Cosmetic and pharmaceutical composition comprising PDRN with single-stranded DNA forms

Abstract

The present invention relates to a cosmetic or pharmaceutical composition containing PDRN in a single-stranded form as an active ingredient. Specifically, the present invention relates to a cosmetic composition containing PDRN in a single-stranded DNA form as an active ingredient, and furthermore, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases and skin pigmentation diseases containing PDRN in a single-stranded DNA form as an active ingredient. The composition containing PDRN in a single-stranded form according to the present invention has excellent DPPH radical scavenging ability, ABTS radical scavenging ability, superoxide scavenging ability, nitrite scavenging ability, tyrosinase activity inhibition ability, elastase activity inhibition ability, or collagenase activity inhibition ability. Therefore, it can be usefully used as a cosmetic composition having antioxidant, anti-inflammatory, skin whitening, or skin wrinkle improvement functions, as well as being usefully used as a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases and skin pigmentation diseases.

Description

Technology Field

[0001] The present invention relates to cosmetic and pharmaceutical compositions comprising PDRN in a single-strand form as an active ingredient. Background Technology

[0002] PDRN( p oly d eoxy r ibo n A ucleotide refers to a mixture of low-molecular-weight DNA fragments or polydisperse deoxyribonucleotide heteropolymers with various chain lengths and nucleotide sequences. PDRN isolated from the human placenta has been found to exhibit therapeutic effects on various diseases, particularly tissue damage; however, there have been difficulties in securing large quantities of PDRN due to cost and ethical reasons regarding its isolation from the human placenta. As a solution to this, PDRN from rainbow trout ( Oncorhynchus mykiss ) and salmon( Oncorhynchus keta PDRN has been isolated from sperm cells and used, and furthermore, recently, research is being conducted to obtain PDRN not only from marine organisms such as seaweed, sea cucumber sperm, and starfish, but also from plants such as aloe, ginseng, and roselle.

[0003] PDRN is primarily 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 some tissues 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 foot ulcers, scars, vascular insufficiency, and female pattern hair loss.

[0004] Currently, two mechanisms of action related to the pharmacological efficacy of PDRN are known. First, PDRN is known to modulate various pharmacological properties by selectively stimulating adenosine A2A receptors as an agonist. PDRN exhibits wound healing and anti-inflammatory effects through adenosine A2A receptor activation, as well as therapeutic effects for mesenchymal stem cell-based osteoarthritis. It is also known to inhibit Janus kinase / signaling factors and transcriptional pathway activators mediated by adenosine A2A receptor expression, and to suppress neuronal apoptosis in ischemia / reperfusion injury models. Second, another mechanism of action of PDRN is that its pharmacological properties are mediated by nucleotide salvage pathways. PDRN is known to be cleaved by cell membrane enzymes to supply purines and pyrimidines to various tissues, and then converted into deoxyribonucleotides to bind to DNA, thereby activating the proliferation and growth of various cells including fibroblasts, adipocytes, osteoblasts, and chondrocytes.

[0005] However, there has been no research on whether the actual active ingredient that directly influences the pharmacological efficacy of PDRN is double-stranded DNA or single-stranded DNA, or specifically which form it is. Prior art literature

[0006] Republic of Korea Published Patent 10-2023-0161776 The problem to be solved

[0007] Accordingly, the inventors confirmed for the first time that the single-stranded DNA form of PDRN produced by heat treatment, i.e., heat denaturation, has significantly superior antioxidant activity, anti-inflammatory activity, skin whitening activity, and skin wrinkle improvement activity compared to the double-stranded DNA form of PDRN, thereby identifying that the various physiological activities of PDRN are in the single-stranded DNA form rather than the double-stranded DNA form, and by identifying that a composition containing the single-stranded DNA form can be usefully used in the manufacture of functional cosmetics and pharmaceuticals, the present invention was completed.

[0008] Therefore, the object of the present invention is to provide a cosmetic composition having PDRN containing a high proportion of single-stranded DNA form as an active ingredient.

[0009] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases using PDRN containing a high proportion of single-stranded DNA as an active ingredient.

[0010] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of skin pigmentation disorders, comprising PDRN containing a large amount of a single-stranded DNA form as an active ingredient. means of solving the problem

[0011] Accordingly, the present invention provides a cosmetic composition comprising an active ingredient including PDRN containing a single-stranded DNA form.

[0012] In one embodiment of the present invention, the PDRN containing a single-stranded DNA form may be obtained by heat denaturation or the like.

[0013] In one embodiment of the present invention, the heat treatment may be performed at 90 to 100°C for 20 to 60 minutes.

[0014] In one embodiment of the present invention, the cosmetic composition may have antioxidant activity, anti-inflammatory activity, skin whitening activity, or skin wrinkle improvement activity.

