KR102144839B1 - Manufacturing method of Cosmetic composition for whitening comprising sparassis crispa extract - Google Patents

Abstract

The method for producing beta-glucan derived from flower mushrooms according to the present invention is a method for producing beta-glucan derived from flower mushrooms having an effect of inhibiting tyrosinase activity, wherein the powder is produced by drying and pulverizing the fruiting body of flower mushrooms. A manufacturing step, a swelling step of swelling the powder by putting the powder in purified water to prepare a powder solution, a fermentation step of adding lactic acid bacteria to the powder solution and fermenting for 22 to 28 hours in an environment of 28 to 32 degrees to generate a fermentation solution; Nanoknife step of adding insoluble tungsten carbide having a diameter of 10 to 1,000 nm as a nanoknife as a nanoknife in a proportion of 20% of the weight of the powder solution and emulsifying it with a high-speed emulsifier for 40 minutes and pulverizing it into a nano size to create a nano solution, and the It characterized in that it comprises an extraction step of adding purified water to the nano solution, extracting it at 95°C, centrifuging to extract beta-glucan, and freeze-drying it to prepare beta-glucan powder derived from flower mushrooms.

Description

Method for producing beta-glucan derived from blossom mushroom {Manufacturing method of Cosmetic composition for whitening comprising sparassis crispa extract}

The present invention relates to a method for producing beta-glucan derived from flower mushrooms by extracting and preparing beta-glucan, an active ingredient, from flower mushrooms having tyrosinase activity and thus the inhibitory effect on melamine synthesis by research experiments.

Melanin, which is present in the base layer of the epidermis, plays the most important role in determining the color of human skin, and is synthesized from melanosomes, a special form of brown intracellular organelles within melanocytes. The synthesized melanin moves to keratinocytes through dendrites and is deposited in the stratum corneum, causing blackening.

Melanocytes contain tyrosinase, tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2), which are specific enzymes necessary for synthesizing melanin. Among them, the most important enzyme is tyrosinase, a kind of polymerase that controls the rate-determining step. Using tyrosine, one of the amino acids as a substrate, is oxidized by tyrosinase to 3,4-dihydroxyphenylalanine (DOPA) and DOPA quinone to synthesize pheomelanin showing mainly red and yellow color3), and DOPA quinone is automatically oxidized to DOPA chrome. It is converted to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by TRP-2, and DHICA is oxidized to indole-5,6-quinone-2-carboxylic acid by TRP-1, resulting in a dark brown color. Produces eumelanin. Therefore, if you inhibit the action of tyrosinase, which is absolutely necessary for the synthesis of pheomelanin and eumelanin, you can effectively inhibit melanin synthesis.

Efforts have been made to develop extracts having tyrosinase inhibitory activity from natural products. Kojic acid, hydroquinone, oxyresveratrol, and p-hydroxybenzyl alcohol isolated from yeast fungus, which are well-known tyrosinase inhibitors, have been reported to inhibit melanin synthesis, and Korean Patent Publication No. 10-2011-0091342 A number of patents have been disclosed, including'whitening cosmetic composition using', Korean Patent No. 10-1715194'Expression of kojic acid and concentrated extraction method from yeast'. However, these materials are difficult to use due to problems such as decomposition, coloring, off-flavor, unclear effect, and safety due to unstable physical properties. Accordingly, research is being actively conducted to find a whitening material from natural products for stably and effectively reducing melanin synthesis without affecting cells.

On the other hand, Sparassis crispa is a mushroom belonging to the Aphyllophorales and Sparassidaceae family, and is distributed in Korea, Japan, China, Europe, North America, and Australia. It grows naturally near the root or in the stump. The fruiting body is 10~25cm high, fleshy, the bottom part is a thick stem, has a common sack, the upper part is flat, the edge is wavy and formed in the shape of a white cauliflower, and it is valuable for food because of its soft scent and chewy texture. . In addition, the content of β-1,3-D-glucan is significantly higher than that of other mushrooms, so various physiological activities such as anti-cancer and anti-angiogenic effects, anti-tumor effects, antibacterial and antifungal effects, and anti-allergic effects, diabetes and acute liver damage. It is known to be effective, and related to Korean Patent Registration No. 10-1693745,'Health supplementary food using matsutake mushroom extract' has been disclosed, but there is no research report on whitening activity.

