KR102566401B1 - a composition comprising Fomitella fraxinea mycelium, Ganoderma applanatum mycelium and Glycyrrhizae uralensis extract for inhibitory effects on melanogenesis of B16F10 cells - Google Patents

Abstract

The present invention relates to a composition having whitening functionality by inhibiting melanin formation using longevity mushroom mycelium culture medium, Zannabibulocho mushroom mycelium culture medium, and licorice extract.

Description

A whitening composition comprising Fomitella fraxinea mycelium, Ganoderma applanatum mycelium and Glycyrrhizae uralensis extract for inhibitory effects on melanogenesis of B16F10 cells}

The present invention relates to a composition having whitening functionality by inhibiting melanin formation using longevity mushroom mycelium culture medium, Zannabibulocho mushroom mycelium culture medium, and licorice extract.

Mushrooms are a variety of species distributed throughout the world, and are higher fungi that form fruiting bodies among basidiomycetes and ascomycetes. Mushrooms contain abundant nutrients evenly, have unique fragrance and taste, and are widely used for food and medicine. is one of

Mushrooms belong to fungi biologically, and spores (seeds) germinate to become mononuclear hyphae. It grows into a fruit body by biochemical changes and takes the form of a mushroom, and the fruit body repeats the growth process of forming spores.

Mushrooms do not photosynthesize because they do not have chlorophyll. They are fungi that absorb nutrients from the dead bodies of plants or animals or are heterotrophic. They produce fruiting bodies composed of fibers called hyphae and reproduce sexually.

The growth of mushrooms is divided into vegetative growth and reproductive growth. The mycelium performs vegetative growth and the fruiting body performs reproductive growth.

Currently, there are 300,000 species of mushrooms on earth, 5,000 species in the United States and 1,554 species in Korea. It is known that there are 100 kinds of edible mushrooms, 50 kinds of poisonous mushrooms, and 162 kinds of medicinal mushrooms.

Mushrooms are rich in carbohydrates, proteins, vitamins and minerals, and during growth, they produce various hydrolytic enzymes such as fiber, protein, and lipolytic enzymes. It has been reported to have excellent effects in preventing various adult diseases such as activity, blood sugar lowering action, blood cholesterol lowering, cardiotonic action, blood pressure lowering action, and heart disease or stroke.

Cellulase secreted by mushrooms is β-1, 4-endoglucanase (EC3.2.1.4), cellobiohydrolase (EC3.2.1.176), cellobiohydrolase (nonreducing end, EC3.2.1.91), It can be classified into β-1,4-glucosidase (EC3.2.1.21) and cellobiose dehydrogenase (EC1.1.99.18), and these enzymes are industrially useful because they can decompose compounds with β-glycoside bonds. It can be said that there is

Mushroom mycelium has physiological activities similar to fruiting bodies, and various functional substances can be effectively obtained depending on the medium. It can be mass-produced cheaply and is easy to supply raw materials, so it is very suitable for industrialization, but its application in the actual bio field is insufficient.

The cell wall of elongated hyphae is composed of chitin, glucan, and protein. Glucan accounts for more than half of the cell wall at 50~60%, and 65~90% is β-(1,3)-Glucan as a structural polysaccharide. Consists of. Protein consists of 20-30% of oligosaccharide-modified glycoproteins, and 10-20% of chitin allows cell walls to remain intact.

The main medicinal efficacy of beta-glucan is to enhance and regulate immunity to have anticancer action, suppress inflammation, prevent allergy and atopy, have antibacterial, antiviral, and wound healing effects.

Fomitella fraxinea is a white wood-decaying basidiomycete belonging to the family Holobasidiomycetidae, Aphyllophorales, and Poly-poraceae. It is called "Black Swan Butterfly Mushroom", "Turtle Shell Lumberjack Mushroom", " Longevity Mushroom " or " Acacia Tongji" . ., Fomitopsis cytisina (Berk.) Bond. Et Sing.

It is morphologically similar to Ganoderma lucidum, but has no stipe, and is characterized by occurring in groups near the roots of broad-leaved trees such as cherry trees, acacia trees, and apple trees.

