Cosmetic composition comprising extracted DNA from Radish sprouts for skin anti-oxidation and skin barrier repair

Abstract

The radish sprout-derived PDRN of the present invention strengthens the skin basement membrane and barrier, protects the skin from free radicals, and enhances skin defense capabilities by further activating the NRF-2 pathway upon oxidative stimulation. Accordingly, a composition containing the radish sprout-derived PDRN can be usefully utilized to maintain a healthy and firm skin environment and can be widely applied as a cosmetic for antioxidant and skin barrier protection purposes.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition for skin antioxidant and skin barrier strengthening containing DNA extracted from radish sprouts. Background Technology

[0002] DNA contains genetic information and is a substance that exhibits effects such as skin regeneration, wound healing, and anti-inflammatory action by activating adenosine receptors on cell membranes. Recently, polydeoxyribonucleotide (hereinafter referred to as 'PDRN'), manufactured from DNA extracted from salmon sperm, is being actively used in the pharmaceutical and cosmetic fields for the purpose of skin regeneration and tissue repair. PDRN has been confirmed to have effects such as promoting growth factor secretion, angiogenesis, improving blood flow, and anti-inflammatory action through A2 receptor stimulation, and is utilized in the treatment of various conditions including diabetic foot ulcers, scars, vascular insufficiency, and hair loss. However, since PDRN is primarily manufactured from salmon sperm, an animal-derived ingredient, it is not suitable for vegan or cruelty-free cosmetics, and there are limitations such as the use of organic solvents in the existing extraction process. Accordingly, research is underway to extract DNA from natural plant resources such as seaweed; however, plants and seaweed contain many impurities such as chlorophyll, and there is a concern that the DNA may be broken down into small molecules during the extraction process, thereby losing its function. Therefore, it is important to develop technologies for extracting and utilizing DNA from non-animal sources that is low in impurities and minimized in damage.

[0003] Meanwhile, skin cells accumulate waste products when exposed to external stimuli such as ultraviolet rays and reactive oxygen species (ROS), leading to a decline in cellular function and a weakening of the skin barrier. To revitalize these skin cells, the development of cosmetic compositions that activate various physiological functions by incorporating non-animal DNA remains insufficient. Therefore, there is a need to develop technologies that can stably secure functional DNA from novel raw material resources and utilize the aforementioned mechanisms in a complex manner. Prior art literature

[0004] Republic of Korea Registered Patent No. 10-2237543 (Title of Invention: Method for preparing DNA derived from Aloe species, and composition for anti-aging and anti-inflammatory purposes containing DNA derived from Aloe species as an active ingredient, Applicant: Moacam Co., Ltd., Registration Date: April 1, 2021) The problem to be solved

[0005] The objective of the present invention is to provide a cosmetic composition for skin antioxidant and skin barrier strengthening containing DNA extracted from radish sprouts. means of solving the problem

[0006] The present invention may relate to a cosmetic composition for skin antioxidant use containing DNA extracted from radish sprouts.

[0007] The above PDRN has the ability to scavenge reactive oxygen species and can enhance the activation of NRF-2 (nuclear factor erythroid 2-related factor 2) induced by oxidative stress.

[0008] In addition, the above PDRN has skin barrier strengthening efficacy and can enhance the protein expression of Laminin 5 or Occludin, or enhance the gene expression of Filaggrin.

[0009] 이하, 본 발명을 상세하게 설명한다.

[0010] In the present invention, the radish sprout may be a sprout grown by germinating a radish seed. That is, at the microgreen stage before 1 to 2 true leaves emerge from the radish seed. It is a radish sprout or radish microgreen. In this case, a sprout grown from a radish seed for 1 to 7 days, preferably 3 to 7 days, can be used, and the length of the radish sprout is not significantly limited. The cultivation method for the radish sprout is not restricted, whether hydroponic or soil cultivation. The radish sprout is distinguished from the radish greens, which are the leaves of a mature radish.

[0011] The radish PDRN of the present invention at 50 to 250 μg / ml increases laminin 5 protein expression by 2.7 to 4.0 times. In addition, Occludin (OCDN) protein expression is enhanced by 1.6 to 4.2 times at radish PDRN at 25 to 100 μg / ml. At this time, Occludin (OCDN) expression is restored to a normal state prior to cell damage with only 25 μg / ml of the radish PDRN. At the same time, the expression of the filaggrin (FLG) gene is also restored to a state similar to that of a normal cell group after cell damage induced by oxidative stress due to radish PDRN at 25 to 100 μg / ml, and it is shown that filaggrin gene expression increases by approximately 1.4 to 1.7 times compared to the oxidative stress-induced group.