[0015] In one embodiment of the present invention, the PDRN containing the single-stranded DNA form may have DPPH radical scavenging ability; ABTS radical scavenging ability; superoxide scavenging ability; nitrite scavenging ability; tyrosinase activity inhibitory ability; elastase activity inhibitory ability; or collagenase activity inhibitory ability.

[0016] In one embodiment of the present invention, the cosmetic composition may have any one formulation selected from softening lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, eye cream, cleansing cream, cleansing foam, cleansing water, lotion, emulsion, spray, or powder.

[0017] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, comprising PDRN containing a single-stranded DNA form as an active ingredient.

[0018] In one embodiment of the present invention, the inflammatory skin disease may be selected from the group consisting of contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, contact urticaria syndrome, autosensitization dermatitis, stagnant dermatitis, acne, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, eczema, and psoriasis.

[0019] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of skin pigmentation disorders, comprising PDRN containing a single-stranded DNA form as an active ingredient.

[0020] In one embodiment of the present invention, the skin pigmentation disorder may be selected from the group consisting of melasma, freckles, lentigo, age spots, moles, café au lait spots, nevus of Ota, blue nevus, hyperpigmented spots, hyperpigmentation after drug use, gravidic chloasma, and post-inflammatory hyperpigmented spots due to wounds or dermatitis. Effects of the invention

[0021] In the present invention, it was first discovered that PDRN containing a single-stranded DNA form produced by heat treatment of PDRN has superior DPPH radical scavenging ability, ABTS radical scavenging ability, superoxide scavenging ability, nitrite scavenging ability, tyrosinase activity inhibition ability, elastase activity inhibition ability, and collagenase activity inhibition ability compared to PDRN containing a double-stranded DNA form. As a result, a composition containing PDRN containing a single-stranded DNA form can be usefully used in the manufacture of cosmetics having antioxidant, anti-inflammatory, skin whitening, or skin wrinkle improvement activities, and furthermore, it has the effect of being usefully used in the manufacture of pharmaceuticals for the prevention or treatment of inflammatory skin diseases and skin pigmentation diseases. Brief explanation of the drawing

[0022] Figure 1 shows the absorbance measurement results at 260 nm for PDRN and PDRN-H (A), and also the concentration of dsDNA measured (B). Figure 2 shows that the amount of ssDNA in PDRN-H obtained by heat treatment was increased compared to PDRN, as confirmed by electrophoresis. (A) shows the results of EtBr staining after agarose gel electrophoresis, (B) shows the results of measuring densitometric patterns for PDRN and PDRN-H lanes using Image J, and (C) shows the results of comparing the areas of the PDRN and PDRN-H profiles obtained in (B). Figure 3 shows the results of analyzing the DPPH radical scavenging activity (A) and ABTS radical scavenging activity (B) for PDRN and PDRN-H, where 1 μg / ml of ascorbic acid (AA) was used as a positive control. *** indicates that P < 0.001 compared to the untreated control (PDRN). Figure 4 shows the results of analyzing the superoxide scavenging activity (A) and nitrite scavenging activity (B) for PDRN and PDRN-H, and 20 μg / ml of ascorbic acid (AA) was used as a positive control. *** indicates P < 0.001 compared to the untreated control (PDRN). Figure 5 shows the tyrosinase inhibitory activity of PDRN and PDRN-H, with results using L-tyrosine as a substrate (A) and L-dopa as a substrate (B). As positive controls, 2 μg / ml kojic acid (KA) and 10 μg / ml kojic acid (KA) were used (A) and (B) respectively, and *** indicates that P < 0.001 compared to the untreated control (PDRN). Figure 6 shows the results of (A) elastase and (B) collagenase inhibitory activities of PDRN and PDRN-H, with 20 μg / ml of EGCG used as a positive control. *** indicates P < 0.001 compared to the untreated control (PDRN). Specific details for implementing the invention

[0023] The present invention is characterized by providing a cosmetic or pharmaceutical composition comprising PDRN containing a single-stranded DNA form as an active ingredient.

[0024] While conducting research on the actual active ingredients that induce various physiological activities of PDRN, the inventors experimentally demonstrated that PDRN containing a single-stranded DNA form produced by heat treatment (thermal denaturation) of PDRN exhibits significantly superior DPPH radical scavenging activity, ABTS radical scavenging activity, superoxide scavenging activity, nitrite scavenging activity, tyrosinase activity inhibition activity, elastase activity inhibition activity, and collagenase activity inhibition activity compared to PDRN containing a double-stranded DNA form. By doing so, they identified for the first time that PDRN containing a single-stranded DNA form, rather than PDRN containing a double-stranded DNA form, is the key ingredient that directly influences various pharmacological effects.

[0025] According to one embodiment of the present invention, the antioxidant activity, superoxide scavenging activity, and nitrite scavenging activity were compared and analyzed for heat-treated PDRN (PDRN-H), in which the proportion of single-stranded DNA helical forms was significantly increased by heat treatment, and unheat-treated PDRN.

