Method for producing PDRN from gynostemma pentaphyllum plant cells

Abstract

The present invention relates to: a method for producing polydeoxyribonucleotide (PDRN) from Gynostemma pentaphyllum plant cells; PDRN produced by the method; and a cosmetic material composition, a pharmaceutical composition, a quasi-drug composition, a food composition, a health functional food, and a feed composition, each comprising the PDRN as an active ingredient for skin improvement. According to the present invention, high-purity and high-concentration PDRN can be produced from Gynostemma pentaphyllum plant cells, and the PDRN thus produced has effects of inhibiting skin inflammation, strengthening the skin barrier, moisturizing, improving wrinkles, regenerating skin, or healing wounds, and thus can be effectively used as a cosmetic material composition, a pharmaceutical composition, a quasi-drug composition, a food composition, a health functional food, and a feed composition for skin improvement.

Description

Method for producing PDRN from Gynostemma pentaphyllum plant cells

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

[0002]

[0003] Polydeoxyribonucleotide (hereinafter 'PDRN'), recently used as a key ingredient in cosmeceuticals and bio-cosmetics, is a DNA-derived polymer that acts as a tissue regeneration activator to promote the self-regeneration of damaged cells and tissues. PDRN consists of DNA fragments cut into small sizes using various physical or chemical methods. When administered into the body, it is known to stimulate adenosine A2A receptors on the surface of tissue cells, thereby promoting cell regeneration, accelerating wound healing, and reducing pain.

[0004] PDRN is manufactured primarily from DNA extracted from salmon sperm and is mainly used for the treatment of wounds and tissue repair resulting from skin grafts, but it is also used at the discretion of medical professionals for the regeneration of certain tissues or the treatment of inflammation for which there is no suitable treatment, and is known to be used in the treatment of a wide range of disorders such as diabetic ulcers, scars, vascular insufficiency, and female pattern hair loss (Republic of Korea Registered Patent No. 10-1722181).

[0005] PDRN is an animal-derived ingredient extracted from salmon sperm. Although its efficacy has been proven, it is not suitable for "vegan" products that do not contain animal-derived ingredients or for "cruelty-free" cosmetics that have not undergone animal testing. In particular, since vegan certification is granted only to products that do not use any ingredients obtainable from animals, it offers the advantage of being less irritating to the skin as well as protecting animals. Therefore, methods to extract PDRN using plant-based sources instead of animals are being attempted, but tangible results have been insufficient so far.

[0006] Against this backdrop, the inventors, through diligent research efforts on a method for producing PDRN from plant materials, developed a method for producing high-purity PDRN from Gynostemma pentaphyllum plant cells and confirmed that the PDRN produced by this method has effects of inhibiting skin inflammation, moisturizing, strengthening the skin barrier, improving wrinkles, skin regeneration, and wound healing, thereby completing the present invention.

[0007]

[0008] The present invention aims to solve the aforementioned problem and other related problems.

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

[0010] (a) A step of inducing plant cells by culturing *Gynostemma pentaphyllum* in a medium containing sucrose;

[0011] (b) a step of obtaining genomic DNA after i) primary crushing the plant cells through physical crushing and ii) secondary crushing through high-frequency treatment; and

[0012] (c) A step of obtaining PDRN by ultrasonically treating the above genomic DNA.

[0013] Another exemplary objective of the present invention is to provide PDRN produced by the above production method.

[0014] Another exemplary objective of the present invention is to provide a cosmetic composition for skin improvement comprising the PDRN as an active ingredient.

[0015] Another exemplary objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of any one skin disease selected from the group consisting of psoriasis, skin wounds, skin scars, and skin inflammation, comprising the PDRN as an active ingredient.

[0016] Another exemplary objective of the present invention is to provide a quasi-drug composition for skin improvement comprising the PDRN as an active ingredient.

[0017] Another exemplary objective of the present invention is to provide a food composition for skin improvement comprising the PDRN as an active ingredient.

[0018] Another exemplary objective of the present invention is to provide a feed composition for skin improvement comprising the PDRN as an active ingredient.

[0019] The technical problems to be solved according to the technical concept of the invention disclosed in this specification are not limited to those for solving the problems mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0020]

[0021] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0022] As one embodiment for achieving the above objective, the present invention provides a method for producing PDRN (polydeoxyribonucleotide) from a plant cell, comprising the following steps.

[0023] (a) A step of inducing plant cells by culturing *Gynostemma pentaphyllum* in a medium containing sucrose;

[0024] (b) a step of obtaining genomic DNA after i) primary crushing the plant cells through physical crushing and ii) secondary crushing through high-frequency treatment; and

[0025] (c) A step of obtaining PDRN by ultrasonically treating the above genomic DNA.

