Method for producing PDRN from plant

Abstract

The present invention relates to a method for producing polydeoxyribonucleotide (PDRN) from a plant, PDRN produced by the method, and a cosmetic composition, a pharmaceutical composition, a quasi-drug composition, a food composition, a health functional food, and a feed composition, each comprising PDRN as an active ingredient for improving skin conditions. According to the present invention, high-purity and high-concentration PDRN can be produced from a plant, and the plant-derived PDRN produced thereby has skin protection against ultraviolet radiations, skin barrier enhancement, moisturization, wrinkle alleviation, skin regeneration, wound healing, or anti-aging effects, and thus can be advantageously used as a cosmetic composition for improving skin conditions, a pharmaceutical composition, a food composition, a health functional food, a quasi-drug composition, and a feed composition.

Description

Method for producing PDRN from plants

[0001] The present invention relates to a method for producing PDRN (polydeoxyribonucleotide) from a plant, PDRN produced by the method, a cosmetic composition for improving skin, a pharmaceutical composition, a quasi-drug composition, a food composition, a health functional food, and a feed composition containing the PDRN as an active ingredient.

[0002]

[0003] Polydeoxyribonucleotide (PDRN), recently used as a key ingredient in cosmeceuticals and bio-cosmetics, is a DNA-derived polymer that promotes the self-regeneration of damaged bodies and tissues. PDRN is a tissue regeneration active substance. PDRN is a DNA fragment made by chopping DNA into small pieces using various physical or chemical methods. When administered in the body, it stimulates the adenosine A2A receptor on the surface of tissues, promoting body regeneration, accelerating wound healing, and reducing pain.

[0004] PDRN is manufactured primarily from DNA extracted from salmon semen, and is primarily used for wound treatment and tissue repair caused by skin grafting, but is also used for tissue regeneration or inflammation treatment for which there is no suitable treatment, depending on the medical professional's judgment (Korean Patent No. 10-1722181). It is known to be used for the treatment of a wide range of disorders such as diabetic ulcers, scars, vascular insufficiency, and female pattern hair loss.

[0005] PDRN, an animal-derived ingredient extracted from salmon semen, has proven its effectiveness. However, it is not suitable for "vegan" products, which do not contain animal ingredients, or "cruelty-free" cosmetics, which are not tested on animals. In particular, vegan certification is granted only to products that do not use any animal-derived ingredients, which not only protects animals but also minimizes skin irritation. Therefore, attempts are being made to extract PDRN from plants, but tangible results have been limited to date.

[0006] Against this backdrop, the inventors of the present invention have diligently studied methods for producing PDRN from plant materials, developed a method for producing high-purity PDRN from plants, and confirmed that PDRN produced by this method has effects of improving wrinkles, healing wounds, protecting the skin from ultraviolet rays, strengthening the skin barrier, moisturizing, and anti-aging, thereby completing the present invention.

[0007]

[0008] The present invention aims to solve the above-mentioned problems and other problems related thereto.

[0009] An exemplary object of the present invention is to provide a method for producing PDRN (polydeoxyribonucleotide) from a plant, comprising the following steps.

[0010] (a) A step of freezing a plant by mixing sodium chloride and a surfactant;

[0011] (b) a step of crushing the frozen plant body and then culturing it;

[0012] (c) a step of obtaining genomic DNA after the above culturing; and

[0013] (d) A step of obtaining PDRN by sonicating the above genomic DNA.

[0014] Another exemplary object of the present invention is to provide PDRN produced by the above production method.

[0015] Another exemplary object of the present invention is to provide a cosmetic composition for improving skin, comprising the PDRN as an active ingredient.

[0016] Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating any skin disease selected from the group consisting of ultraviolet-induced skin diseases, psoriasis, skin wounds, skin scars, and skin inflammation, comprising the PDRN as an active ingredient.

[0017] Another exemplary object of the present invention is to provide a pharmaceutical composition for improving skin, comprising the PDRN as an active ingredient.

[0018] Another exemplary object of the present invention is to provide a food composition for improving skin, comprising the PDRN as an active ingredient.