Korean Patent Publication No. 10-2011-0091342'Whitening cosmetic composition using yeast fungus' Korean Registered Patent No. 10-1715194'Cojic acid expression and concentrated extraction method from yeast' Korean Registered Patent No. 10-1693745'Health supplement food using pine mushroom extract'

The present invention is to extract and utilize a whitening material for stably and effectively reducing melanin synthesis without affecting cells from natural substances.

In addition, the activity of tyrosinase against the blossom mushroom extract, which is known to exhibit various physiologically active effects due to its high beta-glucan content, and thus the inhibitory effect of melamine synthesis are confirmed, and this is applied to a cosmetic composition.

In order to achieve the above object, as a method for producing beta-glucan derived from flower mushrooms having an effect of inhibiting tyrosinase activity, a powder production step of drying and pulverizing a flower mushroom fruiting body to form a powder, and the powder is purified water A swelling step of swelling the powder by preparing a powder solution, and a fermentation step of generating a fermentation solution by fermenting for 22 to 28 hours in an environment of 28 to 32 degrees by adding lactic acid bacteria to the powder solution; Nanoknife step of adding insoluble tungsten carbide having a diameter of 10 to 1,000 nm as a nanoknife as a nanoknife in a proportion of 20% of the weight of the powder solution and emulsifying it with a high-speed emulsifier for 40 minutes and pulverizing it into a nano size to create a nano solution, and the It characterized in that it comprises an extraction step of adding purified water to the nano solution, extracting it at 95°C, centrifuging to extract beta-glucan, and freeze-drying it to prepare beta-glucan powder derived from flower mushrooms.

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The cosmetic composition for whitening comprising beta-glucan derived from flowering mushrooms prepared by the production method according to the present invention has an effect of reducing tyrosinase expression and thus inhibiting melamine production.

The beta-glucan derived from flowering mushrooms is considered to have high industrial applicability as a natural whitening material for stably and effectively reducing melanin synthesis without affecting cells.

Figure 1 is a flow chart showing a method for producing beta-glucan derived from flowering mushrooms of the present invention.
Figure 2 is a graph showing the melanin production inhibitory effect of beta-glucan derived from flowering mushrooms of the present invention.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail.

However, the present invention is not limited to the embodiments disclosed below, but may be implemented in a variety of different forms, only the present embodiments are intended to complete the disclosure of the present invention, and are common in the technical field to which the present invention pertains. It is provided to fully inform those skilled in the art of the scope of the invention, and the invention is only defined by the scope of the claims.

In addition, terms used in the present specification are intended to describe exemplary embodiments and are not intended to limit the present invention.

In this specification, the singular form also includes the plural form unless otherwise specified in the phrase. As used herein, "comprises" and/or "comprising" do not exclude the presence or addition of elements other than the mentioned elements.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification may be used with meanings that can be commonly understood by those of ordinary skill in the art to which the present invention belongs.

Sparassis crispa, a mushroom belonging to the Aphyllophorales and Sparassidaceae family, has a significantly higher content of β-1,3-D-glucan than other mushrooms and has anticancer and antiangiogenic effects. , Anti-tumor effect, antibacterial and antifungal effect, and anti-allergic effect, diabetes and acute liver damage, etc. are known to have various physiologically active effects, but there is no research report on whitening activity.

Accordingly, the present applicant tried to confirm the whitening efficacy by treating B16F1 Mouse melanoma cells with beta-glucan extracted from flower mushrooms.

1 is a flow chart showing a method for producing beta-glucan derived from flowering mushrooms of the present invention.

First, referring to FIG. 1, a method for producing beta-glucan derived from blossom mushrooms will be described. The manufacturing method is a powder manufacturing step of sequentially drying the blossom mushroom fruiting bodies to have a moisture content of 4% and then pulverizing to produce a powder ( S1), and a swelling step of swelling the powder by preparing a powder solution by putting 100 g of the powder in 2L of purified water (S2), fermentation solution by fermenting for 22 to 28 hours in an environment of 28 to 32 degrees by adding lactic acid bacteria to the powder solution In the fermentation step (S3) of generating, adding 20 g of an insoluble tungsten carbide having a diameter of 10 to 1,000 nm as a nanoknife to the fermentation solution, which is a proportion of 20% of the weight of the powder, and emulsifying it with a high-speed emulsifier for about 40 minutes, pulverizing into nano-size Nanoknife step (S4) of generating a sea nano solution, and after extracting for 8 hours, 15 hours, and 30 hours at 95°C by adding purified water to the nano solution, then centrifuging to extract beta-glucan and freeze-drying it It includes an extraction step (S5) to prepare the derived beta-glucan powder.