[0026] The term "PDRN" in the present invention refers to a polydeoxyribonucleotide, which is a mixture of short deoxyribonucleotides. That is, it is a low molecular weight DNA complex made by fractionating a DNA chain into a certain size, and the 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 nucleic acid fragment.

[0027] The term "Gynostemma pentaphyllum" in this invention refers to a dicotyledonous plant of the Cucurbitaceae family, a perennial vine. In Korea, it grows mainly in the forests of mountains and fields on Jeju Island and Ulleungdo.

[0028] The term "plant cell" in the present invention refers to a mass of cells formed by culturing tissue cut from a plant body in a culture medium. In a broad sense, it includes a callus, which is a mass of cells that has lost the stress causing normal organ formation or tissue differentiation, or a plant tumor tissue formed by infection with Agrobacterium or the like. The callus is an unorganized mass of parenchyma cells of a plant, which is tissue formed when a plant body is wounded. In the present invention, the callus may be a callus derived from a plant stem, root, leaf, fruit, flower, or a combination thereof, but is not limited thereto. The method for inducing the callus in the present invention is not particularly limited and can be extracted according to methods commonly used in the relevant technical field. Specifically, in the present invention, to induce Gynostemma pentaphyllum plant cells, a method was used in which a medium containing sucrose was used in a basic MS medium and induced through dark culture.

[0029] In the present invention, the plant cells are obtained by culturing a part of *Gynostemma pentaphyllum*, such as a leaf, stem, root, or a part thereof, and comprise a culture medium in which a part of the *Gynostemma pentaphyllum* plant body has been cultured. Specifically, in the present invention, the plant cells may be obtained from the leaves of *Gynostemma pentaphyllum*, but are not limited thereto.

[0030] In the present invention, step (a) is a step of inducing plant cells by culturing Gynostemma pentaphyllum in a medium containing sucrose.

[0031] In step (a) above, the culture may be dark culture at 21°C to 25°C, but is not limited thereto. In the present invention, when the plant cells are cultured in light, the production of chloroplasts may increase or organization may occur, so dark culture is preferred for smooth cell induction.

[0032] In step (a) above, the medium may be MS (Murashige and Skoog), B5, GD, MS, N6, or SH medium, and specifically may be MS medium.

[0033] In step (a) above, the concentration of sucrose may be 20 g / L to 40 g / L, specifically 25 g / L to 35 g / L, and more specifically 30 g / L, but is not limited thereto.

[0034] In the present invention, step (b) is a step of obtaining genomic DNA after first crushing the plant cells through i) physical crushing and ii) secondarily crushing them through high-frequency treatment.

[0035] In the present invention, the primary crushing of step (b) may be performed using a mixer or a mortar and pestle, but is not limited thereto.

[0036] In the present invention, the secondary crushing of step (b) may be performed at a high frequency of 150 to 200 kHz, and may be performed by treating at the frequency for 30 minutes to 1 hour, but is not limited thereto.

[0037] In the present invention, step (b) may involve culturing the plant cells at room temperature after secondary lysis to obtain genomic DNA, but is not limited thereto.

[0038] In the present invention, step (b) may further include, but is not limited to, a step of removing proteins by adding one or more selected from endoprotease and exoprotease after secondary lysis of the plant cells. Specifically, the endoprotease may be Alcalase and the exoprotease may be Flavorzyme, but is not limited thereto.

[0039] In the present invention, step (b) may further include, but is not limited to, a step of removing RNA by adding RNase A after secondary lysis of the plant cells.

[0040] In the present invention, step (b) may further include a step of adding a lysis buffer before lysing the plant cells, and the lysis buffer may include sodium chloride and a surfactant, but is not limited thereto.

[0041] In the present invention, step (b) may further include a step of culturing at 60°C to 70°C after obtaining the genomic DNA, and specifically may further include a step of culturing at 65°C.

[0042] In the present invention, step (c) is a step of obtaining PDRN by ultrasonically treating the genomic DNA.

[0043] In step (c) above, the ultrasonic treatment may be performed at 20 to 30 KHz, specifically at 25 to 30 KHz, but is not limited thereto.

[0044]

[0045] In another embodiment for achieving the above objective, the present invention provides PDRN produced by the above production method.

[0046] PDRN derived from Gynostemma pentaphyllum plants produced by the production method of the present invention may have a purity of an A260 / 280 ratio of 1.8 to 2.0, but is not limited thereto.

[0047] In another embodiment for achieving the above objective, the present invention provides a cosmetic composition for skin improvement comprising the PDRN as an active ingredient.