[0019] Another exemplary object of the present invention is to provide a feed composition for improving skin, comprising the PDRN as an active ingredient.

[0020] The technical problem to be achieved according to the technical idea of ​​the invention disclosed in this specification is not limited to the problem to solve the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0021]

[0022] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0023] As one aspect for achieving the above object, the present invention provides a method for producing PDRN (polydeoxyribonucleotide) from a plant, comprising the following steps.

[0024] (a) A step of freezing a plant by mixing sodium chloride and a surfactant;

[0025] (b) a step of crushing the frozen plant body and then culturing it;

[0026] (c) a step of obtaining genomic DNA after the above culturing; and

[0027] (d) A step of obtaining PDRN by sonicating the above genomic DNA.

[0028] The term "PDRN" of the present invention refers to polydeoxyribonucleotide, which is a mixture of short deoxyribonucleotides. That is, it is a low molecular weight DNA complex made by fragmenting a DNA chain into a certain size. PDRN used in the present invention may have a size of 2000 bp or less and a molecular weight of 74 kDa or less. The term "PDRN" may be a type of the nucleic acid fragment.

[0029] In the present invention, the plant body is a concept including the whole plant or a part of the plant body, for example, a leaf, stem, root or a part thereof, and includes a culture solution in which the whole or a part of the plant body is cultured.

[0030] In the present invention, the culturing in step (b) may be performed at 60°C to 70°C, and specifically, may be performed at 65°C, but is not limited thereto.

[0031] In the present invention, the freezing may be performed at -90°C to -70°C, specifically at -85°C to -75°C, and more specifically at -80°C.

[0032] In the present invention, the crushing may be performed by i) first crushing the frozen plant body by thawing it at room temperature, and ii) secondarily crushing it through physical crushing.

[0033] Plants contain 70-80% moisture inside, so cells can be naturally destroyed during the thawing process after the freezing step as described above.

[0034] In the present invention, the concentration of the sodium chloride may be 0.1 M to 1 M, specifically, 0.3 M to 0.8 M, more specifically, 0.4 M to 0.6 M, and even more specifically, 0.5 M, but is not limited thereto.

[0035] In the present invention, the PDRN production method can be applied to all plants, regardless of species. As an embodiment of the present invention, it was confirmed through examples that PDRN was produced by applying the production method to hibiscus (Hibiscus sabdariffa) and Houttuynia cordata, which are of different genus and species.

[0036] As another aspect for achieving the above object, the present invention provides PDRN produced by the above production method.

[0037] As another aspect for achieving the above purpose, the present invention provides a cosmetic composition for skin improvement comprising the PDRN as an effective ingredient.

[0038] In the present invention, “skin improvement” means any act of improving or benefiting the skin by using PDRN produced by the production method of the present invention.

[0039] In the present invention, the skin improvement may be, for example, skin protection from ultraviolet rays, skin barrier strengthening, moisturizing, wrinkle improvement, skin regeneration, wound healing, or anti-aging.

[0040] The term "comprising as an effective ingredient" in the present invention means including an effective amount capable of exhibiting various skin improvement effects as described above as a composition.

[0041] The cosmetic composition of the present invention can be manufactured in various forms. For example, the cosmetic composition can be manufactured in any formulation commonly manufactured in the art, and can be formulated as, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. In addition, specifically, the cosmetic composition can have a formulation selected from the group consisting of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisture cream, a hand cream, an essence, a nutrition essence, a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, an emulsion, a lipstick, a makeup base, a foundation, a pressed powder, and a loose powder, but is not limited thereto.

[0042] The cosmetic composition of the present invention may include, in addition to its active ingredient, a carrier acceptable for cosmetic formulations. Here, the term "acceptable carrier for cosmetic formulations" refers to a compound or composition already known and used, or a compound or composition to be developed in the future, that can be included in cosmetic formulations and that does not exhibit toxicity, instability, or irritation beyond what the human body can tolerate when in contact with the skin. The carrier may be included in the cosmetic composition of the present invention in an amount of about 1 wt % to about 99.99 wt %, preferably about 5 wt % to about 99 wt % of the total weight of the composition.

