KR20230041197A - Composition for Anti-oxidant Comprising Fractions of Polyporus parvovarius Culture Extract, and Uses thereof - Google Patents

Abstract

The present invention relates to an antioxidant composition containing a culture fraction of a Polyporus parvovarius NIBRFGC000500557 strain (accession number: KACC 83044BP) as an active ingredient. The culture fraction of the Polyporus parvovarius NIBRFGC000500557 strain according to the present invention is capable of being easily produced by fractionating a culture produced through liquid and solid culture of the Polyporus parvovarius, contains 3,4-dihydroxybenzaldehyde as an active ingredient, and exhibits excellent antioxidant activity. In addition, the composition of the present invention can be usefully used for antioxidant purposes in various fields such as cosmetic compositions, food compositions, etc.

Description

Antioxidant composition containing cultured fractions of small yellow pit bull as an active ingredient

The present invention relates to an antioxidant composition comprising a culture fraction of Polyporus parvovarius NIBRFGC000500557 strain (Accession Number: KACC 83044BP) as an active ingredient.

Fungi are eukaryotic organisms and include molds, mushrooms, yeasts, and lichens. It is taxonomically composed of eight phylums, and based on molecular biology techniques, it is estimated that about 2.5 million species inhabit the earth worldwide.

However, only about 140,000 species have been identified so far, less than 6% of the total (Hawksworth, 2017). About 25,000 species of fungi are estimated to inhabit in Korea, but based on taxonomic studies based on molecular biology, it is estimated that more than 50,000 species can inhabit. So far, 5,616 species have been reported to inhabit in Korea, and only 22.5% of them have been identified compared to the previously estimated number of 25,000 species. This reality is evidence of the need for continuous research on new species habitats.

Higher fungi, which include Basidiomycota and some Ascomycota, which form fruiting bodies that can be observed with the naked eye, are decomposers that help nutrient circulation in the forest ecosystem and symbiotic with plants, playing an important role in maintaining forest diversity. . In particular, wood-decaying fungi decompose hard-to-decompose materials such as wood, and these material decomposition properties are also used to decompose pollutants such as phenol, dyes, and dioxins generated from industrial activities (Barr & Aust, 1994). In addition, as people's interest in animal welfare and environmental pollution has recently increased, a lot of attention has been paid to deokdari mushrooms, which are wood-decaying fungi, as raw materials for alternative meat that can reduce environmental pollution (Southey, 2019).

In addition, since some wood-decaying fungi (such as Ganoderma lucidum ) form mycelium with high tensile strength and tear strength during cultivation, research on artificial leather manufacturing using these mycelium is being conducted (Jones et al., 2020). . In addition, ectomycorrhizal mushrooms, including edible mushrooms with high added value such as pine mushrooms and truffles, are used for afforestation in degraded areas as they help seedlings to settle in a new environment as helpers for growth (Hawkins et al., 2015).

In addition, various species of mushrooms are known to produce natural compounds with antiviral, anticancer, antibiotic, and antipsychotic functions (El-Mekkawy et al., 1998; Yuen & Gohel, 2005; El Dine et al., 2008 (Hetland et al., 2008; Ross et al., 2016; Kombrink et al., 2019).

Therefore, mushrooms are a major food resource and can be used in various ways in high value-added industries, and under the current ABS system in which biological resources are used as a national sovereignty, the need for national research and management of fungi is very high. Until now, research on the living organisms in the burrowing mushroom is incomplete.

The present inventors continuously conducted research related to the discovery of candidates for new and unrecorded species for native fungi in Korea. Among them, the physicochemical characteristics of soil samples were analyzed for 40 grids, and the work of continuously confirming the diversity of fungal species in the soil on a national scale is in progress. At the same time, physiological activities of discovered fungi are analyzed to verify their usefulness, and conditions for artificial propagation (liquid culture, sawdust culture) are established and usefulness analysis is being performed to increase their utilization as biological resources.

In this process, the present inventors found that Polyporus parvovarius NIBRFGC000500557 strain, belonging to the genus Polyporus, can easily proliferate through liquid culture or solid culture, and ascorbic acid, which is widely used as an antioxidant, By comparison, it was confirmed that it had an equivalent level of DPPH radical scavenging activity or ABTS radical scavenging activity, and thus had an excellent antioxidant effect, and the present invention was completed.

KR No. 10-2013-0140154 (2013.11.18): Sialic acid binding-specific recombinant lectin derived from pit bull KR No. 10-2018-0023212 (2018.03.07): Production method of sesquiterpene from winter umbrella mushroom

Accordingly, a main object of the present invention is to provide an antioxidant composition comprising a culture fraction of Polyporus parvovarius strain NIBRFGC000500557 (accession number: KACC 83044BP) as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides an antioxidant composition comprising a cultured fraction of Polyporus parvovarius strain NIBRFGC000500557 (Accession Number: KACC 83044BP) as an active ingredient.