[0012] The scavenging activity of reactive oxygen species (ROS) generated by oxidative stress can be 25–27% at 250 μg / ml of radish PDRN. In particular, 50–250 μg / ml of the radish PDRN of the present invention can enhance the activation of NRF-2 induced by oxidative stress by 1.3–1.5 times. At this time, oxidative stress causes NRF-2 activation, and the PDRN further enhances NRF-2 activation and transports it into the nucleus, thereby upregulating the expression of various antioxidant factors.

[0013]

[0014] Accordingly, the present invention provides a pharmaceutical composition for antioxidant or skin barrier strengthening containing radish PDRN and a pharmaceutical excipient. The PDRN may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 100 weight%, preferably 0.001 to 30 weight%.

[0015] The above pharmaceutical composition 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, and sterile injectable solutions, according to conventional methods. Carriers, excipients, and diluents that may be included in the above pharmaceutical composition may include 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. When formulating, the preparation is made using diluents or excipients such as commonly used fillers, volume expanders, binders, humectants, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules; these solid dosage forms are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract of the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, for example, humectants, sweeteners, flavorings, and preservatives, may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspending agents, 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, cocoa paste, laurin paste, glycerogelatin, etc. may be used.

[0016] The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to treatment, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. The determination of the dosage based on these factors is within the level of a person skilled in the art, and generally, the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is from 1 mg / kg / day to 500 mg / kg / day. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0017] The pharmaceutical composition of the present invention can be administered to mammals, such as rats, livestock, and humans, via various routes. Any mode of administration is expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, dura mater, or intracerebrovascular injection. Since the composition of the present invention has minimal toxicity and side effects, it is a drug that can be safely used for long-term preventive purposes.

[0018] The formulation of the cosmetic composition of the present invention may be any formulation conventionally manufactured in the art, and may be an essence, lotion, cream, emulsion, pack, hand cream, foot cream, body lotion, lip balm, lipstick, eyeshadow, eyeliner, eyebrow pencil, blusher, highlighter, general cosmetic water, skin toner, cream, serum, beauty soap, softening cosmetic water, medicinal cosmetic water, body cleanser, cleansing foam, cleansing lotion, gel, cleansing oil, cleansing cream, cleansing tissue, cleansing water, mask pack, and other hair products. In particular, when it is a various cosmetic product for hair growth, improvement of hair loss, improvement of scalp, etc., the formulation is preferably not particularly limited, and more specifically, hair soap, hair shampoo, hair rinse, hair serum, hair treatment, hair essence, hair water, hair tonic, hair lotion, hair emulsion, It can be selected from any formulation such as hair cream, hair massage cream, hair wax, hair pack, hair oil, hair dryer, hair preservative, hair dye, hair bleach, hair gel, hair glaze, hair mousse, hair spray, hair ampoule, etc.

[0019] More specifically, when the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder 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. In the case where the formulation of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a solvating agent, or an emulsifying agent is 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, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan. In the case where the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol or polyoxyethylene sorbitol ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanthera may be used as a carrier component. In the case where the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.The cosmetic composition of the present invention may additionally contain excipients including fluorescent substances, fungicides, hydrotropism-inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunblocks, vitamins, plant extracts, etc. Depending on the formulation or purpose of use, the amount of the above ingredients added may be selected within a range that does not impair the inherent effects of the cosmetic composition. The amount of the above ingredients added may be, for example, 0.1 to 10 weight%, preferably 0.1 to 6 weight%, based on the total weight of the composition, but is not limited thereto. Effects of the invention

[0020] The present invention relates to a cosmetic composition comprising PDRN extracted and purified from radish sprouts. The PDRN effectively eliminates reactive oxygen species (ROS) while simultaneously enhancing the expression of NRF-2 induced by oxidative stress, thereby precisely regulating the intracellular antioxidant defense system. Furthermore, the radish sprout-derived PDRN exhibits excellent biological activity by enhancing the structural stability of the cell basement membrane and significantly improving skin barrier function. Accordingly, the radish sprout PDRN of the present invention can be easily applied to various cosmetic compositions requiring skin antioxidant, skin barrier protection, and regeneration, and provides excellent efficacy in maintaining healthy skin against external stimuli. Brief explanation of the drawing

