KR20250080307A - Skin whitening composition using Polyporus arcularius culture broth - Google Patents

Abstract

The skin whitening composition of the present invention contains a culture solution of Polyporus arcularius as an active ingredient. In addition, the method for producing the skin whitening active substance of the present invention includes a step of culturing Polyporus arcularius in a liquid medium.

Description

{Skin whitening composition using Polyporus arcularius culture broth}

The present invention relates to a skin whitening composition using a culture solution of the Korean native mushroom, and more specifically, the present invention relates to a skin whitening composition using a mushroom native to Korea, which has low cytotoxicity and exhibits excellent skin whitening effects such as melanin synthesis inhibition activity of skin cells, tyrosinase activity inhibition activity, and melanin formation related factor (MITF) expression inhibition activity, and a method for producing a substance exhibiting the above-mentioned excellent skin whitening activity using a Korean native mushroom.

Whitening is a topic that is of great interest in the field of functional cosmetics as it makes the skin beautiful and white, and accordingly, the discovery of substances that can exhibit this whitening effect is being actively conducted. In general, it is important for the whitening effect to inhibit the formation of melanin. Therefore, it is possible to search for substances with excellent whitening effects by investigating the effect of regulating the formation of melanin, and the whitening effect of the candidate substance can be confirmed by investigating the effect on the activity and/or expression of tyrosinase, an enzyme involved in the initial stage of melanin production as a related factor. In addition, since other factors known to be involved in the production of melanin, such as TRP-1 (tyrosinase related protein 1), TRP-2 (tyrosinase related protein 2), and MITF (microphthalmia associated transcription factor), are known, a method of investigating the effect on these can also be used.

Meanwhile, efforts are being made to utilize natural resources instead of synthetic compounds as functional materials due to environmental pollution problems and the possibility of harm to the human body. Mushrooms are also one of these natural resources and have a very high potential for utilization. Accordingly, studies on the physiological activity of mushrooms are being conducted, but related research is still insufficient. Accordingly, the inventors of the present invention studied the possibility of exhibiting an excellent skin whitening effect by effectively controlling the skin whitening-related activity described above from various mushrooms, especially mushrooms native to Korea.

Korean Patent No. 10-1437059 Korean Patent Registration No. 10-1402193

The main purpose of the present invention is to provide a skin whitening composition that can exhibit an excellent skin whitening effect by utilizing mushrooms, which are natural resources, particularly mushrooms native to Korea.

Another object of the present invention is to provide a method for producing a substance having excellent skin whitening activity by utilizing the above mushroom.

In one aspect, the present invention provides a skin whitening composition according to the following items:

1. A skin whitening composition containing a culture solution of Polyporus arcularius as an effective ingredient.

2. A composition for skin whitening, wherein the honeycomb pore mushroom of item 1 is a NIBRFGC 000143418 strain.

3. A composition for skin whitening, wherein in item 1 or item 2, the culture medium is obtained by culturing in DY, MY, MEB or PDB medium.

4. A composition for skin whitening, wherein in any one of items 1 to 3, the culture solution is obtained by shaking and culturing in a liquid medium at 20 to 30°C and 100 to 250 rpm for 20 to 100 days.

In another aspect, the present invention provides a method for producing a skin whitening active substance according to the following items:

5. A method for producing a skin whitening active substance, comprising: a step of culturing Polyporus arcularius in a liquid medium.

6. A method for producing a skin whitening active substance according to item 5, wherein the honeycomb pore mushroom is a NIBRFGC 000143418 strain.

7. A method for producing a skin whitening active substance according to item 5 or item 6, wherein the liquid medium is DY, MY, MEB or PDB medium.

8. A method for producing a skin whitening active substance according to any one of items 5 to 7, wherein the culturing is a shaking culture at 20 to 30°C and 100 to 250 rpm for 20 to 100 days.

In another aspect, the present invention provides a strain of Polyporus arcularius NIBRFGC 000143418 deposited under the accession number KACC 93418P.

According to the present invention, a skin whitening composition is provided, which uses mushrooms native to Korea and has low cytotoxicity and exhibits excellent skin whitening effects such as melanin synthesis inhibition activity of skin cells, tyrosinase activity inhibition activity, and melanin formation related factor (MITF) expression inhibition activity. In addition, a method for producing a substance capable of exhibiting the above-described excellent skin whitening activity is provided using mushrooms native to Korea.

