Method for preparing mushroom-derived highly functional DNA fragment mixture, and functional composition comprising DNA fragment mixture prepared thereby

Abstract

The present invention relates to a method for preparing a mushroom-derived highly functional DNA fragment mixture, and a functional composition comprising a DNA fragment mixture prepared thereby. Specifically, the present invention relates to preparing a DNA fragment mixture having a uniform size within a predetermined range by using a specific restriction enzyme in mushrooms. According to the present invention, it is possible to provide a functional composition which, by having the DNA fragment mixture as a main component thereof, is highly effective in filling a scaffold between tissue, improving the state of skin, regenerating tissue, healing wounds, moisturizing, promoting ECM synthesis, inhibiting cellular senescence, forming new blood vessels, reducing inflammation, relieving pain, treating dermatitis such as atopy, inhibiting hair loss, enabling hair growth, and promoting stem cell activity.

Description

Method for producing a mushroom-derived high-functionality DNA fragment mixture and a functional composition comprising the DNA fragment mixture produced thereby

[0001] The present invention relates to a method for producing a highly functional DNA fragment mixture derived from mushrooms and a functional composition comprising the DNA fragment mixture produced thereby.

[0002] Specifically, the present invention relates to the production of a mixture of DNA fragments having a uniform size within a certain range using a specific restriction enzyme in mushrooms. In addition, the present invention relates to a functional composition using the DNA fragment mixture produced thus as a main component, which has excellent effects of filling inter-tissue support, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix (ECM) synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, anti-inflammation, inhibiting hair loss, promoting hair growth, or promoting stem cell differentiation.

[0003] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0189897, filed December 22, 2023, the entire contents of which are incorporated herein by reference.

[0004] Generally, mushrooms can be divided into two parts: the mycelium and the fruiting body. Functionally, the mycelium is the vegetative organ, comparable to roots, stems, or leaves, while the fruiting body is the reproductive organ, comparable to flowers or fruits.

[0005] Most mushrooms belong to the order Agaricales of the Basidiomycetes, and as mushrooms mature, the hymenium is exposed to the air, making it ideal for spore dispersal. Taxonomically, most mushrooms belong to the Basidiomycetes, but some belong to the Ascomycetes. The main taxonomic groups of mushrooms are Agaricales, Aphyllophorales, Gasteromycetes, Wood Ears, and Ascomycetes. Currently, mushrooms are used in various ways, including for food and medicine. Mushrooms are high in protein and low in calories, and are rich in dietary fiber, vitamins, iron, zinc, and other minerals. Mushrooms contain a whopping 40% of dietary fiber, which helps excrete harmful substances, waste, and carcinogens from the intestines and purify the blood. Mushroom cell walls are composed of a substance called beta-glucan. Glucan is a fluorinated polysaccharide recognized for its anti-cancer and immune-boosting properties. The beta-glucan found in mushrooms is known to be highly effective. Furthermore, it helps improve blood circulation and contains bioactive substances, contributing significantly to the promotion and maintenance of health.

[0006] Among them, Tremella fuciformis, a representative edible and medicinal mushroom, belongs to the Tremellaceae family, Tremellales order, Heterobasidiomycetes class. It is a wood-rotting mushroom that grows on old trees, dry branches, and stems of deciduous trees from spring to fall. It is an aerobic basidiomycete distributed all over the world, including Korea, Japan, China, Europe, and America. This mushroom fungus is divided into mycelia and fruiting bodies, and exists in the form of dikaryotic hyphae in which the mycelia that germinate from basidiospores become vegetative organs and form monokaryotic hyphae and fruiting bodies. The mushroom is 6 to 12 cm in diameter and 3 to 6 cm in height, and it splits into the shape of petals, but shrinks when dry and unfolds again when wet.

[0007] Today, white oyster mushrooms are used for various purposes, including food and medicine. They are effective in treating chronic bronchitis, pulmonary and heart disease, postpartum care, constipation, and asthma. They are also known to possess strong anticancer properties, prevent arteriosclerosis, and provide nutritional support. In particular, white oyster mushrooms are known to have high polysaccharide content, which enhances the body's immune system, delays aging, suppresses tumors, aids in weight loss, and suppresses cholesterol levels.

[0008] Meanwhile, active research is being conducted on cosmetics and pharmaceutical compositions using mushroom cultures and extracts. However, there is currently no research on producing a mixture of DNA fragments from mushrooms or applying them to fillers, cosmetics, foods, and pharmaceutical compositions. Domestic Patent No. 10-1516384 discloses a method for producing a white oyster mushroom extract. However, this prior art differs from the present invention, which produces a mixture of DNA fragments from white oyster mushrooms by extracting them with water or an organic solvent at temperatures above 100°C.

[0009] Polynucleotides, which are included in the DNA fragment mixture, are biopolymers composed of phosphate, sugar, and base. They have viscoelastic properties and exhibit tissue repair effects when injected subcutaneously. In particular, polynucleotides form a complex with the adenosine A2 receptor, a skin regeneration signaling molecule, to promote the secretion of various growth factors and stimulate the secretion of extracellular matrix (ECM) by fibroblasts that make up skin tissue. This makes them the main component of a new concept of tissue repair biomaterial that can induce skin tissue regeneration, rather than a simple skin filler.

[0010] Domestic Patent No. 10-2402360 discloses a method for producing polynucleotides, but the prior art differs from the present invention in that it produces polynucleotides derived from fish. Furthermore, the prior art differs from the present invention in that it uses a salting-out method (high salt) and buffers to obtain low-molecular-weight polynucleotides of 5 kDa or less, and uses a specific restriction enzyme in mushrooms to obtain a mixture of highly functional DNA fragments having a uniform size within a certain range.

[0011] Polynucleotides, commonly used as core ingredients in cosmeceuticals and biocosmetics, are primarily derived from animal-derived raw materials derived from fish such as flounder, trout, and salmon. Mixtures of animal-derived DNA fragments pose a risk of contamination with viruses originating from animal tissues, and securing a consistent supply of raw materials presents challenges. Furthermore, the nature of aquaculture makes management cumbersome and expensive, and feed waste and aquatic organism excrement from aquaculture farms can accelerate pollution of the marine ecosystem.

[0012] Against this backdrop, research has been conducted to extract polynucleotides from various plants or to manufacture functional compositions containing them as active ingredients. Furthermore, active research is being conducted to manufacture polynucleotides from various non-fish sources, such as seaweed.

[0013] However, as mentioned above, no research has been conducted to date on extracting and manufacturing a mixture of DNA fragments from mushrooms, and no research has been conducted on manufacturing or confirming the efficacy of medical device fillers, cosmetics, food, and pharmaceutical compositions.

[0014] Accordingly, the present invention provides a method for producing a highly functional DNA fragment mixture having a uniform size within a certain range by utilizing a specific restriction enzyme in a fruiting body or mycelium of mushrooms, and provides a functional composition comprising the DNA fragment mixture obtained by the method for producing the same.

[0015] The problem to be solved by the present invention is to provide a method for producing a mixture of highly functional DNA fragments derived from mushrooms.

[0016] Another object of the present invention is to provide a functional composition comprising a mixture of DNA fragments prepared according to the above-described method.

[0017] In order to solve the above problem, the present invention provides a composition for filling interstitial scaffolds, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, preventing inflammation, inhibiting hair loss, promoting hair growth or promoting stem cell differentiation, comprising a mixture of DNA fragments separated and extracted from mushrooms and having a low molecular size of less than 1,000 bp or less than 37 kDa.