[0021] Figure 1 shows the electrophoresis results of the radish sprout-derived PDRN of the present invention. Figure 2 shows the results of confirming the cytotoxicity of radish sprout PDRN. Figure 3 shows the results confirming the efficacy of radish sprout PDRN in enhancing Laminin 5 protein expression. Figure 4a shows the fluorescent immunohistochemistry imaging and quantification results confirming the efficacy of radish sprout PDRN in enhancing Occludin (OCDN) protein expression, and Figure 4b shows the results confirming the efficacy of radish sprout PDRN in enhancing Filaggrin (FLG) gene expression. Figure 5 shows the results of confirming the ROS scavenging ability of unsprouted PDRN. Figure 6 shows the results confirming the efficacy of radish sprout PDRN in enhancing NRF-2 protein expression. Specific details for implementing the invention

[0022] Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein is provided to fully convey the concept of the present invention to those skilled in the art, so that it may be thorough and complete.

[0023] <실시예 1. 무순 유래 PDRN 제조>

[0024] To obtain radish sprout PDRN (DNA), the radish sprouts were first washed and then finely ground.

[0025] Tris(hydroxymethyl)aminomethane hydrochloride 500mM, potassium phosphate 100mM, ethylenediaminetetraacetic acid 170mM, sodium chloride 0.1M, sodium acetate 0.1M, sodium hydroxide 100mM The raw radish sprouts were mixed into a buffer solution containing 10 mM ascorbic acid and 0.5 wt% sodium dodecyl sulfate. At this time, purified water for preparing the buffer solution was added in an amount equal to 40 times the weight of the raw radish sprouts. Afterward, the mixture was heated to 60°C for 1 hour while stirring.

[0026] Once the raw radish tissue and DNA were separated, impurities other than DNA, such as the tissue, were removed. Additionally, impurities were primarily removed by centrifuging at 4°C and 7500 rpm for 10 minutes to remove the precipitated solids. Furthermore, to the solution remaining after most impurities had been removed, 99.9% ethanol was added to precipitate the radish-derived DNA within the solution, and the ethanol concentration in the solution was adjusted to 50 (v / v)%. Centrifugation was performed again to discard the ethanol, and only the precipitate containing DNA was obtained. The obtained precipitate was dissolved in purified water, precipitated again with ethanol (purity 99.99% or higher), centrifuged, and the process of rehydrating only the precipitate in purified water was repeated 2 to 4 times to remove the ethanol component from the precipitate and purify only the DNA component, thereby obtaining high-purity radish PDRN.

[0027] <실시예 2. 무순 유래 PDRN의 유전자 서열 분석>

[0028] The extracted PDRN derived from radish sprouts was quantified using Nanodrop, and the presence or absence of DNA was confirmed by performing DNA electrophoresis on 10 µl of DNA in a 0.5% agarose gel. As a result, as shown in Figure 1, it was confirmed that the PDRN was in good condition and had an average size of 400–700 bp and a molecular weight of 250–650 kDa.

[0029] Subsequently, Metagenome Amplicon Sequencing was performed by Macrogen Co., Ltd. using the obtained unsprouted PDRN. This assay is a community genomics analysis method capable of identifying the species composition of a specific DNA sample; it involves creating an amplicon library by amplifying specific regions of marker genes and then analyzing the distribution and diversity through sequencing.

[0030] For library preparation for sequencing analysis, rbcl2-rbcLa and ITS2F-ITS4R were amplified according to the illuminine 16s metagenomics sequencing library protocol. As a result, the DNA sample was ' Raphanus sativus’ It was analyzed as follows. Buffer for PCR amplification was added to 5 ng (rbcL2-rbcLa) and 10 ng (ITS2F-ITS4R) gDNA, along with 1 mM dNTP mix and 500 nM F / R PCR primers. The primer sequence information is as follows.

[0031] Primer Sequence rbcL2 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 rbcLa gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55 ITS2F tcgtcggcag cgtcagatgt gtataaga cagatgcgat acttggtgtg aat 53 ITS4R gtctcgtggg ctcggagatg tgtataagag acagtcctcc gcttattgat atgc 54

[0032] Metagenome Amplicon Sequencing analysis results showed that the radish sprout PDRN obtained in this invention is 99.9919% at the species level Raphanus Analysis revealed that it typically possesses the following genes.