[0026] As a result, the heat-treated PDRN (PDRN-H) group, in which the proportion of single-stranded DNA helical forms increased due to heat treatment (thermal denaturation), was found to have significantly superior scavenging ability against DPPH and ABTS, known causative factors of oxidative stress, compared to untreated PDRN, which is mostly composed of double-stranded DNA helical forms; furthermore, it was found to have significantly superior scavenging ability against superoxide and nitrite.

[0027] Specifically, the heat-treated PDRN group was found to have DPPH and ABTS scavenging abilities 10.4 times and 1.6 times higher, respectively, than the non-heat-treated PDRN, the superoxide scavenging ability was found to be about 2.2 times higher in the heat-treated PDRN group compared to the non-heat-treated PDRN, and the nitrite scavenging ability was found to be about 4.7 times higher in the heat-treated PDRN group compared to the non-heat-treated PDRN.

[0028] Through these results, the inventors found that PDRN in the form of single-stranded random coiled DNA generated by heat treatment, rather than PDRN composed mostly of double-stranded DNA helices, is a substantial active ingredient capable of inducing antioxidant activity, superoxide scavenging activity, and nitrite scavenging activity.

[0029] In addition, according to another embodiment of the present invention, the degree of tyrosinase activity inhibition was analyzed for a heat-treated PDRN group and a non-heat-treated PDRN group, and as a result, it was confirmed that the heat-treated PDRN group was about 2.2 times higher than the non-heat-treated PDRN.

[0030] Through this, it was found that PDRN containing single-stranded DNA random coils generated by heat treatment, rather than PDRN composed mostly of double-stranded DNA helix, is a substantial active ingredient capable of inhibiting tyrosinase activity, and that a composition containing PDRN containing single-stranded DNA random coils can be used as a composition for skin whitening.

[0031] Furthermore, according to another embodiment of the present invention, the degree of inhibition of elastase and collagenase activity was analyzed for a heat-treated PDRN group and a non-heat-treated PDRN group. As a result, the heat-treated PDRN group showed approximately 3.9 times higher elastase inhibitory activity and approximately 2.2 times higher collagenase inhibitory activity compared to the non-heat-treated PDRN group.

[0032] Through this, it was found that PDRN containing a single-stranded DNA random coil generated by heat treatment, rather than PDRN composed mostly of a double-stranded DNA helix, is a substantial active ingredient capable of inhibiting the activity of elastase and collagenase, and that a composition containing PDRN containing a single-stranded DNA random coil can be used as a composition for improving skin wrinkles.

[0033] Therefore, the present invention may provide a cosmetic composition comprising PDRN containing a single-stranded DNA form as an active ingredient, wherein the PDRN containing a single-stranded DNA form may be PDRN containing a single-stranded DNA random coil obtained by heat-treating PDRN.

[0034] In addition, the heat treatment can be performed under conditions of heating to a temperature capable of inducing thermal denaturation of double-stranded DNA into single-stranded DNA, and preferably, it can be performed at 90°C to 100°C for 20 to 60 minutes.

[0035] In this invention, "PDRN" refers to a mixture of polydeoxyribonucleotides, composed of relatively short polydeoxyribonucleotides. That is, it refers to a low molecular weight DNA complex produced by fractionating DNA chains into specific sizes.

[0036] Furthermore, the “PDRN containing single-stranded DNA form” of the present invention refers to a PDRN containing a single-stranded DNA random coil or a PDRN containing a significant amount of a single-stranded DNA random coil form by heat-treating the PDRN to denature the double-stranded DNA form into a single-stranded DNA form.

[0037] In the present invention, "active ingredient" refers to a component that exhibits the intended activity alone, or can exhibit activity together with a carrier that is inactive itself.

[0038] As previously described, the composition containing PDRN in the form of single-stranded DNA according to the present invention has been confirmed to have excellent antioxidant activity, anti-inflammatory activity, skin whitening activity, and skin wrinkle improvement activity through experiments of the examples. Therefore, the composition can be used as a cosmetic composition having antioxidant activity, anti-inflammatory activity, skin whitening activity, or skin wrinkle improvement activity.

[0039] In addition, the cosmetic composition of the present invention may be prepared in any one formulation selected from softening lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, eye cream, cleansing cream, cleansing foam, cleansing water, lotion, emulsion, spray, or powder, but is not limited thereto.

[0040] The cosmetic composition according to the present invention may be composed of, in addition to the PDRN containing the single-stranded DNA form of the present invention, various ingredients generally used in cosmetic compositions, such as water-soluble ingredients, powder ingredients, oils, surfactants, moisturizers, viscosity modifiers, preservatives, antioxidants, fragrances, colorants, etc., as needed and within a range that does not reduce the effect of the present invention.