[0048] In the present invention, "skin improvement" means any act of using PDRN produced by the production method of the present invention to improve or benefit the skin.

[0049] In the present invention, the skin improvement may be, for example, inhibition of skin inflammation, strengthening of the skin barrier, moisturization, improvement of wrinkles, skin regeneration, or wound healing.

[0050] The meaning of the term "containing as an active ingredient" in the present invention refers to including an effective amount in a composition sufficient to exhibit various skin improvement effects as described above.

[0051] The cosmetic composition of the present invention can be manufactured in various forms, for example, the cosmetic composition can be manufactured in any formulation conventionally manufactured in the art, for example, it can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. In addition, specifically, it may have a formulation selected from the group consisting of skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, essence, nourishing essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, pressed powder, and loose powder, but is not limited thereto.

[0052] The cosmetic composition of the present invention may include a carrier acceptable in a cosmetic formulation in addition to its active ingredient. Here, "a carrier acceptable in a cosmetic formulation" refers to a compound or composition that is already known and in use or will be developed in the future, which can be included in a cosmetic formulation and does not have toxicity, instability, or irritation beyond what the human body can tolerate when in contact with the skin. The said carrier may be included in the cosmetic composition of the present invention in an amount of about 1% by weight to about 99.99% by weight, preferably about 5% by weight to about 99% by weight of the composition, based on the total weight thereof.

[0053] However, since the above ratio varies depending on the aforementioned form in which the cosmetic of the present invention is manufactured, as well as on its specific application site (face or hand) or its preferred application amount, the above ratio should not be understood as limiting the scope of the present invention in any aspect.

[0054] Meanwhile, the above carrier may include alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV blocker, colorant, fragrance, etc. Since compounds or compositions that can be used as alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV blocker, colorant, fragrance, etc., which can be used as the above carrier, are already known in the art, a person skilled in the art may select and use an appropriate substance or composition.

[0055] In another embodiment for achieving the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of any one skin disease selected from the group consisting of psoriasis, skin wounds, skin scars, and skin inflammation, comprising the above PDRN as an active ingredient.

[0056] In the present invention, "prevention" refers to any act of improving a skin disease by administering the pharmaceutical composition of the present invention, and "treatment" refers to any act of improving, alleviating, or beneficially altering an onset skin disease by administering the pharmaceutical composition of the present invention. In the present invention, "improvement" refers to any act of improving a skin disease by administering the said composition.

[0057] In the present invention, "pharmaceutical composition" refers to a composition prepared for the purpose of preventing or treating a disease, and may be formulated into various forms according to conventional methods. For example, depending on the route of administration, it may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated into topical preparations and sterile injectable solutions. Specifically, the route of administration may be any suitable route including local, oral, intravenous, intramuscular, and direct absorption through mucosal tissues, and may be used in combination of two or more routes. An example of a combination of two or more routes is a case where two or more drug formulations according to the route of administration are combined, for example, when one drug is administered intravenously as a primary and another drug is administered locally as a secondary.

[0058] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers, excipients, or diluents according to conventional methods. Pharmaceutically acceptable carriers are known in the art according to the route of administration or formulation, and specifically, reference may be made to 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, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, 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.

[0059] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, or sterile injectable solutions, according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, and surfactants that are commonly used. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, and such solid formulations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above compounds. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cacao gelatin, laurin gelatin, glycerogelatin, etc., may be used. Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to literature such as [Remington's Pharmaceutical Sciences (19th ed. 1995)]. The said literature is considered to be part of this specification.

[0060] 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 with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, 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.

[0061] The pharmaceutical composition of the present invention may be administered to mammals such as rats, dogs, cats, cattle, horses, pigs, and humans via various routes, and may be preferred in humans. Any mode of administration may be anticipated and may be administered, for example, orally, intravenously, intramuscularly, or by subcutaneous injection, but is not limited thereto.

[0062] In the present invention, the meaning of "containing as an active ingredient" refers to including an effective amount sufficient to exhibit a preventive or therapeutic effect on skin diseases as a pharmaceutical composition.

[0063]

[0064] In another embodiment for achieving the above objective, the present invention provides a method for preventing, improving, or treating any one of the skin diseases selected from the group consisting of psoriasis, skin wounds, skin scars, and skin inflammation, comprising the step of administering the PDRN to an individual.

[0065] The terms "PDRN" and "prevention," "improvement," and "treatment" of skin diseases of the present invention are as described above.

[0066] In the present invention, the PDRN may be administered to an individual requiring it in an effective therapeutic amount for skin diseases. In this case, the effective therapeutic amount refers to an amount of the PDRN of the present invention described above that is sufficient to achieve efficacy in preventing, improving, or treating skin diseases.