Longevity mushroom has an excellent ability to secrete polyphenol oxidase, such as laccase, which can oxidize phenolic compounds, and has the potential to be used industrially. Longevity mushroom is one of the mushrooms that are attracting increasing interest because of its effectiveness in anticancer, adult disease, and blood pressure lowering.

Research on immunoactive polysaccharides extracted from longevity mushrooms, isolation and regeneration of protoplasts, antioxidant activity and structure analysis of steroid compounds, research on optimal production and enzymatic properties of fibrin-degrading enzymes from mycelium, and research on mass production of laccase etc. have been reported.

Ganoderma applanatum is taxonomically classified into Basidiomycota, Basidiomycetes , Aphyllophorales, Ganodermataceae, and Ganoderma.

It is a decaying fungus that occurs on the trunks of dead broad-leaved trees in summer and autumn and is distributed all over the world including Korea.

The length of the fruit body is about 10 cm, the shape is sedentary or sometimes semi-sessile, and the cap is flat semicircular to somewhat horseshoe-shaped, and the surface is flat and cut into cuticles. It is yellowish brown to grayish brown, sometimes grayish white, with a somewhat rugged structure with deep concentric rings, and occasionally superficial radial cracks are observed over time.

It has been widely used as a traditional folk remedy and herbal medicine in Korea and China, and has been used as a raw material for alcohol in China and elsewhere because of its good flavor.

It has been reported that fruiting bodies of Zannabibulocho mushroom ( G. applanatum ) have various physiological actions such as anticancer, antiviral, antibacterial, immunomodulatory effects and whitening.

As active substances of Zannabibulocho mushroom, ergostane-based steroids, triterpenoid-type compounds, lanostane-type triterpenes, and heterogalactans have been reported. Recently, ergosterol, β-amyrinacetate, 2-hydroxy-hexacosanoimacid, β-amyrenone, β-amyrenone, 2,5-dihydroxybenzoic acid, ganoderenicacids A, B, and G, and ganodermaaldehyde were also isolated.

Cereproside, 5-dihydroergosterol, 2-methoxy fatty acid or cerevisterol compounds isolated from the ethyl acetate fraction or methylene chloride fraction of Zannabibulocho inhibit the accumulation of glycation end-products by their inhibitory activity against aldosereductase. It has been reported to be useful for the prevention and treatment of diabetes-related diseases.

It was reported that two types of β-D-glucans from hot water extracts of fruiting bodies of Zannabibulocho have strong anticancer activity. It was reported that the liquid cultured mycelia of Zannabibulocho contain useful anti-cancer polysaccharides, and the mycelial polysaccharides had significant anti-cancer activity as a result of investigating Sarcoma180/mice by the ip or po method.

Zannabibulocho mushroom has been recognized as having a very high potential as a variety of functional food materials and nutraceuticals.

Licorice ( Glycyrrhizae Radix , Licorice, Glycrrhiza) is known by its scientific name as Glycyrrhizae uralensis Fisch Et, DC, and is a perennial herb belonging to the leguminous family, Glycyrrhizae uralensis Fisch., Glycyrrhizae inlata Bat., Glycyrrhizae glabra L .etc. Licorice has been mainly used for the treatment of anti-inflammatory ulcers, hepatitis C and lung and skin diseases, and is known to have anti-inflammatory, anti-viral, anti-bacterial, antioxidant, anti-cancer activity, immune regulation, liver protection and heart protection effects. .

The present applicant devised a composition having whitening functionality by inhibiting melanin formation using longevity mushroom mycelium culture medium, zannabibulocho mushroom mycelium culture medium, and licorice extract.

Looking at the prior art literature, Korean Registered Patent Publication No. 10-1072133 "Cosmetic Composition for Increasing Skin Whitening Efficacy" describes a cosmetic composition containing licorice extract, red ginseng extract, birch extract, and papain enzyme. In addition, Korean Patent Publication No. 10-2018-0106155 "Whitening and moisturizing functional composition containing a fermented material of natural mixture" is a cosmetic for whitening containing fermented lactic acid bacteria of a mixture of horsetail, dermis, licorice, gardenia and nut gall as an active ingredient. The composition is described.