In order to confirm the whitening activity using the beta-glucan derived from flowering mushrooms, mushroom tyrosinase assay was performed primarily to determine the degree of direct whitening activity of beta-glucans extracted from flowering mushrooms. Mushroom tyrosinase inhibition is involved in the change of the state of copper ions at the active site of tyrosinase and can regulate the oxidation and reduction processes of tyrosinase.Thus, in vitro tyrosinase activity inhibition experiments using mushroom tyrosinase are a useful primary in the development of whitening agents. It is recognized as an evaluation method. In the case of kojic acid, a positive control group that has already been identified as inhibiting the activity of tyrosinase, it inhibited the activity of mushroom tyrosinase in a concentration-dependent manner, but beta-glucan extracted from blossom mushrooms inhibited the activity of mushroom tyrosinease even when the concentration increased. There was no change in activity, that is, in an in vitro experiment, beta-glucan extracted from flower mushrooms did not inhibit tyrosinase activity.

Therefore, the effect of beta-glucan directly inhibiting the activity of tyrosinase, which is involved in the early stages of melanin synthesis, was low, but its effect at the cell level of B16F1 melanoma to confirm the possibility of inhibiting melanin pigment by affecting the metabolic process of cells. I recognized it. That is, it was determined that it would inhibit intracellular tyrosinase activity, not cytotoxicity such as apoptosis, and thus the tyrosinase activity of beta-glucan extracted from blossom mushrooms on the B16F1 melanoma cell level and the efficacy on melanin synthesis were measured. The results will be described sequentially together with FIGS. 2 to 5.

Figure 2 is a graph showing the melanin production inhibitory effect of beta-glucan derived from blossom mushrooms of the present invention.

In order to examine the effect of beta-glucan extracted from flower mushrooms on the melanogenesis of B16F1 melanoma cells according to the method for producing beta-glucan derived from flowering mushrooms, beta-glucans were used at a concentration of 0, 10, 100, 1,000 μg/mL. After 72 hours, the amount of melanin production was measured.

The B16F1 melanoma cells used in this process were obtained from the American Type Culture Collection (ATCC), and the medium used for cell culture was DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Incubated in a 37°C, 5% CO2 incubator.

To measure the amount of melanin produced in B16F1 melanoma cells, B16F1 melanoma cells were inoculated into a 60mm culture dish at 500,000 cells/dish, and then cultured in a 5% CO2 incubator at 37°C for one day. The medium was removed, and beta-glucan extracted from the flower mushrooms was replaced with a medium diluted to 0, 10, 100, 1,000 μg/mL, and cultured for an additional 72 hours. Thereafter, the cells from which the medium was removed were washed twice with PBS, and the pellet obtained by centrifugation at 12,000 rpm for 30 minutes was used for melanin quantification.

In order to cause the liquefaction of melanin, which is insoluble in water, an appropriate amount of 1N NaOH containing 10% DMSO was added and reacted at 65° C. for 1 hour, and the absorbance was measured at 405 nm using a microplate reader (BioTek Instruments).

As a result, the amount of melanin production was significantly decreased by 15.0% and 22.0%, respectively, compared to the control group at concentrations of 100 and 1,000 μg/mL of beta-glucan extracted from flower mushrooms.

3 is a graph showing the inhibitory effect of tyrosinase activity in B16F1 melanoma cells of the beta-glucan derived from flowering mushrooms of the present invention.

In order to confirm the effect of beta-glucan extracted from flower mushrooms on the tyrosinase activity of B16F1 melanoma cells according to the method for producing beta-glucan derived from flower mushrooms, beta-glucans of 0, 10, 100, 1,000 μg/mL After 72 hours of treatment at the concentration, the activity of tyrosinase was measured.