[0043] However, since the above ratio varies depending on the form in which the cosmetic of the present invention is manufactured as described above, as well as on its specific application site (face or hands) and its desired application amount, the above ratio should not be understood as limiting the scope of the present invention in any way.

[0044] Meanwhile, the carrier may include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, UV blocker, colorant, fragrance, etc. Compounds or compositions that can be used as the carrier, such as alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, UV blocker, colorant, fragrance, etc. are already known in the art, so a person skilled in the art can select and use an appropriate corresponding material or composition.

[0045] As another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating any skin disease selected from the group consisting of skin diseases caused by ultraviolet rays, psoriasis, skin wounds, skin scars, and skin inflammation, comprising the PDRN as an active ingredient.

[0046] In the present invention, the skin disease caused by ultraviolet rays may be at least one selected from the group consisting of sunburn, erythema, edema, pigmentation, skin inflammation, skin keratinization, photoallergy, phototoxicity, photosensitivity, and photoaging.

[0047] In the present invention, "prevention" means any action that improves a skin disease by administering the pharmaceutical composition of the present invention, and "treatment" in the present invention means any action that improves, alleviates, or beneficially alters a skin disease that has developed by administering the pharmaceutical composition of the present invention. In the present invention, "improvement" means any action that improves a skin disease by administering the composition.

[0048] In the present invention, the "pharmaceutical composition" means a composition manufactured for the purpose of preventing or treating a disease, and each can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., depending on the route of administration, and can be formulated and used in the form of topical preparations and sterile injectable solutions. Specifically, the administration route can be any appropriate route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes can also be used in combination. An example of a combination of two or more routes is a case where two or more drug formulations are combined according to the route of administration, for example, a case where one drug is administered first via the intravenous route and another drug is administered secondarily via the topical route.

[0049] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specific examples thereof may be found in the pharmacopoeias of each country, including the 'Korean Pharmacopoeia'. Carriers, excipients, and diluents that may be included in the composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, carriers, excipients, and diluents that may be included in the composition of the present invention may be non-natural carriers, but are not limited thereto.

[0050] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the above compound with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. Specific formulations of pharmaceutical compositions are known in the art, and reference may be made, for example, to Remington's Pharmaceutical Sciences (19th ed. 1995), which is incorporated herein by reference.

[0051] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, type and severity of the disease, drug activity and sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent drug use, and other factors well known in the medical field. The dosage and frequency of administration do not limit the scope of the present invention in any way.

[0052] The pharmaceutical composition of the present invention can be administered to mammals, including rats, dogs, cats, cows, horses, pigs, and humans, via various routes, preferably to humans. Any route of administration is conceivable, including, but not limited to, oral, intravenous, intramuscular, or subcutaneous injection.

[0053] In the present invention, the meaning of “comprising as an effective ingredient” means including an effective amount capable of exhibiting a preventive or therapeutic effect on a skin disease as a pharmaceutical composition.

[0054] As another aspect for achieving the above purpose, the present invention provides a pharmaceutical composition for skin improvement comprising the PDRN as an active ingredient.

[0055] The terms “PDRN” and “skin improvement” of the present invention are as described above.

[0056] The quasi-drug composition of the present invention may include, in addition to the PDRN, ingredients commonly used in quasi-drug compositions, such as, but not limited to, an abrasive, a humectant, a binder, a foaming agent, a sweetener, a preservative, a medicinal ingredient, a flavoring agent, a pigment, a solvent, a whitening agent, a solubilizer, or a pH adjuster.

[0057] The quasi-drug composition of the present invention may be exemplified as an external preparation, powder, sterilizing disinfectant, toothpaste, ointment, lotion, internal preparation (vitamin / mineral preparation, tonic), wet tissue, spray patch, band or patch, etc., but is not particularly limited thereto, and the formulation method, dosage, method of use, components, etc. of the quasi-drug may be appropriately selected by a person skilled in the art from conventional techniques known in the art.

[0058] As another aspect for achieving the above purpose, the present invention provides a food composition for improving skin, comprising the PDRN as an effective ingredient.