The term of the present invention, " Polyporus parvovarius ) NIBRFGC000500557 (accession number: KACC 83044BP)" is a new strain of the yellow mushroom collected in the Nari Basin of Ulleungdo and newly isolated and identified by the present inventors. , It was named the strain NIBRFGC000500557 of the small yellow mushroom ( Polyporus parvovarius ), deposited with the Rural Development Administration Agricultural Genetic Resources Center (KACC) on April 08, 2021, and was given the accession number KACC 83044BP.

In the present invention, "Little Yellow Ponytail Mushroom NIBRFGC000500557 Strain (Accession Number: KACC 83044BP)", "Little Yellow Ponytail Mushroom NIBRFGC000500557 Strain", "NIBRFGC000500557 Strain" or "NIBRFGC000500557" are strains deposited under Accession No. KACC 83044BP They are used with the same meaning, and may be used interchangeably in the present specification.

In the present invention, the term "culture" is a material obtained as a result of culturing the strain, and may include all substances secreted from a medium, a cultured strain, and a cultured microorganism. For example, inorganic salt components, amino acids, vitamins, nucleic acids and / or other components that can be generally contained in a culture medium may be included in addition to nutrient sources necessary for culturing cells, such as carbon sources and nitrogen sources.

As used herein, the term "extract" refers to an extract obtained by extraction of a culture, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or a mixture thereof, It includes the extract itself and extracts of all formulations that can be formed using the extract. The extract of the present invention may be preferably prepared and used in the form of a dry powder after extraction.

In the extraction of the culture of the strain NIBRFGC000500557, the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods.

The type of extraction solvent used to extract the culture of the strain NIBRFGC000500557 is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; or mixtures thereof may be used.

In addition, the term "fraction" used in the present invention refers to a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

The cultured fraction of the Polyporus parvovarius NIBRFGC000500557 strain (accession number: KACC 83044BP) exhibits very good DPPH radical scavenging activity or ABTS radical scavenging activity. In a preferred embodiment of the present invention, the "Little Yellow Amanita mushroom NIBRFGC000500557" strain (accession number: KACC 83044BP) can be easily propagated through liquid and solid culture, and the extract of the strain culture is compared with ascorbic acid, which is widely used as an antioxidant. It has been confirmed that there is an equivalent level of excellent antioxidant activity (see Figs. 1 to 4).

In the antioxidant composition of the present invention, the cultured fraction is at least one composed of ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose) and SDA (Sabouraud dextrose) It may be a fraction of a culture solution of a strain of Polyporus parvovarius NIBRFGC000500557 cultured in a culture medium.

In the present invention, the fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. A non-limiting example of the fractionation method is to obtain a fraction from the extract by treating the extract obtained by extracting the culture of Polyporus parvovarius strain NIBRFGC000500557 (accession number: KACC 83044BP) with a predetermined solvent way can be In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of two or more. When alcohol is used among the fractionation solvents, C1 to C4 alcohols may be specifically used.

Specifically, in the antioxidant composition of the present invention, the culture fraction is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethanol extract of the culture medium is 30% methanol, 70% methanol, 100% methanol, and a 70% methanol solvent fraction produced by sequential elution with acetone.

In addition, in the antioxidant composition of the present invention, the culture fraction is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethanol extract of the culture medium is 30% methanol, 70% methanol, 100% methanol, and a 70% methanol solvent fraction produced by sequential elution with acetone and a culture fraction obtained by distribution and extraction with ethyl acetate.

In addition, in the composition for antioxidant of the present invention, the cultured fraction is characterized in that it contains 3,4-dihydroxybenzaldehyde as an active ingredient.

In a preferred embodiment of the present invention, the ethanol extract of the culture medium of Polyporus parvovarius strain NIBRFGC000500557 (accession number: KACC 83044BP) is sequentially eluted with 30% methanol, 70% methanol, 100% methanol, and acetone It has been confirmed that the resulting 70% methanol solvent fraction exhibits excellent antioxidant activity. In addition, the culture fraction obtained by distributing and extracting the 70% methanol solvent fraction with ethyl acetate was dissolved in hloroform-MeOH (100: 1, v/v) solution to obtain silica gel column chromatography; elution solvent: chloroform-MeOH = 100: 1 → 0: 100, v / v), NMR spectrum analysis and mass spectrum analysis were performed, and the active ingredient showing antioxidant activity contained in the fraction was 3,4-dihydroxybenzaldehyde (3,4-dihydroxybenzaldehyde).

The cultured fraction of the strain Polyporus parvovarius NIBRFGC000500557 (accession number: KACC 83044BP) of the present invention exhibits excellent antioxidant activity, so it can be usefully used as a cosmetic composition for antioxidant. The effects on the antioxidant activity of the Polyporus parvovarius NIBRFGC000500557 strain and its cultured fractions of the present invention are as described in the antioxidant composition.