[0067] In the present invention, "individual" includes, for example, mammals such as rats, dogs, cats, cows, horses, pigs, and humans, but is not particularly limited, and preferably means humans.

[0068] In the present invention, "administration" means providing a specific substance to an individual by any appropriate method, and the route of administration may be oral or parenteral through any general route capable of reaching the target tissue, and may also be administered using any device capable of delivering the active ingredient to a target cell, tissue, or organ.

[0069]

[0070] As another embodiment for achieving the above objective, the present invention provides a quasi-drug composition for skin improvement comprising the above PDRN as an active ingredient.

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

[0072] The quasi-drug composition of the present invention may include ingredients commonly used in quasi-drug compositions in addition to the PDRN, such as abrasives, wetting agents, binders, foaming agents, sweeteners, preservatives, active ingredients, flavoring agents, coloring agents, solvents, whitening agents, solubilizers, or pH adjusters, but is not limited thereto.

[0073] Examples of the quasi-drug compositions of the present invention include external preparations, powders, disinfectants, toothpastes, ointments, lotions, internal preparations (vitamin-mineral preparations, nutritional tonics), wet wipes, spray patches, bandages, or patches, but are not particularly limited thereto. The formulation method, dosage, method of use, and components of the quasi-drug may be appropriately selected by a person skilled in the art from the ordinary technology known in the relevant technical field.

[0074] In another embodiment for achieving the above objective, the present invention provides a food composition for skin improvement comprising the PDRN as an active ingredient.

[0075] The terms "PDRN" and "skin improvement" of the present invention are as described above.

[0076] The food composition of the present invention can be manufactured in various forms of formulations, and unlike general pharmaceuticals, it has the advantage of not causing side effects that may occur during long-term use of pharmaceuticals by using food as a raw material. The food composition of the present invention can be manufactured in any form, and specifically, it may be 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, and bars, as well as beverages, gums, and candies, but is not particularly limited thereto.

[0077] In addition, the food composition of the present invention may include food additives in addition to its active ingredients. Food additives can generally be understood as substances added to, mixed with, or permeated into food during the manufacture, processing, or preservation of food; since they are consumed daily and over a long period along with food, their safety must be guaranteed. The food additives are classified in terms of function into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc., and are not particularly limited as long as they meet the purpose to be achieved by the food composition of the present invention. Furthermore, in addition to the above food additives, the food composition of the present invention may include physiologically active substances or minerals known in the art for the purpose of functionality and nutritional supplementation, and whose safety as food additives is guaranteed. The physiologically active substances or minerals are not particularly limited as long as they meet the purpose to be achieved by the food composition of the present invention.

[0078] The food composition of the present invention may include the aforementioned food additives in an effective amount capable of achieving the purpose of their addition depending on the product type, and regarding 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.

[0079] In the present invention, the term “active ingredient” includes a component that exhibits the intended activity alone or can exhibit activity together with a carrier that is inactive itself.

[0080] In addition to PDRN, which is an active ingredient, the composition of the present invention may include compounds or natural extracts known to have skin-improving effects to enhance or reinforce the skin-improving effect.

[0081] In the present invention, the active ingredient may be included in any amount (effective amount) depending on the use, formulation, purpose of combination, etc., as long as it exhibits skin improvement activity. A typical effective amount may be included within the range of 0.001% by weight to 99.99% by weight based on the total weight of the composition. Here, "effective amount" refers to the amount of the active ingredient capable of inducing a skin improvement effect. Such effective amount may be determined experimentally within the ordinary capacity of a person skilled in the art.

[0082] In another embodiment for achieving the above objective, the present invention provides a health functional food for skin improvement comprising the above PDRN as an active ingredient.

[0083] The terms "PDRN" and "skin improvement" of the present invention are as described above.

[0084] In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body. The "functional properties" mean obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding 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 industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry during manufacturing.

[0085] In addition, the formulation of the above-mentioned health functional food may be manufactured without restriction as long as it is a formulation recognized as a health functional food, and non-limiting examples of the above-mentioned formulation may be 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, and bars, as well as beverages, gums, and candies, but are not particularly limited thereto.

[0086] In another embodiment for achieving the above objective, the present invention provides a feed composition for skin improvement comprising the PDRN as an active ingredient.

[0087] The terms "PDRN" and "skin improvement" of the present invention are as described above.

[0088] The above feed composition may include a feed additive. The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act.

[0089] In the present invention, the term "feed" may specifically refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, for or suitable for animals to eat, consume, and digest.