[0018] According to another embodiment of the present invention, a composition for filling interstitial scaffolds, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, preventing inflammation, inhibiting hair loss, promoting hair growth or promoting stem cell differentiation is provided, comprising a mixture of DNA fragments isolated and extracted from mushrooms and having a molecular size of 1,000 bp or more and less than 5,000 bp or 37 kDa or more and less than 185 kDa.

[0019] According to another embodiment of the present invention, a composition for filling interstitial scaffolds, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, preventing inflammation, inhibiting hair loss, promoting hair growth or promoting stem cell differentiation is provided, comprising a mixture of DNA fragments isolated and extracted from mushrooms and having a polymerized size of 5,000 bp or more or 185 kDa or more.

[0020] According to one embodiment, the mushrooms used in the present invention may be selected from medicinal mushrooms or edible mushrooms including white oyster mushrooms, mulberry mushrooms, shiitake mushrooms, oyster mushrooms, sang-hwang mushrooms, deer antler mushrooms, pine mushrooms, ling-i mushrooms, enoki mushrooms, truffles, reishi mushrooms, chaga mushrooms, cordyceps sinensis, chitin mushrooms, yellow oyster mushrooms, cloud mushrooms, oyster mushrooms, net mushrooms, horseshoe mushrooms, poria cocos, chanterelle mushrooms, oyster mushrooms, schisandra chinensis, schisandra chinensis, oyster mushrooms, oyster mushrooms, chanterelle ...

[0021] According to one embodiment, the DNA fragment in the composition of the present invention may be at least one selected from the group consisting of polydeoxyribonucleotides and polynucleotides.

[0022] In one embodiment, the tissue may be selected from the group consisting of skin, cartilage, muscle and ligament.

[0023] According to one embodiment, the mixture of DNA fragments having a low molecular size

[0024] (S1) A step of adding the mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours;

[0025] (S2) A step of centrifuging the lysate obtained in the step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant;

[0026] (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield;

[0027] (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Alu1, Bsn1, Cfo1 (Hha1), Mal1, Nla3, Mbo2, Rsa1, Hae3, and Fok1;

[0028] (S5) a step of inactivating the above restriction enzyme; and

[0029] (S6) It may be manufactured by a manufacturing method including a step of secondary fragmentation of a mixture of DNA fragments by sonication.

[0030] According to one embodiment, the mixture of DNA fragments having the above-described intermediate size is

[0031] (S1) A step of adding the mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours;

[0032] (S2) A step of centrifuging the lysate obtained in the step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant;

[0033] (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield;

[0034] (S4) A step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2;

[0035] (S5) a step of inactivating the above restriction enzyme; and

[0036] (S6) It may be manufactured by a manufacturing method including a step of secondary fragmentation of a mixture of DNA fragments by sonication.

[0037] According to one embodiment, the mixture of DNA fragments having the polymerized size

[0038] (S1) A step of adding the mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours;

[0039] (S2) A step of centrifuging the lysate obtained in the step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant;

[0040] (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield;

[0041] (S4) a step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2; and

[0042] (S5) It may be manufactured by a manufacturing method including a step of inactivating the above restriction enzyme.

[0043] According to one embodiment, the DNA fragment mixture of the present invention may be manufactured by a manufacturing method comprising shaking and culturing mycelia of mushrooms and then performing the steps below (S1).

[0044] According to one embodiment, the shaking culture is prepared by sterilizing a liquid medium containing 97 to 98 wt% of malt extract, 1 wt% of yeast, and 1 to 2 wt% of dry corn extract at 110 to 130°C for 10 to 20 minutes; and

[0045] It may include a step of adding mushroom mycelia to the liquid medium obtained in the above step and culturing them under conditions of 20 to 30°C and 150 to 200 rpm for about 48 to 96 hours.

[0046] According to one embodiment, the composition of the present invention may be a cosmetic composition.

[0047] According to one embodiment, the composition of the present invention may be a food composition.

[0048] According to one embodiment, the composition of the present invention may be a medical device filler composition.

[0049] Specific details of other implementation examples according to the present invention are included in the detailed description below.

[0050] According to the composition of the present invention, a mixture of mushroom-derived DNA fragments can be made uniformly sized within a certain range by selectively grinding them with specific restriction enzymes. Accordingly, a functional composition with excellent effects of inter-tissue support filling, improving skin condition, promoting tissue regeneration, wound healing, moisturizing, promoting extracellular matrix (ECM) synthesis, inhibiting cell aging, promoting neovascularization, alleviating pain, anti-inflammation, inhibiting hair loss, promoting hair growth, and promoting stem cell differentiation can be provided.

[0051] Figure 1 is a schematic diagram showing a method for producing a mushroom-derived high-functionality DNA fragment mixture according to the present invention.

[0052] Figure 2 shows the results of confirming the size of a mixture of multi-segmented DNA fragments derived from the fruiting body and mycelia of the white oyster mushroom using gel electrophoresis.

[0053] Figure 3 shows the results of confirming the size of a mixture of high-functionality, low-molecular-weight DNA fragments derived from the fruiting body of the white oyster mushroom using gel electrophoresis.

[0054] Figure 4 shows the results of confirming the size of a mixture of high-functionality middle-molecular DNA fragments derived from the fruiting body of the white oyster mushroom using gel electrophoresis.

[0055] Figure 5 shows the results of confirming the size of a mixture of high-functionality polymer DNA fragments derived from the fruiting body of the white oyster mushroom using gel electrophoresis.

[0056] Figure 6 shows the results of confirming the size of a mixture of high-functionality low-molecular-weight DNA fragments derived from white oyster mushroom mycelia using gel electrophoresis.

[0057] Figure 7 shows the results of confirming the size of a mixture of high-functionality middle-molecular DNA fragments derived from white oyster mushroom mycelia using gel electrophoresis.

[0058] Figure 8 shows the results of confirming the size of a mixture of high-functionality polymer DNA fragments derived from white oyster mushroom mycelia using gel electrophoresis.

[0059] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced herein are provided to ensure that the disclosure is thorough and complete and to sufficiently convey the spirit of the present invention to those skilled in the art.

[0060] The term "fruiting body" of the present invention refers to a cellular structure for sexual reproduction of fungi.

[0061] The term "mycelium" of the present invention means hyphae (singular: hypha, plural: hyphae) that grow in a densely intertwined state.

[0062] The term "DNA fragment complex" of the present invention means one or more groups selected from polydeoxyribonucleotides and polynucleotides.

[0063] The "DNA fragment" used in the present invention may have a size of 20 bp to 300,000 bp. Specifically, for example, the molecular weight may be 1 to 10,000 kDa, for example, 1 to 37 kDa, 37 to 185 kDa, or 185 kDa to 1,000 kDa.

[0064] The term "low-molecular-weight DNA fragment mixture" of the present invention means a DNA fragment mixture having a size of less than 1,000 bp or 37 kDa. In order to prepare a mushroom-derived high-functional low-molecular-weight DNA fragment mixture, the present invention may use one or more of restriction enzymes Alu1, Bsn1, Cfo1 (Hha1), Mal1, Nla3, Mbo2, Rsa1, Hae3, and Fok1, for example, Alu1. Specifically, for example, the method for preparing a low-molecular-weight DNA fragment mixture comprises the steps of: (S1) adding mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract, and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours; (S2) a step of centrifuging the lysate obtained in the step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) a step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield; (S4) a step of treating the solution obtained in the step (S3) with one or more restriction enzymes selected from the group consisting of Alu1, Bsn1, Cfo1 (Hha1), Mal1, Nla3, Mbo2, Rsa1, Hae3, and Fok1 at 5 U / μL to 15 U / μL under conditions of 37°C for 5 to 7 hours to primarily fragment the mixture of DNA fragments; (S5) a step of inactivating the restriction enzyme; And (S6) a step of secondary fragmentation by sonicating the first fragmented DNA fragment mixture under conditions of 20 to 40 kHz and 30 to 50% Amp for 10 to 15 minutes.

