Method for extracting polydeoxyribonucleotide from plant, plant callus, or mushroom mycelium, polydeoxyribonucleotide prepared thereby, and composition comprising polydeoxyribonucleotide

Abstract

The present invention provides a method for extracting polydeoxyribonucleotide from plant callus, polydeoxyribonucleotide prepared thereby, and a composition comprising said polydeoxyribonucleotide, the method comprising the steps of: 1) adding a cetrimonium bromide (CTAB) buffer or a sodium dodecyl sulfate (SDS) buffer to a crushed product of a plant, a plant callus, or a mushroom mycelium, and stirring the mixture; 2) maintaining the mixture at a temperature of 20°C to 70°C for 0.1 to 2 hours; 3) centrifuging the mixture at a speed of 8,000 to 15,000 rpm; 4) after the centrifuging, isolating a supernatant; 5) mixing the mixture with a mixed solution in which phenol, chloroform, and isoamyl alcohol are mixed in a ratio of 20-30:20-30:1 by volume; 6) centrifuging the mixture at a speed of 8,000 to 15,000 rpm; 7) after the centrifuging, isolating a supernatant; 8) admixing 50 to 200 parts by weight of isopropyl alcohol with respect to 100 parts by weight of the isolated supernatant; 9) centrifuging the mixture at a speed of 8,000 to 15,000 rpm; 10) after the centrifuging, isolating the precipitate polydeoxyribonucleotide; and 11) washing the isolated polydeoxyribonucleotide with ethanol and drying same.

Description

Method for extracting polydeoxyribonucleotides from plants, plant calli or mushroom mycelia, polydeoxyribonucleotides produced by the method, and compositions comprising the polydeoxyribonucleotides

[0001] The present invention relates to a method for extracting polydeoxyribonucleotides from plants, plant calli or mushroom mycelia, polydeoxyribonucleotides produced by the method, and compositions comprising the polydeoxyribonucleotides.

[0002] In modern society, rising economic standards and a steadily increasing elderly population have led to a growing interest in health across all age groups. Numerous pharmaceuticals, health functional foods, and functional cosmetics are being developed to promote health and delay the aging process. In particular, active research is underway to achieve various physiological benefits, such as promoting self-regeneration of damaged cells and tissues, anti-inflammation, improving skin, preventing hair loss, and promoting hair growth.

[0003] As part of these studies, a technique for obtaining polydeoxyribonucleotides (PDNUs) from human placenta has been disclosed. PDNUs are known to possess various physiological activities, including promoting self-regeneration of damaged cells and tissues, anti-ischemia, and anti-inflammation.

[0004] However, human placenta is limited in supply, making it difficult to supply in sufficient quantities, expensive, and subject to ethical issues.

[0005] To address these issues, methods for obtaining deoxyribonucleotides from fish such as salmon and trout have been developed. However, since these deoxyribonucleotides are also obtained from the semen and testes of salmon and trout, securing a sufficient supply remains challenging.

[0006] Therefore, there is a need for the development of a method for producing deoxyribonucleotides that is free from these problems.

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] (Patent Document 1) Republic of Korea Patent No. 10-2056144

[0010] The present invention has been devised to solve the above problems of the prior art.

[0011] The purpose of the present invention is to provide a method for extracting polydeoxyribonucleotides by extracting polydeoxyribonucleotides from plants, plant calli or mushroom mycelia, which enables sufficient supply and supply at an appropriate price and is free from ethical issues, a polydeoxyribonucleotide produced by the method, and a composition comprising the polydeoxyribonucleotide.

[0012] In addition, the present invention aims to provide a method for extracting polydeoxyribonucleotide, which provides various physiological effects such as promotion of self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth, a polydeoxyribonucleotide prepared by the method, and a composition comprising the polydeoxyribonucleotide.