[0090] The types of the above feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of the above feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, oils and fats, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0091] As another embodiment for achieving the above objective, the present invention provides a skin improvement method in which an effective amount of PDRN for skin improvement is administered to a subject in need.

[0092] In the present invention, "effective amount" refers to an amount of the composition of the present invention described above that is sufficient to achieve skin improvement efficacy.

[0093] In this specification, the term "subject" includes, but is not specifically limited to, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, and preferably means mammals, more preferably humans.

[0094] In this specification, the term "administration" means providing a specific substance to a subject by any appropriate method.

[0095] The administration route of the composition of the present invention may be oral or parenteral, as long as it can reach the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells, tissues, or organs.

[0096] Another aspect of the present invention for achieving the above objective provides a use of PDRN for skin improvement.

[0097] Another aspect of the present invention for achieving the above objective provides the use of PDRN for the manufacture of a drug for the prevention, improvement, or treatment of skin diseases.

[0098]

[0099] According to the present invention, high-purity and high-concentration PDRN can be produced from Gynostemma pentaphyllum plant cells, and the PDRN derived from Gynostemma pentaphyllum plant cells produced accordingly has effects of inhibiting skin inflammation, strengthening the skin barrier, moisturizing, improving wrinkles, regenerating skin, or healing wounds, so it can be usefully used as a cosmetic composition for skin improvement, a pharmaceutical composition, a food composition, a health functional food, a quasi-drug composition, and a feed composition.

[0100]

[0101] Figure 1 is the result of confirming PDRN obtained from Gynostemma pentaphyllum plant cells.

[0102] Figure 2 shows the results of confirming the wound healing effect by treating HaCaT cells with PDRN derived from Gynostemma pentaphyllum plant cells.

[0103] Figure 3 is a graph showing the wound healing area after treating HaCaT cells with PDRN derived from Gynostemma pentaphyllum plant cells.

[0104]

[0105] The present invention will be explained in more detail below through the following examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0106]

[0107] Example 1. Extraction of PDRN from Gynostemma pentaphyllum plant cells

[0108] 1-1. Plant Cell Induction and Culture

[0109] After immersing the leaves of the *Gynostemma pentaphyllum* plant in 70% ethanol for 30 seconds, washing with sterile water, disinfecting again with a disinfectant solution (30% bleach + Tween 20) for 20 minutes, and rinsing three times with sterile water, a single incision was made using a sharp knife, and cells were induced by dark culture under growth conditions of 25°C and 70% humidity in basic MS medium (Murashige and Skoog 1962, Duchefa, Cat No. M0221) containing 1 mg / L Zeatin, 3% Sucrose, and 8 g / L agar.

[0110] Plant cells of *Gynostemma pentaphyllum* induced in solid medium were subcultured at 2-week intervals, and liquid mass culture was performed in a 20 L bioreactor (purchased from: Samsung Science & Technology Co., Ltd.) with an air supply of approximately 0.1 vvm for 5 weeks under culture room conditions of 23±2 ℃ and 70% humidity, and harvested for use in PDRN isolation.

[0111]

[0112] 1-2. DNA Extraction and PDRN Acquisition from Plant Cells

[0113] In Example 1-1, 100 g of Gynostemma pentaphyllum plant cells were mixed with a lysis buffer (composition: 0.5 M sodium chloride, vegan-certified surfactant) and finely ground in a mixer or mortar to perform primary crushing, and then treated at a high frequency of 150 to 200 kHz for 30 minutes to 1 hour to perform secondary crushing.

[0114] Subsequently, to remove RNA, RNase A (20 μg / g FW) was added, and to remove proteins, 0.5% Alcalase (Endo-Protease) and 1% Flavourzyme (Exo-Protease) were added. The mixture was allowed to react sufficiently at room temperature for 30 to 60 minutes. After sufficient reaction, byproducts were removed by centrifugation, and only the supernatant was collected. Then, an equal volume of 95% ethanol was added, and the mixture was precipitated at -20 to -80°C for 12 hours. Afterward, the mixture was centrifuged at 4,500 rpm for 20 minutes to secure the settled DNA pellet, and the supernatant was discarded. Next, 80% ethanol was added, and the mixture was centrifuged at 4,500 rpm for 10 minutes, leaving only the DNA pellet and discarding the supernatant. The DNA pellet was completely dried at 65°C for at least one hour, and then purified water was added to obtain the genomic DNA.