[0065] The term "middle-molecule DNA fragment mixture" of the present invention means a DNA fragment mixture having a size of 1,000 bp or more and less than 5,000 bp or 37 kDa or more and less than 185 kDa. In order to prepare a highly functional middle-molecule DNA fragment mixture derived from mushrooms, the present invention may use at least one of restriction enzymes Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1 and Avr2, for example, Kpn1. Specifically, for example, the method for preparing a middle-molecule DNA fragment mixture comprises the steps of: (S1) adding mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours; (S2) a step of centrifuging the lysate obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) a step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and placing it in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield; (S4) a step of first fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2 at 5 U / μL to 15 U / μL of the restriction enzyme under conditions of 37°C for 5 to 7 hours; (S5) a step of inactivating the restriction enzyme; And (S6) a step of secondary fragmentation by sonicating the first fragmented DNA fragment mixture under conditions of 20 to 40 kHz and 20 to 30% Amp for 10 to 15 minutes.

[0066] The term "polymer DNA fragment mixture" of the present invention means a DNA fragment mixture having a size of 5,000 bp or more or 185 kDa or more. In order to prepare a mushroom-derived high-functional polymer DNA fragment mixture, the present invention may use one or more of restriction enzymes Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2, for example, Kpn1. Specifically, for example, the method for preparing a polymer DNA fragment mixture comprises the steps of: (S1) adding mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours; (S2) centrifuging the lysate obtained in the step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) a step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing the supernatant in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield; (S4) a step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with 5 U / μL to 15 U / μL of one or more restriction enzymes selected from the group consisting of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2 under conditions of 37°C for 5 to 7 hours; and (S5) a step of inactivating the restriction enzyme.

[0067] Because higher molecular weights slow degradation and persist longer in the body, molecular weight can be adjusted depending on the intended use. Furthermore, when used as a filler for medical devices, PDRN requires different molecular weights depending on the application site, necessitating the use of an appropriate molecular weight. The present invention facilitates the adjustment of molecular weight according to the intended use.

[0068] The term "multi-segmented DNA fragment mixture" of the present invention means that the size of the DNA fragment mixture is mixed in the range of 1,000 bp to 10,000 bp.

[0069] The term "functional composition" of the present invention may be a composition having the effects of, but is not limited to, inter-tissue support filling, tissue regeneration, wound healing, moisturizing, promoting extracellular matrix (ECM) synthesis, inhibiting cell aging, angiogenesis, pain relief, anti-inflammation, inhibiting hair loss, promoting hair growth, and promoting stem cell differentiation. For example, the tissue may be selected from the group consisting of skin, cartilage, muscle, and ligament.

[0070] The cosmetic composition according to the present invention can be manufactured into any formulation according to a conventional method. For example, the cosmetic composition can be manufactured into a formulation selected from the group consisting of solutions, external ointments, creams, foams, nourishing toners, emulsifying toners, packs, emulsions, makeup bases, foundations, essences, soaps, liquid cleansers, bath products, sunscreens, sun oils, suspensions, gels, lotions, powders, surfactant-containing cleansers, patches, and sprays, but is not limited thereto.

[0071] The pharmaceutical composition according to the present invention can be prepared in any dosage form using conventional methods. For example, it can be formulated and used in the form of oral dosage forms such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, and sterile injectable solutions, but is not limited thereto.

[0072] The food composition according to the present invention can be manufactured into one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, effervescent tablets, and beverages, but is not limited thereto.

[0073] As described above, the present invention provides a functional composition comprising a mushroom-derived DNA fragment mixture as an active ingredient, which has excellent nucleic acid stability and excellent effects of filling inter-tissue support, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix (ECM) synthesis, inhibiting cell aging, forming new blood vessels, alleviating inflammation, relieving pain, anti-inflammation, inhibiting hair loss, promoting hair growth, and promoting stem cell differentiation. The DNA fragment mixture manufactured according to the present invention has excellent binding affinity for the A2 receptor and thus has an excellent cyclic AMP signaling activation effect.

[0074] According to one embodiment, the composition of the present invention may include cationic polysaccharides and cationic amino acids to enhance stability with a mixture of isolated and purified mushroom-derived DNA fragments. By including polysaccharides and cationic amino acids, a composition with excellent nucleic acid stability can be provided.

[0075] According to one embodiment, the present invention enables efficient separation and purification of a mixture of DNA fragments from mushroom mycelia or fruiting bodies by using a dissolution buffer containing a plum extract or the like. For example, a method for preparing a plum extract may include a step of drying 500 g of plum pulp at 65°C for 30 to 60 minutes and grinding the pulp with a grinder to a mesh size of 80 to 320 to prepare a ground product. Thereafter, water of 5 times the volume is added to the ground product, hot water extraction and filtration are performed at 90°C, and the extract is concentrated under reduced pressure to obtain an extract. This extract can be dissolved in purified water and used as the plum extract of the present invention.

[0076] The present invention may include the steps of: placing mycelia or fruiting bodies of mushrooms in a lysis buffer containing 200 to 300 μg / mL of plum extract, and performing lysis and homogenization at 50 to 60°C for 1 to 3 hours to separate and purify a high-purity DNA fragment mixture from mushrooms; centrifuging the lysate at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; and adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to degrade RNA and increase DNA yield, in order to isolate and purify a mixture of highly pure DNA fragments from mushrooms. At this time, the content of the plum extract may be 100 to 500, for example, 150 to 300 μg / mL, per 1 L of the lysis buffer.

[0077] According to one embodiment, the shaking cultivation method of mushroom mycelia according to the present invention can increase the yield of mycelia by using a culture medium containing dried corn extract or the like. Specifically, the shaking cultivation method of the present invention may include (1) a step of preparing a liquid medium containing 97 to 98 wt% of malt extract, 1 wt% of yeast, and 1 to 2 wt% of dried corn extract for increasing the growth rate of mycelia by sterilizing at 110 to 130°C for 10 to 20 minutes; (2) a step of adding mycelia of a white oyster mushroom to the liquid medium prepared in (1) and culturing at 20 to 30°C and 150 to 200 rpm for 48 to 96 hours, for example, about 72 hours.

[0078] According to one embodiment, the mushrooms used in the present invention are Tremella fuciformis, Armillaria mellea, Lentinula edodes, Pleurotus ostreatus, Phellinus linteus, Hericium erinaceus, Tricholoma matsutake, Sarcodon imbricatus, Flammulina filiformis, Rhizopong (Rhizopogon rubescens), Ganoderma lucidum, Chaga mushroom (Inonotus obliquus), Cordyceps militaris, Schizophore mushroom (Schizophyllum commune), Pleurotus citrinopileatus, Trametes versicolor, It may include medicinal mushrooms or edible mushrooms including Fistulina hepatica, Boletus edulis, Fomes fomentarius, Wolfiporia extensa, Ganoderma applanatum, Cryptoporus volvatus, Pseudohydnum gelatinosum, Auricularia auricula-judae, Ramaria botrytis, Umbilicaria esculenta, Amanita hemibapha, Lactarius piperatus, Sparassis crispa, Cantharellus cibarius, or a combination of one or more of these. Specifically, for example, the mushrooms of the present invention may include white oyster mushrooms, mulberry mushrooms, shiitake mushrooms, oyster mushrooms, or a combination of one or more thereof.