[0013] In order to achieve the above purpose, the present invention,

[0014] 1) A step of adding CTAB buffer (cetrimonium bromide buffer) or SDS buffer (sodium dodecyl sulfate buffer) to a pulverized plant, plant callus, or mushroom mycelium (callus) and stirring the mixture;

[0015] 2) A step of maintaining the mixture at a temperature of 20 to 70°C for 0.1 to 2 hours;

[0016] 3) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm;

[0017] 4) After the centrifugation, a step of separating the supernatant;

[0018] 5) A step of mixing the above mixture with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20 to 30: 20 to 30: 1;

[0019] 6) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm;

[0020] 7) After the centrifugation, a step of separating the supernatant;

[0021] 8) A step of mixing 50 to 200 parts by weight of isopropyl alcohol with 100 parts by weight of the separated supernatant;

[0022] 9) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm;

[0023] 10) A step of separating the precipitate, polydeoxyribonucleotide, after the centrifugation; and

[0024] 11) A method for extracting polydeoxyribonucleotide from plant callus is provided, comprising the steps of washing the separated polydeoxyribonucleotide with ethanol and drying it.

[0025]

[0026] In addition, the present invention

[0027] A polydeoxyribonucleotide manufactured by the above method is provided.

[0028]

[0029] In addition, the present invention

[0030] A composition comprising the above polydeoxyribonucleotide is provided.

[0031] The composition may be a pharmaceutical composition, a cosmetic composition, or a food composition.

[0032] The method of extracting polydeoxyribonucleotides from plants, plant calli or mushroom mycelia of the present invention enables a sufficient supply of polydeoxyribonucleotides having various physiological activities and supplying them at an appropriate price, and provides the effect of being able to supply polydeoxyribonucleotides without ethical issues.

[0033] In addition, polydeoxyribonucleotides produced by the above method provide various physiological effects such as promotion of self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth.

[0034] In addition, the composition containing the polydeoxyribonucleotide provides various physiological effects such as promotion of self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth.

[0035] Figure 1 is a photograph showing the results of analyzing PDRN derived from white dandelion callus using an electrophoresis method.

[0036] Figure 2 is a photograph showing the cell regeneration efficacy of PDRN derived from white dandelion callus.

[0037] Figure 3 is a graph showing the cell regeneration, moisturizing, and hair growth effects of PDRN derived from white dandelion callus.

[0038] Figure 4 is a photograph showing the results of analyzing PDRN derived from soybean leaves using an electrophoresis method.

[0039] Figure 5 is a photograph showing the cell regeneration efficacy of PDRN derived from soybean leaves.

[0040] Figure 6 is a photograph showing the results of analyzing PDRN derived from winter oyster mushrooms using an electrophoresis method.

[0041] Figure 7 is a photograph showing the cell regeneration efficacy of PDRN derived from winter oyster mushrooms.

[0042] Figure 8 is a photograph showing the results of analyzing PDRN derived from peanut sprouts using an electrophoresis method.

[0043] Figure 9 is a photograph showing the cell regeneration efficacy of PDRN derived from peanut sprouts.

[0044] Figure 10 is a photograph showing the results of analyzing PDRN derived from the cap of the scorpionfish using an electrophoresis method.

[0045] Figure 11 is a photograph showing the cell regeneration efficacy of PDRN derived from the hair of the snail.

[0046] The present invention is described in detail below.

[0047] The method for extracting polydeoxyribonucleotide from a plant, plant callus or mushroom mycelium of the present invention is characterized by comprising the following steps.

[0048] 1) A step of adding CTAB buffer (cetrimonium bromide buffer) or SDS buffer (sodium dodecyl sulfate buffer) to a pulverized plant, plant callus, or mushroom mycelium (callus) and stirring the mixture;

[0049] 2) A step of maintaining the mixture at a temperature of 20 to 70°C for 0.1 to 2 hours;

[0050] 3) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm;

[0051] 4) After the centrifugation, a step of separating the supernatant;

[0052] 5) A step of mixing the above mixture with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20 to 30: 20 to 30: 1;

[0053] 6) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm;

[0054] 7) After the centrifugation, a step of separating the supernatant;

[0055]

[0056] *8) A step of mixing 50 to 200 parts by weight of isopropyl alcohol with respect to 100 parts by weight of the separated supernatant;

[0057] 9) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm;

[0058] 10) A step of separating the precipitate, polydeoxyribonucleotide, after the centrifugation; and

[0059] 11) A step of washing the separated polydeoxyribonucleotide with ethanol and drying it.

[0060]

[0061] After the above step 10), a step of reducing the molecular weight of the separated polydeoxyribonucleotide may be further included. At this time, the reducing molecular weight may be performed, for example, using an ultrasonic processor.