[0033] 서열번호 sequence No.1 ctcaacccgg agttccacct gaagaagcag gggctgcggt agctgctgaa tcttctactg 60gtacatggac aactgtgtgg accgatgggc ttaccagcct tgaccgttac aaaggccgat 120gctaccacat cgagcccgtt ccaggagaagt aagattcacctat gtagccttac 180ccttagacct tttgaagaa gggtctgtta ctaacatgtt tacctcaatt gtgggtaacg 240tatttgggtt caaagccctg gctgctctac gtctagagga tctgcgaatc cctccggctt 300atactaaaac tttccagggga ccaccagcat tgaccacagatt tgacagatt agatt 360agtatggacg tcccctatta ggatgtacta ttaaacctaa gttggggtta tccgcgaaga 420actatggtag agcagtttat gaatgtcta 449 No.2 ctcaacccgg agttccacct gaagaagcag gggctgcggt agctgctgaa tcttctactg 60gtacatggac aaaactgtgt ggaccgatgg gcttaccagc cttgatcgtt acaaaggacg 120atgctaccac atcgagcccg ttccaggagata agagaattcaa atgtagctct 180ccctttagac cttttttagaag aaggttcggt tactaacatg tttacctcga ttgtgggtaa 240tgtatttggg ttcaaagccc tggctgctct acgtcttagag gatctgcgaa tccctcctgc 300ttatactcaa actttccagg gaccacct tcaattcgagatc 360caagtatgga cgtcccctat taggagtc tattaaacct aaattggggt tatccgcgaa 420gaactgtggt agagcagttt atgaatgtct a 451

[0034] Raphanus As for plants of the genus Raphanus sativus ; Raphanus raphanistrum It may include, and the above Raphanus sativus Depending on the characteristics of climate or topography, they are bred or cultivated into numerous horticultural types, varieties, subspecies, and variants; representatively, Raphanus sativus var. longipinnatus ; Raphanus sativus var. sativus ; Raphanus sativus var. caudatus ; Raphanus sativus var. oleiformis ; Raphanus sativus var. raphanistroides This includes the back.

[0035] < 실시예 3. 무순 PDRN의 세포 독성 확인>

[0036] Cytotoxicity tests were conducted on Human keratinocytes (HaCaT cells), A431 (Keratinocyte-like; Human epidermoid carcinoma), and Fibroblasts (Human Dermal Fibroblast, CCD-1064Sk; HDF) by treating with radish-infused PDRN at various concentrations. Cell cultures were performed according to standard methods, and basically, all were carried out in a 37°C, 5% CO2 incubator.

[0037] The experimental method for confirming cytotoxicity is as follows. Cells were inoculated into a 24-well plate and cultured for 24 hours. Subsequently, the culture medium was replaced with serum-free medium, and PDRN samples were added at various concentrations and cultured for another 24 hours. After the culture was complete, the medium was replaced with a 10-fold diluted 2.5 mg / mL MTT solution to allow the reaction to occur. The supernatant was removed, and 1 mL of DMSO was added to dissolve the formed MTT-formazan crystals. The absorbance was then measured at 570 nm using an ELISA reader. For the control group, DMSO (Dimethyl Sulfoxide) was used as the dissolution solvent.

[0038] Cytotoxicity was expressed as a percentage of absorbance compared to the control, and the results are shown in Figures 2a to 2c.

[0039] Fibrobalst Sample Conc. (μg / ml) Other.(%) Stdev. p-value Control 100.0 1.29 1.0000 Radish sprout PDRN 1 103.0 4.33 0.3146 5 102.3 4.91 0.4778 10 104.0 3.45 0.1472 25 108.4 2.89 0.0100 50 101.9 1.42 0.1549 100 109.2 3.27 0.0104 250 111.8 9.23 0.0935

[0040] A431 Sample Conc. (μg / ml) Other.(%) Stdev. p-value Control 100.0 4.73 1.0000 Radish sprout PDRN 1 99.5 1.41 0.8771 5 102.2 5.78 0.6316 10 104.7 1.83 0.1810 25 107.2 2.88 0.0860 50 100.1 6.27 0.9812 100 102.5 4.36 0.5334 250 103.8 2.15 0.2731