[0115] The obtained genomic DNA was treated with an ultrasonic cleaner (28 KHz) to obtain low-molecular-weight PDRN between 100 and 200 bp. As shown in Fig. 1, the purity of the obtained PDRN was measured to be between 1.8 and less than 2.0. The purity was determined by the ratio of the absorbance value at 260 nm to the absorbance value at 280 nm (A260 / 280 ratio). If the A260 / 280 ratio is less than 1.8, it is judged that the DNA concentration is very low or there is protein contamination; if it is between 1.8 and 1.9, it is judged that the purity is good; and if it is 2.0 or higher, it is considered that the DNA concentration is high or that RNA contamination or DNA fragmentation has occurred.

[0116]

[0117] Example 2. Evaluation of PDRN's skin improvement efficacy

[0118] 2-1. Confirmation of Non-irritating to Skin Cells

[0119] An experiment was performed to determine whether the PDRN obtained in Example 1 causes irritation to skin cells.

[0120] Specifically, HaCaT (Human Keratinocyte) and Detroit 551 (human fibroblast) were seeded into a 96-well plate with DMEM (Dulbecco Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum) and 1% Antibiotic-Antimycotic, and cultured for 24 hours in an incubator at 37°C under conditions of 5% CO2. Subsequently, a control group treated with purified water and experimental groups treated with PDRN derived from Gynostemma pentaphyllum plants at different concentrations (0.5%, 1%, 5%) were cultured for an additional 24 hours. After removing the culture medium, the D-Plus™ CCK cell viability assay solution was diluted 10-fold with FBS-free medium, and 100 µl was added to each well and reacted for 3 hours. Subsequently, absorbance was measured at 450 nm.

[0121] As a result, as shown in Table 1 below, when PDRN derived from Gynostemma pentaphyllum plant cells was treated at a concentration of 0.5 to 5 ppm, it did not show toxicity in keratinocytes (HaCaT) and fibroblasts (Detroit 551), confirming that it is a safe substance with a low potential to cause skin irritation.

[0122] PDRN Treatment Concentration (ppm) Cell Viability %HaCaTDetroit 551 Control Group 0100100 Experimental Group 0.5101.3115.01.0107.6107.05.0102.698.7

[0123]

[0124] 2-2. Confirmation of Inhibitory Effect on Skin Inflammation by COX-2 Gene Expression

[0125] COX-2 (Cyclooxygenase-2) is a gene encoding an enzyme that promotes the production of prostaglandins, which play an important role in physiological responses such as inflammation, pain, and fever. COX-2 is an inducible enzyme whose expression increases in response to specific stimuli; its expression is activated when cells are exposed to inflammatory stimuli, growth factors, cellular stress, etc., and overexpression of COX-2 is observed particularly in pathological conditions related to inflammatory responses and pain. To confirm whether the PDRN derived from Gynostemma pentaphyllum obtained in Example 1 has an inhibitory effect on skin inflammation, changes in the expression levels of the COX-2 (Cyclooxygenase-2) gene, which is involved in inflammatory responses, were investigated.

[0126] Specifically, HaCaT cells were seeded into a 96-well plate and cultured for 24 hours under cell culture conditions. After removing the medium, 50 mJ / cm² 2 Under the conditions, UVB was irradiated using UVP® CL-1000® Ultraviolet Crosslinkers to induce an inflammatory response. Subsequently, cells were treated with the control group and the test composition, and cultured for an additional 4 hours. Real-time PCR was performed to confirm expression at the gene level, and the test procedure is as follows.

[0127] SuperPrep for RNA isolation and cDNA synthesis TMA cell lysis & RT Kit for qPCR (TOYOBO, Cat. SCQ-101) was used. Cells from which the medium had been removed were washed once with PBS, 50 μL of cell lysis mixture was added and reacted for 5 minutes, after which 10 μL of stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template, and Real-time PCR analysis was performed using KAPA SYBR FAST UNI (KAPA biosystems, SIGKK4601-SIAL). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, COX-2; Cat. QT00040586), and the gene expression levels of the samples were quantified using GADPH. The real-time qPCR conditions were as follows: first, a reaction at 95°C for 5 minutes, followed by a total of 40 cycles at 94°C for 15 seconds, 60°C for 15 seconds, and 72°C for 20 seconds per cycle.

[0128] As a result, when the PDRN of the present invention was treated to skin cells at a concentration of 0.5 ppm, the COX-2 gene expression was reduced compared to the control group irradiated with ultraviolet light, confirming the anti-inflammatory effect (Table 2).