[0062] For example, by using an ultrasonic processor, ultrasonic waves can be applied for 10 seconds at a time at 30% output, and this can be repeated 5 to 20 times to perform low molecular weight polymerization.

[0063] The present invention may further include a step of placing the polydeoxyribonucleotide obtained in step 11) into an elution buffer.

[0064]

[0065] In the above step 1), the plant, plant callus, or mushroom mycelium (callus) can be crushed using, for example, liquid nitrogen or a bead beater. The liquid nitrogen can be continuously injected into the plant, plant callus, or mushroom mycelium (e.g., dandelion callus) and repeated until the plant, plant callus, or mushroom mycelium is completely crushed. When the bead beater is used, the plant, plant callus, or mushroom mycelium (e.g., dandelion callus) and a stainless steel bead (e.g., 5 mm in diameter) can be placed in a vial and stirred to crush. At this time, the crushing can be performed by repeating the process twice at 3,000 rpm for 45 seconds, for example.

[0066] In step 1), plant callus can be produced by disinfecting plant tissue in 70% ethanol for 1 minute, 2-3% sodium hypochlorite for 20 minutes, and then inducing the sterilized tissue on MS medium containing various plant growth regulators. Alternatively, commercially available callus can be purchased and used, or provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Biological Resource Center.

[0067] In the above step 1), the CTAB buffer (cetrimonium bromide buffer) may include EDTA, Tris-HCl, saline, and CTAB. For example, it may include 6 to 10 parts by weight of a 0.3 to 0.8 M EDTA (pH 7 to 9) aqueous solution; 10 to 30 parts by weight of a 0.5 to 2 M Tris-HCl (pH 7 to 9) aqueous solution; 40 to 70 parts by weight of a 3 to 7 M saline solution; and 2 to 8 parts by weight of CTAB.

[0068] Additionally, the CTAB buffer may further contain 90 to 130 parts by weight of sterile water.

[0069] Additionally, the CTAB buffer may further contain 2-mercaptoethanol at 0.1 to 3% (v / v).

[0070] Specifically, it may contain 8 mL of 0.5 M EDTA (pH 8.0), 20 mL of 1 M Tris-HCl (pH 8.0), 56 mL of 5 M saline solution, and 4 g of CTAB, 114 mL of sterile water, and 2 mL of 2-mercaptoethanol.

[0071] The SDS buffer (sodium dodecyl sulfate) may include EDTA, Tris-HCl, saline, and SDS. For example, it may include 3 to 8 parts by weight of a 0.3 to 0.8 M EDTA (pH 7 to 9) aqueous solution; 10 to 30 parts by weight of a 0.5 to 2 M Tris-HCl (pH 7 to 9) aqueous solution; 40 to 70 parts by weight of a 3 to 7 M saline solution; and 1 to 4 parts by weight of SDS.

[0072] Additionally, the SDS buffer may further contain 90 to 130 parts by weight of sterile water.

[0073] Additionally, the SDS buffer may further contain 2-mercaptoethanol at 0.1 to 3% (v / v).

[0074] Specifically, it may contain 2 mL of 0.1 M EDTA (pH 8.0), 20 mL of 1 M Tris-HCl (pH 8.0), 56 mL of 5 M saline solution, 2 g of SDS, 114 mL of sterile water, and 2 mL of 2-mercaptoethanol.

[0075] In the above step 1), CTAB buffer or SDS buffer may be added in an amount of 500 to 1000 parts by weight based on 100 parts by weight of the crushed plant, plant callus or mushroom mycelium (callus).

[0076]

[0077] In the above step 2), it may be more preferable to maintain the mixture at a temperature of 25 to 60°C for 20 to 40 minutes.

[0078] In addition, in the above step 2), it may be more preferable to first additionally mix 1 to 3 times the CTAB buffer or SDS buffer added to the mixture in step 1), preferably 1.5 to 2.5 times the CTAB buffer, and then perform the remaining process.

[0079]

[0080] In the above step 5), a mixed solution of phenol, chloroform, and isoamyl alcohol in a volume ratio of 23 to 27: 22 to 26: 1 can be more preferably used.

[0081] In addition, the mixture obtained in step 4) and the above phenol, chloroform, and isoamyl alcohol mixture can be mixed in a volume ratio of 1:0.5 to 1.5, or a volume ratio of 1:0.7 to 1.2.