The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the cultured fraction of the Polyporus parvovarius NIBRFGC000500557 strain (Accession Number: KACC 83044BP) as active ingredients, such as antioxidants and stable conventional adjuvants such as agents, solubilizers, vitamins, pigments and flavors, and carriers.

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be formulated into a formulation of softening lotion (skin), nourishing lotion (milk lotion), nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder. .

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier components. It can be.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar or tragacantha and the like may be used.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Specific examples of the cosmetics of the present invention include face wash cream, face wash foam, cleansing cream, cleansing milk, cleansing lotion, massage cream, cold cream, moisture cream, lotion, toner, pack, after-saving cream, sun protection cream, suntan oil, soap , body shampoo, hair shampoo, hair conditioner, hair treatment, hair conditioner, hair conditioner, hair cream, hair liquid, set lotion, hair spray, hair bridge, color rinse, color spray, permanent wave liquid, press powder, loose powder , eye shadow, hand cream, lipstick, and the like.

According to another aspect of the present invention, the present invention provides a food composition for antioxidant comprising a cultured fraction of Polyporus parvovarius strain NIBRFGC000500557 (Accession Number: KACC 83044BP) as an active ingredient.

The cultured fraction of Polyporus parvovarius strain NIBRFGC000500557 (accession number: KACC 83044BP) of the present invention exhibits excellent antioxidant activity and can be usefully used as a food composition for improving skin conditions. The effects on the cultured fraction and antioxidant activity of Polyporus parvovarius NIBRFGC000500557 strain (accession number: KACC 83044BP) of the present invention are as described in the antioxidant composition. The food composition may be used in the form of health functional food, but is not limited thereto.

The food composition of the present invention may be included in the form of a cultured fraction of Polyporus parvovarius NIBRFGC000500557 strain (Accession Number: KACC 83044BP) or a processed product thereof. In addition, the composition may include a food additive that is acceptable in food science in addition to the active ingredient.

In the present invention, "food supplement additive" refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

The food composition of the present invention may include health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation.

In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using plant extracts as raw materials, has excellent portability, and is effective in skin care. It can be taken as an adjuvant to increase the water content, suppress the generation of oxidative free radicals or enhance the effect of removing the generated radicals.

There is no limit to the form that the health functional food of the present invention can take, and it may include all foods in a conventional sense, and may be used interchangeably with terms known in the art, such as functional foods. In addition, the health functional food of the present invention may be prepared by mixing suitable other auxiliary ingredients and known additives that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes, etc., which can be prepared by adding the cultured fraction of the Polyporus parvovarius NIBRFGC000500557 strain (accession number: KACC 83044BP) according to the present invention to juice, tea, jelly and juice prepared as a main component can Also included are foods used as feed for animals.

According to another aspect of the present invention, the present invention is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose), and SDA (Sabouraud dextrose); recovering the culture solution by culturing the inoculated strain at 20° C. to 30° C. for 7 to 60 days; And sequentially eluting the ethanol extract of the recovered culture medium with 30% methanol, 70% methanol, 100% methanol and acetone to obtain a 70% methanol solvent fraction.

In the method for preparing the antioxidant composition of the present invention, distributing and extracting the obtained 70% methanol solvent fraction with ethyl acetate; may further include.

In a preferred embodiment of the present invention, ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose) and SDA (Sabouraud dextrose) at a temperature of 20 ° C to 30 ° C ) It was confirmed that the medium was inoculated and cultured with the strain Polyporus parvovarius NIBRFGC000500557 to grow normally, and it was confirmed that the cultured fraction of the strain exhibited excellent antioxidant activity.

When culturing the small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP), a medium additionally containing a suitable carbon source or nitrogen source may be used. Carbon sources are mainly CO ₂ and carbonate, and mixed sugars of glucose and xylose can be used as carbon sources. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose, soybean oil, sunflower oil, Oils and fats such as castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid may be included. These materials may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, malt extract, corn steep liquor, casein hydrolysate, fish or decomposition products thereof, defatted soybean cakes or decomposition products thereof may be used. These nitrogen sources may be used alone or in combination. The medium may include amino acids, vitamins, phosphorus, and inorganic compounds.

According to another aspect of the present invention, the present invention is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose), and SDA (Sabouraud dextrose) medium consisting of inoculation step; recovering the culture solution by culturing the inoculated strain at 20° C. to 30° C. for 7 to 60 days; Obtaining a 70% methanol solvent fraction by sequentially eluting the ethanol extract of the recovered culture medium with 30% methanol, 70% methanol, 100% methanol and acetone; Distributing and extracting the obtained 70% methanol solvent fraction with ethyl acetate; and purifying 3,4-dihydroxybenzaldehyde from the distribution-extracted ethyl acetate fraction using column chromatography. A method for producing 3,4-dihydroxybenzaldehyde is provided.