Figure 1 shows a phylogenetic tree based on alignment of ITS sequences of the new honeycomb pore mushroom strains provided in the present invention.
Figure 2 shows the results of a cytotoxicity experiment of a culture solution of the honeycomb pore mushroom (Sample) according to one embodiment of the present invention targeting B16F10 melanoma cells. The results are expressed as a percentage compared to the value obtained in the control group.
Figure 3 shows the results of an experiment on the melanin synthesis inhibitory activity of a culture solution of the honeycomb pore mushroom (Sample) according to one embodiment of the present invention targeting B16F10 melanoma cells. The results are expressed as a percentage compared to the value obtained in the control group. The results are expressed as the mean ± standard deviation of three repeated experiments (*p<0.05, **p<0.01, ***p<0.001).
Figure 4 shows the results of an experiment on the tyrosinase activity inhibition activity of the Pa_MY culture solution of the honeycomb pore mushroom according to one embodiment of the present invention targeting B16F10 melanoma cells. The results are expressed as a percentage compared to the value obtained in the control group. The results are expressed as the mean ± standard deviation of three repeated experiments (*p<0.05;**p<0.01).
Figure 5 shows the results of an experiment on the MITF expression inhibition activity of the Pa_MY culture solution of the honeycomb pore mushroom according to one embodiment of the present invention targeting B16F10 melanoma cells. The results are expressed as a percentage compared to the value obtained in the control group. The results are expressed as the mean ± standard deviation of three repeated experiments (*p<0.05, **p<0.01, ***p<0.001).

The skin whitening composition of the present invention is characterized by containing a culture solution of Polyporus arcularius as an effective ingredient.

The honeycomb pore mushroom belongs to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Polyporaceae, and genus Polyporus. In the present invention, for the effect aimed at in the present invention, it is preferable to use a strain native to Korea or isolated therefrom, and more preferably, NIBRFGC 000143418 strain provided as a new strain in the present invention is used.

For example, the NIBRFGC 000143418 strain of the present invention can be obtained from the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Sciences, Rural Development Administration. The accession number of this strain is KACC 93418P.

In the present invention, the culture solution can be obtained by culturing the honeycomb pore mushroom under typical mushroom culture conditions. For the effect aimed at in the present invention, DY (Dextrose & Yeast extract), MY (Malt extract & Yeast extract), MEB (Malt extract broth) or PDB (Potato dextrose broth) medium is preferably used, and MY medium is more preferably used.

The culture temperature is preferably 20 to 30°C, more preferably 21 to 29°C, more preferably 22 to 28°C, more preferably 23 to 27°C, and more preferably 24 to 26°C, for the effect targeted by the present invention. The shaking culture is preferably performed for the effect targeted by the present invention, and the shaking culture is preferably performed at 100 to 250 rpm, more preferably 150 to 200 rpm, and more preferably 160 to 180 rpm. The culture time is preferably 20 to 100 days, more preferably 30 to 90 days, more preferably 40 to 80 days, more preferably 50 to 70 days, and more preferably 55 to 65 days, for the effect targeted by the present invention.

In the present invention, the culture solution of the honeycomb pore mushroom can be obtained by a method of filtering after the above-mentioned culture process. In addition, the culture solution of the present invention can be obtained by additionally undergoing a process such as concentration, reduced pressure drying, or freeze drying after filtering, for example, as a freeze-dried powder.

According to the research results, the culture solution of the present invention may exhibit little cytotoxicity to skin cells even at high concentrations. For example, when treated at a concentration of 400 ㎍/㎖ to B16F10 cells, which are skin cells, it may exhibit a cell viability of 85% or more compared to the control B16F10 cells. In addition, the culture solution of the present invention may inhibit melanin synthesis in skin cells in a concentration-dependent manner, inhibit tyrosinase activity in a concentration-dependent manner, and suppress the expression of melanin formation-related factor (MITF) in a concentration-dependent manner.

The above factors, namely melanin, tyrosinase and MITF, are important factors for skin whitening, and the honeycomb pore mushroom culture solution of the present invention can exhibit an excellent skin whitening effect by effectively controlling these factors.

The skin whitening composition of the present invention may contain the culture solution of the honeycomb pore mushroom of the present invention in various amounts depending on various purposes such as the form and use of the composition. For example, the composition may contain the culture solution of the honeycomb pore mushroom of the present invention in an amount of 0.01 to 100 wt%, but is not limited thereto.