[0082] In the above step 9), it is more preferable to mix 70 to 130 parts by weight of isopropyl alcohol with respect to 100 parts by weight of the separated supernatant, and it may be even more preferable to mix 90 to 110 parts by weight.

[0083] In step 11), the ethanol washing can be performed by adding ethanol to polydeoxyribonucleotide and centrifuging. However, the present invention is not limited to this method, and may be performed using a method known in the art.

[0084] In the above step 11), the ethanol washing may be performed two or more times while changing the ethanol concentration from a low concentration to a high concentration. It may be preferable to select the ethanol concentration in the range of 70 to 100%.

[0085]

[0086] In the present invention, the plant, plant callus or mushroom mycelium may be dandelion callus, soybean leaves, Polyporus brumalis, peanut sprouts, or Sargassum horneri.

[0087] The above white dandelion callus can be manufactured by, for example, sterilizing plant tissue in 70% ethanol for 1 minute and 2-3% sodium hypochlorite for 20 minutes, and then inducing the sterilized tissue in MS medium containing various plant growth regulators, or can be supplied and used from the Korea Research Institute of Bioscience and Biotechnology.

[0088] Polydeoxyribonucleotide obtained from the above white dandelion callus provides excellent effects on cell regeneration, moisturizing, and hair growth.

[0089] Polydeoxyribonucleotides obtained from the above soybean leaves provide excellent effects on cell regeneration, etc.

[0090] The above-mentioned Polyporus brumalis is, for example, a strain isolated from a fruiting body, and a culture stored in the National Institute of Biological Resources' Biological Materials Bank is received, and pre-cultured on a Potato Dextrose Agar (BD Difco Laboratories, Detroit, MI, USA) medium at 25℃ for 7 days, and the solid medium on which P. brumalis grew is sectioned with a 5 mm cork border (KA-0049, Korea Ace Scientific, Seoul, Korea), and 7 pieces are inoculated onto a DY medium (20.0 g Dextrose, 2.0 g Yeast extract, BD Difco Laboratories, Detroit, MI, USA), and then cultured with shaking at 170 rpm for 60 days at 25℃. It can be manufactured or supplied by the National Institute of Biological Resources.

[0091] Polydeoxyribonucleotides obtained from the above-mentioned winter hole mushroom provide excellent effects on cell regeneration, etc.

[0092]

[0093] *The above peanut sprouts can be manufactured by cultivating peanuts in liquid and sprouting them.

[0094] Polydeoxyribonucleotides obtained from the above peanut sprouts provide excellent effects on cell regeneration, etc.

[0095] The above-mentioned sea squirt hat can be supplied and used from the Jeju Island coast or the National Institute of Biological Resources.

[0096] Polydeoxyribonucleotide obtained from the above-mentioned scutellaria baicalensis provides excellent effects on cell regeneration, etc.

[0097]

[0098] In addition, the present invention provides polydeoxyribonucleotides extracted from plants, plant calli, or mushroom mycelia produced by the above method.

[0099] The above polydeoxyribonucleotide provides various physiological effects such as promoting self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth.

[0100]

[0101] The present invention also provides a composition comprising the polydeoxyribonucleotide. The composition provides various physiological effects, such as promoting self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth.

[0102] The composition may further comprise a carrier, which may be a pharmaceutically acceptable carrier.

[0103] The composition may be a pharmaceutical composition, a cosmetic composition, or a food composition.

[0104] The pharmaceutical composition, cosmetic composition, or food composition may contain 0.1 to 100 wt% of the polydeoxyribonucleotide, and may also contain various carriers and additives known in the art.

[0105]

[0106] The above pharmaceutical composition can be prepared as a composition in a conventional dosage form by selecting one or more types of pharmaceutically acceptable conventional carriers or one or more types of additives in the effective amount of the polydeoxyribonucleotide.

[0107] The above pharmaceutical composition provides various physiological effects such as promotion of self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth.

[0108] The above anti-inflammatory effect can be applied to the treatment of inflammatory diseases. The inflammatory diseases can be one or more diseases selected from the group consisting of inflammatory bowel disease, non-alcoholic chronic hepatitis, chronic hepatitis, Crohn's disease, pancreatitis, esophagitis, gastritis, colitis, ulcerative colitis, pneumonia, bronchitis, pharyngitis, myocardial infarction, heart failure, arthritis, psoriatic arthritis, rheumatoid arthritis, renal failure, psoriasis, anemia, diabetes, fibrosis, multiple sclerosis, systemic lupus erythematosus, ankylosing spondylitis, asthma, chronic obstructive pulmonary disease, periodontitis, neuropathic pain, and idiopathic inflammatory myopathy.