The features and advantages of the present invention are summarized as follows:

(1) The present invention provides an antioxidant composition comprising a culture fraction of Polyporus parvovarius strain NIBRFGC000500557 (accession number: KACC 83044BP) as an active ingredient.

(2) The culture fraction of the strain of Polyporus parvovarius NIBRFGC000500557 of the present invention can be easily produced by fractionating the culture produced through liquid and solid culture of the small yellow hole mushroom, and the active ingredient It contains 3,4-dihydroxybenzaldehyde and shows excellent antioxidant activity.

(3) In addition, the composition of the present invention can be usefully used for antioxidant purposes in various fields such as cosmetic compositions and food compositions.

1 is a graph showing the growth (day 7) of mycelial growth (day 7) by temperature and medium of 8 strains (DYA: Dextrose & yeast extract Agar, MEA: Malt extract Agar, MYA: Malt extract & yeast extract Agar, PDA: Potato dextrose Agar, SDA: Sabouraud dextrose Agar).
Figure 2 is a photograph of liquid culture of 5 strains of the genus Foramina and comparing the culture solutions for each strain (A: Polyporus arcularius , B: Polyporus brumalis , C: Polyporus parvovarius , D: Polyporus tuberaster , E: Polyporus ulleungus ).
Figure 3 is a graph showing the DPPH radical scavenging ability measured by treating medium extracts of 5 types of mushrooms of the genus Polyporus at a concentration of 10 ⁴ mg/L for each sample.
4 is a graph showing ABTS radical scavenging ability after treatment with medium extracts of 5 types of mushrooms of the genus Polyporus at a concentration of 10 ⁴ mg/L for each sample.
Figure 5 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) to silica gel column chromatography ( The ABTS radical scavenging activity of six fractions (PP3-1 to PP3-6) produced by silica gel column chromatography) (elution solvent: chloroform-MeOH=100:1→0:100, v/v) was summarized. It is a table.
Figure 6 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) to silica gel column chromatography ( This is the HPLC profile of the PP32-1 fraction produced by silica gel column chromatography) (elution solvent: chloroform-MeOH=100:1→0:100, v/v).
Figure 7 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) silica gel column chromatography ( This is the HPLC profile of the PP34-1 fraction produced by silica gel column chromatography) (elution solvent: chloroform-MeOH=100:1→0:100, v/v).
8 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) to silica gel column chromatography ( This is the ODS MPLC profile of the PP34-2 fraction produced by silica gel column chromatography) (elution solvent: chloroform-MeOH = 100:1→0:100, v/v).
9 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) and silica gel column chromatography ( These are the HPLC profiles of the PP342-1 and PP342-2 fractions obtained by silica gel column chromatography) (elution solvent: chloroform-MeOH = 100:1→0:100, v/v).
Figure 10 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) to silica gel column chromatography ( These are the preparative HPLC profiles of the PP342-1 and PP342-2 fractions obtained by silica gel column chromatography) (elution solvent: chloroform-MeOH = 100:1→0:100, v/v).
11 is a small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) ethyl acetate fraction of the culture extract was dissolved in chloroform-MeOH (100: 1, v / v) and silica gel column chromatography ( These are the HPLC profiles of the PP32-1, PP33-1, and PP34-1 fractions obtained by silica gel column chromatography) (elution solvent: chloroform-MeOH = 100:1 → 0:100, v/v).
12 is a picture summarizing the fractionation and purification process of the culture extract of Polyporus parvovarius strain NIBRFGC000500557 (accession number: KACC 83044BP).
13 is a ¹ H NMR spectrum analysis graph of a compound present singly in the PP34-1 fraction (PP34-1).
14 is a ¹³ C NMR spectrum analysis graph of a single compound present in the PP34-1 fraction.
15 is a graph of ¹ H- ¹ H COZY spectrum analysis of a single compound present in the PP34-1 fraction.
16 is a diagram showing the chemical structure (left) of a single compound present in the PP34-1 fraction and the assignment of ¹ H (red) and ¹³ C (blue) peaks (right).
17 is an HMQC spectrum analysis graph of a single compound present in the PP34-1 fraction.
18 is an HMBC spectrum analysis graph of a single compound present in the PP34-1 fraction.
19 is a spectrum analysis graph (top: positive mode, bottom: negative mode) of a single compound present in a PP34-1 fraction.

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

Example 1. Obtaining fungal samples through artificial propagation

1-1. Selection of strains of the genus Mushroom Mushroom

8 strains of 5 species of Polyporus were selected (Table 1).

The 8 selected strains belong to Polyporales and Polyporaceae, and a total of 17 strains, including P. parvovarius , are currently on the national species list managed by the National Institute of Biological Resources. Species Polyporus strains are listed.

Among the 8 selected strains, the P. brumalis strain is known to produce various secondary metabolites such as terpenoids in previous studies, and the Polyporus ulleungus strain is known in 2017 As a new strain identified through the National Institute of Biological Resources discovery project in 2018, it was determined that additional characterization studies were needed, and it was selected as a test subject strain (Lee et al., 2016; Tibpromma et al., 2017; Lee et al., 2017).