Tyrosinase plays a very important role in the production of skin melanin, and it acts as DOPA oxidase that oxidizes tyrosine by oxidizing tyrosine in the melanosome and oxidizes DOPA again to create DOPA chrome to synthesize melanin polymer. It acts as a key enzyme. Therefore, since tyrosinase activity inhibitors can effectively inhibit the synthesis of melenin polymer in the skin, the L-DOPA oxidase method was adopted to confirm the degree of inhibition of DOPA chrome, a reaction product produced by tyrosinase in the intermediate production step. .

After inoculation at 500,000 cells/dish in a 60 mm culture dish, culture was performed in a 37°C, 5% CO2 incubator for one day. The medium was removed, and beta-glucan extracted from the flower mushrooms was replaced with a medium diluted to 0, 10, 100, 1,000 μg/mL, and cultured for 72 hours. Thereafter, the cells from which the medium was removed were washed twice with PBS, cell lysis buffer was added, the cells were lysed at -80°C for 30 minutes, and then centrifuged at 12,000 rpm for 30 minutes. Then, 40 μL of the obtained supernatant and 160 μL of 2 mg/mL L-DOPA dissolved in 100 mM sodium phosphate buffer (pH 6.8) were added and incubated in an incubator at 37° C. for 1 hour. After the reaction was over, the amount of DOPA chrome formed from DOPA by tyrosinase was measured at 490 nm and expressed as relative tyrosinase activity.

As a result, beta-glucan extracted from flower mushrooms decreased tyrosinase activity in a concentration-dependent manner, 4.3% at a concentration of 10 μg/mL, 22.9% at a concentration of 100 μg/mL, and a final concentration of 1,000 μg/mL In comparison with the control group, the activity of tyrosinase was significantly reduced by 26.5%.

Figure 4 is a graph showing the melanin production inhibitory effect of beta-glucan derived from flowering mushrooms after induction of melanin production by α-MSH.

As shown in FIG. 4, the applicant of the present invention observed the production of melanin in B16F1 melanoma cells stimulated with α-MSH in order to confirm whether beta-glucan extracted from flowering mushrooms is effective at the cell level producing melanin.

That is, α-MSH was used as an external stimulator to find out what effect beta-glucan extracted from flower mushrooms has on the overproducing melanin activity in B16F1 melanoma cells against external stimulation. In this way, after inoculation at 500,000 cells/dish in a 60 mm culture dish, culture was performed in a 5% CO2 incubator at 37°C for one day. After removing the medium, 200 nM of α-MSH and beta-glucan extracted from flowering mushrooms were replaced with a medium diluted at concentrations of 0, 10, 100, and 1,000 μg/mL, and cultured for an additional 72 hours, after which the experimental method was B16F1. It was performed in the same manner as in the experimental method for the amount of melanin produced in melanoma cells.

As a result of the experiment, the group induced with 200 nM α-MSH in B16F1 melanoma cells increased the amount of melanin production by 207.5% compared to the control group. Here, when beta-glucan extracted from flower mushrooms was administered at concentrations of 10, 100,1,000 μg/mL, the amount of melanin production decreased by 13.9%, 18.7%, and 39.5%, respectively, compared to the α-MSH-inducing group, which was statistically significant.

Figure 5 is a graph showing the inhibitory effect of tyrosinase activity of beta-glucan derived from flowering mushrooms after α-MSH administration.

After treatment with α-MSH on B16F1 melanoma cells, the effect of beta-glucan extracted from flowering mushrooms on tyrosinase activity was confirmed. When 200 nM of α-MSH was administered to B16F1 melanoma cells, tyrosinase activity was increased by 218.8% compared to the control group, and α-MSH without beta-glucan when 10 μg/mL of beta-glucan extracted from flower mushroom was administered. Compared to the induction group, tyrosinase activity decreased by 15.6%, 26.9% when treated with 100 μg/mL, and 43.2% at the final concentration of 1,000 μg/mL, indicating statistically significant activity of tyrosinase overproduced by α-MSH. Was significantly suppressed.

6 is a graph showing the effect of beta-glucan derived from flowering mushrooms on the expression of tyrosinase, TRP-1, and TRP-2 proteins after α-MSH administration.