[0109] The above carrier may be selected from one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and the additives may be selected from one or more of flavorings, vitamins, and antioxidants.

[0110] In the present invention, the carrier and additive are not limited in type, and specifically, the diluent may be selected from lactose monohydrate, trehalose, corn starch, soybean oil, microcrystalline cellulose, or D-mannitorl, the lubricant may be selected from magnesium stearate or talc, and the binder may be selected from polyvinylpyrrolidone (PVP) or hydroxypropylcellulose (HPC). In addition, the disintegrant can be selected from carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone; the sweetener can be white sugar, fructose, sorbitol or aspartame; the stabilizer can be selected from carboxymethylcellulosesodium (Na-CMC), beta-cyclodextrin, white bee's wax or xanthan gum; and the preservative can be selected from methyl p-hydroxy benzoate (methhlparaben), propyl p-hydroxybenzoate (propylparaben) or potassium sorbate. Can be.

[0111] The dosage of the pharmaceutical composition may vary depending on the age, sex, and weight of the patient or animal to be treated, and above all, it will depend on the condition of the subject to be treated, the specific category or type of the disease to be treated, the route of administration, and the properties of the therapeutic agent used. The pharmaceutical composition is appropriately selected depending on the absorption and excretion rate of the active ingredient in the body, the age and weight, sex, and condition of the patient or animal to be treated, the severity of the disease to be treated, etc., but it is generally preferable to administer 0.1 to 1,000 mg / kg per day. The unit dosage form preparation formulated in this way can be administered several times at regular intervals as needed.

[0112] The above pharmaceutical composition may be administered individually as a preventive or therapeutic agent, or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0113] The above pharmaceutical compositions can be formulated and used as oral dosage forms such as powders, granules, tablets, capsules, troches, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods. When formulating, they can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included.

[0114]

[0115] In one embodiment of the present invention, the composition may be a cosmetic composition. The cosmetic composition provides various physiologically active effects, such as promoting self-regeneration of damaged cells and tissues, improving skin, preventing hair loss, and promoting hair growth. The term "skin improvement" includes antioxidant, anti-wrinkle, and whitening effects.

[0116] The above cosmetic composition can be manufactured in any formulation commonly manufactured in the art, and for example, it can be any one selected from among a base cosmetic formulation selected from among a solution, a suspension, an emulsion, a paste, a toner, a gel, a water-soluble liquid, a cream, an essence, a surfactant-containing cleanser, an oil, an oil-in-water (O / W) type and a water-in-oil (W / O) type; a skin; a lotion; an eye cream; a soothing gel; an ointment; a formulation for a mask pack; a formulation for a body wash; a peeling gel; an oil-in-water and water-in-oil type makeup base; a foundation; a skin cover; a color cosmetic formulation selected from among a lipstick, a lip gloss, a face powder, a two-way cake, an eye shadow, a cheek color and an eyebrow pencil; and a formulation for the scalp.

[0117] In addition, the cosmetic composition may contain adjuvants commonly used in the cosmetic field, such as hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blocking agents, pigments, deodorants, dyes, etc. The amount of these various adjuvants is an amount commonly used in the field, and is, for example, 0.001 to 30 wt% based on the total weight of the composition. However, in any case, the adjuvants and their proportions will be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.

[0118] In the above cosmetic composition, when the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0119] In addition, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoate, propylene glycol, 1,3-butylene glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0120] In addition, when the formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0121] Additionally, when the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and especially in the case of a spray, a propellant such as chlorofluorohydrocarbon, protane / butane or dimethyl ether may be additionally included.

[0122] In addition, when the formulation is a surfactant-containing cleanser, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0123] In addition, if the formulation is a surfactant-containing cleanser formulation, a surfactant-free cleanser formulation, or soap, it can be applied to the skin and then wiped off, removed, or washed off with water. For example, the soap is liquid soap, powder soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is cleansing foam, cleansing water, cleansing towel, and cleansing pack, and the surfactant-free cleansing formulation is cleansing cream, cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.