[Table 1]

1-2. Artificial propagation of selected Mushroom fungus strains

The growth characteristics and optimal culture conditions of the strains of the genus Mushrooms, which are the species to be studied, were confirmed, and artificial propagation was carried out through liquid culture.

The growth characteristics of 8 strains of 5 types for artificial propagation were investigated by temperature and medium. First, the optimal culture temperature for each strain was confirmed by culturing under temperature control conditions of 20 ° C, 25 ° C, 30 ° C, and 35 ° C, and DYA (Dextrose & yeast extract Agar), MEA (Malt extract Agar) ), MYA (Malt extract & yeast extract Agar), PDA (Potato dextrose Agar), and SDA (Sabouraud dextrose Agar) were used for culture.

[Table 2]

Each experiment was repeated three times. One inoculum of the same size was placed on each medium using a 5 mm cork borer (KA00-49, Korea Ace, Korea). The temperature of the incubator was continuously measured with a digital thermometer, and the medium was checked daily for contamination.

The diameter and characteristics of mycelia were recorded from the 3rd to the 11th day, and based on the measurement results on the 7th day, the optimum temperature and medium for the growth of the strain were determined.

Prism (GraphPad Software, Ver 7.04, USA) was used for two-way ANOVA analysis and graphing (Kim et al., 2018).

Based on the mycelial length data measured on the 7th day, the optimal temperature and optimal medium conditions for 8 strains of Polyporus were confirmed.

As a result of the experiment, referring to [Figure 1], the two strains belonging to Polyporus arcularius showed almost the same growth tendency, and the optimum temperature for growth was predicted to be between 25 ° C and 35 ° C, and the corresponding temperature In the range, mycelia reached the edge of the plate on the 11th day after inoculation. By medium, all showed similar levels of growth except for SDA medium.

The two strains belonging to the winter hole mushroom ( Polyporus brumalis ) also showed almost the same growth trend. On the 7th day graph, it can be interpreted as showing a similar level of growth in all temperature ranges, but at 30℃ and 35℃, the 5th day It was observed that the hyphae grew to the edge of the plate, and through this, the optimum growth temperature of winter hole mushroom was confirmed to be between 30 ° C and 35 ° C, and the growth rate was the fastest in DYA and MEA media It became.

For Polyporus parvovarius and Polyporus tuberaster, testable strains were obtained within 1 week, respectively, so differences between different strains within the same species could not be compared. However, as a result of comparison with the small bee nest mushroom ( Polyporus arcularius ) or winter hole mushroom ( Polyporus brumalis ), it was confirmed that the growth rate was remarkably slow, and the optimum growth temperature was 25℃, and it could grow between 20℃ and 30℃. It was confirmed that it could hardly grow at 35℃.

In the case of the small yellow hole mushroom ( Polyporus parvovarius ), all media except for the PDA medium showed a similar level of growth rate, and the hole hole mushroom ( Polyporus tuberaster ) showed the fastest growth in the MEA medium.

On the other hand, it was confirmed that Polyporus ulleungus had the slowest growth rate among the five mushroom strains and could not grow at all at 35 ° C. The optimal growth temperature of Polyporus ulleungus was 25 ° C, and it was possible to grow between 20 ° C and 30 ° C, and the growth medium was confirmed to show rapid growth in the order of MYA, MEA, and DYA medium.

1-3. Artificial growth and sample pretreatment through liquid culture

Based on the results of the growth characteristics test, strains with the best growth rate for each species were selected as shown in Table 3 below and liquid culture was performed under optimal culture conditions.

[Table 3]

Strain inoculation was carried out by fragmenting the strain whose activity was confirmed in PDA (Potato dextrose agar) medium with a cork borer, and inoculated into four types of liquid medium, excluding SDA medium, among the five types of medium for which growth characteristics were confirmed. Shaking incubation was performed at a temperature of 25° C. for 60 days at 170 rpm.

The cultured liquid culture sample was separated into mycelium and culture filtrate using sterile gauze and a glass filter (Porosity 100 ~ 160㎛, DURAN), freeze-dried, and stored in a cryogenic freezer (-70 ° C) until further analysis.

As a result of the culture, referring to [Fig. 2], the color of the culture solution showed a difference depending on the species rather than the type of medium, and the culture solution of Polyporus parvovarius showed the darkest color. On the other hand, as a result of comparing the drying amount after freeze-drying the culture filtrate sample, it was found to be inversely proportional to the growth rate of each strain.

It was confirmed that the drying amount of Polyporus arcularius and Winter hole mushroom ( Polyporus brumalis ), which had the fastest growth rate, was smaller than that of Polyporus ulleungus , which had the slowest growth rate. Depending on the culture rate, it seems to vary depending on how much medium components are used. That is, it was found that the strains with a fast growth rate consumed more medium components, and the dry weight of the culture filtrate was relatively small. By medium, the drying amount of the culture filtrate cultured in the PDB medium was generally high (see Table 4).