The skin whitening composition of the present invention may contain only the honeycomb pore mushroom culture solution of the present invention as an active ingredient, and may also contain other skin whitening active substances.

In one embodiment, the skin whitening composition of the present invention contains only the honeycomb pore mushroom culture of the present invention as an active ingredient, and does not contain any other skin whitening active substance, for example, any other compound and/or extract known to have skin whitening activity.

In another embodiment, the skin whitening composition of the present invention further contains, together with the culture solution of the honeycomb pore mushroom of the present invention, another skin whitening active substance, for example, another compound and/or extract known to have skin whitening activity.

The skin whitening composition of the present invention may be a skin whitening cosmetic composition.

The skin whitening composition of the present invention may be the honeycomb pore mushroom culture solution of the present invention itself, or a composition mixed with a pharmaceutically, food-wise or cosmetically acceptable carrier.

The skin whitening composition of the present invention can be manufactured into a cosmetic formulation commonly manufactured in this field. For example, it can be formulated into a solution, suspension, emulsion, powder, paste, gel, cream, etc., but is not limited thereto. More specifically, it can be manufactured into a formulation of a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

The skin whitening composition of the present invention may be used alone or in combination with other skin products.

The method for producing a skin whitening active substance of the present invention is characterized by including a step of culturing a honeycomb pore mushroom in a liquid medium.

The above culturing step may be a step of inoculating the honeycomb pore mushroom into a liquid medium and culturing under conditions that allow the mushroom to grow. For the effect aimed at in the present invention, the honeycomb pore mushroom is preferably cultured in a DY, MY, MEB or PDB medium. Accordingly, the skin whitening active substance can be produced more efficiently. More preferably, it is cultured in an MY medium.

In addition, the culturing step is preferably a step of shaking culture at 100 to 250 rpm for 20 to 100 days at a temperature condition of 20 to 30°C for the effect targeted by the present invention. The temperature condition is more preferably 21 to 29°C, more preferably 22 to 28°C, more preferably 23 to 27°C, and more preferably 24 to 26°C. The shaking culture condition is more preferably 150 to 200 rpm, and more preferably 160 to 180 rpm. The culture period is more preferably 30 to 90 days, more preferably 40 to 80 days, more preferably 50 to 70 days, and more preferably 55 to 65 days. According to the conditions as described above, a skin whitening active substance can be produced more efficiently.

In addition, the method for producing the skin whitening active substance of the present invention may further include a filtering step, a concentrating step, a depressurizing drying step, a freeze-drying step, and/or a powdering step after the culturing step, and may further include a step of further purifying the skin whitening active substance.

The strain of the present invention is a strain that was purified by separating clean tissue from the fruiting body of the oyster mushroom collected from a tree in Mt. Odae, spreading it on a PDA (Potato dextrose agar) medium, and identifying it by amplifying the ITS (internal transcribed spacer) section using ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') primers. The oyster mushroom can be used in the skin whitening composition of the present invention and the method for producing the skin whitening active substance of the present invention, and in this case, a more excellent skin whitening composition can be provided, and a more excellent skin whitening active substance can be efficiently produced. The strain of the present invention has been deposited with the Microbial Bank (KACC) of the National Institute of Agricultural Sciences, Rural Development Administration under the accession number KACC 93418P.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

Example 1. Isolation of the strain NIBRFGC 000143418 of the honeycomb-hole mushroom

Clean tissue was separated from the fruiting body of the Pleurotus eryngii collected from Mt. Odae, plated on PDA (Potato dextrose agar) medium, and the purified strain was identified by amplifying the ITS (internal transcribed spacer) section using ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') primers. The phylogenetic tree by alignment of ITS sequences is as shown in Figure 1.

Example 2. Preparation of a culture solution of the honeycomb pore mushroom

The honeycomb pore mushroom (NIBRFGC 000143418 strain, KACC 93418P) was cultured in DY (Dextrose & Yeast extract), MY (Malt extract & Yeast extract), MEB (Malt extract broth), and PDB (Potato dextrose broth) media at 25°C and 170 rpm for 60 days. After culture was completed, the mycelia were removed with Miracloth (Calbiochem, La Jolla, CA, USA), and the culture filtrate was freeze-dried and powdered.