[0041] Keratinocytes Sample Conc. (μg / ml) Other.(%) Stdev. p-value Control 100.0 4.17 1.0000 Radish sprout PDRN 1 105.4 4.43 0.2003 5 104.7 3.43 0.2096 10 104.2 2.21 0.2006 25 107.6 2.74 0.0574 50 107.4 1.21 0.0413 100 106.0 1.13 0.0725 250 109.4 4.10 0.0496

[0042] As a result, it was confirmed that the radish sprout PDRN extracted in the present invention has no cytotoxicity to human keratinocytes, A431, and fibroblasts, as it showed viability of over 99.5% in all cells and all sample treatment groups up to 250 μg / ml of radish sprout PDRN.

[0043] < 실시예 4. 무순 PDRN의 세포기저막 및 피부장벽 강화 효능>

[0044] Example 4-1. Efficacy of enhancing Laminin 5 protein expression

[0045] A431 cells were seeded into 60 mm plates and cultured in MEM medium supplemented with 10% FBS at 37°C in a 5% CO2 incubator for 24 hours. Afterward, the medium was removed from the plates, washed once with 1X HBSS (Hanks' Balanced Salt Solution), and UVB 50 mJ / cm² was applied before treatment with radish sprout PDRN. 2 ...was investigated. Subsequently, the medium was replaced with serum-free medium, and PDRN was added at various concentrations and cultured for 24 hours. After the culture was finished, the culture medium was removed and washed once with PBS (phosphate buffered saline) to remove the medium components. Then, the cells were lysed using pro prep (iNtRON) buffer, proteins were extracted and quantified from the cells, and Western blot was performed. Subsequently, the expression of Laminin 5 protein, a factor related to skin barrier improvement, was confirmed. The results are shown in Table 6 and Figure 3.

[0046] Sample Conc. (µg / ml) Aver. Stdev. p-value Control 170.4 10.85 0.0012 UVB 50 mJ / ㎠ 100.0 10.02 1.0000 UVB + Radish sprout PDRN 50 277.9 10.96 0.0003 100 395.2 10.58 0.0000 250 298.1 20.49 0.0001 UVB + Vitamin C 75 161.7 8.96 0.0060

[0047] When the Western blot results were analyzed, it was found that the expression of Laminin 5 protein was very low in cells damaged by UVB treatment, but Laminin 5 expression increased again by 2.7 to 4.0 times with treatment with 50 to 250 μg / ml of unsprouted PDRN.

[0048] Example 4-2. Occludin (OCDN) Enhanced Efficacy

[0049] The protein expression level of Occludin, a skin barrier factor, was directly verified intracellularly. To this end, cells were cultured using the sample treatment as per the Laminin 5 experimental conditions. Primary normal human keratinocytes (NHEK cells) were seeded into 4-well slides, and UVB 50 mJ / cm² 2 First, the cells were investigated and treated with PDRN at various concentrations, and cultured for 24 hours. Afterward, the cells were fixed and permeated, then immunostained by sequentially treating with an anti-OCDN antibody and a secondary fluorescent antibody, and images were obtained using a fluorescence microscope.

[0050] Sample Conc. (µg / ml) Aver. Stdev. p-value Control 157.2 1.06 0.0001 UVB 50mJ / cm 2 (Damaged skin) 100.0 6.81 1.0000 UVB + Radish sprout PDRN 25 166.1 0.61 0.0001 50 303.9 2.96 0.0000 100 419.2 2.97 0.0000

[0051] As a result, as shown in Table 7 and Figure 4a, when examining the fluorescence staining images and the results quantified therefrom, it can be confirmed that when treated with unsprouted PDRN, the protein expression level of Occludin (OCDN) increases, and the cell is restored to a state prior to cell damage, preferably increasing by 1.6 to 4.2 times.

[0052] Example 4-3. Filaggrin (FLG) Enhancement Efficacy

[0053] Primary normal human keratinocytes (NHEK cells) were inoculated into 6-well plates and cultured. The medium was replaced with serum-free medium, and the sample was added. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After treatment with acutely oxidative stress (H2O2 0.1 mM), cells were collected using QIAzol™ Lysis Reagent (Qiagen), and RNA was extracted according to the manufacturer's instructions. After quantifying the extracted RNA, cDNA was synthesized, and Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System) was performed to confirm gene expression (Filaggrin).

