 7296) 5 parts by weight of inoculation and fermentation at 25 to 35°C for 60 to 80 hours;

   Sterilizing the fermented powder liquid at 110 to 130° C. for 10 to 20 minutes; And

   A method for producing an anti-aging composition comprising a fermented mulberry mushroom comprising the step of centrifuging and filtering the sterilized fermented powder liquid to obtain a supernatant.
2. The method of claim 1,

   The composition is fermented or non-fermented woolly shield mushroom ( Albatrellus ovinus (Schaeff.) Kotl. & Pouz.), flower shield mushroom ( Albatrellus dispansus (Lloyd) Canf. & Gilb.), green shield mushroom ( Albatrellus caeruleoporus (Peck)) Pouz.), gray-blue shield mushroom ( Albatrellus yasudae (Lloyd) Pouzar), long legs shield mushroom ( Albatrellus pes- caprae), Albatrellus cristatus and Albatrellus ellisii , which further comprises one or more extracts selected from the group consisting of, anti-aging Method of making the composition.
3. The method of claim 1, wherein the ethanol extract of mulberry mushroom,

   Adding 70% ethanol of 10 times the weight of the shield mushroom powder;

   Stirring for 2 to 4 hours at 30 to 50 ℃;

   Centrifuging at 6000rpm for 20 minutes and then filtering with 0.45um; And

   The method for producing an anti-aging composition, which is produced by a method comprising the step of producing a powder by concentration.
4. The method of claim 1,

   The method for producing an anti-aging composition, wherein the anti-aging includes one or more uses selected from the group consisting of anti-heat aging, skin elasticity, skin barrier protection, antioxidant, moisture retention and antibacterial.
5. An anti-aging composition prepared by the manufacturing method according to any one of claims 1 to 4.
6. A cosmetic composition containing the anti-aging composition according to claim 5.
7. A health functional food composition containing the anti-aging composition according to claim 5.

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