[0059] The terms “PDRN” and “skin improvement” of the present invention are as described above.

[0060] The food composition of the present invention can be manufactured into various dosage forms, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long period of time by using food as a raw material. The food composition of the present invention can be manufactured into any form, and specifically, it can be one or more dosage forms selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, beverages, gums, and candies, but is not particularly limited thereto.

[0061] In addition, the food composition of the present invention may contain food additives in addition to the effective ingredients. Food additives can generally be understood as substances added to, mixed with, or infiltrated into food during the manufacturing, processing, or preservation of food, and their safety must be guaranteed because they are consumed daily and over a long period of time with food. The food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. in terms of function, and are not particularly limited as long as they are consistent with the purpose to be achieved by the food composition of the present invention. In addition, the food composition of the present invention may contain, in addition to the food additives, physiologically active substances or minerals known in the art for the purpose of functionality and nutritional supplementation and guaranteed for safety as food additives. The physiologically active substances or minerals are not particularly limited as long as they are consistent with the purpose to be achieved by the food composition of the present invention.

[0062] The food composition of the present invention may include the aforementioned food additives in an effective amount that can achieve the purpose of addition depending on the product type, and with respect to other food additives that may be included in the food composition of the present invention, reference may be made to the food codes or food additive codes of each country.

[0063] In the present invention, the term “active ingredient” includes an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0064] In addition to the active ingredient PDRN, the composition of the present invention may contain a compound or natural extract known to have a skin improvement effect so as to increase or enhance the skin improvement effect.

[0065] In the present invention, the effective ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit skin improvement activity. Typically, the effective amount may be included within the range of 0.001 wt % to 99.99 wt % based on the total weight of the composition. Here, "effective amount" refers to the amount of the effective ingredient capable of inducing a skin improvement effect. This effective amount can be experimentally determined within the normal ability of a person skilled in the art.

[0066] As another aspect for achieving the above purpose, the present invention provides a health functional food for improving skin, comprising the PDRN as an effective ingredient.

[0067] The terms “PDRN” and “skin improvement” of the present invention are as described above.

[0068] The term "health functional food" in the present invention refers to a food manufactured and processed using raw materials or ingredients with functionality beneficial to the human body. The term "functionality" refers to obtaining a beneficial effect for health purposes, such as regulating nutrients or physiological functions in relation to the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during manufacturing.

[0069] In addition, the formulation of the above health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food, and non-limiting examples of the formulation include one or more formulations selected from the group consisting of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, beverages, gums, and candies, but are not particularly limited thereto.

[0070] As another aspect for achieving the above purpose, the present invention provides a feed composition for improving skin, comprising the PDRN as an effective ingredient.

[0071] The terms “PDRN” and “skin improvement” of the present invention are as described above.

[0072] The above feed composition may include feed additives. The feed additives of the present invention fall under the category of supplementary feed under the Feed Management Act.

[0073] In the present invention, the term "feed" may mean any natural or artificial diet, meal, etc. or a component of said meal, especially for eating, ingesting, and digesting by an animal or suitable therefor.

[0074] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant-based feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal-based feed such as proteins, inorganic substances, oils, minerals, oils, simple proteins, zooplankton, or food. These may be used alone or in combination of two or more types.

[0075] As another aspect for achieving the above object, the present invention provides a skin improvement method comprising a step of administering or applying a composition containing the PDRN to a subject in need thereof in an effective amount for skin improvement.

[0076] The terms “PDRN” and “skin improvement” of the present invention are as described above.

[0077] In the present invention, “effective amount” means an amount sufficient for the composition of the present invention described above to achieve skin improvement efficacy.

[0078] The term "subject" as used herein is not particularly limited to, but may include, for example, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit or a guinea pig, and is preferably a mammal, and more preferably a human.

[0079] As used herein, the term "administration" refers to providing a given substance to a subject by any suitable method. Furthermore, the term "application" as used herein refers to any method of contacting a composition according to the present invention with the skin of a subject by any suitable method, with the aim of allowing the composition to be absorbed into the skin.

