A composition containing DNA extracted from camellia, possessing anti-aging and antioxidant properties.

Abstract

This invention provides a composition containing DNA extracted from camellia flowers that has anti-aging and antioxidant properties. [Solution] The present invention relates to a composition containing DNA (PDRN) extracted from camellia. The camellia PDRN improves and activates the state of mitochondria and enhances anti-glycation efficacy. It also expresses various cell-stimulating factors or skin-improving factors that restore aged or damaged cells to a youthful state. For this reason, the camellia PDRN of the present invention can be widely applied as a cosmetic composition or pharmaceutical composition for anti-aging or skin condition improvement.

Description

Technical Field

[0001] The present invention relates to a composition having anti-aging and antioxidant effects containing DNA extracted from Camellia sp.

Background Art

[0002] Aging originates from functional changes at the cellular level prior to the decline in the functions of organs and tissues. Individual aging phenomena, such as the reduction of subcutaneous fat and the decline in melanin production ability, are closely related to the process of cellular aging. Cellular senescence is defined as a state of cell cycle arrest due to various stimuli such as telomere shortening, oxidative stress, DNA damage, and abnormal signal activity. In actual human skin, as such aging-related changes are observed, the search for substances that regulate aging at the cellular level has been positioned as an important research field for the prevention and improvement of diseases related to individual aging. Furthermore, mitochondrial dysfunction related to aging is accompanied by an obvious decline in the division and proliferation ability of young cells, and substances that actually activate ATP synthase may help restore the function of the inner mitochondrial membrane and improve aging. In such an aging process, cell function declines, oxidative stress increases, and the autophagy function, which plays an important role in maintaining intracellular homeostasis, also declines. Autophagy is an important mechanism for removing damaged proteins and organelles to maintain cell quality, and the activation of autophagy contributes to the enhancement of cell regenerative ability, the maintenance of mitochondrial quality (mitophagy), and the delay of aging. In this process, TFEB (Transcription Factor EB), an important transcription factor for lysosome and autophagy regulation, plays an important role in regulating autophagy activity. However, abnormal TFEB expression and regulation can be observed during the aging process, which induces abnormal activity or burden of the intracellular digestive system. Therefore, the restoration of the regulatory balance of TFEB, the promotion of autophagy activity, and the antioxidant promotion reaction have attracted attention as important strategies for delaying cell aging.

[0003] On the other hand, DNA is a double-helix polymer composed of phosphate, bases (adenine, thymine, guanine, cytosine), and deoxyribose. It contains genetic information and activates adenosine receptors in cell membranes, which are used for skin regeneration, wound and scar treatment, and arthritis improvement. In recent years, PDRN (polydeoxyribonucleotide), which has attracted attention as an important material in the cosmetics field, is mainly produced from DNA derived from salmon semen and contains a deoxyribonucleotide polymer of 50 to 2000 bp. PDRN is widely used for diabetic foot ulcers, scars, vascular insufficiency, and female pattern hair loss due to its various effects such as tissue regeneration and anti-inflammatory properties. However, because PDRN is based on animal-derived raw materials, it is not suitable for vegan and cruelty-free cosmetics, and it has the limitation that chemical solvents such as chloroform and phenol must be used during extraction. To address this, research is being conducted on techniques for obtaining DNA derived from natural materials such as plants and seaweed. However, these raw materials contain many impurities such as chlorophyll, pectin, and cellulose, making advanced impurity removal technology essential for the commercial extraction of high-purity DNA. [Prior art documents] [Patent Documents]

[0004] (Patent Document 1) Korean Registered Patent No. 10-2249241 (Title of Invention: Polydeoxyribonucleotides and Polynucleotide Extraction Method Derived from Seaweed Having Novel Angiogenesis and Cell Regeneration Effects, Applicant: Han Ji-seong, Registration Date: November 20, 2020) [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a composition containing DNA extracted from camellia that has anti-aging and antioxidant properties. [Means for solving the problem]

[0006] The present invention relates to an anti-aging and antioxidant composition containing PDRN (polydeoxyribonucleotide) extracted from camellia. The PDRN has the effect of suppressing the production of ROS (Reactive Oxygen Species). The PDRN may also have anti-glycation effects. Preferably, the PDRN can inhibit the expression of RAGE (Receptor for Advanced Glycation End products) genes.