[0079] According to one embodiment, the present invention can provide a medical device filler, cosmetic, food composition or pharmaceutical composition comprising the composition as described above.

[0080] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0081]

[0082] Comparative Example 1-1: Preparation of a mixture of multi-segmented DNA fragments derived from the fruiting body of the white oyster mushroom.

[0083] 1) The fruiting bodies of the white oyster mushroom were frozen using liquid nitrogen and then ground in a grinder. After that, 5 mL of lysis buffer was added per 1 g of the white oyster mushroom fruiting bodies and the mixture was homogenized vigorously to obtain a lysate. The composition of the lysis buffer was 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 2% PVPP (Polyvinylpolypyrrolidone), 0.5% IGEPAL-CA630, 0.5% Sodium deoxycholate, and 1% SDS per 1 L, and the dissolution and homogenization were performed for 2 hours under conditions of 56℃.

[0084] 2) The above lysate was centrifuged, and a mixture of DNA fragments was extracted from the supernatant. The supernatant was obtained after centrifugation at 11,000 rpm for 20 minutes.

[0085] 3) The supernatant was sonicated for 10 cycles (pulse; 30 / 30 sec) under conditions of 20 kHz and 25% Amp to fragment the DNA fragment mixture.

[0086] 4) A precipitate was obtained from the supernatant containing the above-mentioned mixture of fragmented DNA using alcohol. At this time, alcohol was added in an amount twice the volume of the supernatant, and the precipitate was allowed to settle overnight at 4°C. The precipitate was then centrifuged at approximately 7,000 rpm for 1 hour using a centrifuge.

[0087] 5) The purified product was dried to obtain a DNA fragment mixture. At this time, the precipitate was dried under conditions of 25-30℃ for 1-3 hours to obtain a DNA fragment mixture. The concentration of the DNA fragment mixture thus obtained was confirmed through DNA quantification, and the decomposition was confirmed through DNA electrophoresis. The DNA fragment mixture prepared from Comparative Example 1-1 contained randomly multi-fragmented DNA of 1,000-10,000 bp. For the experiment, it was made into a 1 mg / mL stock and used at a final concentration of 10 μg / mL, etc.

[0088]

[0089] Comparative Example 1-2: Preparation of a mixture of multi-segmented DNA fragments derived from white oyster mushroom mycelia.

[0090] 1) The mycelia of the white oyster mushroom were cultured in a culture medium by shaking, and the mycelia of the white oyster mushroom were obtained using a centrifuge. At this time, 97 to 98 wt% of malt extract and 1 wt% of yeast were added based on 1 L of purified water to prepare a liquid medium. The solution was uniformly mixed using a magnetic bar, sterilized at 120°C for 20 minutes, and then used. It was cultured for about 24 hours under conditions of 25°C and 180 rpm until the OD600 nm value became 1.0. Thereafter, the mycelia were obtained by centrifugation at 4°C and 15,000 rpm. Thereafter, 5 mL of lysis buffer was added per 1 g of the white oyster mushroom mycelia, and the lysate was obtained using a homogenizer. At this time, the composition of the dissolution buffer was 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 2% PVPP (Polyvinylpolypyrrolidone), 0.5% IGEPAL-CA630, 0.5% Sodium deoxycholate, and 1% SDS per 1 L, and dissolution and homogenization were performed for 2 hours under conditions of 56°C.

[0091] 2) The above lysate was centrifuged, and a mixture of DNA fragments was extracted from the supernatant. The supernatant was obtained after centrifugation at 11,000 rpm for 20 minutes.

[0092] 3) The supernatant was sonicated for 10 cycles (pulse; 30 / 30 sec) under 20 kHz 25% AMP conditions to fragment the DNA fragment mixture.

[0093] 4) A precipitate was obtained using alcohol from the supernatant containing the above-mentioned mixture of fragmented DNA. At this time, alcohol was added in an amount twice the volume of the supernatant, and the mixture was precipitated overnight at 4°C, and then centrifuged at approximately 7,000 rpm for 1 hour using a centrifuge to obtain a precipitate. The DNA fragment mixture prepared from Comparative Example 1-2 contained randomly fragmented DNA of 1,000 to 10,000 bp. The concentration of the DNA fragment mixture thus obtained was confirmed through DNA quantification, and the decomposition was confirmed through DNA electrophoresis. For the experiment, a 1 mg / mL stock was prepared and used at a final concentration of 10 μg / mL, etc.

[0094]

[0095] Comparative Example 2-1: Preparation of a mixture of multi-segmented DNA fragments derived from the fruiting body of the mulberry mushroom.

[0096] In the manufacturing method of Comparative Example 1-1 above, the fruiting body of a mulberry mushroom was used instead of a white oyster mushroom, and the remaining processes were manufactured using the same method.

[0097]

[0098] Comparative Example 2-2: Preparation of a mixture of multi-segmented DNA fragments derived from mulberry mushroom mycelia.

[0099] In the manufacturing method of Comparative Example 1-2 above, mulberry mushroom mycelia were used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0100]

[0101] Comparative Example 3-1: Preparation of a mixture of multi-segmented DNA fragments derived from the fruiting body of shiitake mushrooms.

[0102] In the manufacturing method of Comparative Example 1-1 above, the fruiting body of shiitake mushrooms was used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0103]

[0104] Comparative Example 3-2: Preparation of a mixture of multi-segmented DNA fragments derived from shiitake mushroom mycelia.

[0105] In the manufacturing method of Comparative Example 1-2 above, shiitake mushroom mycelia were used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0106]

[0107] Comparative Example 4-1: Preparation of a mixture of multi-segmented DNA fragments derived from the fruiting body of oyster mushrooms.

[0108] In the manufacturing method of Comparative Example 1-1 above, the fruiting body of oyster mushrooms was used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0109]

[0110] Comparative Example 4-2: Preparation of a mixture of multi-segmented DNA fragments derived from oyster mushroom mycelia.

[0111] In the manufacturing method of Comparative Example 1-2 above, oyster mushroom mycelia were used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0112]

[0113] Comparative Example 5-1: Preparation of a multi-segmented DNA fragment mixture derived from a mushroom fruiting body mixture.

[0114] In the manufacturing method of Comparative Example 1-1, instead of the white oyster mushroom, the mushroom fruiting bodies used in Comparative Examples 1 to 4 of the present invention were all mixed in the same ratio and used. That is, the fruiting bodies of the white oyster mushroom, mulberry mushroom, shiitake mushroom, and oyster mushroom were mixed in a weight ratio of 1:1:1:1 and used, and the remaining process was manufactured in the same manner as Comparative Example 1-1.

[0115]

[0116] Comparative Example 5-2: Preparation of a multi-segmented DNA fragment mixture derived from a mushroom mycelia mixture

[0117] In the manufacturing method of Comparative Example 1-2, instead of the white oyster mushroom, the mushroom mycelia used in Comparative Examples 1 to 4 of the present invention were all mixed in the same ratio and used. That is, the fruiting bodies of the white oyster mushroom, mulberry mushroom, shiitake mushroom, and oyster mushroom were mixed in a weight ratio of 1:1:1:1 and used, and the remaining process was manufactured in the same manner as Comparative Example 1-2.