[0080] The composition of the present invention may be administered orally or parenterally via any conventional route capable of reaching the target tissue. Furthermore, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells, tissues, or organs.

[0081] Another aspect of the present invention for achieving the above object provides a skin improvement use of the PDRN of the present invention.

[0082] Another aspect of the present invention for achieving the above object provides the use of PDRN for the preparation of a cosmetic composition for improving skin.

[0083] Another aspect of the present invention for achieving the above object provides the use of PDRN for the preparation of a pharmaceutical composition for preventing or treating skin diseases.

[0084] The terms “PDRN” and “skin improvement” of the present invention are as described above.

[0085]

[0086] According to the present invention, high-purity and high-concentration PDRN can be produced from a plant, and the plant-derived PDRN thus produced has effects of protecting the skin from ultraviolet irradiation, strengthening the skin barrier, moisturizing, improving wrinkles, regenerating the skin, healing wounds, or anti-aging, and can be usefully used as a cosmetic composition for improving the skin, a pharmaceutical composition, a food composition, a health functional food, a quasi-drug composition, and a feed composition.

[0087]

[0088] Figure 1 shows the results of confirming PDRN obtained from the plants of Eoseongcho and Hibiscus.

[0089]

[0090] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0091]

[0092] Example 1. PDRN extraction from plants

[0093] 10 g of each plant of Houttuynia Cordata and Hibiscus sabdariffa were frozen at -80℃ (deepfreezer) with 2 parts by weight of lysis buffer per 1 part by weight of the total sample. The lysis buffer contained 37.5 mL of distilled water, 1.16 g of sodium chloride (NaCl, final concentration 0.5 M), and 1.75 mL of surfactant (Alpicare gl 612). The frozen samples were thawed at room temperature with the lysis buffer to induce cell rupture, and the cells were ground finely using a blender to perform secondary cell disruption. 100 ㎍ / mL RNase A was added and incubated in a 65℃ water bath for 1 hour to ensure sufficient lysis of the samples. Afterwards, by-products were removed using a Miracloth filter (22-25 ㎛), and the same volume of 100% ethanol was added to the liquid passing through the filter, and the precipitate was precipitated at -80℃ for 12 hours. After centrifugation at 13,000 rpm for 10 minutes, only the supernatant was collected, and the surface of the precipitated DNA was washed by mixing the same volume of 80% ethanol with the supernatant, and then centrifuged at 13,000 rpm for 2 minutes. The ethanol was discarded, the pellet was completely air-dried, and purified water was added to obtain genomic DNA. The obtained genomic DNA was sonicated 4 or 8 times to obtain PDRN with a small molecular weight of 100-300 bp (Fig. 1). The purity of the obtained PDRN was measured to be 1.8 or more and less than 2.0 (Table 1). The above purity was determined by the ratio of the absorbance value at 260 nm / the absorbance value at 280 nm (A260 / 280 ratio). If the A260 / 280 ratio is less than 1.8, the DNA concentration is considered very low or there is protein contamination. If it is 1.8 to 1.9, the purity is considered good, and if it is 2.0 or higher, the DNA concentration is considered high, or there is RNA contamination or DNA fragmentation.

[0094] Plant Concentration (ng / ㎕) Purity (260A / 280A) gDNA Houttuynia cordata_1318.71.86 gDNA Houttuynia cordata_2302.51.85 PDRN Houttuynia cordata_1 (ultrasonic treatment 4 times) 328.51.88 PDRN Houttuynia cordata_1 (ultrasonic treatment 8 times) 312.51.87 gDNA Hibiscus_1107.61.85 gDNA Hibiscus_2101.81.88 PDRN Hibiscus_1 (ultrasonic treatment 4 times) 109.51.86 PDRN Hibiscus_1 (ultrasonic treatment 8 times) 101.51.88

[0095]

[0096] Example 2. Evaluation of the skin improvement efficacy of PDRN

[0097] 2-1. Confirmation of non-irritation of skin cells

[0098] An experiment was conducted to determine whether the PDRN obtained in Example 1 causes irritation to skin cells.