[0041] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., as conventional ingredients, but is not limited thereto.

[0042] Cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

[0043] When the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more types.

[0044] When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto. These may be used alone or in a mixture of two or more types.

[0045] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc., may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan may be used, but are not limited thereto. These may be used alone or in a mixture of two or more.

[0046] When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more.

[0047] When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more types.

[0048] In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, comprising PDRN containing a single-stranded DNA form as an active ingredient.

[0049] As described above, in one embodiment of the present invention, it was confirmed that the group treated with PDRN containing a helical form including a single-stranded DNA form by heat treatment had a significantly superior scavenging ability against nitrites that induce an inflammatory response compared to PDRN that was not heat-treated.

[0050] Therefore, a composition containing PDRN in the form of single-stranded DNA as an active ingredient can be used as a pharmaceutical composition capable of preventing or treating inflammatory skin diseases caused by excessive inflammatory responses.

[0051] In the present invention, the inflammatory skin disease may be selected from the group consisting of contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, contact urticaria syndrome, autosensitization dermatitis, stagnant dermatitis, acne, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, eczema, and psoriasis.

[0052] In addition, the present invention may provide a pharmaceutical composition for the prevention or treatment of skin pigmentation disorders, comprising PDRN containing a single-stranded DNA form as an active ingredient.

[0053] As described above, in one embodiment of the present invention, it was confirmed that the group treated with PDRN containing a helical form including a single-stranded DNA form by heat treatment had a significantly superior ability to inhibit tyrosinase activity compared to PDRN that was not heat-treated.

[0054] In the present invention, "skin pigmentation disease" refers to any disease or condition of the skin in which discoloration of the skin or proliferation of abnormally pigmented cells occurs, and skin pigmentation may include excessive or unwanted pigmentation.

[0055] The types of skin pigmentation disorders mentioned above are not limited thereto, but may be selected from a group consisting of melasma, freckles, lentigo, age spots, moles, café au lait spots, nevus of Ota, blue nevus, hyperpigmented spots, hyperpigmentation after drug use, gravidic chloasma, and post-inflammatory hyperpigmented disorders due to injury or dermatitis.

[0056] In addition, skin pigmentation disorders can be caused by factors such as aging, environmental stress, and UV exposure. The skin pigmentation of the present invention may be attributed to an increase in one or more different types of melanin that are biosynthesized in the skin and / or hair follicles and deposited in the hair or skin compared to the patient's underlying pigmentation, and may be, but is not limited to, one or more selected from the group consisting of, for example, melasma, freckles, lentigo, age spots, moles, café au lait spots, nevus of Ota, blue nevus, hyperpigmented spots, hyperpigmentation after drug use, gravidic chloasma, and hyperpigmentation after inflammation due to wounds or dermatitis.

[0057] Melanin is a collective term for black or brown pigments found in tissues such as the skin and eyes of various animals. In particular, melanin is a crucial factor in determining human skin color; melanin-expressing genes differ by race, and consequently, the amount of melanocytes is regulated, thereby determining skin color. While melanin functions as a UV-blocking agent that maintains body temperature and protects the skin from ultraviolet rays, excessive melanin production causes the formation of melasma, freckles, and skin spots, and further contributes to the development of skin cancer.

[0058] Tyrosinase (TYR) is a copper-containing enzyme that catalyzes the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) and the oxidation of L-DOPA to its corresponding o-quinone derivative in the melanin synthesis pathway, and can be a useful target for inhibiting unwanted skin pigmentation. Therefore, inhibiting tyrosinase activity can inhibit melanin synthesis.

[0059] In this regard, PDRN containing a helical form including a single-stranded DNA form can effectively inhibit tyrosinase activity through heat treatment of the present invention, thereby inducing a skin whitening effect and preventing or treating skin pigmentation disorders.

[0060] In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier, wherein "pharmaceutically acceptable" means not significantly stimulating a living organism and not inhibiting the biological activity and properties of the administered active substance.

[0061] The term "prevention" as used in this invention refers to any act of suppressing the symptoms of a target disease or delaying its progression through the administration of the composition of this invention.

[0062] The term "treatment" as used in this invention refers to any act of improving or beneficially altering the symptoms of a target disease through the administration of the composition of this invention.

[0063] The term "improvement" as used in the present invention refers to any action that at least reduces parameters related to the condition being treated, such as the degree of symptoms.

[0064] The term "administration" as used in the present invention means providing a specific composition of the present invention to an individual by any appropriate method. In this case, the individual refers to any animal, such as a human, monkey, dog, goat, pig, or rat, having a disease in which symptoms of an inflammatory skin disease or skin pigmentation disease may be improved by administering the composition of the present invention.