[0007] The aforementioned PDRN may be one that activates autophagy, thereby inducing lysosome biosynthesis and removing intracellular waste products.

[0008] The aforementioned PDRN preferably has the efficacy of enhancing the expression of LC3-II (Microtubule-associated protein 1A / 1B-light chain 3B) and activating autophagy.

[0009] The aforementioned PDRN enhances the expression of the transcription factor TFEB (Transcription Factor EB), thereby inducing lysosomal biosynthesis. This lysosomal biosynthesis induces intracellular digestion, i.e., waste removal and cellular detoxification functions.

[0010] Furthermore, the PDRN is characterized by its ability to enhance mitochondrial vitality and promote the regeneration of damaged cells. When intracellular mitochondria are activated via the PDRN of the present invention, it can be confirmed that cell damage is minimal or reduced, and recovery from such damage proceeds rapidly, both before and after treatment with a cell stimulant such as UV.

[0011] Furthermore, the aforementioned PDRN is highly effective in reducing CPD (Cyclobutane pyrimidine dimer) generated by DNA damage.

[0012] In addition to these anti-aging effects, the PDRN may also have the following effects in skin cells or subcutaneous vascular cells: suppression of skin pigmentation by reducing the expression of the SDF-1 (Stromal Cell-Derived Factor 1) gene, whitening effect by suppressing melanin production, suppression of skin erythema by suppressing the expression of the EDNRb (Endothelin receptor B) gene, and improvement of wrinkles by suppressing the expression of the MMP-1 (Matrix Metalloproteinase-1) gene.

[0013] Therefore, the PDRN of the present invention can be applied as a cosmetic composition having the above-mentioned various effects, including anti-aging and antioxidant functions.

[0014] The present invention will be described in detail below.

[0015] The present invention reduces RAGE gene expression by 20-30% at 1-10 μg / mL of camellia PDRN. Furthermore, the PDRN reduces MMP-1 gene expression by 20-50% at 1-10 μg / mL and by 40-50% at 5-10 μg / mL. LC3-II expression is increased 1.9-3.8 times at 1-10 μg / mL, 2.4-3.8 times at 5-10 μg / mL, and 3.5-3.8 times at 10 μg / mL. Lipofuscin accumulation is suppressed by 80-90% at 1-10 μg / mL. TFEB is reduced by 20-45% at 5-10 μg / mL and by 40-45% at 10 μg / mL. Mitochondrial function is enhanced 1.4 to 1.5 times at 1 μg / mL of camellia PDRN. SDF-1 gene expression is enhanced 1.2 to 1.6 times at 1 to 10 μg / mL of camellia PDRN, and 1.4 to 1.6 times at 5 to 10 μg / mL. In addition, melanin production rate per unit cell decreases by 20 to 30% at 1 to 10 μg / mL of camellia PDRN, and melanin secretion rate per unit cell also decreases by 20 to 30%. ROS scavenging ability is confirmed to be approximately 20 to 25% at 10 μg / mL of camellia PDRN. It is observed that 30 to 50% of CPD generated by DNA damage is scavenged at 1 to 10 μg / mL of camellia PDRN, and cells are activated. EDNRb (Endothelin receptor B) expression decreases by 20 to 25% at 5 to 10 μg / mL of camellia PDRN, returning to a normal state.

[0016] The PDRN of the present invention is characterized by being extracted from Camellia sp.

[0017] The aforementioned PDRN can be extracted by the following method.

[0018] (Step 1) The raw camellia material is finely ground, and a buffer solution containing salt, ascorbic acid, and SDS (Sodium Dodecyl Sulfate) is added and stirred at 40-60°C for 1-24 hours to soften the material. (Step 2) To extract the effective DNA component from the softened camellia, only the liquid phase is obtained after centrifugation, and the precipitated impurities are removed. (Step 3) Adding alcohol to the obtained DNA eluate, which is the liquid phase, and immersing the solid content containing DNA. (Step 4) Obtaining the precipitated DNA-containing solid content. (Step 5) It can include the step of dissolving and purifying the obtained DNA solid content.

[0019] The salts in the buffer solution include 100 - 1000 mM of tris(hydroxymethyl)aminomethane hydrochloride, 50 - 500 mM of potassium phosphate, 50 - 500 mM of ethylenediaminetetraacetic acid, 0.01 - 1.0 M of sodium chloride, 0.01 - 1.0 M of sodium acetate, and 50 - 500 mM of sodium hydroxide. Ascorbic acid can be included at 5 - 50 mM, and sodium dodecyl sulfate can be included at 0.1 - 3.0 wt%.