The above documents have some similarities with the present invention in that licorice is used in a whitening composition, but are different in that they do not additionally use longevity mushroom mycelium culture solution and Zannabibulocho mushroom mycelium culture solution.

Republic of Korea Registered Patent Publication No. 10-1072133 (registered on October 4, 2011) Republic of Korea Patent Publication No. 10-2018-0106155 (published on October 1, 2018)

The problem to be solved is to provide a composition having whitening functionality by inhibiting melanin formation using longevity mushroom mycelium culture medium, zannabibulocho mushroom mycelium culture medium and licorice extract as a technical problem.

A means for solving the problem is a whitening composition containing longevity mushroom ( Fomitella fraxinea ) mycelium culture medium and licorice ( Glycyrrhizae uralensis ) extract as active ingredients.

The longevity mushroom mycelium culture solution,

It can be replaced with one or more of longevity mushroom mycelium culture medium and zannabibulocho mushroom ( Ganoderma applanatum ) mycelium culture medium.

In the above composition, the longevity mushroom mycelium culture medium or the zannabibulocho mushroom mycelium culture medium is characterized in that it is composed to be mixed with the licorice extract at 1: 1 (v / v).

A means for solving the problem is a whitening cosmetic composition using the composition described above.

The composition according to the present invention inhibits melanin formation and tyrosinase activity, so it has a whitening effect.

1 to 7 are graphs and photographs showing experimental results of experimental examples of the present invention.

The application of the present invention is not limited by the configuration and arrangement of components described in the embodiments or shown in the drawings. The invention is capable of implementation in other embodiments and of being carried out in various ways. In addition, expressions and predicates used in the embodiments with respect to terms such as the orientation of devices or elements are merely used to simplify the description of the present invention, and do not indicate or imply that related devices or elements should simply have a specific orientation. . For example, when terms such as "first" and "second" are used in the embodiments and claims describing the present invention, these terms are not intended to indicate or imply a relative significance or meaning.

In addition, the terms or words used in this specification and claims should not be construed as being limited to a conventional or dictionary meaning, and the inventors should not use the technical spirit of the present invention to explain the terms and concepts of the invention in the best way. It should be interpreted as written based on the corresponding meaning and concept.

Therefore, the present invention is not limited to the presented embodiments, and the equivalent scope of the technical idea of the present invention and the technical idea described in the claims to be described below by those skilled in the art to which the present invention belongs Various modifications and changes are possible within.

Although the present invention has been described based on preferred embodiments with reference to the accompanying drawings, it is clear that various modifications are possible without departing from the technical scope from these descriptions by those skilled in the art. Accordingly, the scope of the present invention may include many such variations.

Experiment preparation and method

1. Reagents

Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS) were products of Gibco (NY, USA), tyrosinase, TRP-1, TRP-2, and MITF were products of Santa Cruz (CA, USA), MAPKs, pCREB, pMEK, anti-Goat, anti-Rabbit, anti-Mouse IgG HRP conjugate antibody was a product of Cell Signaling (CA, USA), hybond-ECL nitrocellulose membrane was a product of Amersham Biosciences (Buckinghanshire, England), western blotting Detection reagent was iNtRON (Seongnam, Korea), non-fat skim milk was Becton (Le Pont de Claix, France), dimethylsulfoxide (DMSO), phosphatase inhibitor and protase inhibitor cocktail, N,N,N' ,N'tetrametylethylenediamine (TEMED), thiazolyl blue tetrazolium bromide (MTT), L-3,4-dihydroxyphenyl alanine (L-DOPA) were from Sigma, and protein quantification reagents (Bicinchoninic acid (BCA) kit) were from Pierce (Rockford) , IL, USA) was used.

2. Cell line culture

B16F10 cells (melanoma cells) were grown at 37°C using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Incubated in 5% CO2.