[Table 4]

Example 2. Antioxidant activity assay

2-1. DPPH radical scavenging activity

The lyophilized culture filtrate in Example 1-3 was extracted with 70% ethanol and used as an analysis sample, and antioxidant of each sample with DPPH (2.2-Diphenyl-1-picrylhydrazyl, Sigma-Aldrich Co., USA) radical scavenging activity Activity was measured (Blois, 1958).

The DPPH compound is a stable free radical and is characterized in that it changes color from purple to yellow by reacting with substances having antioxidant activity. Samples with a concentration of 10 ⁴ mg/L dissolved in the same solvent as the DPPH compound dissolved in ethanol at a concentration of 0.2 mM were mixed at a ratio of 5:1 (v/v), reacted for about 30 minutes in the dark, and microplate reader ( The absorbance was measured at a wavelength of 517 nm using SpectraMax190, Molecular devices). As a control, ascorbic acid at a concentration of 10 ⁴ mg/L was used. The activity was measured three times repeatedly, and the activity was calculated based on [Equation 1] below.

[Equation 1]

Referring to [Figure 3], as a result of analyzing the antioxidant activity of the culture filtrate for each strain confirmed through the DPPH assay, the culture medium sample of the Polyporus parvovarius strain cultured in the DY medium showed the best antioxidant activity. Observed. In addition, the sample cultured with the small yellow hole mushroom ( Polyporus parvovarius ) strain in the MEB medium showed the next highest antioxidant activity.

For the small yellow hole mushroom ( Polyporus parvovarius ) strain, the culture filtrate samples cultured in 4 types of media (DY, MEB, MY, PDB) showed more than 50% antioxidant activity compared to the positive control group, and the DY medium and MEB Samples cultured in the medium were analyzed to exhibit antioxidant activity close to that of the positive control.

In addition to the small yellow hole mushroom ( Polyporus parvovarius ) strain, the samples of the little honeycomb hole mushroom ( Polyporus arcularius ), winter hole hole mushroom ( Polyporus brumalis ), and hole hole mushroom ( Polyporus tuberaster ) showed generally similar levels of antioxidant activity. , the antioxidant activity of Polyporus ulleungus was significantly lowered.

2-2. ABTS radical scavenging activity

Antioxidant activity was measured using an ABTS assay kit from Sigma-Aldrich. ABTS [2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] is a substance used to measure the activity of peroxidase or laccase enzymes and has high reducing power. It has a dark blue-green color and becomes transparent when it reacts with antioxidants (Miller & Rice-Evans, 1997).

The lyophilized culture filtrate in Example 1-3 was extracted with 70% ethanol and used as an analysis sample. After reacting the sample and myoglobin dissolved at a concentration of 10 ⁴ mg/L according to the manufacturer's manual, Phosphate-Citrate buffer 150 μl of the melted ABTS (Sigma-Aldrich Co., USA) compound was mixed and reacted for 10 minutes in the dark, and the absorbance was measured at a wavelength of 405 nm using a Microplate reader (SpectraMax190, Molecular devices). As a positive control group, ascorbic acid at a concentration of 10 ⁴ mg/L was used. The activity was measured three times repeatedly, and the activity was calculated based on [Equation 2] below.

[Equation 2]

Referring to [Figure 4], the antioxidant activity confirmed by the ABTS assay showed a similar trend to that of the DPPH assay.

The antioxidant activity of the culture filtrate samples cultured in DY and MEB medium of Polyporus parvovarius strains was 93%, and this value was higher than that of Ascorbic acid treated as a positive control. It is an activity comparable to that of antioxidant activity.

Through the results of the DPPH assay and the ABTS assay, it was confirmed that the Polyporus parvovarius strain produced antioxidants on its own, and the culture medium of the Polyporus parvovarius strain was ascorbic It was confirmed that the acid (Ascorbic acid) has an excellent antioxidant activity similar to that of the antioxidant activity.

Example 3. Isolation and Purification of Antioxidant Active Ingredients

3-1. 70% methanol solvent fraction (PP3 fraction)

Column chromatography analysis to identify antioxidant active components in the culture filtrate of Polyporus parvovarius strain, which was analyzed to have the highest antioxidant activity through the DPPH assay and ABTS assay results of Example 2 was performed.

2L of Diaion HP-20 resin was added to about 16L of the culture medium cultured in the DY medium of the Polyporus parvovarius strain, which was shown to have the best antioxidant activity, and stirred and adsorbed for 3 hours. After filling the column with the adsorbed Diaion HP-20 resin, the non-adsorbed pass fraction was separated by sequential elution with 30%, 70%, and 100% methanol aqueous solution and acetone.

As a result of measuring the antioxidant activity of the fraction by the ABTS radical scavenging method of Example 2-2, the 70% methanol solvent fraction (PP3 fraction) showed high antioxidant activity.