[0129] UVB Treatment Concentration (ppm) COX-2 Gene Expression Rate (% of Control) Control Group --0.35 Control Group ±1.00 Positive Control Group + Dexamethasone 5 μM 0.47 Experimental Group +0.5 0.23

[0130]

[0131] 2-3. Confirmation of moisturizing effect by AQP3 gene expression

[0132] AQP3 (Aquaporin 3) is known as a membrane transporter that is present in large quantities in the epidermis of human skin and transports water and small molecules to maintain fluid homeostasis. To confirm whether the PDRN derived from Gynostemma pentaphyllum plant cells obtained in Example 1 has moisturizing-improving efficacy, changes in the expression levels of the AQP3 gene involved in moisturization were investigated in HaCaT cells.

[0133] Specifically, HaCaT cells were seeded into a 96-well plate and cultured under cell culture conditions for 24 hours. Subsequently, the control group and the composition serving as the test substance were treated to the cells, and cultured for an additional 24 hours. Real-time PCR was performed to confirm results at the genetic level, and the test procedure is as follows.

[0134] SuperPrep for RNA isolation and cDNA synthesis TMA cell lysis & RT Kit for qPCR (TOYOBO, Cat.SCQ-101) was used. Cells from which the medium had been removed were washed once with PBS, 50 μL of cell lysis mixture was added and reacted for 5 minutes, after which stop solution was added. 8 μL of the previously extracted lysate was added to 32 μL of RT reaction mixture, and cDNA was synthesized using PCR under conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 95°C for 5 minutes. To compare and analyze gene expression, the cDNA synthesized above was used as a template, and Real-time PCR analysis was performed using KAPA SYBR FAST UNI (KAPA biosystems, SIGKK4601-SIAL). The primers used in the experiment were Qiagen's QuantiTect primer assays (GAPDH; Cat. QT01192646, AQP3; Cat. QT00212996, FLG; Cat. QT00092218, PCOLCE; Cat. QT01005725), and the gene expression levels of the samples were quantified using GADPH. The real-time qPCR conditions were as follows: first, a reaction at 95°C for 5 minutes, followed by a total of 40 cycles with 94°C for 15 seconds, 60°C for 15 seconds, and 72°C for 20 seconds per cycle.

[0135] As a result, when the PDRN of the present invention was treated to skin cells at a concentration of 0.5 ppm, the AQP3 gene expression increased compared to the control group, confirming the moisturizing improvement effect (Table 3).

[0136] Treatment Concentration (ppm) AQP-3 Gene Expression Rate (% of Control) Control Group 0 1.00 Positive Control Glyceryl glucoside 1 % 1.70 Experimental Group 0.5 1.53

[0137]

[0138] 2-4. Confirmation of skin barrier strengthening effect by FLG gene expression

[0139] Filaggrin (FLG) is known to be an important protein that affects the structure and function of the skin barrier. The skin barrier strengthening effect of PDRN derived from Gynostemma pentaphyllum plants obtained in Example 1 was evaluated by measuring the expression level of the Filaggrin gene.

[0140] Specifically, HaCaT cells were seeded into a 96-well plate and cultured under cell culture conditions for 24 hours. Subsequently, the control group and the composition of the test substance were treated to the cells, and cultured for an additional 24 hours. Real-time PCR was performed to confirm at the genetic level, and the gene expression measurement method is the same as in Examples 2-3.

[0141] As a result, when the PDRN of the present invention was treated to skin cells at a concentration of 0.5 ppm, FLG gene expression increased compared to the control group, confirming the skin barrier strengthening effect (Table 4).

[0142] Treatment Concentration (ppm) FLG Gene Expression Rate (% of control) Control Group 0.00 Positive Control Group Glyceryl glucoside 1.00 % 1.70 Experimental Group 0.5 1.71

[0143]

[0144] 2-5. Confirmation of wrinkle improvement effect by PCOLCE gene expression

[0145] Collagen exists in the human body in various forms, and in the skin, collagen type 1 is particularly abundant and plays an important role in maintaining skin elasticity and structure. Procollagen C-endopeptidase enhancer (PCOLCE) is a gene that activates enzymes acting during the procollagen degradation process, and it assists in the process of converting procollagen into collagen, thereby participating in the production of collagen in the skin. To confirm whether the PDRN derived from Gynostemma pentaphyllum obtained in Example 1 has wrinkle-improving efficacy, changes in the expression levels of the PCOLCE gene, which is involved in wrinkle improvement, were investigated in fibroblasts.

[0146] Specifically, Detroit 551 cells were seeded into a 96-well plate and cultured under cell culture conditions for 24 hours. Subsequently, the control group and the composition of the test substance were treated to the cells, and cultured for an additional 24 hours. Real-time PCR was performed to confirm at the genetic level, and the gene expression measurement method is the same as in Examples 2-3.