Example 3. Evaluation of whitening activity

3-1. Method

3-1-1. Experimental materials and cell culture

B16F10 melanoma cells used in the experiment were obtained from ATCC (American Type Cell Culture, USA), and α-MSH (α-melanocyte stimulating hormone) and L-DOPA (L-3,4-dihydroxyphenylalanin), reagents used in the experiment, were purchased from Sigma-Aldrich (St. Louis, MO, USA). B16F10 melanoma cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum), 100 U/㎖ penicillin, and 100 ㎍/㎖ streptomycin at 37℃ in a 5% _CO2 incubator. Cells were subcultured every 3 days and used in the experiment.

3-1-2. Cytotoxicity evaluation

For cytotoxicity evaluation, B16F10 melanoma cells were seeded at a density of 1.0 × 10 ⁴ cells/well in a 24-well plate and cultured for 24 h. Afterwards, the sample and α-MSH (200 nM) were treated simultaneously and reacted for 72 h. After 72 h, 2 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) reagent prepared using 1× PBS buffer (Sigma-Aldrich) as a solvent was added and reacted for 2 h to form formazan blue. Afterwards, formazan blue was dissolved in DMSO and transferred to a 96-well plate. The absorbance was measured at a wavelength of 570 nm using an ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA), and the cytotoxicity was evaluated by comparing it with the absorbance measurement value of the control group.

3-1-3. Measurement of melanin synthesis inhibition activity

To evaluate the inhibitory activity of melanin synthesis, B16F10 melanoma cells were seeded at a density of 4.0 × 10 ⁴ cells/well in 6-well plates using DMEM medium (10% FBS, 1% penicillin-streptomycin) and cultured for 24 h in an incubator at 37°C and 5% CO _2. Then, the cells were simultaneously treated with the sample and α-MSH (200 nM) and cultured for 72 h. Afterwards, the cells were washed once with 1× PBS buffer and harvested by treating with 0.5× trypsin-EDTA (Gibco, Grand Island, NY, USA), and then centrifuged at 800 rpm for 3 min. The pellet from which the supernatant was removed was dissolved by adding 100 ㎕ of 1× RIPA buffer (1× radioimmunoprecipitation assay buffer, Bioworld, Seongnam, Korea) containing 1% protease inhibitor (Sigma-Aldrich), and centrifuged at 13,000 rpm for 30 min. After that, 500 ㎕ of 1 N NaOH (10% DMSO) was added to the recovered pellet, heat-treated in a heating block at 90°C for 1 h, and then transferred to a 96-well plate and the absorbance was measured at a wavelength of 405 nm using an ELISA reader.

3-1-4. Measurement of tyrosinase inhibition activity

To evaluate tyrosinase inhibitory activity, B16F10 cells were seeded in 6-well plates at 4.0 × 10 ⁴ cells/well and cultured for 24 h in a 5% CO ₂ incubator at 37°C. After that, the sample and α-MSH (200 nM) were treated simultaneously and cultured for 72 h under the same conditions. After lysis with 1× RIPA buffer, the proteins present in the recovered supernatant were quantified using a BCA kit (Thermo Fisher Scientific). 2 mg/mL L-DOPA (Sigma-Aldrich) was added to the quantified protein, and the reaction was performed at 37°C for 2 h. Then, the absorbance was measured at a wavelength of 490 nm using an ELISA reader.

3-1-5. Western Blot Analysis

B16F10 cell-well plates were seeded at 4.0 × 10 ⁴ cells/well and pre-cultured for 72 h in an incubator at 37°C, 5% CO₂. The samples were then co-treated with α-MSH (200 nM), and 100 μl of lysis buffer [1×RIPA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na ₃ VO ₄ , and protease inhibitor] was added to the harvested cells 24 h later to lyse the cells, and centrifuged at 4°C, 13,000 rpm for 30 min to separate the supernatant. Proteins were quantified using a BCA kit, mixed with an equal volume of 2× Laemmli sample buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue), electrophoresed on a 10% SDS-PAGE gel, and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-rad). After blocking with 5% skim milk for 1 hour at room temperature, the membrane was immersed in the primary antibody MITF (1:500, Cell signaling, USA) and reacted overnight at 4°C. After the reaction, the slides were washed four times with 1× TBST and reacted with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 1 hour and 30 minutes. The slides were washed again four times with 1× TBST and measured using an imaging densitometer (model GS-700, Bio-rad) using an ECL kit (Bio-Rad). After measurement, the area value for MITF protein expression compared to β-actin was quantified using the ImageJ program (1.52v, NIH, Bethesda, MD, USA) and then presented in a graph.