[0124]

[0125] The above food composition provides various physiological effects such as promotion of self-regeneration of damaged cells and tissues, anti-inflammation, skin improvement, hair loss prevention, and hair growth.

[0126] The above food composition may be a health functional food.

[0127] The food composition may be tea, jelly, juice, extract, beverage, etc., and there is no limitation on the processing form as long as it contains the extract of the purslane. In addition, the food composition may be selected from the group consisting of general foods, such as ice cream, dairy products including milk, soy milk, and cheese, soy milk products, beverages, meat, sausage, bread, chocolate candies, snacks, confectionery, pizza, ramen, other noodles, gum, various soups, beverages, tea, drinks, alcoholic beverages, and lactic acid bacteria preparations in capsule or stick packaging.

[0128] The above beverage composition may contain various flavoring agents, sweeteners, or natural carbohydrates as additional ingredients, as in conventional beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener may be a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame.

[0129]

[0130] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to further illustrate the present invention, and the scope of the present invention is not limited by these examples. Those skilled in the art may appropriately modify or alter the following examples within the scope of the present invention.

[0131]

[0132] Example 1: Preparation of polydeoxyribonucleotides from white dandelion callus

[0133] Plant callus was prepared by sterilizing plant tissue in 70% ethanol for 1 minute and 2-3% sodium hypochlorite for 20 minutes, and then using MS medium containing plant growth regulators of 1 mg / L 2,4-dichlorophenoxyacetic acid, 30,000 mg / L sucrose, 100 mg / mL myo-inositol, and 0.4 mg / mL thiamine HCl.

[0134] The above-mentioned white dandelion callus was crushed, SDS buffer was added, and the mixture was stirred. The mixture was maintained at a temperature of 25℃ for 0.5 hour. The mixture was centrifuged at a speed of 12,000 rpm, and the supernatant was separated. The supernatant was mixed with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20-30:20-30:1. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and 100 parts by weight of the supernatant was mixed with 100 parts by weight of isopropyl alcohol. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and the precipitate, polydeoxyribonucleotide, was isolated. The separated polydeoxyribonucleotide was washed with ethanol, dried, and then placed in an elution buffer to prepare polydeoxyribonucleotide.

[0135]

[0136] Example 2: Preparation of polydeoxyribonucleotides from soybean leaves

[0137] Soybean leaves were crushed, SDS buffer was added, and the mixture was stirred. The mixture was maintained at a temperature of 25℃ for 0.5 hours. The mixture was centrifuged at a speed of 12,000 rpm, and the supernatant was separated. The supernatant was mixed with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20-30:20-30:1. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and 100 parts by weight of the supernatant was mixed with 100 parts by weight of isopropyl alcohol. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and the precipitate, polydeoxyribonucleotide, was isolated. The separated polydeoxyribonucleotide was washed with ethanol, dried, and then placed in an elution buffer to prepare polydeoxyribonucleotide.

[0138]

[0139] Example 3: Preparation of polydeoxyribonucleotides from winter oyster mushroom fungi

[0140] The strain was isolated from the fruiting body collected from Gayasan Mountain in Hapcheon-gun, Gyeongnam Province. The culture was stored in the National Institute of Biological Resources' Biological Materials Bank and pre-cultured on Potato Dextrose Agar (BD Difco Laboratories, Detroit, MI, USA) for 7 days at 25℃. The solid medium on which P. brumalis was grown was sectioned with a 5 mm cork border (KA-0049, Korea Ace Scientific, Seoul, Korea), and 7 pieces were inoculated onto DY medium (20.0 g Dextrose, 2.0 g Yeast extract, BD Difco Laboratories, Detroit, MI, USA). The mycelia of P. brumalis were prepared by shaking and culturing at 170 rpm at 25℃ for 60 days.

[0141] The above-mentioned winter hole mushroom cells were crushed, SDS buffer was added, and stirred. The mixture was maintained at a temperature of 25℃ for 0.5 hours. The mixture was centrifuged at a speed of 12,000 rpm, and the supernatant was separated. The supernatant was mixed with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20-30:20-30:1. The mixture was centrifuged under the same conditions as above, and the supernatant was separated. 100 parts by weight of the supernatant was mixed with 100 parts by weight of isopropyl alcohol. The mixture was centrifuged under the same conditions as above, and the supernatant was separated, and the precipitate, polydeoxyribonucleotide, was isolated. The separated polydeoxyribonucleotide was washed with ethanol, dried, and placed in an elution buffer.