[0020] The buffer solution is preferably prepared at 5 - 50 times the weight of the original camellia.

[0021] The centrifugation in Step 2 is performed at 4 - 30 °C, 5,000 - 10,000 × g for 10 minutes - 2 hours.

[0022] While DNA is eluted in the second step, other components constituting camellia other than the DNA can be separated as impurities. These can be various inorganic components, lipids, fatty acids, proteins, fibrous substances, and amino acids that constitute camellia.

[0023] When adding alcohol in the third step, it is often better to slowly add the cooled alcohol at a rate of 0.1 - 10 mL / sec so that the sample containing alcohol and DNA is well mixed. At this time, the DNA-containing solid content can also be maintained in a state of immersion / sedimentation for 1 - 72 hours so that the precipitation and solidification of DNA are better performed.

[0024] Furthermore, the alcohol used in all steps of the present invention may be a C1-C4 alcohol, preferably selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. The alcohol may also be a mixed solution with water at a concentration of 10-90% (v / v) if necessary.

[0025] In the DNA purification steps described in steps 3 to 5 above, once the DNA-containing solid settles, the precipitate can be separated by centrifugation to obtain only the solid. The obtained DNA-containing solid can be dissolved in purified water or an ionized buffer solution, treated with alcohol to cause it to settle as a solid, then centrifuged again, and the solid can be recovered again. This process of redissolving the solid repeatedly can be continuously repeated to further increase the purity of the DNA.

[0026] The DNA (PDRN) production method described above is characterized by the extraction and purification of high-purity DNA using only environmentally friendly substances such as ascorbic acid, without using any substances harmful to the human body (chloroform, phenol).

[0027] Furthermore, the present invention relates to a cosmetic composition containing camellia DNA (PDRN) produced by the method described above.

[0028] The camellia DNA (PDRN) of the present invention is characterized by comprising one or more polynucleotide sequences selected from SEQ ID NOs: 1 to 3, and having a molecular weight of 75 to 300 bp and 50 to 200 kDa.

[0029] The camellia-derived DNA (PDRN) extracted from this invention is light brown or light lilac in color, has no distinctive odor, and has a pH of 5.5 to 8.5.

[0030] In this invention, all parts of the camellia plant, including the flowers, stems, branches, fruits, roots, and seeds, can be used as the raw material for camellia PDNR.

[0031] Furthermore, the present invention provides an anti-aging and antioxidant pharmaceutical composition containing PDRN derived from camellia and a pharmaceutical excipient. The PDRN can be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 30% by weight.

[0032] The aforementioned pharmaceutical compositions can be prepared by conventional methods in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and solutions for sterile injection. Examples of carriers, excipients, and diluents that may be included in the pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. Such solid formulations are prepared by mixing the extract of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suitable suppository bases include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0033] The dosage of the pharmaceutical composition of the present invention varies depending on the age, sex, weight, specific disease or condition to be treated, severity of the disease or condition, route of administration, and the prescriber's judgment. Determining the dosage based on such factors is within the realm of those skilled in the art, and generally, the dosage ranges from 0.01 mg / kg / day to about 2000 mg / kg / day. A more preferred dosage is 1 mg / kg / day to 500 mg / kg / day. The drug may be administered once daily or in several divided doses. The aforementioned dosages do not limit the scope of the present invention in any way.

[0034] The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans via various routes. All methods of administration are predictable, but for example, it can be administered orally, rectally, intravenously, intramuscularly, subcutaneously, intrauterine, dura mater, or intravascularly. Because the composition of the present invention has virtually no toxicity or side effects, it is a drug that can be safely used even when taken long-term for preventive purposes.

[0035] The cosmetic composition of the present invention can be manufactured in any dosage form commonly produced in the art, including essences, lotions, creams, emulsions, masks, hand creams, foot creams, body lotions, lip balms, lipsticks, eyeshadows, eyeliners, eyebrow pencils, blushes, highlighters, general toners, skin lotions, creams, serums, beauty soaps, softening toners, medicated toners, body washes, cleansing foams, cleansing lotions, gels, cleansing oils, cleansing creams, cleansing tissues, cleansing waters, and mask packs. Other hair products may also be used, and in particular, in the case of various hair cosmetic products for hair growth, hair loss improvement, scalp improvement, etc., the dosage form is preferably not particularly limited, but more specifically, it can be selected from any dosage form such as hair soap, hair shampoo, hair rinse, hair serum, hair treatment, hair essence, hair water, hair tonic, hair lotion, hair emulsion, hair cream, hair massage cream, hair wax, hair pack, hair oil, hair drying agent, hair preservation treatment agent, hair dye, hair bleach, hair gel, hair glaze, hair mousse, hair spray, hair ampoule, etc.