[0099] Specifically, HaCaT cells (keratinocytes) and Detroit cells (fibroblasts) were seeded in 96-well plates with DMEM (Dubelcco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% Antibiotic-Antimycotic, respectively, and cultured in an incubator at 5% CO2 and 37°C for 24 hours. Afterwards, the control group with purified water added and the experimental group treated with PDRN derived from the plant of Eoseongcho at 0.5%, 1%, and 5% by weight of each cell were cultured for an additional 24 hours, respectively.

[0100] 4 μL of 5 mg / mL MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution was added to each well and incubated for 4 hours to react. After removing the culture medium, 100 μL of DMSO (Dimethyl sulfoxide) solution was added to dissolve the sieve, and the absorbance was measured at 540 nm.

[0101] As a result, when PDRN derived from the plant of Eoseongcho was treated to HaCaT cells and Detroit cells, the cell viability increased compared to the control group, confirming that it was harmless and did not cause toxicity in the skin (Table 2).

[0102] Control group PDRN0.5% PDRN1% PDRN5% Cell viability (%) HaCaT cells 100103112119 Detroit cells 100106110113

[0103]

[0104] 2-2. Check the wrinkle improvement effect

[0105] In order to examine the wrinkle improvement effect of PDRN obtained in Example 1, the biosynthetic amount of procollagen (Procollagen Type I C-Peptide, PIP), a collagen synthesis precursor, was measured.

[0106] Specifically, Detroit551 cells were seeded on a 48-well plate and cultured for 24 hours under the same conditions as in Example 3-1. Thereafter, the control group containing purified water, the experimental group treated with 0.5%, 1%, and 5% by weight of PDRN derived from the plant of Eoseongcho, and the positive control group treated with 5 ng / ml TGF-β1 were cultured for an additional 48 hours. Then, 20 μL of the diluted supernatant of the cultured medium was placed into each antibody-coated microtiter plate along with 20 μL of the PIP standard substance in the PIP EIA Kit (Takara, Cat. MK101), and 100 μL of the peroxidase-labeled antibody solution was also added to each well, and the cells were reacted at 37°C for 3 hours. The medium was removed, washed four times with 200 μL of PBS, and 100 μL of the substrate solution was added to each well, followed by reaction at room temperature for 15 minutes in a light-shielded state. Afterwards, 100 μL stop solution (1 N H2SO4) was added, and the absorbance at 450 nm was measured.

[0107] As a result, it was confirmed that the amount of PIP synthesis increased in cells treated with PDRN derived from the plant of Eoseongcho at different concentrations compared to the control group, indicating that there would be a wrinkle improvement effect (Table 3).

[0108] Control group Positive control group PDRN0.5% PDRN1% PDRN5% PIP Content(%)100125104121131

[0109]

[0110] 2-3. Confirmation of wound healing and anti-aging effects

[0111] In order to examine the skin cell regeneration effect of PDRN obtained in Example 1, a wound healing assay was used to observe whether the test substance promoted cell proliferation and migration, thereby evaluating the anti-aging efficacy.

[0112] Specifically, HaCaT cells were seeded on a 24-well plate and cultured for 24 hours under the same conditions as in Example 2-1. Thereafter, the control group with purified water added, the experimental group treated with 0.5%, 1%, or 2% by weight of PDRN derived from the plant of Eoseongcho, and the positive control group treated with 100 ng / ml EGF were additionally cultured for 18 hours at 37°C and 5% CO2. Cell images were taken under a microscope before the additional culture and cultured. After 16 hours of culture, the medium was removed, a fixative solution (4% paraformaldehyde) was added, and the cells were incubated at room temperature for 15 minutes and washed three times with PBS. The fixed cells could be stored at 4°C for one month. The degree of wound healing was calculated using a program called Image J after taking microscopic photographs.

[0113] As a result, the healing area was confirmed to have increased in cells treated with PDRN derived from the plant of Eoseongcho at various concentrations compared to the control group, confirming the skin cell regeneration effect (Table 4).