[0142] Polydeoxyribonucleotides were prepared.

[0143]

[0144] Example 4: Preparation of polydeoxyribonucleotides from peanut sprouts

[0145] Peanut sprouts were prepared by sprouting peanuts through liquid culture.

[0146] The peanut sprouts were ground, SDS buffer was added, and the mixture was stirred. The mixture was maintained at 25°C for 0.5 hours. The mixture was centrifuged at 12,000 rpm, and the supernatant was separated. The supernatant was mixed with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20-30:20-30:1. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and 100 parts by weight of the supernatant was mixed with 100 parts by weight of isopropyl alcohol. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and the precipitate, polydeoxyribonucleotide, was isolated. The separated polydeoxyribonucleotide was washed with ethanol, dried, and then placed in an elution buffer to prepare polydeoxyribonucleotide.

[0147]

[0148] Example 5: Preparation of polydeoxyribonucleotide from the plant of the genus Amurense

[0149] The National Institute of Biological Resources provided a snail-shaped hat.

[0150] The above-mentioned spore-forming substance was ground, CTAB buffer was added, and the mixture was stirred. The mixture was maintained at a temperature of 25℃ for 0.5 hours. The mixture was centrifuged at a speed of 12,000 rpm, and the supernatant was separated. The supernatant was mixed with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20-30:20-30:1. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and 100 parts by weight of the supernatant was mixed with 100 parts by weight of isopropyl alcohol. The mixture was centrifuged under the same conditions as above, the supernatant was separated, and the precipitate, polydeoxyribonucleotide, was isolated. The separated polydeoxyribonucleotide was washed with ethanol, dried, and then placed in an elution buffer to prepare polydeoxyribonucleotide.

[0151]

[0152] Comparative Example 1: Trout-derived polydeoxyribonucleotide

[0153] Polydeoxyribonucleotide obtained from trout semen was purchased from Noah Biotech Co., Ltd. and used as a comparative example.

[0154]

[0155] Experimental Example 1: Confirmation of the shape of polydeoxyribonucleotide

[0156] The forms of the polydeoxyribonucleotides prepared in Examples 1 to 5 and the trout polydeoxyribonucleotides of Comparative Example 1, extracted using CATB buffer or SDS buffer, were analyzed and compared by electrophoresis.

[0157] The above confirmation results are shown in Fig. 1 (PDRN derived from white dandelion callus), Fig. 4 (PDRN derived from soybean leaves), Fig. 6 (PDRN derived from winter oyster mushroom), Fig. 8 (PDRN derived from peanut sprouts), and Fig. 10 (PDRN derived from spore-bearing moss).

[0158]

[0159] Experimental Example 2: Evaluation of Cell Regeneration Performance

[0160] The cell regeneration performance of the polydeoxyribonucleotides prepared in Examples 1 to 5 above was measured by the following method.

[0161] Human keratinocyte HaCaT cells were seeded at 2Х10 cells / well in a 12-well plate, cultured for 24 hours, replaced with medium not containing FBS, and each well was scratched with a 200p tip and the sample was processed. After sample processing, the medium was removed and the well was placed in a fixing solution (4% paraformaldehyde), incubated for 15 minutes at room temperature, washed three times with PBS, and fixed. The degree of wound healing was photographed under a microscope to evaluate the cell regeneration performance of polydeoxyribonucleotides.

[0162] The above confirmation results are shown in Figs. 2 and 3 (PDRN derived from white dandelion callus), Fig. 5 (PDRN derived from soybean leaf), Fig. 7 (PDRN derived from winter oyster mushroom callus), Fig. 9 (PDRN derived from peanut sprout callus), and Fig. 11 (PDRN derived from spore-bearing moss callus).

[0163] As confirmed in the above drawing, the polydeoxyribonucleotide of the present invention exhibited an equivalent or superior cell regeneration effect at the same concentration compared to the polydeoxyribonucleotide obtained from trout.

[0164]

[0165] Experimental Example 3: Measurement of the moisturizing effect of PDRN derived from white dandelion callus.