3-2. Column Fractionation of Ethyl Acetate Distribution Extract

The 70% methanol solvent fraction showing the highest antioxidant activity was concentrated, distributed and extracted with ethyl acetate, and then the ethyl acetate layer was concentrated under reduced pressure to remove the solvent. The concentrated antioxidant fraction was dissolved in chloroform-MeOH (100:1, v/v) solution and subjected to silica gel column chromatography (eluent: chloroform-MeOH=100:1→0:100, v/v) was performed and divided into 6 fractions (PP3-1→PP3-6).

The ABTS radical scavenging activity of these 6 fractions was measured and summarized in [Fig. 5].

Referring to [FIG. 5], it was observed that the PP32 fraction, the PP33 fraction, and the PP34 fraction showed excellent antioxidant activity overall.

First, the PP32 fraction was subjected to Sephadex LH-20 column chromatography (eluent: methanol) to separate the purified PP32-1 (9.9 mg) compound.

The HPLC analysis conditions are as follows, and the analysis result graph is shown in [Figure 6].

[HPLC system] HITACHI EzchromElite, HITACHI, Japan

Pump L2130, Autosampler L2200

PDA Detector L2455

[Analysis conditions]

-Detector: UV detector DAD

- Column: TSK-gel 100V (4.6mm×250mm, 5㎛), TOSOH, Japan

-Injection vol: 10μL

- Mobile phase:

In addition, the fraction PP3-3 was subjected to Sephadex LH-20 column chromatography (eluent: methanol) to obtain a purified PP33-1 (8.5 mg) compound.

And, fraction PP3-4 was divided into PP34-1 (19.6 mg) and PP34-2 fractions by Sephadex LH-20 column chromatography (eluent: methanol) (see FIG. 7), and PP34-2 was analyzed by ODS MPLC was performed and divided into PP342-1 and PP342-2 (28.2 mg) (see FIG. 8).

Fraction PP342-1 was subjected to preparative HPLC (eluent: 70% methanol aqueous solution) to separate a PP3421-1 (3.1 mg) fraction and a PP3421-2 (4.2 mg) compound (see FIGS. 9 and 10).

Preparative HPLC conditions are as follows.

-Detector: UV detector DAD

- Column: Cosmosil, 5C18-MS-II, 10×150 mm, Nacalai tesque

- Solvent condition: 45% methanol aqueous solution

- Flow rate: 3 mL/min

Compounds PP32-1, PP33-1 and PP34-1 exhibited strong ABTS radical scavenging activity, and as a result of HPLC analysis, it was confirmed that these compounds are all the same compounds. The HPLC analysis results are summarized in [Figure 11].

In addition, ABTS radical scavenging activities for the four purified compounds (PP34-1, PP342-2, PP3421-1 and PP3421-2) are as follows.

[Table 5]

The separation and purification process described above is summarized and summarized in [Figure 12].

Example 4. Chemical Structure Analysis of Antioxidant Active Ingredients

4-1. Chemical structure of compound PP34-1

4-1-1. Measurement and interpretation of NMR spectrum of compound PP34-1

In order to identify the chemical structure of the compound PP34-1, after dissolving it in CD ₃ OD, measure and analyze 1-dimensional NMR such as ¹ H NMR and ¹³ C NMR and 2-dimensional NMR spectrum such as ¹ H- ¹ H COSY, HMQC, and HMBC did

(Measurement and interpretation of 1H NMR spectrum)

As a result of measuring ¹ H NMR spectrum by dissolving in CD ₃ OD, aldehyde proton at 9.68 ppm, 7.30 (1H, dd, J = 8.0, 1.5 Hz), 7.28 (1H, br. s), 6.90 (1H , d, J = 8.0 Hz) at ppm, three aromatic methine protons were observed. From this ¹ H NMR spectrum, the PP34-1 compound was estimated to be a phenolic compound composed of “1,2,4-trisubstituted benzene” and “aldehyde” groups (see FIG. 13).

(Measurement and interpretation of ¹³ C NMR spectrum)

As a result of measuring the ¹³ C NMR spectrum, one "aldehyde carbonyl carbon" at 193.0 ppm, two "oxygenated sp ² quaternary carbon" at 153.9 ppm and 147.2 ppm, one "sp2 quaternary carbon" at 130.8 ppm, 126.4 ppm, Three "sp2 methine carbon" were observed at 116.2 ppm and 115.3 ppm. ^{The 13} C NMR spectrum also showed that the PP34-1 compound was a “phenolic compound” having an “aldehyde” group (see FIG. 14).

(Measurement and interpretation of ^{1H- 1H} COZY spectrum)

In order to identify the chemical structure of the compound PP34-1, the ¹ H- ¹ H COZY spectrum, which can identify the correlation ( ³ J _HH ) between neighboring hydrogens, was measured and analyzed (FIG. 15). As a result of the analysis, one partial structure (indicated by a thick line) represented by [Formula 1] below was identified.