3. Preparation of mushroom mycelium culture medium

After inoculating the mushroom mycelium in a solid medium, the culture is prepared by culturing at 20 to 32 ° C for 5 to 10 days. After preparing a liquid medium containing culture nutrients, the culture medium is inoculated and then cultured to prepare a culture medium. It is preferable to prepare a culture solution by culturing at 20 to 32 ° C. for 3 to 5 days.

Experimental Example 1. Inhibition of melanin synthesis

Measurement of melanin production was performed by culturing the B16F10 cell line, dispensing at a concentration of 1Х10 ⁴ cells/well in a 24-well plate per well, and cultured for 24 hours for attachment and stabilization in a 37°C, 5% CO ₂ incubator.

After that, a mixture of arbutin, kojic acid, Uzbek licorice hot water extract, domestic licorice hot water extract, longevity mushroom mycelium culture medium, licorice hot water extract and longevity mushroom mycelium culture medium mixed at 1:1 (v/v) After adding α-MSH (200nM) to the cells treated with a mixed material of 1:1 (v/v) mixture of Zannabibulocho mushroom mycelium culture medium, licorice hot water extract and Zannabibullocho mushroom mycelium culture medium at 1:1 (v/v), 37℃ , and cultured in a 5% CO ₂ incubator for 1 to 3 days. After incubation, each well was washed with PBS, and 400 μl of 1 N NaOH solution was added, dissolved at 80° C. for 1 hour, and then measured at 475 nm using a Microplate Reader.

1 to 6 are graphs and photos of the experimental group melanin synthesis inhibition test results.

Arbutin and kojic acid were tested in order to contrast with the present experimental groups, which are widely used as whitening ingredients. (Fig. 1)

The domestic licorice hot water extract showed a higher inhibitory effect than the Uzbek licorice hot water extract. (Fig. 2, using domestic licorice from the subsequent experiments)

As a result of experimenting with a mixture of licorice hot-water extract, longevity mushroom mycelium culture medium and licorice hot-water extract and longevity mushroom mycelium culture medium mixed at a ratio of 1:1 (v/v), the melanin formation inhibitory effect of longevity mushroom mycelium culture medium and mixed material was confirmed. could (See Fig. 3)

As a result of testing a mixed material that mixed 1:1 (v/v) of licorice hot-water extract, Zannabibulocho mushroom mycelium culture medium, licorice hot-water extract and Zannabibulocho mushroom mycelium culture medium, melanin formation was inhibited by the mixture of Zannabibulocho mushroom mycelium culture medium and mixed material effect could be confirmed. (See Fig. 4)

As a result of a separate test of the mixed material in which the hot water extract of licorice root and the mycelium culture medium of longevity mushroom were mixed at a ratio of 1:1 (v/v), the melanin formation inhibitory effect was found to be about 27% at 8 ul/ml. (See Fig. 5)

As a result of testing again with a mixed material in which hot-water extract of licorice root and mycelium culture medium of Zannabibulocho mushroom were mixed at a ratio of 1:1 (v/v), the melanin formation inhibitory effect was about 23% at 4 ul/ml. (See Fig. 5)

Experimental Example 2. Intracellular Tyrosinase Activity Measurement

Intracellular tyrosinase activity was measured by dispensing 1×10 ⁴ /mL of B16F10 cells into a 6-well culture container and incubating them for 24 hours. α-MSH in a container treated with a mixture of mycelium culture medium in a ratio of 1:1 (v/v), a mixture of licorice hot-water extract and a mixture of mycelium culture medium in a ratio of 1:1 (v/v) to each concentration. (200 nM) and incubated for 1-3 days, washed twice with PBS, and 1% (V/V) Triton X- After dispensing 200 μL of lysis buffer mixed with 100 and 0.1% (V/V) of 0.1 M PMSF, the cells were collected, left on ice for 30 minutes, and centrifuged at 4℃ 15,000 rpm for 30 minutes. The resulting supernatant was tyrosinase active used for measurement.

For protein quantification, the same amount of protein was calculated by measuring the absorbance at 575 nm with BCA reagent. The reaction was carried out at 37 ° C. for 1 hour by dispensing, and the change in absorbance at 475 nm was measured at 30-minute intervals.