[0118]

[0119] Example 1-1: Preparation of a mixture of DNA fragments of different sizes derived from the fruiting bodies of the white oyster mushroom.

[0120] 1) The fruiting bodies of the white oyster mushroom were frozen using liquid nitrogen and then ground in a grinder. After that, 5 mL of lysis buffer was added per 1 g of the white oyster mushroom fruiting bodies and the mixture was homogenized vigorously to obtain a lysate. The composition of the lysis buffer was 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 2% PVPP (Polyvinylpolypyrrolidone), 0.5% IGEPAL-CA630, 0.5% Sodium deoxycholate, 1% SDS per 1 L, and 250 μg / mL plum extract. Dissolution and homogenization were performed for 2 hours under conditions of 56℃. Plum extract was prepared by drying 500 g of plum pulp at 65°C for 60 minutes and grinding it to a particle size of 200 mseh using a grinder. Five times the volume of water was added to the extracted sample, and hot water extraction and filtration were performed at 90°C. The extract was concentrated under reduced pressure to obtain an extract. The extract was dissolved in purified water and used as a plum extract.

[0121] 2) The above lysate was centrifuged, and a mixture of DNA fragments was extracted from the supernatant. The supernatant was obtained after centrifugation at 11,000 rpm for 20 minutes.

[0122] 3) 5 μL of RNase A at a concentration of 10 mg / mL was added to the supernatant, and then placed in an incubator at 37°C for 20 to 30 minutes to degrade RNA and increase DNA yield.

[0123] 4-1) To extract a mixture of low-molecular-weight DNA fragments, the solution obtained in step 3 was treated with a specific restriction enzyme, and the mixture of DNA fragments was fragmented. At this time, Alu1 was used as the restriction enzyme, and 10 U / μL of the restriction enzyme was treated for 6 hours under conditions of 37°C. Thereafter, the restriction enzyme was inactivated for 1 hour under conditions of 65°C. The mixture of DNA fragments was fragmented a second time through 10 cycles (pulse; 30 / 30 sec) of ultrasonication under conditions of 30 kHz and 40% Amp. The specific restriction enzyme for preparing a mixture of low-molecular-weight DNA fragments derived from enoki mushrooms may include one or more of Alu1, Bsn1, Cfo1 (Hha1), Mal1, Nla3, Mbo2, Rsa1, Hae3, and Fok1, but is not limited thereto.

[0124] 4-2) To extract a mixture of middle-molecule DNA fragments, the solution obtained in step 3 was treated with a specific restriction enzyme, and the DNA fragment mixture was fragmented. At this time, Kpn1 was used as the restriction enzyme, and 10 U / μL of the restriction enzyme was treated for 6 hours under conditions of 37°C. After that, the restriction enzyme was inactivated for 1 hour under conditions of 65°C. Afterwards, the DNA fragment mixture was fragmented a second time through 10 cycles (pulse; 30 / 30 sec) of ultrasonication under conditions of 30 kHz and 25% Amp. The specific restriction enzyme for the production of a mixture of middle-molecule and high-molecule DNA fragments derived from the white oyster mushroom may include one or more of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2, but is not limited thereto.

[0125] 4-3) To extract a mixture of high-molecular-weight DNA fragments, the solution obtained in Step 3 was treated with a specific restriction enzyme, and the DNA fragment mixture was fragmented. At this time, Kpn1 was used as the restriction enzyme, and 10 U / μL of the restriction enzyme was treated for 6 hours under conditions of 37°C. Thereafter, the restriction enzyme was inactivated for 1 hour under conditions of 65°C.

[0126] 5) A precipitate was obtained from the supernatant containing the above-mentioned mixture of fragmented DNA using alcohol. At this time, alcohol was added in an amount twice the volume of the supernatant, and the precipitate was allowed to settle overnight at 4°C. The precipitate was then centrifuged at approximately 7,000 rpm for 1 hour using a centrifuge.

[0127] 8) The purified product was dried to obtain a DNA fragment mixture. At this time, the precipitate was dried under conditions of 25-30℃ for 1-3 hours to obtain a DNA fragment mixture. The concentration of the DNA fragment mixture thus obtained was confirmed through DNA quantification, and the decomposition was confirmed through DNA electrophoresis. For the experiment, a 1 mg / mL stock was prepared and used at a final concentration of 10 μg / mL, etc.

[0128]

[0129] Example 1-2: Preparation of a mixture of DNA fragments of different sizes derived from white oyster mushroom mycelia

[0130] 1) The mycelia of the white oyster mushroom were cultured in a culture medium by shaking, and the mycelia of the white oyster mushroom were obtained using a centrifuge. At this time, 97 to 98 wt% of malt extract and 1 wt% of yeast were added to 1 L of purified water, and 1 wt% of dried corn mash was added to increase the growth rate of the mycelia to prepare a liquid medium. The solution was uniformly mixed using a magnetic bar, sterilized at 120°C for 20 minutes, and then used. It was cultured for about 72 hours under conditions of 25°C and 180 rpm until the OD600 nm value became 1.0. Thereafter, the mycelia were obtained by centrifugation at 4°C and 15,000 rpm. After that, 5 mL of lysis buffer was added per 1 g of the white oyster mushroom mycelia and the lysate was obtained by grinding strongly through a homogenizer. At this time, the composition of the lysis buffer was 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 2% PVPP (Polyvinylpolypyrrolidone), 0.5% IGEPAL-CA630, 0.5% Sodium deoxycholate, 1% SDS, and 250 μg / mL plum extract per 1 L, and dissolution and homogenization were performed for 2 hours under 56 ℃ conditions. The plum extract was prepared by drying 500 g of plum flesh at 65 ℃ for 60 minutes and grinding it to 200 mesh size with a grinder and using it as an extraction sample. Five times the volume of water was added to the extracted sample, hot water extraction was performed at 90°C, filtration was performed, and the extract was concentrated under reduced pressure to obtain an extract. The extract was dissolved in purified water and used as a plum extract.

[0131] 2) The remaining processes were manufactured in the same manner as in Example 1-1.

[0132]

[0133] Example 2-1: Preparation of a mixture of DNA fragments of different sizes derived from the fruiting bodies of the mulberry mushroom.

[0134] In the manufacturing method of Example 1-1 above, the fruiting body of a mulberry mushroom was used instead of a white oyster mushroom, and the remaining processes were manufactured using the same method.

[0135]

[0136] Example 2-2: Preparation of a mixture of DNA fragments of different sizes derived from mulberry mushroom mycelia

[0137] In the manufacturing method of the above Example 1-2, mulberry mushroom mycelia were used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0138]

[0139] Example 3-1: Preparation of a mixture of DNA fragments of different sizes derived from the fruiting bodies of shiitake mushrooms.

[0140] In the manufacturing method of Example 1-1 above, the fruiting body of shiitake mushrooms was used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0141]

[0142] Example 3-2: Preparation of a mixture of DNA fragments of different sizes derived from shiitake mushroom mycelia

[0143] In the manufacturing method of the above Example 1-2, shiitake mushroom mycelia were used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0144]

[0145] Example 4-1: Preparation of a mixture of DNA fragments of different sizes derived from the fruiting bodies of oyster mushrooms.