[Formula 1]

(Measurement and interpretation of HMQC and HMBC spectra)

The HMQC spectrum was measured and analyzed in order to investigate the hydrogen-carbon correlation ( ¹ J _CH ) of the compound PP34-1. As a result of the analysis, the correlation between hydrogen and carbon constituting this compound was identified.

In addition, in order to identify the chemical structure of the compound PP34-1, the HMBC spectrum was measured and analyzed to determine the 2-bond or 3-bond bonding relationship between hydrogen and carbon ( ² J _CH , ³ J _CH ).

As a result of the analysis, 193.0, 153.9, and 115.3 ppm of carbon from 7.30 ppm of "methine proton" and 193.0, 153.9, and 126.4 ppm of carbon from "methine proton" of 7.28 ppm ( carbon), a “long-range correlation” was observed from “methine proton” at 6.90 ppm to carbon at 147.2 and 130.8 ppm.

In addition, "long-range correlation" was observed from "aldehyde proton" at 9.68 ppm to carbon at 130.8, 126.4, and 115.3 ppm, and the substitution positions of the aldehyde groups were determined.

From the above results, the chemical structure of this compound was identified as "3,4-dihydroxybenzaldehyde" (FIG. 16). The attribution of each proton and carbon peak is shown in [Fig. 17] and [Fig. 18].

4-1-2. Measurement and interpretation of mass spectrum of compound PP34-1

From NMR measurement, compound PP34-1 was identified as "3,4-dihydroxybenzaldehyde". To confirm this, ESI-mass analysis was performed in positive and negative modes. As a result of measurement in positive mode, [M+H] ⁺ peak was observed at m/z 139.10, and as a result of measurement in negative mode, [MH] ^- peak was observed at m/z 137.10, confirming the molecular weight as 138 (FIG. 19). reference).

This exactly matches the molecular weight of 138 of "3,4-dihydroxybenzaldehyde".

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Agricultural Biotechnology Research Institute KACC83044 20210408

Claims (10)

A composition for antioxidant comprising a cultured fraction of the small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) as an active ingredient. According to claim 1,
The culture fraction is a culture solution cultured in one or more culture media consisting of ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose) and SDA (Sabouraud dextrose) media Antioxidant composition, characterized in that the fraction. According to claim 1,
The culture fraction is 70% produced by sequentially eluting the ethanol extract of the culture medium of Polyporus parvovarius NIBRFGC000500557 strain (Accession No.: KACC 83044BP) with 30% methanol, 70% methanol, 100% methanol and acetone Antioxidant composition, characterized in that the methanol solvent fraction. According to claim 1,
The culture fraction is 70% produced by sequentially eluting the ethanol extract of the culture medium of Polyporus parvovarius NIBRFGC000500557 strain (Accession No.: KACC 83044BP) with 30% methanol, 70% methanol, 100% methanol and acetone Antioxidant composition, characterized in that the culture fraction obtained by distributing and extracting the methanol solvent fraction with ethyl acetate. According to claim 1,
The cultured fraction is an antioxidant composition, characterized in that it contains 3,4-dihydroxybenzaldehyde as an active ingredient. According to claim 1,
The composition is an antioxidant composition, characterized in that the cosmetic composition. According to claim 1,
The composition is an antioxidant composition, characterized in that the food composition. The small yellow hole mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) is ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose) and SDA (Sabouraud dextrose) step of inoculating any medium consisting of the medium;
recovering the culture solution by culturing the inoculated strain at 20° C. to 30° C. for 7 to 60 days; and
Step of sequentially eluting the ethanol extract of the recovered culture medium with 30% methanol, 70% methanol, 100% methanol and acetone to obtain a 70% methanol solvent fraction; Method for producing an antioxidant composition comprising a. According to claim 8,
Distributing and extracting the obtained 70% methanol solvent fraction with ethyl acetate (ethyl acetate); Method for producing an antioxidant composition, characterized in that it further comprises. Small yellow mushroom ( Polyporus parvovarius ) NIBRFGC000500557 strain (accession number: KACC 83044BP) was ME (Malt extract), DY (Dextrose & yeast extract), MY (Malt extract & yeast extract), PD (Potato dextrose), and Inoculation into any one medium composed of SDA (Sabouraud dextrose);
recovering the culture solution by culturing the inoculated strain at 20° C. to 30° C. for 7 to 60 days;
Obtaining a 70% methanol solvent fraction by sequentially eluting the ethanol extract of the recovered culture medium with 30% methanol, 70% methanol, 100% methanol and acetone;
Distributing and extracting the obtained 70% methanol solvent fraction with ethyl acetate; and
Purifying 3,4-dihydroxybenzaldehyde from the distribution-extracted ethyl acetate fraction using column chromatography; Method for producing hydroxybenzaldehyde (3,4-dihydroxybenzaldehyde).

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