[0065] In the present invention, the term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit-or-risk ratio applicable to medical treatment, which may be determined by factors including the type and severity of the individual's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0066] The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient of the present invention, and the adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

[0067] The formulation forms of the above pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops, or injectable liquids. For example, for formulation into the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, trackercanth, or sodium oleate; sodium stearate; magnesium stearate; sodium benzoate; sodium acetate; sodium chloride; etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using the method disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA, USA) as an appropriate method in the field.

[0068] In one embodiment of the present invention, the active ingredient of the present invention may be included in an amount of 0.001 to 99 weight% based on the total weight of the composition.

[0070] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0072] <Preparation Example>

[0073] Reagent preparation

[0074] Ethidium bromide (EtBr), ascorbic acid (AA), DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], ammonium persulfate, sodium nitrite, NADH, nitroblue tetrazolium, phenazine methosulfate, L-tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), mushroom tyrosinase type I, kojic acid (KA), grease reagent, porcine pancreatic elastase, collagenase type I, N-succinyl-(L-Ala)3-p-nitroanilide (STANA), epigallocatechin gallate (EGCG), and N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) are Sigma-Aldrich Chemical Co. It was purchased and used from (St. Louis, Missouri, USA). The QuantiFluor® dsDNA system used was from Promega (Madison, WI, USA). In the experiments of this invention, PDRN products labeled as PDRN were purchased from a reliable commercial chemical company, and additional reagents were purchased directly from a high-end commercial chemical company.

[0076] Preparation of heat-treated PDRN (PDRN-H)

[0077] The PDRN solution was heat-treated at 94°C for 30 minutes, and the heat-treated PDRN solution (PDRN-H) was diluted with physiological saline and stored in ice water, and frozen in a refrigerator when necessary.

[0079] Characterization of heat-treated PDRN (PDRN-H)

[0080] After electrophoresis of heat-treated PDRN (PDRN-H) solutions on an agarose gel, comparison of dsDNA levels at 260 nm absorbance and EtBr-stained DNA regions confirmed that heat-treated PDRN contained significantly more single-stranded DNA fragments than PDRN. The dsDNA concentrations of PDRN and PDRN-H were quantified using the QuantiFluor® dsDNA System. The EtBr-stained regions of PDRN and PDRN-H were compared after 2% agarose gel electrophoresis, followed by EtBr staining and image analysis using ImageJ version 1.54h (NIH, Bethesda, MD, USA).

[0082] DPPH radical scavenging activity analysis

[0083] The DPPH radical scavenging activity of PDRN and PDRN-H was analyzed as follows. A reaction mixture of 250 μL was prepared by mixing 120 μL of 1.8 mg / ml PDRN or PDRN-H with 130 μL of 0.1 mM DPPH, and the mixture was reacted in a dark room at room temperature for 30 minutes. Ascorbic acid (AA, 50 μg / ml, 30 μL) was used as a positive control. Absorbance was measured at 517 nm using a microplate reader, and the DPPH scavenging rate (%) was calculated using the following formula.

[0084] Elimination rate (%) = [(Control group - Test group) / Control group] x 100.

[0086] ABTS radical scavenging activity analysis

[0087] The ABTS radical scavenging activity analysis for PDRN and PDRN-H was conducted based on methods known in the art, incorporating slight modifications. Specifically, ABTS radical cations (ABTS) generated by reacting an ABTS stock solution (0.07 mM) with 0.12 mM ammonium persulfate were ·+ ) was stored in a dark room at room temperature for 16 hours until use. 100 μL of 1.8 mg / ml PDRN or PDRN-H solution ABTS ·+ A total of 300 μL of reaction mixture was prepared by mixing with 200 μL of solution, and the mixture was reacted in a dark room at room temperature for 15 minutes. Ascorbic acid (AA) (1 μg / ml) was used as a positive control. Absorbance was measured at 745 nm, and the radical scavenging rate was calculated using the above DPPH scavenging rate formula.

[0089] Superoxide radical scavenging activity analysis

[0090] The superoxide radical scavenging activity of PDRN and PDRN-H was analyzed in vitro. For this purpose, 20 μL of 1.2 mg / ml PDRN or PDRN-H was mixed with 180 μL of 1 mM Tris buffer (pH 8.0) (containing 100 μM nitroblue tetrazolium, 156 μM NADH, and 20 μM phenazine methosulfate) to prepare a total reaction mixture of 200 μL, which was then left at room temperature for 5 minutes. Ascorbic acid (AA) was used as a positive control. Absorbance at 560 nm was measured using a microplate reader. The scavenging rate was calculated as described above.