[0036] More specifically, when the dosage form of the cosmetic composition of the present invention is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide can be used as the carrier component. When the dosage form of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier component, and especially in the case of a spray, propellants such as chlorofluorohydrocarbon, propane-butane, or dimethyl ether may be further included. When the dosage form of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent, or emulsifier can be used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, or sorbitan fatty acid esters. When the dosage form of the cosmetic composition of the present invention is a suspension, the carrier component can be 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 tracant. When the dosage form of the cosmetic composition of the present invention is a surfactant-containing cleanser, the carrier component can be aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinate monoester, acetylate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic acid derivative, or ethoxylated glycerol fatty acid ester. The cosmetic composition of the present invention may further contain excipients such as fluorescent substances, fungicides, hydrophrenic agents, humectants, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, UV-blocking agents, vitamins, and plant extracts.The amount of these components added can be selected according to the dosage form or intended use, within a range that does not impair the inherent effects of the cosmetic composition. The amount of these components added may be, for example, 0.1 to 10% by weight, preferably 0.1 to 6% by weight, relative to the total weight of the composition, but is not limited to these amounts. [Effects of the Invention]

[0037] This invention relates to a composition containing DNA (PDRN) extracted from camellia. The camellia PDRN improves and activates the state of mitochondria and enhances anti-glycation efficacy. Furthermore, it is excellent in expressing various cell-stimulating factors or skin-improving factors that restore aged or damaged cells to a youthful state. [Brief explanation of the drawing]

[0038] [Figure 1] This image shows the electrophoretic results of the Camellia PDRN of the present invention. [Figure 2] This graph shows the results of confirming the RAGE gene expression suppression efficacy of camellia PDRN. [Figure 3] This graph shows the results of confirming the MMP-1 gene expression suppression efficacy of camellia PDRN. [Figure 4] This image shows a Western blot band image confirming the efficacy of Camellia PDRN in increasing LC3-II protein expression, along with a graph of the resulting numerical values. [Figure 5] This graph shows the results of confirming the lipofuscin accumulation inhibitory effect of camellia PDRN. [Figure 6] This graph shows the results of confirming the efficacy of Camellia PDRN in suppressing TFEB gene expression. [Figure 7] This graph shows the results of confirming the efficacy of camellia PDRN in restoring mitochondria in damaged cells. [Figure 8] This graph shows the results of confirming the efficacy of Camellia PDRN in suppressing SDF-1 gene expression. [Figure 9] This graph shows the results of examining the melanin production rate per unit cell in camellia PDRN. [Figure 10]This graph shows the results of confirming the efficacy of camellia PDRN in suppressing ROS generation. [Figure 11a] This image shows the results of confirming the CPD production inhibitory effect of camellia PDRN by cell fluorescence staining. [Figure 11b] This graph shows the results of confirming the CPD production inhibitory effect of camellia PDRN using cell fluorescence staining, quantified numerically. [Figure 12] This graph shows the results of confirming the anti-redness effect of suppressing EDNRb gene expression in Camellia PDRN. [Modes for carrying out the invention]

[0039] 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 embodiments presented herein are provided so that the concept of the present invention may be fully conveyed to those skilled in the art, so that the disclosed content may be thorough and complete. <Cell preparation>

[0040] For this experiment, senescent human dermal fibroblast cells (ATCC PCS-201-010) with a doubling time of 12 days or more after subculturing were used. The experimental results are simply labeled "Adult." (The typical doubling time for normal and cancer cells is at least 72 hours, and as fast as 16 hours.)

[0041] As the control group for the aforementioned senescent cells, young human dermal fibroblast cells (ATCC CRL-2076, CCD-1064Sk, neonatal) with a doubling time of 2 days or less were used, and the experimental results were simply described as "Young."