As confirmed in FIGS. 2 to 5, the applicant of the present invention confirmed that beta-glucan extracted from flower mushrooms affects melanin synthesis and reduction of tyrosinase activity at the cellular level, so further overexpression by α-MSH To investigate the effect of beta-glucan extracted from blossom mushrooms on the protein expression of tyrosinase and TRP-1, TRP-2, 200 nM of α-MSH and beta-glucan, which are external stimulants, were 0 and 0 in B16F1 melanoma cells. After administration at concentrations of 10, 100, and 1,000 μg/mL, the expression levels of tyrosinase and TRP-1 and TRP-2 proteins were confirmed after 72 hours of culture.

The α-MSH-induced group treated with 200 nM tyrosinase and TRP-1. It was confirmed that the activity of TRP-2 was increased, and the expression of tyrosinase, TRP-1 and TRP-2 proteins decreased as the concentration of beta-glucan extracted from flower mushroom increased.

7 is a graph showing the effect of the beta-glucan derived from flowering mushrooms on the MITF activity.

In order to investigate the effect of beta-glucan extracted from flower mushrooms on the expression of MITF protein, a potent transcriptional regulator of tyrosinase and TRP, the present applicant has 200 nM of α-MSH and beta-extracted from flower mushrooms in B16F1 melanoma cells. Glucan was simultaneously administered at concentrations of 0, 10, 100, and 1,000 μg/mL, and cultured for 72 hours to confirm the level of MITF protein expression (Fig. 8). As a result, it was confirmed that the α-MSH-induced group treated with 200 nM increased MITF activity compared to the control group that was not treated with anything. As the concentration of beta-glucan extracted from flowering mushrooms increased, the expression of MITF protein was remarkable. Decreased significantly.

In summary, as a result of conducting an experiment to evaluate the possibility of use as a skin whitening agent by treating B16F1 mouse melanoma cells with beta-glucan extracted from blossom mushrooms and confirming the efficacy of inhibiting melanin biosynthesis and tyrosinase activity, the result of an experiment was basically extracted from blossom mushrooms. When beta-glucan was administered, the amount of melanin production and tyrosinase activity decreased, and when beta-glucan extracted from flower mushrooms was administered together compared to the group administered with α-MSH alone, the amount of melanin production decreased and the tyrosinase activity was significantly reduced. Was suppressed. As a result, it was confirmed that beta-glucan extracted from flower mushrooms inhibited the expression of MITF in B16F1 melanoma cells, thereby reducing the expression of tyrosinase and suppressing melanin production.

Therefore, it was proved that beta-glucan extracted from flower mushrooms is valuable as a whitening agent. As an example, the blossom mushroom extract, that is, the beta-glucan powder extracted from the blossom mushroom may be contained in an amount of 0.0001 to 20% by weight with respect to the total weight of the cosmetic composition for whitening, and the detailed weight ratio may include the formulation of the cosmetic composition, purpose of use, etc. It can be individually judged accordingly.

As described so far, the composition and action of the cosmetic composition for whitening containing beta-glucan derived from blossom mushrooms according to the present invention and the method for producing beta-glucan derived from blossom mushrooms were described in the above examples, but this However, the spirit of the present invention is not limited to the above embodiments, and various changes and changes are possible without departing from the technical spirit of the present invention.

Claims (4)

delete delete delete As a method for producing beta-glucan derived from flower mushrooms having an inhibitory effect on tyrosinase activity,
A powder manufacturing step of drying and pulverizing the flowering mushroom mushrooms to generate powder;
A swelling step of swelling the powder by preparing a powder solution by putting the powder in purified water;
Fermentation step of generating a fermentation solution by adding lactic acid bacteria to the powder solution and fermenting it for 22 to 28 hours in a 28 to 32 degree environment;
A nanoknife step of adding insoluble tungsten carbide having a diameter of 10 to 1,000 nm as a nanoknife as a nanoknife and emulsifying it with a high-speed emulsifier for 40 minutes to generate a nano solution by adding insoluble tungsten carbide having a diameter of 10 to 1,000 nm in a proportion of the weight of the powder solution;
Extracting step of adding purified water to the nano solution, extracting it at 95° C., and then centrifuging to extract beta-glucan and freeze-drying it to prepare a beta-glucan powder derived from flower mushroom; characterized in that it comprises, derived from flower mushroom. Method for producing beta-glucan.

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