[0147] As a result, when the PDRN of the present invention was treated to skin cells at a concentration of 5 ppm, PCOLCE gene expression increased compared to the control group, confirming the wrinkle improvement effect (Table 5).

[0148] Treatment Concentration (ppm) PCOLCE Gene Expression Rate (% of control) Control -1.00 Positive Control TGF-β1 5ng / mL 1.59 Experimental Group 0.5 0.79 10.9 75 1.71

[0149]

[0150] 2-6. Confirmation of Wound Healing Effect

[0151] When the skin is wounded, skin cells divide to fill the loss of tissue in that area and regenerate the damaged skin tissue back to its original state. To confirm whether the PDRN derived from Gynostemma pentaphyllum obtained in Example 1 has wound healing efficacy, a scratch was made in a cell monolayer, and the test sample was treated; the skin regeneration efficacy was evaluated by measuring cell motility after a period of time.

[0152] Specifically, HaCaT cells were seeded into a 24-well plate and cultured for 24 hours. Subsequently, scratches were created using the SPLScar™ Scratcher, and cell images at the 0-hour mark were captured under a microscope. Then, the control group and the test composition were treated and cultured for an additional 16 to 20 hours. Afterward, the cell profiles at the 16 to 20-hour mark were photographed under a microscope, and the degree of wound healing was calculated by measuring the cell migration area using the Image J program. The healing area (%) was calculated as follows: Healing area (%) = (T16~20 surface area / T0 surface area) x 100.

[0153] As a result, when the composition of the present invention was applied to skin cells at concentrations of 0.5 ppm and 1 ppm, the recovery area increased compared to the control group, confirming the wound healing and skin regeneration effects (Table 6, Figs. 2 and 3).

[0154] Treatment Concentration (ppm) Wound Healing Area (%) Control Group (C) -5 2.86 Positive Control Group (PC) EGF 100 ng / mL 76.45 Experimental Group 0.56 0.56 17 1.68

[0155]

[0156] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

A method for producing PDRN (polydeoxyribonucleotide) from Gynostemma pentaphyllum plant cells, comprising the following steps: (a) A step of inducing plant cells by culturing *Gynostemma pentaphyllum* in a medium containing sucrose; (b) a step of obtaining genomic DNA after i) primary crushing the plant cells through physical crushing and ii) secondary crushing through high-frequency treatment; and (c) A step of obtaining PDRN by ultrasonically treating the above genomic DNA. In paragraph 1, A method for producing PDRN, wherein the culture in step (a) above is dark culture at 21°C to 25°C. In paragraph 1, A method for producing PDRN in which the medium of step (a) above is MS (Murashige and Skoog) medium. In paragraph 1, The above step (b) is a method for producing PDRN, wherein the plant cells are lysed a second time and then cultured at room temperature to obtain genomic DNA. In paragraph 1, A method for producing PDRN, wherein step (b) further comprises the step of removing proteins by adding one or more selected from endoprotease and exoprotease after secondary lysis of the plant cells. In paragraph 1, A method for producing PDRN, wherein step (b) further comprises the step of removing RNA by adding RNase A after secondary lysis of the plant cells. In paragraph 1, A method for producing PDRN, wherein the primary crushing in step (b) above is performed using a mixer or a mortar and pestle. In paragraph 1, A method for producing PDRN, wherein the secondary crushing in step (b) above is crushed by treating at a high frequency of 150 to 200 kHz for 30 minutes to 1 hour. PDRN produced by the production method of any one of paragraphs 1 through 8. A cosmetic composition for skin improvement comprising PDRN of claim 9 as an active ingredient. In Paragraph 10, The above-mentioned skin improvement is a cosmetic composition that inhibits skin inflammation, strengthens the skin barrier, moisturizes, improves wrinkles, regenerates skin, or heals wounds. A pharmaceutical composition for the prevention or treatment of any one skin disease selected from the group consisting of psoriasis, skin wounds, skin scars, and skin inflammation, comprising PDRN of claim 9 as an active ingredient. A quasi-drug composition for skin improvement comprising PDRN of claim 9 as an active ingredient. In Paragraph 13, The above-mentioned skin improvement is a quasi-drug composition that inhibits skin inflammation, strengthens the skin barrier, moisturizes, improves wrinkles, regenerates skin, or heals wounds. A food composition for skin improvement comprising PDRN of claim 9 as an active ingredient. A health functional food for skin improvement containing PDRN of claim 9 as an active ingredient. A feed composition for skin improvement comprising PDRN of claim 9 as an active ingredient. A method for preventing or treating any one of the skin diseases selected from the group consisting of psoriasis, skin wounds, skin scars, and skin inflammation, comprising the step of administering PDRN of claim 9 to an individual.

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