[0114] After 16 hours of incubation, control group positive control group PDRN0.5% PDRN1% PDRN2% wound healing ability (%) 18.1640.9924.9339.5330.72

[0115]

[0116] 2-4. Skin protection against UV radiation

[0117] The skin protective effect of PDRN obtained in Example 1 against UV irradiation was evaluated.

[0118] Specifically, the light source for UV irradiation was Ultraviolet crosslinker CL-1000 (Ultra-Violet Products, CA), which emits ultraviolet light with a wavelength of 302 nm. Human keratinocytes (HaCaT) were cultured at a density of 1x10 5Cells / ml were placed in a 24-well plate and cultured in an incubator for 24 hours. The medium was removed and washed with phosphate buffer. 500 μL of phosphate buffer was added again, irradiated with UV light at 10 mJ / cm², replaced with fresh cell culture medium without FBS, and treated with PDRN derived from the plant of Eoseongcho obtained in Example 1 at various concentrations (0.5%, 1%, 5%), followed by additional culture for 24 hours. Afterwards, 5 mg / ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) was added and cultured in an incubator for 3 hours. The formed fomazan was dissolved in DMSO, transferred to a 96-well plate, and the absorbance was measured at 595 nm using an ELISA reader to confirm cell viability. As a control group, HaCaT cells (negative control) were prepared without PDRN treatment after UV irradiation, and a normal control group was prepared without UV or PDRN treatment in HaCaT cells.

[0119] As a result, it was confirmed that cell viability increased in a concentration-dependent manner when PDRN derived from the plant was treated compared to the negative control group (Table 5). Therefore, it was confirmed that the PDRN obtained in Example 1 can protect skin cells from ultraviolet rays.

[0120] Whether or not UV irradiation was performed PDRN treatment concentration (%) Cell survival rate (% of Control) Normal control group - 100 Negative control group + - 58 Experimental group + 0.56 2 1.07 8 5.07 3

[0121]

[0122] 2-5. Moisturizing effect

[0123] The skin moisturizing effect of PDRN obtained in Example 1 was evaluated.

[0124] Specifically, human keratinocyte cell line (HaCaT) was cultured in 100 mm / 60.1 cm² culture dishes with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fatal bovine serum (FBS), and 1% Antibiotic-Antimycotic (GIBCO, Cat.# 15240-062) at 37℃ and 5% CO2. When human keratinocyte cells reached more than 80% confluence, they were seeded at a concentration of 1 × 10⁴ cells / well in a 96-well plate and cultured until the confluence reached more than 80%. Once the confluence reached more than 80%, the medium was replaced with DMEM free medium (without FBS) and then treated with PDRN derived from the plant of Houttuynia cordata obtained in Example 1 at various concentrations (0.5%, 1%, 5%). Human keratinocyte cells not treated with PDRN were prepared as a control. The effect on the expression of genes related to moisturizing (Aquaporin-3, AQP-3) was confirmed by additional culture for 24 hours.

[0125] Gene expression rates were measured using the following Real-time PCR method.

[0126] Specifically, RNA isolation was performed using the FastLane Cell one-step buffer set (QIAGEN Cat.# 216213). Cells from which the medium had been removed were washed with FCW (cell wash buffer), and 50 μl of cell treatment mixture (a mixture of FCPL buffer and gDAN Wipeout buffer) was added. The mixture was incubated at room temperature for 5 minutes, transferred to a new Eppen tube, and reacted at 75°C for 5 minutes. The extracted RNA was used as a template and the PCR was performed using a Rotor Gene Q Real-time PCR Machine (Qiagen) according to the manual of 2X QuantiTect SYBR Green RT-PCR Master mix. The primers used in the experiment were normalized with Qiagen's QuantiTect® primerassays (AQP-3, Cat.# QT00212996).

[0127] As a result, it was confirmed that the expression rate of the AQP-3 gene related to moisturizing increased as the concentration of PDRN derived from the plant obtained in Example 1 increased (Table 6).

[0128] PDRN treatment concentration (%) AQP-3 gene expression rate (% of Control) Control group 01.00 Experimental group 0.5 1.3 1.02.9 5.03.1

[0129]

[0130] 2-6. Anti-aging effect

[0131] The anti-aging effect of PDRN derived from the plant of ...