[0042] Other cells used included B16-F1 (mouse melanoma cells), keratinocytes (human keratinocytes, HaCaT), and HUVEC (human umbilical vein endothelial cells). All cell cultures were basically performed in a 37°C, 5% CO2 incubator.

[0043] Furthermore, depending on the cell type, IMDM (Iscove's Modified Dulbecco's Medium, Welgene, South Korea), EpiLife (Gibco, USA), and DMEM (Dulbecco's Modified Eagle's Medium, Welgene, South Korea) were used, mixed with fetal bovine serum (FBS) at appropriate concentrations. <Example 1. Production of Camellia-derived PDRN> To obtain camellia PDRN (DNA), the original camellia plant was first finely ground.

[0044] Camellia extract was mixed with a buffer solution containing 500 mM tris(hydroxymethyl)aminomethane hydrochloride, 100 mM potassium phosphate, 170 mM ethylenediaminetetraacetic acid, 0.1 M sodium chloride, 0.1 M sodium acetate, and 100 mM sodium hydroxide, along with 10 mM ascorbic acid and 0.5% by weight sodium dodecyl sulfate. At this time, 40 times the weight of the camellia extract was added as purified water for preparing the buffer solution. The mixture was then heated at 60°C for 1 hour with stirring.

[0045] After separating the camellia tissue from the DNA, impurities other than DNA, such as the aforementioned tissue, were removed. Further, impurities were removed by centrifuging at 4°C and 7500 rpm for 10 minutes and removing the precipitated solids. In addition, to the solution remaining after most of the impurities had been removed, 99.9% ethanol was added to adjust the ethanol concentration in the solution to 50 (v / v)% in order to precipitate the camellia-derived DNA in the solution. Further centrifugation was performed, and the ethanol was discarded, leaving only the precipitate containing DNA. The obtained precipitate was dissolved in purified water, precipitated again in ethanol (purity 99.99% or higher), centrifuged, and the precipitate was rehydrated in purified water. This process was repeated 2 to 4 times, and the ethanol component was removed from the precipitate, purifying only the DNA component and obtaining high-purity camellia PDRN. <Example 2. Gene sequence analysis of PDRN derived from camellia>

[0046] Extracted PDRNs from camellia were quantified using nanodrop, and the presence or absence of DNA was confirmed by electrophoresis of 10 μL of DNA on a 0.5% agarose gel. As shown in Figure 1, the PDRNs were found to be in good condition, with an average size of 75-300 bp and a molecular weight of 50-200 kDa.

[0047] Subsequently, we commissioned Macrogen Co., Ltd. to perform metagenome amplicon sequencing using the obtained Camellia PDRN. This method is a community dielectric analysis method that can determine which species make up a particular DNA sample. It involves amplifying a specific region of a marker gene to create an amplicon library, and then analyzing its distribution and diversity via sequencing.

[0048] For library preparation for sequence analysis, rbcl2-rbcLa and ITS2F-ITS4R were amplified according to the Illumine 16s metagenomics sequencing library protocol. The DNA sample was analyzed as "Camellia sp." 5 ng (rbcL2-rbcLa) and 10 ng (ITS2F-ITS4R) of gDNA were mixed with buffer solution for PCR amplification, followed by 1 mM dNTP mix and 500 nM F / R PCR primers. The primer sequence information is as follows:

[0049] [Table 1]

[0050] Metagenomic amplicon sequencing analysis revealed that the Camellia PDRN obtained in this invention was analyzed at the genetic level as belonging to the genus Camellia, and was confirmed to possess the following representative genes. These genes may include Camellia elongate, Camellia honkonggensis, and Camellia reticulate.

[0051] [Table 2] <Example 3. Evaluation of the anti-glycation efficacy of Camellia PDRN - Confirmation of RAGE gene expression>

[0052] RAGE (Receptor for Advanced Glycation End Products) is a gene that encodes a receptor that recognizes and binds to advanced glycation end products (AGEs). The RAGE protein is located on the surface of the cell membrane and is highly expressed, especially in immune cells, nerve cells, and vascular endothelial cells. When AGEs bind to the RAGE receptor on the cell surface, strong inflammatory and oxidative stress signals are activated within the cell, accelerating aging and various diseases while promoting inflammation and tissue damage. Therefore, inhibiting RAGE is considered very important in anti-aging, and we confirmed the relationship of camellia PDRN.