[0092] Nitrite Scavenging Activity Analysis

[0093] The nitrite scavenging activity of PDRN and PDRN-H was analyzed in vitro as follows. 60 μL of 1.2 mg / ml PDRN or PDRN-H was mixed with 30 μL of 0.1 mM citric acid buffer (pH 3.0), 6 μL of 50 μg / ml sodium nitrite, and 54 μL of distilled water to prepare a total reaction mixture of 150 μL. This mixture was incubated at 37°C for 60 minutes, after which an equal amount of grease reagent was added and reacted for 10 minutes. Subsequently, the absorbance at 538 nm was measured using a microplate reader. 20 μg / ml of ascorbic acid (AA) was used as a positive control. The scavenging rate was calculated as described above.

[0095] Analysis of Tyrosinase Inhibitory Activity

[0096] The inhibitory activities of PDRN and PDRN-H on the L-tyrosine hydroxylase and L-DOPA oxidase activities of tyrosinase were analyzed. To measure L-tyrosine hydroxylase activity, 80 μl of 0.04 mM phosphate buffer (pH 6.8), 40 μl of 0.16 mg / ml PDRN or PDRN-H, 40 μl of 0.5 mM L-tyrosine dissolved in 0.04 mM phosphate buffer (pH 6.8), and 40 μl of 6 units / ml mushroom tyrosinase type 1 were added to each reaction mixture (200 μl), and the mixture was reacted at 37°C for 10 minutes. Subsequently, the amount of L-DOPA produced was quantified by absorbance at 475 nm using a microplate reader. In addition, 0.5 mM L-DOPA was used as a substrate instead of L-tyrosine to measure L-DOPA oxidase activity, and the generated dopaquinone was quantified by absorbance at 450 nm. Kojic acid (KA, 10 μg / ml) was used as a positive control. The inhibition rate was calculated using the following formula.

[0097] Inhibition rate (%) = [(Control group - Test group) / Control group] x 100.

[0099] Analysis of Elastase Inhibitory Activity

[0100] Elastase inhibitory activity was analyzed by measuring the degree of inhibition of elastase activity in the presence of PDRN or PDRN-H. Elastase activity was confirmed by measuring the amount of p-nitroaniline produced from STANA, which was used as a chromogenic substrate. A reaction mixture of 100 μl of 0.2 M Tris buffer (pH 8.0) and 50 μl of 1.2 mg / ml PDRN or PDRN-H was mixed with 100 μl of STANA (0.8 mM) and pre-reacted at 37°C for 20 minutes, after which 50 μl of porcine pancreatic elastase (0.1 U / ml) was added to 0.2 M Tris buffer (pH 8.0) to start the enzymatic reaction. After the reaction was completed, the absorbance at 410 nm was measured using a microplate reader, EGCG (2 mg / ml) was used as a positive control, and the inhibition rate was calculated as described above.

[0102] Analysis of Collagenase Inhibitory Activity

[0103] To confirm the collagenase inhibitory activity of PDRN and PDRN-H, the degree of collagenase activity reduction was measured using a slightly modified known spectrophotometric method. For the collagenase activity analysis, FALGPA was used as the synthetic substrate, utilizing the characteristic of reduced absorbance between 324 nm and 345 nm due to the proteolytic degradation of FALGPA. Clostridium histolyticum type 1 collagenase (1 mg / ml, 20 μl) was reacted with 20 μl of 1.8 mg / ml PDRN or PDRN-H in a 96-well microtiter plate containing 20 μl of 50 mM Tris buffer (pH 7.4) and 0.36 mM CaCl2, and incubated for 20 minutes in a dark room at 37°C. After the pre-culture was completed, 40 μl of FALGPA (2.4 mM) was added to each well, and the mixture was reacted for an additional 30 minutes in a dark room at 37°C. Then, the absorbance was measured at 335 nm using a microplate reader. EGCG (0.25 mM) was used as a positive control, and the inhibition rate was calculated as described above.

[0105] Statistical processing

[0106] The experimental results were expressed as mean ± standard deviation. Differences between experimental groups were analyzed using the Student t-test. A P-value of less than 0.05 was considered statistically significant.

[0108] <Example 1>

[0109] Confirmation of increased single-stranded DNA (ssDNA) in heat-treated PDRN solution

[0110] The levels of dsDNA and ssDNA contained in the heat-treated PDRN-H solution were analyzed for the PDRN-H solution prepared by heat-treating PDRN.

[0111] As a result, as shown in Fig. 1, the UV light absorbance for PDRN and PDRN-H at 260 nm showed that PDRN-H exhibited approximately 10% higher absorbance than PDRN (Fig. 1A). This result implies that the PDRN solution contains the majority of the dsDNA form. Meanwhile, the dsDNA content in the heat-treated PDRN-H solution was found to have decreased to 71.6% compared to PDRN, which means that approximately 28.4% of the original dsDNA in PDRN was converted to ssDNA by heat treatment (Fig. 1B).