[0146] In the manufacturing method of Example 1-1 above, the fruiting body of oyster mushrooms was used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0147]

[0148] Example 4-2: Preparation of a mixture of DNA fragments of different sizes derived from oyster mushroom mycelia

[0149] In the manufacturing method of the above Example 1-2, oyster mushroom mycelia were used instead of white oyster mushrooms, and the remaining processes were manufactured using the same method.

[0150]

[0151] Example 5-1: Preparation of a mixture of DNA fragments by size derived from a mixture of mushroom fruiting bodies.

[0152] In the manufacturing method of the above Example 1-1, instead of the white oyster mushroom, the mushroom fruiting bodies used in Examples 1 to 4 were all mixed in the same ratio and used. That is, the fruiting bodies of the white oyster mushroom, mulberry mushroom, shiitake mushroom, and oyster mushroom were mixed in a weight ratio of 1:1:1:1 and used, and the remaining process was manufactured in the same manner as in Example 1-1.

[0153]

[0154] Example 5-2: Preparation of a mixture of DNA fragments by size derived from a mushroom mycelia mixture

[0155] In the manufacturing method of the above Example 1-2, instead of the white oyster mushroom, the mushroom mycelia used in Examples 1 to 4 were all mixed in the same ratio and used. That is, the fruiting bodies of the white oyster mushroom, mulberry mushroom, shiitake mushroom, and oyster mushroom were mixed in a weight ratio of 1:1:1:1 and used, and the remaining process was manufactured in the same manner as in Example 1-2.

[0156]

[0157] The low-molecular DNA fragment mixture prepared in the above example was denoted as A (Process 4-1), the medium-molecular DNA fragment mixture was denoted as B (Process 4-2), and the high-molecular DNA fragment mixture was denoted as C (Process 4-3).

[0158]

[0159] Experimental Example 1: Quality Control of DNA Fragment Mixtures

[0160] 1) 1 mL of purified water was added to the DNA fragment mixtures prepared from Comparative Examples 1 to 5 and Examples 1 to 5, and then the purity was measured and the concentration quantified using Nanodrop. 1 μg of the quantified product was used as a sample and electrophoresed on a 1% agarose gel to confirm the range and size of the DNA fragment mixture. Pure double-stranded DNA has an OD 260 / OD 280 of 1.8, and when contaminated with protein, the OD 260 / OD 280 value is lower than 1.8, and when contaminated with RNA, the OD 260 / OD 280 value is greater than 1.8.

[0161] 2) The results of confirming the purity of the DNA fragment mixtures manufactured in Comparative Examples 1, 2, Example 1, and Example 2 are as shown in Table 1 below. As shown in Table 1, it can be confirmed that the DNA fragment mixtures manufactured in Examples 1 to 5 had lower protein and impurity contents and were extracted and manufactured as higher-purity DNA fragment mixtures compared to Comparative Examples 1 to 5. In particular, it was confirmed that the DNA fragment mixture manufactured in Example 1 had the best purity with a purity close to 1.8.

[0162] 3) In addition, the results of confirming the size of the DNA fragment mixture manufactured in Comparative Example 1 are as shown in Figure 2 below. As shown in Figure 2, it was confirmed that the size of the multi-segmented DNA fragment mixture manufactured in Comparative Example 1 was randomly mixed in the range of 1,000 to 10,000 bp.

[0163] 4) On the other hand, it was confirmed that the low-molecular DNA fragment mixture manufactured in Example 1 was fragmented into uniform sizes within a certain range, with sizes of 1,000 bp or less, the medium-molecular DNA fragment mixture between 1,000 and 5,000 bp, and the high-molecular DNA fragment mixture being fragmented into sizes of 5,000 bp or more.

[0164] In all the tables below, the low-molecular-weight DNA fragment mixture manufactured in the present invention is indicated as A, the medium-molecular-weight DNA fragment mixture as B, and the high-molecular-weight DNA fragment mixture as C.

[0165]

[0166]

[0167] Experimental Example 2: Evaluation of in vivo degradation rate and volume change

[0168] The present inventors investigated whether the mushroom-derived DNA fragment mixture of the present invention exhibits sustained performance in living tissue.

[0169] 1) The experiment was conducted using 20 female SKH1 hairless mice.

[0170] 2) 100 μg / mL of the DNA fragment mixtures prepared in Comparative Examples 1 to 5 and Examples 1 to 5 were injected intradermally at 0.5 ml per animal at 3 sites, and 5 animals were euthanized at 4, 8, and 16 weeks after injection. An autopsy was performed, the injection site was removed, the size of the injection site was measured, and the volume change was analyzed immediately after injection and after 4, 8, and 16 weeks.

[0171] 3) The experimental results are shown in Table 2 below. Among them, the group treated with the polymer DNA fragment mixture manufactured in Examples 1 to 5 showed the smallest change in volume and maintained the initial injection volume for a long period of time. In particular, the group treated with the polymer DNA fragment mixture manufactured in Example 1 maintained the initial injection volume for the longest period of time, confirming that the inter-tissue support filling effect was the most excellent.

[0172]

[0173]

[0174] Experimental Example 3: Adenosine A2 Receptor Binding Affinity Analysis

[0175] 1) Protein lysates were obtained from 293T cells overexpressing adenosine A2 receptor protein.

[0176] 2) The DNA fragment mixtures prepared in Comparative Examples 1 to 5 and Examples 1 to 5 were labeled using the Pierce Biotin 3' End DNA labeling kit from ThermoFisher Scientific.

[0177] 3) EMSA assays were performed using the Thermo Scientific LightShift Chemiluminescent EMSA kit. Details are as follows.

[0178] 4) The DNA fragment mixture prepared in Comparative Examples 1 to 5 and Examples 1 to 5 was added to 200 μL of a solution rich in A2 receptor protein dissolved from cells to induce a binding reaction at 100 μg / mL.

[0179] 5) After electrophoresis using a polyacrylamide gel, detection is performed on a membrane that has undergone transfer, crosslinking, and blocking processes. At this time, 200 μL of HRP-conjugated solution that binds to biotin is treated, and then luminescence is emitted through a substrate such as Luminol.

[0180] 6) The size of the emitted band was then quantified using the ImageJ program (National Institutes of Health, NIH) and shown in Table 3.

[0181] 7) As a result of the experiment, the binding affinity of the low-molecular-weight and medium-molecular-weight DNA fragment mixtures prepared in Examples 1 to 5 to the A2 receptor increased compared to the DNA fragment mixtures prepared in Comparative Examples 1 to 5, and among them, the low-molecular-weight DNA fragment mixture prepared in Example 1 showed the highest binding affinity. The following experimental example was performed using only Comparative Example 1 to compare it with the DNA fragment mixture prepared in Example 1, which had the highest A2 receptor binding affinity.

[0182]

[0183]

[0184] Experimental Example 4: Analysis of collagen and extracellular matrix (ECM) gene expression

[0185] 1) A 100pi dish containing 70-80% human dermal fibroblasts (HDFs) was cultured with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and cultured for an additional 6 hours.

[0186] 2) Total RNA was extracted and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as a template.

[0187] 3) qRT-PCR was performed using primers for COL1A1, COL3A1, MMP1, and Elastase1 using the generated cDNA.

[0188] 4) As shown in Table 4, human dermal fibroblasts (HDFs) showed increased collagen synthesis enzyme (COL1A1, COL3Al) mRNA expression in the group treated with the mixture of low-molecular and medium-molecular DNA fragments prepared in Example 1 compared to Comparative Examples 1 and 2, and among them, the group treated with the mixture of low-molecular DNA fragments prepared in Example 1 showed the highest COL1A1 and COL3Al mRNA expression.