[0166] The moisturizing effects of PDRN derived from white dandelion callus and PDRN derived from trout were measured using the following method, and the results are shown in Fig. 3.

[0167] HaCaT cells were seeded at 2.0 x 104 cells / well in 96-well plates and cultured at 37°C in 5% CO2. After 24 hours, the cells were washed with PBS and added to DMEM medium (serum-free medium) without FBS but with only 1% P / S, and cultured for 24 hours. After adding the sample and reacting for 24 hours, the amount of hyaluronic acid produced in the culture supernatant was measured using a human hyaluronan ELISA set (R&D Systems, Minneapolis, MN, USA).

[0168]

[0169] Experimental Example 4: Measurement of the hair growth effect of PDRN derived from white dandelion callus.

[0170] The hair growth effect of PDRN derived from white dandelion callus and PDRN derived from trout was measured using the following method, and the results are shown in Fig. 3.

[0171] Human dermal papilla cells (HFDPC) were cultured in a 5% CO2 incubator using DMEM medium containing 10% FBS and 1% p / s, and seeded at 4 x 103 cells / well in a 24-well plate. After 24 hours of culture, untreated control wells and positive control wells treated with 10 μM minoxidil were prepared, and samples were treated in the remaining wells. After 48 hours and 72 hours, the 0.05% MTT assay was performed for 3 hours, the supernatant was removed, and the formed formazan was dissolved in 1 mL of DMSO, transferred to a 96-well plate, and the absorbance was measured at 570 nm.

Claims

1. 1) A step of adding CTAB buffer (cetrimonium bromide buffer) or SDS buffer (sodium dodecyl sulfate buffer) to the crushed plant, plant callus, or mushroom mycelium (callus) and stirring the mixture; 2) A step of maintaining the mixture at a temperature of 20 to 70°C for 0.1 to 2 hours; 3) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm; 4) After the centrifugation, a step of separating the supernatant; 5) A step of mixing the above mixture with a mixture of phenol, chloroform, and isoamyl alcohol in a volume ratio of 20 to 30: 20 to 30: 1; 6) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm; 7) After the centrifugation, a step of separating the supernatant; 8) A step of mixing 50 to 200 parts by weight of isopropyl alcohol with 100 parts by weight of the separated supernatant; 9) A step of centrifuging the mixture at a speed of 8,000 to 15,000 rpm; 10) A step of separating the precipitate, polydeoxyribonucleotide, after the centrifugation; and 11) A method for extracting polydeoxyribonucleotide from plant callus, comprising the steps of washing the separated polydeoxyribonucleotide with ethanol and drying it.

2. In paragraph 1, A method for extracting polydeoxyribonucleotides from plants, plant calli or mushroom mycelia, characterized in that it further comprises a step of reducing the molecular weight of the separated polydeoxyribonucleotides after the above step 10).

3. In paragraph 1, A method for extracting polydeoxyribonucleotides from plants, plant calli, or mushroom mycelia, characterized in that it further comprises a step of placing the polydeoxyribonucleotides obtained in the above step 11) into an elution buffer.

4. In paragraph 1, A method for extracting polydeoxyribonucleotides from plants, plant calli, or mushroom mycelia, characterized in that step 2) above first additionally mixes 1 to 3 times the amount of CTAB buffer or SDS buffer added to the mixture in step 1), and then performs the remaining processes.

5. In paragraph 1, A method for extracting polydeoxyribonucleotides from plant callus, characterized in that the ethanol washing in step 11) above is performed through a process of adding ethanol to polydeoxyribonucleotides and centrifuging them.

6. In paragraph 5, A method for extracting polydeoxyribonucleotides from plants, plant calli, or mushroom mycelia, characterized in that in the above step 11), ethanol washing is performed twice or more while changing the concentration of ethanol from a low concentration to a high concentration.

7. In paragraph 1, A method for extracting polydeoxyribonucleotides from a plant or callus, characterized in that the plant is dandelion callus, soybean leaf, Polyporus brumalis, peanut sprout, or Sargassum horneri.

8. Polydeoxyribonucleotide extracted from a plant, plant callus or mushroom mycelium manufactured by the method of paragraph 1.

9. A composition comprising the polydeoxyribonucleotide of paragraph 1.

10. In paragraph 9, A composition characterized in that the composition is a pharmaceutical composition, a cosmetic composition, or a food composition.

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