[0113] In addition, the inventors further confirmed the conversion from dsDNA to ssDNA by heat treatment through EtBr staining following agarose gel electrophoresis. As a result, as shown in Fig. 2, the EtBr stained area of ​​heat-treated PDRN-H was found to be smaller and move faster than that of unheat-treated PDRN, indicating that the proportion of ssDNA in PDRN-H increased (Fig. 2A). Furthermore, the results of intensity profile analysis according to gel distance using ImageJ software are shown in Fig. 2B. Additionally, when the profile areas for the PDRN and PDRN-H lanes were quantified by density and compared, the profile area of ​​the PDRN-H lane was found to be 76.4% relative to 100% of the PDRN lane's profile area, indicating that PDRN-H was reduced compared to PDRN (Fig. 2C).

[0114] These results indicate that 24.6% of the dsDNA was converted into ssDNA that was not sufficiently stained with EtBr by heat treatment of PDRN. That is, it means that about 26.4% of the dsDNA present in PDRN was denatured into ssDNA by heat treatment of the present invention.

[0116] <Example 2>

[0117] Antioxidant activity analysis

[0118] To compare the antioxidant activities of PDRN and PDRN-H, the inventors performed total antioxidant activity using DPPH and ABTS scavenging assays.

[0120] As a result, as shown in Figure 3, PDRN and PDRN-H showed scavenging activities of 1.8% and 18.8%, respectively, in the DPPH scavenging activity analysis (Figure 3A). In addition, in the ABTS scavenging activity analysis, the scavenging activities for PDRN and PDRN-H were found to be 8.7% and 14.2%, respectively (Figure 3B). That is, PDRN-H was found to have DPPH and ABTS scavenging activities 10.4 times and 1.6 times higher than PDRN, respectively.

[0121] Through these results, the inventors were able to determine that PDRN-H, in which ssDNA is increased by heat treatment, has significantly superior antioxidant activity compared to PDRN composed mostly of dsDNA, and that the component primarily responsible for antioxidant activity is not dsDNA but ssDNA-containing PDRN (PDRN-H).

[0123] <Example 3>

[0124] Superoxide Scavenging Ability Analysis

[0125] Superoxide radical anion (O2 ·- O2 is a reactive oxygen species generated by the reduction of oxygen via electron transfer in the enzymatic respiration chain, and is known to be converted to hydrogen peroxide by superoxide dismutase and then to hydroxyl radicals. In cells, O2 is converted by superoxide dismutase and superoxide reductase. ·- Although the concentration of these two enzymes is regulated, a deficiency of these two enzymes acts as a cause of oxidative stress in organisms.

[0126] Accordingly, the inventors compared and analyzed the superoxide radical scavenging activity of PDRN and PDRN-H.

[0128] As a result, the superoxide scavenging activity of PDRN and PDRN-H was found to be 23.0% and 51.5%, respectively, confirming that PDRN-H has approximately 2.2 times higher scavenging activity than PDRN (Fig. 4A). These results also indicate that PDRN containing the ssDNA form (PDRN-H) plays a major role in scavenging activity.

[0130] <Example 4>

[0131] Nitrite scavenging ability analysis

[0132] Excessive production of nitric oxide (NO) within cells serves as a key mediator for inflammation induction and is a cause of serious skin diseases such as atopic dermatitis or psoriasis in skin cells. Additionally, the generated NO is converted into nitrite, a stable storage form of NO, through oxidative degradation metabolism. Since nitrite is reverse-circulated back to NO via the nitrate-nitrite-NO axis under inflammatory skin conditions, such as UV exposure or other inflammation-related diseases, the anti-inflammatory properties of a specific substance can be verified by analyzing in vitro nitrite scavenging activity.

[0133] The inventors analyzed the anti-inflammatory activity of PDRN and PDRN-H through an analysis of nitrite scavenging ability.

[0135] As a result, the nitrite scavenging activities for PDRN and PDRN-H were found to be approximately 11.1% and 52.3%, respectively, indicating that PDRN-H, which contains ssDNA due to heat treatment, has a nitrite scavenging ability approximately 4.7 times higher than PDRN, which is composed mostly of dsDNA (Fig. 4B).

[0136] Through these results, the inventors were able to determine that the increase in the ssDNA helical structure of PDRN-H by heat treatment is a key active ingredient that enables excellent nitrite scavenging ability, and through this, it was determined that PDRN-H has significantly superior anti-inflammatory activity compared to PDRN.

[0138] <Example 5>

[0139] Analysis of tyrosinase inhibitory activity

[0140] Next, the inventors compared and analyzed the tyrosinase inhibitory activity of PDRN and PDRN-H. It is known that while melanin protects the skin from harmful effects of ultraviolet rays, DNA damage, and oxidative stress, its overproduction acts as a cause of skin diseases, including skin darkening and aging. The tyrosinase enzyme present in the melanocyte membrane catalyzes the hydroxylation of tyrosine and the oxidation of 3,4-dihydroxy-L-phenylalanine (L-DOPA) to o-dopaquinone, which are rate-limiting reactions of melanin synthesis. Therefore, inhibiting tyrosinase activity can reduce melanin synthesis and induce a skin whitening effect.