[0189] 5) Also, as shown in Table 4, compared to Comparative Example 1, the expression of collagenase (MMP1) and elastinase (Elastase1) mRNA decreased in the group treated with the mixture of low-molecular-weight and medium-molecular-weight DNA fragments prepared in Example 1. Among them, the expression of MMP1 mRNA and Elastase mRNA decreased the most in the group treated with the mixture of low-molecular-weight DNA fragments prepared in Example 1-1.

[0190]

[0191]

[0192] Experimental Example 5: In vitro wound regeneration effect analysis

[0193] 1) A physical scratch was made on the bottom of a 100pi dish in which HaCaT was 100% cultured, and the medium was replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1. The wound area was measured under a microscope after 12 hours, 24 hours, and 48 hours.

[0194] 2) As shown in Table 5, HaCaT cells showed higher wound regeneration efficacy in the group treated with the mixture of low-molecular and medium-molecular DNA fragments prepared in Example 1 compared to Comparative Example 1, and among them, the group treated with the mixture of low-molecular DNA fragments prepared in Example 1-1 showed the highest wound regeneration efficacy.

[0195]

[0196]

[0197] Experimental Example 6: Analysis of Moisturizing-Related Gene Expression

[0198] 1) The medium was replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and further cultured for 6 hours.

[0199] 2) Total RNA was extracted and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as a template.

[0200] 3) qRT-PCR was performed using primers for HAS1, HAS2, and HAS3 using the generated cDNA.

[0201] 4) As shown in Table 6, in HaCaT cells, mRNA expression of the HAS family (HAS1, HAS2, HAS3), an enzyme that synthesizes hyaluronic acid, increased in the group treated with the mixture of low-molecular and medium-molecular DNA fragments prepared in Example 1 compared to Comparative Example 1.

[0202]

[0203]

[0204] Experimental Example 7: Analysis of the efficacy of alleviating inflammatory and atopic cytokines.

[0205] 1) The plate on which Raw264.7 cells were cultured was replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and the medium was further cultured for 6 hours.

[0206] 2) Total RNA was extracted and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as a template.

[0207] 3) qRT-PCR was performed using primers for IL-1β, IL-6, and IL-4 using the generated cDNA.

[0208] 4) As shown in Table 7, in Raw264.7 cells, mRNA expression of inflammatory cytokine and atopy-related cytokine genes (IL-1β, IL-6, IL-4) decreased in the group treated with the mixture of low-molecular and medium-molecular DNA fragments prepared in Example 1 compared to Comparative Example 1.

[0209]

[0210]

[0211] Experimental Example 8: VEGF Gene Expression Analysis

[0212] 1) Human dermal fibroblasts (HDF) were cultured at 70-80% confluence in a 100pi dish, and the medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1 was replaced with the medium, and the medium was further cultured for 6 hours.

[0213] 2) Total RNA was extracted and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as a template.

[0214] 3) qRT-PCR was performed using primers for VEGF and VEGFR using the generated cDNA.

[0215] 4) As shown in Table 8, HDF cells showed increased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGF, VEGFR) mRNA in the group treated with the mixture of low-molecular and medium-molecular DNA fragments prepared in Example 1 compared to Comparative Example 1, and among them, the group treated with the mixture of low-molecular DNA fragments prepared in Example 1-1 showed the highest expression of vascular endothelial growth factor and vascular endothelial growth factor receptor (VEGF, VEGFR) mRNA.

[0216]

[0217]

[0218] Experimental Example 9: Analysis of Cell Aging Alleviation Efficacy

[0219] 1) After culturing human epidermal keratinocytes (HEK) for 24 hours, the medium was replaced with a medium containing 10 ppm of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and pretreated for 4 hours. Thereafter, 200 μM of H2O2 was treated and further cultured for 4 hours.

[0220] 2) After removing the medium from the cultured cells, wash them twice with 1 to 2 mL of PBS.

[0221] 3) Add the same amount of fixation solution as PBS and incubate at room temperature for 5 minutes.

[0222] 4) After removing the fixation solution, repeat step 2).

[0223] 5) After adding 1 to 2 mL of staining solution, seal the plate, wrap it with aluminum foil, and incubate it at 37°C for about 10 hours.

[0224] 6) The degree of staining between the control group and the negative control group treated only with H2O2 was confirmed, and when a significant change was observed, the staining solution was removed, washed with PBS, and 70% glycerol was added.

[0225] 7) The number of stained cells was counted under a microscope.

[0226] 8) As shown in Table 9, the SA-β-gal activity increased by H2O2 was decreased in the low-molecular-weight and medium-molecular-weight DNA fragment mixture treatment group of Example 1 compared to the DNA fragment mixture treatment group of Comparative Example 1. Among them, the group treated with the low-molecular-weight DNA fragment mixture prepared in Example 1-1 showed the highest cell senescence inhibition effect.

[0227]

[0228]

[0229] Experimental Example 10: Analysis of hair growth-related gene expression

[0230] 1) Human dermal papilla cells (HDPC) were cultured at 70-80% confluence in a 100pi dish, and the medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1 was replaced with the medium, and the culture was further cultured for 6 hours.

[0231] 2) Total RNA was extracted and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as a template.

[0232] 3) qRT-PCR was performed using primers for FGF7, FGF10, and NOG using the generated cDNA as a template.

[0233] 4) As shown in Table 10, the mRNA of hair growth factors FGF7, FGF10, and NOG increased in the HDP cells treated with the low-molecular-weight and medium-molecular-weight DNA fragment mixture of Example 1 compared to the treated group with the DNA fragment mixture of Comparative Example 1.

[0234]

[0235]

[0236] Experimental Example 11: Analysis of expression of stem cell potency regulatory factors

[0237] 1) The mesenchymal stem cells (MSC) in culture were replaced with a medium containing 10 μg / mL of the DNA fragment mixture prepared in Comparative Example 1 and Example 1, and further cultured for 6 hours.

[0238] 2) Total RNA was extracted and cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as a template.

[0239] 3) qRT-PCR was performed using primers for OCT4 and SOX2 using the generated cDNA.

[0240] 4) As shown in Table 11, in mesenchymal stem cells, the mRNA of OCT4 and SOX2 increased in the low-molecular-weight and medium-molecular-weight DNA fragment mixture treatment group of Example 1 compared to the DNA fragment mixture treatment group of Comparative Example 1. Among them, the highest mRNA expression levels of OCT4 and SOX2 were observed in the low-molecular-weight DNA fragment mixture treatment group prepared in Example 1-1.

[0241]

[0242]

[0243] Experimental Example 12: Pain Level Assessment

[0244] 1) 1 mL each of 100 μg / mL of the DNA fragment mixture of Comparative Example 1 and Example 1 was injected into the skin of male Sprague-Dawley (SD) rats weighing 200 to 250 g.

[0245] 2) To determine the level of pain immediately after injection and 24 hours later, the 22-27 kHz ultrasonic vocalizations produced by rats in pain, distress, and stress states were measured for 10 minutes each using Sonotrack (Metris).

[0246] 3) It was confirmed that the frequency of ultrasonic generation was lower when the DNA fragment mixture of Example 1 was treated compared to Comparative Example 1 in the USV call measurement values ​​immediately after injection.