Figure 7 is a table showing the experimental results, licorice hot water extract from 156 ug / mL, licorice hot water extract and longevity mushroom mycelium culture medium 1: 1 (v / v) mixed material mixed from 156 ug / mL, licorice It was confirmed that the mixed material, which was a mixture of hot water extract and Zannabibulocho mycelium culture medium at a ratio of 1:1 (v/v), inhibited tyrosinase activity by more than 20% from 78 ug/mL.

Example 1. Whitening composition containing longevity mushroom mycelium, zannabibulocho mushroom mycelium and licorice extract as active ingredients

The composition according to the present invention includes a mixed material in which a longevity mushroom mycelium culture medium and licorice extract are mixed. In addition, a mixed material obtained by mixing the zannabibulocho mushroom mycelium culture medium and licorice extract may be added and included.

Preferably, the licorice extract is a hot water extract, and the mixing ratio of each mixed material is 1:1 (v/v). Depending on the design conditions, the licorice extract may be an ethanol extract, and the mixing ratio may be changed, of course.

In the composition according to the present invention, based on B16F10 melanoma cells 1Х10 ⁴ cells / well, licorice hot water extract is 156 ug / mL or more, when mixing licorice hot water extract and longevity mushroom mycelium culture medium 1: 1 (v / v) The mixed material of contains more than 156 ug/mL, and more than 78 ug/mL of mixed material when mixing the licorice hot water extract and the mycelium culture medium of Zannabibulocho mushroom at a ratio of 1:1 (v/v).

The present invention can provide a preventive and therapeutic agent for immune enhancing and anti-inflammatory diseases formulated in a pharmaceutical unit dosage form by adding the extract and the culture medium as active ingredients and adding a pharmaceutically acceptable carrier, excipient or diluent thereto. there is. Here, the carrier, excipient, and diluent include toze, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, undecided quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set. In addition, when formulating the active ingredient, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, the pharmaceutical dosage form is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions. can

The solid preparation for oral administration may be prepared by mixing the extract with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent.

As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, age, sex, drug type, administration route and period, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the extract of the present invention is preferably administered at 0.001 to 300 mg/kg, and administration may be administered once a day or divided several times. The dosage is not intended to limit the scope of the present invention in any way.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular or subcutaneous administration.

It is possible to provide a whitening health functional food composition that inhibits melanin formation by adding a food additive to the active ingredient of the present invention.

Foods to which the active ingredient can be added include, for example, various foods, beverages, chewing gum, tea, vitamin complexes, health functional foods, and the like.

The amount of the active ingredient in the food or beverage may be added in an amount of 0.01 to 20% by weight of the total weight of the food or beverage, and the health drink composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. there is.

The health functional beverage composition of the present invention is not particularly limited in other ingredients other than containing the extract, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose; disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. In addition to the above, natural flavors (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavoring agents. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protection It may contain a colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like.

In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

It can also be provided as a whitening cosmetic composition containing the active ingredient of the present invention.

The embodiments described in this specification and the configurations shown in the drawings relate to one of the most preferred embodiments of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents and modifications that can replace them It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiments, and within the equivalent scope of the technical idea of the present invention and the technical idea described in the claims to be described below by those skilled in the art to which the present invention belongs There may be embodiments in which various modifications and changes are possible.

Claims (4)

Longevity mushroom ( Fomitella fraxinea ) Mycelium culture medium and Zannabi bullocho mushroom ( Ganoderma applanatum ) One or more of mycelium culture medium and licorice ( Glycyrrhizae uralensis ) Containing extract as an active ingredient,
The content conditions are 156-313 ug/mL of longevity mushroom mycelium culture medium and licorice extract mixed at a 1:1 volume ratio, and 1250-2500 ug of Zannabibulocho mushroom mycelium culture medium and licorice extract mixed at a 1:1 volume ratio Characterized in that it contains / mL,
Whitening cosmetic composition. delete delete delete