[0141] The inhibitory activity of PDRN and PDRN-H on tyrosinase was analyzed for tyrosinase tyrosine hydroxylase and L-DOPA oxidase activities, respectively. As a result, as shown in Figure 5, PDRN and PDRN-H exhibited inhibitory activities of 11.5% and 26.2%, respectively, for tyrosine hydroxylase activity, indicating that PDRN-H showed approximately 2.2 times higher inhibitory activity than PDRN (Figure 5A). Additionally, the analysis of L-DOPA oxidase inhibitory activity showed that PDRN and PDRN-H exhibited inhibitory activities of 11.0% and 19.6%, respectively, indicating that PDRN-H showed approximately 1.8 times higher inhibitory activity than PDRN (Figure 5B).

[0142] Through these results, the inventors were able to determine that the ssDNA of PDRN-H primarily acts to inhibit the tyrosine hydroxylase and L-DOPA oxidase activities of tyrosinase, and through this, it was determined that the ssDNA of PDRN-H can be usefully utilized for skin whitening purposes.

[0144] <Example 6>

[0145] Analysis of Elastase and Collagenase Inhibitory Activity

[0146] Elastin and collagen, produced by fibroblasts, are components that are abundant in the extracellular matrix (ECM) of the skin's dermis. The cleavage and degradation of elastin and collagen proceed through the activation of elastase and collagenase, and the degradation of elastin and collagen caused by the activation of elastase and collagenase is known to result in major characteristics of skin aging such as skin wrinkles, sagging skin, pigmentation, and thinning skin.

[0147] Accordingly, the inventors analyzed whether PDRN and PDRN-H affect the activity of elastase and collagenase.

[0148] As a result, as shown in Fig. 6, PDRN and PDRN-H showed inhibitory activities of 9.0% and 35.1%, respectively, for elastase activity (Fig. 6A), and PDRN and PDRN-H showed inhibitory activities of 10.2% and 22.4%, respectively, for collagenase activity (Fig. 6B).

[0149] In other words, PDRN-H was found to have significantly higher inhibitory activity against elastase and collagenase than PDRN; elastase inhibitory activity was found to be about 3.9 times higher than PDRN, and collagenase inhibitory activity was found to be about 2.2 times higher than PDRN.

[0150] Through these results, the inventors found that the ssDNA of PDRN-H can effectively inhibit the activity of two types of skin wrinkle-forming enzymes, and thus can be usefully used for preventing skin aging.

[0152] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 A cosmetic composition comprising PDRN containing a single-stranded DNA form as an active ingredient. Claim 2 A cosmetic composition according to claim 1, characterized in that the PDRN containing the single-stranded DNA form is obtained by heat-treating PDRN. Claim 3 A cosmetic composition according to claim 2, characterized in that the heat treatment is performed at 90°C to 100°C for 20 minutes to 60 minutes. Claim 4 A cosmetic composition according to claim 1, characterized in that the cosmetic composition has antioxidant activity, anti-inflammatory activity, skin whitening activity, or skin wrinkle improvement activity. Claim 5 A cosmetic composition according to claim 4, wherein the PDRN containing the single-stranded DNA form has DPPH radical scavenging ability; ABTS radical scavenging ability; superoxide scavenging ability; nitrite scavenging ability; tyrosinase activity inhibitory ability; elastase activity inhibitory ability; or collagenase activity inhibitory ability. Claim 6 A cosmetic composition according to claim 1, characterized in that the cosmetic composition has any one formulation selected from softening lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, eye cream, cleansing cream, cleansing foam, cleansing water, lotion, emulsion, spray, or powder. Claim 7 A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases, comprising PDRN containing a single-stranded DNA form as an active ingredient. Claim 8 A pharmaceutical composition for the prevention or treatment of inflammatory skin diseases according to claim 7, wherein the inflammatory skin disease is selected from the group consisting of contact dermatitis, irritant contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, contact urticaria syndrome, autosensitization dermatitis, stagnation dermatitis, acne, atopic dermatitis, allergic dermatitis, seborrheic dermatitis, papular urticaria, eczema, and psoriasis. Claim 9 A pharmaceutical composition for the prevention or treatment of skin pigmentation disorders, comprising PDRN containing a single-stranded DNA form as an active ingredient. Claim 10 A pharmaceutical composition for the prevention or treatment of skin pigmentation disorders according to claim 9, characterized in that the skin pigmentation disorder is selected from the group consisting of melasma, freckles, lentigo, age spots, moles, café au lait spots, nevus of Ota, blue nevus, hyperpigmented spots, hyperpigmentation after drug use, gravidic chloasma, and post-inflammatory hyperpigmented disorders caused by wounds or dermatitis.

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