[0053] For this purpose, human neonatal dermal fibroblast cells were inoculated into 60 mm plates and cultured. Once the cells were stabilized, the medium was changed to serum-free medium, treated with TNF-α (Tumor Necrosis Factor-α) and camellia PDRN samples, and cultured in a 37°C, 5% CO2 incubator. Subsequently, the cells were treated with QIAzol TM RAGE gene expression was confirmed by collecting RNA using Lysis Reagent (Qiagen), extracting it according to the manufacturer's method, quantifying the extracted RNA, synthesizing cDNA, and performing Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0054] [Table 3] As a result, as shown in Figure 2 and Table 3, RAGE gene expression was reduced by 26.5% with 10 μg / mL of camellia PDRN.

[0055] Furthermore, to confirm cytotoxicity, the same cells were cultured with PDRN samples for 24 hours and then treated with MTT solution. The supernatant was removed, DMSO was added to dissolve the resulting MTT-formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader. As a result, no cytotoxicity was observed with Camellia PDRN at concentrations of 1-10 μg / mL. <Example 4. Anti-wrinkle evaluation of Camellia PDRN - Confirmation of MMP-1 (Matrix Metalloproteinase-1) gene expression>

[0056] MMP-1 (Matrix Metalloproteinase-1) specifically cleaves and degrades type I procollagen, which makes up approximately 90% of the dermis layer of the skin. Collagen is a fibrous protein responsible for skin elasticity and structural support, but excessive activation of MMP-1 disrupts the collagen network, causing the skin to break down and lose elasticity. This led to the confirmation of the effect of camellia PDRN on MMP-1 expression.

[0057] Young human dermal fibroblast cells were inoculated into 60 mm plates and cultured. UVA 25 mJ / cm² 2 After irradiation, the medium was changed to serum-free medium, and the camellia PDRN sample was treated and cultured for 24 hours at 37°C in a 5% CO2 incubator. The cultured cells were then treated with QIAzol TM RNA was collected using Lysis Reagent (Qiagen) and extracted according to the manufacturer's method. Next, after quantifying the extracted RNA, cDNA was synthesized, and MMP-1 gene expression was confirmed by Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0058] [Table 4] As a result, as shown in Figure 3 and Table 4, treatment with camellia PDRN5 at 10 μg / mL reduced MMP-1 expression to approximately 43.3% and 43.8%, respectively. <Example 5. Autophagy activation of Camellia PDRN>

[0059] LC3 is an important biomarker of autophagy and a protein essential for the process of autophagosome formation. LC3 is classified into LC3-I (cytoplasmic type) and LC3-II (membrane-bound type) depending on its intracellular morphology. During autophagy activation, LC3-I is converted to LC3-II through lipidation. Of these, LC3-II specifically binds to the autophagosome membrane and is widely used as an indicator reflecting the degree of autophagy activity.

[0060] Adult and young human dermal fibroblasts were inoculated into 60mm plates and cultured. Once the cells stabilized, the culture medium was changed, the PDRN sample was treated, and the cells were cultured for 24 hours at 37°C in a 5% CO2 incubator. Next, Proprep solution (iNtRON) was added, and proteins were extracted according to the manufacturer's method. After quantifying the proteins, SDS-PAGE was performed, the membrane was blocked with 5% skim milk, and the cells were treated with primary antibodies [LC3-II, β-actin]. After primary antibody treatment, the cells were washed with 1×TBST and treated with secondary antibodies. Finally, after washing with 1×TBST again, the cells were treated with Claro Supremo ECL solution, and the bands were confirmed with Chemidoc (ATTO, Japan).

[0061] [Table 5]

[0062] Referring to Figure 4 and Table 5, we can see that LC3-II expression is decreased in adult cells compared to young cells, confirming that autophagy induction is inhibited in the senescent state. However, LC3-II expression increased in a concentration-dependent manner upon treatment with camellia PDRN, and was shown to increase 3.6 times, particularly at 10 μg / mL. <Example 6. Evaluation of waste product accumulation improvement by autophagy activity of Camellia PDRN - Confirmation of lipofuscin accumulation>

[0063] Removing dysfunctional organelles is one of the important processes within cells. Traditionally, cells have removed dysfunctional organelles via the autophagy system. However, as cellular senescence progresses, problems arise with lysosomes and the autophagy system, preventing them from functioning normally. The accumulation of dysfunctional organelles accelerates cellular senescence. Lipofuscin, a macromolecular substance within lysosomes, is mainly composed of cross-linked protein residues formed by iron-catalyzed oxidation processes. Because it is not degraded, it accumulates in cells during the aging process, and therefore, improvement in lipofuscin accumulation can be considered an indicator of aging improvement. For this reason, we investigated whether camellia PDRN inhibits lipofuscin accumulation.