[0132] As a result, it was confirmed that the expression rate of the TERT gene related to anti-aging increased as the concentration of PDRN derived from the plant obtained in Example 1 increased (Table 7).

[0133] PDRN treatment concentration (%)TERT gene expression rate (% of control)Negative control group 01.0Positive control group (1 mM SA) 05.2Experimental group 0.51.41.04.25.03.8

[0134]

[0135] 2-7. Skin barrier strengthening effect

[0136] The skin barrier strengthening effect of PDRN derived from the plant of Houttuynia cordata obtained in Example 1 was evaluated by measuring the expression level of filaggrin, a gene related to the skin barrier function of human keratinocytes. The experimental method, except for the use of QuantiTect® primerassays (Filaggrin, Cat#QT01192646, Qiagen) for gene expression measurement, was the same as in Example 2-5. If the expression of filaggrin in the skin, which is converted into a natural moisturizing factor in the skin, is promoted, the skin barrier function can be improved.

[0137] As a result, it was confirmed that as the concentration of PDRN derived from the plant of the plant obtained in Example 1 increased, the expression rate of the Filaggrin gene related to strengthening the skin barrier increased (Table 8).

[0138] PDRN treatment concentration (%) Filaggrin gene expression rate (% of control) Control group 0 1.0 Experimental group 0.5 1.2 1.0 3.9 5.0 3.4

[0139]

[0140] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and are not limiting.

[0141]

[0142] The scope of the present invention should be interpreted as including within the scope of the present invention all changes or modifications derived from the meaning and scope of the patent claims described below rather than the detailed description above and the equivalent concepts. The present invention was conducted as part of the research project "Design and Commercialization of a Synthetic Biology-Based Plant Cell Culture / Production Biofoundry System" supported by the New Growth Engine Research and Development Support Project of Incheon Technopark, Incheon Metropolitan City (Project No.: 2023-New Growth Engine-3, Research Period: 2023.04.01~2024.03.31).

Claims

1. A method for producing PDRN (polydeoxyribonucleotide) from a plant, comprising the following steps: (a) a step of freezing a plant by mixing sodium chloride and a surfactant; (b) a step of crushing and culturing the frozen plant body; (c) a step of obtaining genomic DNA after the above culturing; and (d) A step of obtaining PDRN by sonicating the above genomic DNA.

2. In paragraph 1, A production method wherein the culturing in step (b) is performed at 60°C to 70°C.

3. In paragraph 1, A production method wherein the above freezing is performed at -90°C to -70°C.

4. In paragraph 1, A production method wherein the above crushing is performed by i) first crushing the frozen plant body by thawing it at room temperature, and ii) secondarily crushing it through physical crushing.

5. PDRN produced by any one of the production methods of clauses 1 to 4.

6. A cosmetic composition for skin improvement, comprising PDRN of clause 5 as an effective ingredient.

7. In paragraph 6, The above skin improvement is a cosmetic composition for protecting the skin from ultraviolet rays, strengthening the skin barrier, moisturizing, improving wrinkles, skin regeneration, wound healing or anti-aging.

8. A pharmaceutical composition for the prevention or treatment of any one skin disease selected from the group consisting of skin diseases caused by ultraviolet rays, psoriasis, skin wounds, skin scars, and skin inflammation, containing PDRN of clause 5 as an active ingredient.

9. A pharmaceutical composition for skin improvement, comprising PDRN of Article 5 as an effective ingredient.

10. In paragraph 9, The above skin improvement is a pharmaceutical composition for protecting the skin from ultraviolet rays, strengthening the skin barrier, moisturizing, improving wrinkles, regenerating the skin, healing wounds or having anti-aging properties.

11. A food composition for improving skin, comprising PDRN of clause 5 as an effective ingredient.

12. A health functional food for skin improvement containing PDRN of Article 5 as an effective ingredient.

13. A feed composition for improving skin, comprising PDRN of Article 5 as an effective ingredient.

14. Use of PDRN for skin improvement produced by any one of the production methods of clauses 1 to 4.

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