[0247] 4) It was confirmed that the frequency of ultrasound generation was lower when the DNA fragment mixture of Example 1 was treated than in Comparative Example 1 in the USV call measurement values ​​24 hours after injection. Among them, the group treated with the low-molecular-weight DNA fragment mixture prepared in Example 1-1 showed the lowest pain level.

[0248]

[0249]

[0250] Formulation Example 1: Filler

[0251] After mixing 5% of the DNA fragment mixture of Examples 1 to 5 of the present invention, 50% of hyaluronic acid, 0.2% of vitamin C, 1% of L-arginine, and 0.01% of chitosan, purified water was added to make up to 100% of the volume, and a filler was manufactured according to a conventional filler manufacturing method.

[0252]

[0253] Formulation Example 2: Tablets

[0254] After mixing 10 mg of the DNA fragment mixture of Examples 1 to 5 of the present invention, 400 mg of lactose, 400 mg of corn starch, 2 mg of magnesium stearate, 0.1 mg of L-arginine, and 0.01 mg of chitosan, tablets were manufactured by compressing the mixture according to a conventional tablet manufacturing method.

[0255]

[0256] Formulation Example 3: Drink

[0257] 10 mg of the DNA fragment mixture of Examples 1 to 5 of the present invention, 10 g of glucose, 0.6 g of citric acid, 25 g of liquid oligosaccharide, 1 mg of L-arginine, and 0.1 mg of chitosan were mixed, and 300 ml of purified water was added, and 200 ml was filled into each bottle. After filling the bottles, the mixture was sterilized at 130°C for 4 to 5 seconds to prepare a drink.

[0258]

[0259] Formulation Example 4: Lotion

[0260] After mixing 1% of the DNA fragment mixture of Examples 1 to 5 of the present invention, 15% of glycerin, 10% of butylene glycol, 1% of cetearyl alcohol, 1% of sorbitan olivate, 3% of caprylic / capric triglyceride, 0.5% of squalane, 2% of 1,2-hexanediol, 3% of cyclopentasiloxane / cyclohexasiloxane, 5% of mineral oil, 0.01% of disodium EDTA, 0.05% of BHT, 0.5% of tocopheryl acetate, 0.2% of ethylhexyl methoxycinnamate, 0.1% of L-arginine, and 0.01% of chitosan, purified water was added to make up to 100% of the volume, and a lotion was prepared according to a conventional lotion preparation method.

Claims

1. A composition for filling inter-tissue support, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, anti-inflammation, inhibiting hair loss, promoting hair growth or promoting stem cell differentiation, comprising a mixture of DNA fragments isolated and extracted from mushrooms and having a low molecular size of less than 1,000 bp.

2. A composition for filling interstitial support, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, anti-inflammation, inhibiting hair loss, promoting hair growth or promoting stem cell differentiation, comprising a mixture of DNA fragments separated and extracted from mushrooms and having a middle molecular size of 1,000 bp or more and less than 5,000 bp.

3. A composition for filling interstitial support, improving skin condition, regenerating tissue, healing wounds, moisturizing, promoting extracellular matrix synthesis, inhibiting cell aging, forming new blood vessels, relieving pain, anti-inflammation, inhibiting hair loss, promoting hair growth or promoting stem cell differentiation, comprising a mixture of DNA fragments separated and extracted from mushrooms and having a polymerized size of 5,000 bp or more.

4. A mixture of DNA fragments according to any one of paragraphs 1 to 3, wherein the mushroom is selected from medicinal mushrooms or edible mushrooms, including white oyster mushrooms, mulberry mushrooms, shiitake mushrooms, oyster mushrooms, sang-hwang mushrooms, deer antler mushrooms, pine mushrooms, lingzhi mushrooms, enoki mushrooms, truffles, reishi mushrooms, chaga mushrooms, cordyceps sinensis, chive mushrooms, yellow oyster mushrooms, cloud mushrooms, small tongue mushrooms, net mushrooms, horseshoe mushrooms, poria cocos, chanterelles, Chinese chive mushrooms, Chinese chive mushrooms, tongue-head mushrooms, wood mushrooms, sari mushrooms, shiitake mushrooms, egg mushrooms, milk mushrooms, flower mushrooms, and magpie mushrooms, or a combination of one or more of these.

5. A composition according to any one of claims 1 to 3, wherein the DNA fragment is at least one selected from the group consisting of polydeoxyribonucleotides and polynucleotides.

6. A composition according to any one of claims 1 to 3, wherein the tissue is selected from the group consisting of skin, cartilage, muscle, and ligament.

7. In the first paragraph, the mixture of DNA fragments having a low molecular size is (S1) A step of adding mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours; (S2) A step of centrifuging the dissolved substance obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to decompose RNA and increase DNA yield; (S4) a step of first fragmenting the mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Alu1, Bsn1, Cfo1 (Hha1), Mal1, Nla3, Mbo2, Rsa1, Hae3, and Fok1; (S5) a step of inactivating the above restriction enzyme; and (S6) A composition manufactured by a manufacturing method including a step of secondary fragmentation of a mixture of DNA fragments by sonication.

8. In the second paragraph, the mixture of DNA fragments having the intermediate molecular size is (S1) A step of adding mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours; (S2) A step of centrifuging the dissolved substance obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to decompose RNA and increase DNA yield; (S4) a step of first fragmenting the mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2; (S5) a step of inactivating the above restriction enzyme; and (S6) A composition manufactured by a manufacturing method including a step of secondary fragmentation of a mixture of DNA fragments by sonication.

9. In the third paragraph, the mixture of DNA fragments having the polymerized size is (S1) A step of adding mycelia or fruiting bodies of mushrooms to a lysis buffer containing 200 to 300 μg / mL of plum extract and performing dissolution and homogenization at 50 to 60°C for 1 to 3 hours; (S2) A step of centrifuging the dissolved substance obtained in step (S1) at 9,000 to 11,000 rpm for 20 to 40 minutes to obtain a supernatant; (S3) A step of adding 2.5 to 7.5 μL of RNase A at a concentration of 5 to 15 mg / mL to the supernatant and then placing it in an incubator at 30 to 40°C for 10 to 30 minutes to decompose RNA and increase DNA yield; (S4) a step of fragmenting a mixture of DNA fragments by treating the solution obtained in step (S3) with one or more restriction enzymes selected from the group consisting of Kpn1, Nhe1, Pml1, Mab1, Bsu36I, SnaB1, Bmt1, and Avr2; and (S5) A composition manufactured by a manufacturing method including a step of inactivating the above restriction enzyme.

10. A composition according to any one of claims 7 to 9, wherein the DNA fragment mixture is produced by a production method comprising shaking and culturing mycelia of a mushroom and then performing the following steps (S1).

11. A composition according to any one of claims 7 to 9, wherein the DNA fragment mixture is prepared by a manufacturing method comprising: performing shaking culture of mushroom mycelia and then performing the following steps (S1), wherein the shaking culture comprises: a step of sterilizing mushroom mycelia in a liquid medium containing 97 to 98 wt% of malt extract, 1 wt% of yeast, and 1 to 2 wt% of dried corn extract at 110 to 130° C. for 10 to 20 minutes; and a step of adding mushroom mycelia to the prepared liquid medium and culturing the mixture at 20 to 30° C. and 150 to 200 rpm for 48 to 96 hours.

12. A composition according to any one of claims 1 to 3, wherein the composition is a cosmetic composition.

13. A composition according to any one of claims 1 to 3, wherein the composition is a food composition.

14. A composition according to any one of claims 1 to 3, wherein the composition is a medical device filler composition.

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