[0064] Adult and young human dermal fibroblasts were inoculated into 4-well slides and cultured. Once the cells stabilized, the medium was changed to serum-free medium, treated with PDRN samples, and cultured for 24 hours at 37°C in a 5% CO2 incubator. After fixing the cultured cells and staining the nuclei with DAPI, autofluorescence and DAPI fluorescence imaging were performed using a Leica fluorescence microscope. Subsequently, quantification was performed using Image J.

[0065] [Table 6]

[0066] As confirmed in Figure 5 and Table 6, camellia PDRN reduced lipofuscin autofluorescence by 83.6% in adult cells, showing the greatest improvement in lipofuscin accumulation at 10 μg / mL. <Example 7. Efficacy of Camellia PDRN to improve the cell digestive system: Suppression of TFEB gene expression>

[0067] TFEB (Transcription Factor EB) is a transcription factor that induces the expression of genes related to intracellular lysosome biogenesis and autophagy, and functions to ensure the efficient removal of damaged organelles. In other words, TFEB activation contributes to the removal of waste products and the maintenance of cellular homeostasis by promoting the intracellular detoxification system.

[0068] To confirm the efficacy of camellia PDRN, adult human dermal fibroblasts and young human dermal fibroblasts were first inoculated into 60 mm plates and cultured. Once the cells stabilized, the culture medium was changed, the PDRN sample was treated, and the cells were cultured for 24 hours at 37°C in a 5% CO2 incubator. Subsequently, the cells were treated with QIAzol TM RNA was collected using Lysis Reagent (Qiagen), extracted according to the manufacturer's method, quantified, and then cDNA was synthesized. TFEB gene expression was confirmed by performing Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0069] [Table 7]

[0070] Figure 6 and Table 7 confirm that TFEB gene expression is increased in adult cells, and that TFEB gene expression is reduced by 41.4% with 10 μg / mL of camellia PDRN. <Example 8. Confirmation of the efficacy of Camellia PDRN in improving mitochondrial function - JC-1 staining>

[0071] To assess intracellular damage through mitochondrial function, JC-1 staining was performed. For this purpose, adult and young human dermal fibroblasts were inoculated into 24-well plates and cultured. Once the cells stabilized, the culture medium was changed, PDRN samples were treated, and the cells were cultured at 37°C in a 5% CO2 incubator. Next, the medium was changed to one containing 1.5 μg / mL of JC-1 dye, and the cells were incubated at 37°C in a 5% CO2 incubator for 1 hour and 30 minutes. Finally, after washing with HBSS, the cells were measured using an ELISA reader (Ex=530nm, Em=590nm). Imaging was then performed using a fluorescence microscope (Leica).

[0072] [Table 8]

[0073] Referring to Figure 7 and Table 8, it can be confirmed that mitochondrial function activation occurs most actively at 1 μg / mL of camellia PDRN, and at this time, adult human dermal fibroblasts were shown to be restored to the state of young human dermal fibroblasts. <Example 9. Efficacy of Camellia PDRN in improving complexion - Measurement of SDF-1 (Stromal Cell-Derived Factor 1) gene expression>

[0074] SDF-1 (Stromal Cell-Derived Factor 1) is a type of chemokine secreted by dermal fibroblasts. It is involved in the migration of epidermal stem cells for skin regeneration, as well as the proliferation and migration of keratinocytes. It also influences the regulation of pigmentation and plays an important role in age spots / dark spots. For this reason, SDF-1 expression is known to decrease in aged skin (exogenous / endogenous aging).