3-1-6. Statistical processing

All experiments were repeated three times, and the results were expressed as the mean ± standard deviation. In addition, statistical analysis was performed using analysis of variance (ANOVA) followed by student's t-test for multiple comparisons to verify the significance of each treatment group (*p<0.05; **p<0.01; ***p<0.001).

3-2. Results

3-2-1. Cell viability

To compare the effects of the culture solutions of the Pleurotus eryngii obtained by culturing in four types of media (MY, DY, MEB, and PDB) on cell survival, the freeze-dried powder samples of each culture filtrate of Example 2 (Pa_MY, Pa_DY, Pa_MEB, and Pa_PDB, respectively) were treated at concentrations of 50, 100, 200, and 400 μg/㎖ and subjected to an MTT assay. As a result, a survival rate of 85% or higher was confirmed in all cases, and in particular, a cell survival rate of 90% or higher was confirmed in Pa_MY and Pa_DY (Fig. 2).

3-2-2. Melanin synthesis inhibitory activity

To investigate the effect of the culture solution of the Pleurotus eryngii obtained by culturing in four kinds of media (MY, DY, MEB, and PDB) on B16F10 melanoma cells induced by α-MSH, the freeze-dried powder sample of each culture filtrate of Example 2 was treated at concentrations of 50, 100, 200, and 400 μg/㎖ and the inhibitory activity on melanin was compared. As a result, it was confirmed that melanin synthesis was inhibited in all samples, and in particular, Pa_MY was confirmed to exhibit the highest activity, inhibiting melanin synthesis by 11.0%, 19.2%, 29.0%, and 57.8%, respectively (Fig. 3). Therefore, Pa_MY, which had the highest melanin synthesis inhibitory activity at a concentration that did not show cytotoxicity, was selected and the following additional experiments were conducted.

3-2-3. Tyrosinase inhibitory activity

Tyrosinase acts in the initial reaction, which is the rate-determining step of melanogenesis, and is one of the major enzymes acting in melanogenesis. Therefore, when α-MSH (200 nM) and Pa_MY (50, 100, 200, 400 μg/㎖) were simultaneously treated with B16F10 melanoma cells, the effect on tyrosinase activity was investigated. As a result, tyrosinase activity was inhibited by 9.5%, 13.1%, 24.3%, and 42.7% at concentrations of 50, 100, 200, and 400 μg/㎖, respectively (Fig. 4).

3-2-4. Inhibitory activity of expression of melanin synthesis related factor (MITF)

The effect of Pa_MY on the expression of MITF, a transcription factor for melanin synthesis, was investigated in B16F10 melanoma cells. As a result, the expression of MITF was inhibited by 40.8%, 47.8%, 60.0%, and 81.5% in B16F10 melanoma cells treated with Pa_MY at concentrations of 50, 100, 200, and 400 μg/㎖, respectively, and at the highest concentration of 400 μg/㎖, it was decreased to a level similar to that of the untreated α-MSH group (Fig. 5).

<Microbial Consignment Certificate>

National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) KACC93418P 20231019

Claims (8)

A skin whitening composition containing a culture solution of Polyporus arcularius as an effective ingredient. A composition for skin whitening, wherein in claim 1, the honeycomb pore mushroom is a NIBRFGC 000143418 strain. A composition for skin whitening, wherein in claim 1, the culture medium is obtained by culturing in DY, MY, MEB or PDB medium. A composition for skin whitening, wherein in claim 1, the culture solution is obtained by shaking and culturing in a liquid medium at 20 to 30°C and 100 to 250 rpm for 20 to 100 days. A method for producing a skin whitening active substance, comprising the step of culturing Polyporus arcularius in a liquid medium. A method for producing a skin whitening active substance, wherein in claim 5, the honeycomb pore mushroom is a NIBRFGC 000143418 strain. A method for producing a skin whitening active substance in claim 5, wherein the liquid medium is DY, MY, MEB or PDB medium. A method for producing a skin whitening active substance, wherein in claim 5, the culturing is a shaking culture at 20 to 30°C and 100 to 250 rpm for 20 to 100 days.