[0054] Sample Conc. (μg / ml) Average Control 131.6 H2O2(0.1 mM) 100.0 H2O2 + RADISH SPROUT PDRN 25 146.8 50 160.5 100 166.2

[0055] In Table 8 and Figure 4b, it can be seen that 100 μg / ml of unsprouted PDRN increases filaggrin gene expression 1.66-fold in cells damaged by H2O2 induction.

[0056] < Examples 5. Radish sprouts PDRN's Antioxidant benefits - reactive oxygen species ( ROS , Reactive Oxygen Species) assay>

[0057] Human keratinocytes (HaCaT cells) were inoculated into a 24-well plate and cultured. The medium was replaced with serum-free medium, and the samples were added and incubated for 24 hours in a 37°C, 5% CO2 incubator. After treatment with H2DcFDA reagent and acutely oxidative stress (H2O2 1 mM), the cells were washed with 1X HBSS and measured using an ELISA reader (Ex=485 nm, Em=535 nm).

[0058] Sample Conc. (µg / ml) Aver. Stdev. p-value Control 74.8 2.38 0.0002 H2O2 1mM 100.0 2.48 1.0000 H2O2 + EGCG 1 81.3 6.44 0.0093 H2O2+ Radish sprout PDRN 50 95.0 9.20 0.4120 100 93.9 5.12 0.1367 250 74.2 4.85 0.0012

[0059] In Table 9 and Figure 5, treatment with 250 μg / ml of unsprouted PDRN reduced the production of ROS (Reactive Oxygen Species) in cells damaged by oxidative stress by 25.8%.

[0060] < Examples 6. Radish sprouts PDRN's Detox Care - NRF -2 Protein expression Confirm

[0061] NRF-2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates the expression of intracellular antioxidant and detoxification (Phase II) enzymes. Under normal levels, it is degraded by the action of Keap1, but upon oxidative stimulation, it translocates to the nucleus and its expression actually increases. Accordingly, we investigated how NRF-2 expression changes upon treatment with unripe PDRN.

[0062] To this end, human keratinocytes (HaCaT cells) were inoculated into a 60 mm dish and cultured. The medium was then replaced with serum-free medium, treated with H2O2 and the sample, and incubated at 37°C in a 5% CO2 incubator. The cells were scraped with a cell scraper, centrifuged to discard the supernatant, and then protein extraction solution (iNtRON) was added and centrifuged to collect the supernatant. Proteins were quantified, transferred to a membrane after SDS-PAGE, and then blocked with 5% BSA or 5% skim milk before treatment with the primary antibody [NRF-2, beta-actin]. After washing with 1X TBST, the secondary antibody was applied, followed by a final wash with 1X TBST and treatment with ECL solution. The bands were then visualized using Chemi-doc.

[0063] Sample Conc. (µg / ml) Aver. Stdev. p-value Control 46.0 5.12 0.0001 H2O2(1 mM) 100.0 0.00 1.0000 H2O2 + EGCG 1.5 161.4 10.97 0.0006 H2O2+ Radish sprout PDRN 50 142.0 4.26 0.0001 100 140.3 1.67 0.0000 250 135.4 3.96 0.0001

[0064] In Table 10 and Figure 6, it can be seen that oxidative stress induced by H2O2 activated the expression of NRF-2 protein, and that NRF-2 production was further increased due to treatment with radish PDRN.

Claims

Claim 1 A cosmetic composition for skin antioxidant use characterized by containing PDRN (polydeoxyribonucleotide) extracted from radish sprouts. Claim 2 A cosmetic composition for skin antioxidants according to claim 1, characterized in that the PDRN has the ability to scavenge reactive oxygen species. Claim 3 A cosmetic composition for skin antioxidant according to claim 1, characterized in that the PDRN further enhances the activation of NRF-2 (nuclear factor erythroid 2-related factor 2) induced by oxidative stress. Claim 4 A cosmetic composition for skin antioxidants according to claim 1, wherein the PDRN has a skin barrier strengthening effect. Claim 5 A cosmetic composition for skin antioxidant according to claim 1, characterized in that the PDRN enhances the protein expression of Laminin 5 or Occludin, or enhances the gene expression of Filaggrin.

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