[0075] To investigate the effect of camellia PDRN on SDF-1 expression, adult human dermal fibroblasts and young human dermal fibroblasts were inoculated into 6-well slides and cultured. Once the cells were stabilized, the culture medium was changed, the PDRN sample was treated, and the cells were cultured for 24 hours at 37°C in a 5% CO2 incubator. The cells were then treated with QIAzol TM RNA was collected using Lysis Reagent (Qiagen) and extracted according to the manufacturer's method. After quantifying the extracted RNA, cDNA was synthesized and SDF-1 gene expression was confirmed by Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0076] [Table 9]

[0077] Referring to Figure 8 and Table 9, SDF-1 mRNA expression was decreased in adult cells, but it was confirmed to increase by approximately 1.5 times after treatment with 10 μg / mL of camellia PDRN. <Example 10. Confirmation of the whitening efficacy of Camellia PDRN: Measurement of melanin production per unit cell>

[0078] Cells were inoculated with B16-F1 in 24-well plates and cultured. After 8 hours, the medium was changed to one containing α-MSH, the PDRN sample was treated, and the cells were cultured for a further 72 hours at 37°C in a 5% CO2 incubator. Next, the cell culture medium was transferred to a 96-well plate and the absorbance (450 nm) was measured using an ELISA reader. Each cell was treated with 1N NaOH, lysed at 60°C for 30 minutes, transferred to a 96-well plate, and the absorbance (450 nm) was measured using an ELISA reader.

[0079] [Table 10]

[0080] Referring to Figure 9 and Table 10, we were able to confirm that the melanin production rate per unit cell decreased by 27% when treated with Camellia PDRN at 10 μg / mL (the melanin secretion rate per unit cell decreased by 26% with Camellia PDRN at 10 μg / mL).

[0081] Furthermore, to confirm cytotoxicity, PDRN samples were cultured in B16-F1 for 72 hours, followed by treatment with MTT solution. The supernatant was removed, DMSO was added to dissolve the resulting MTT-formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader. As a result, no cytotoxicity was observed with Camellia PDRN at concentrations of 1-10 μg / mL. <Example 11. Antioxidant evaluation of Camellia PDRN - ROS (Reactive Oxygen Species) measurement>

[0082] Adult and young human dermal fibroblasts were inoculated into 24-well plates and cultured. The culture medium was changed to serum-free medium, treated with PDRN samples for 24 hours, and cultured at 37°C in a 5% CO2 incubator. H2DcFDA solution was added and the cells were reacted at 37°C in a 5% CO2 incubator for 30 minutes. After washing with HBSS, the cells were measured using an ELISA reader (Ex=485nm, Em=535nm).

[0083] [Table 11]

[0084] Referring to Figure 10 and Table 11, ROS (Reactive Oxygen Species) generation decreased by 22.0% when treated with 10 μg / mL of camellia PDRN. <Example 12. Measurement of UV-induced DNA damage (CPD) formation in Camellia PDRN>

[0085] A typical indicator of DNA damage caused by UVB-induced sunburn is the increased production of CPD (Cyclobutane pyrimidine dimer) in keratinocytes. However, UV-damaged DNA is repaired through the nucleotide excision repair (NER) pathway, which causes CPD to decrease again.

[0086] Therefore, we treated cells damaged by UVB with camellia PDRN to confirm the degree of CPD generation. After inoculating keratinocytes (HaCaT) into 4-well slides, we replaced the medium with serum-free medium, treated the PDRN samples, and cultured them at 37°C in a 5% CO2 incubator. Next, we applied UVB 25 mJ / cm². 2 After irradiation and incubation for 2 hours, fixation was performed, and the cells were reacted with a TDM-2 antibody that recognizes CPD for 30 minutes. Subsequently, they were reacted with Alexa Fluor 594 secondary antibody and DAPI, and then imaged using a fluorescence microscope (Leica).

[0087] [Table 12]

[0088] Referring to Figure 11a, Figure 11b, and Table 12, UVB 25 mJ / cm 2The treatment significantly increased the production of CPD (Cyclobutane pyrimidine dimer) in damaged cells compared to normal cells, but it was confirmed that more than 30% was eliminated / recovered by treatment with camellia PDRN. <Example 13. Confirmation of the vasoconstrictive effect of Camellia PDRN - Erythema suppression (anti-redness)>

[0089] Keratinocytes, upon UVB exposure, induce COX-2 (Cyclooxygenase-2) expression, which increases the production / synthesis of PGE2 (Prostaglandin E2). The synthesized PGE2 acts on dermal endothelial cells to cause vasodilation, resulting in the sunburn erythema response, where the skin becomes red and warm. Therefore, using HUVEC, a type of vascular endothelial cell, we attempted to confirm whether Camellia PDRN suppresses UVB (Ultraviolet B)-induced vasodilation by inhibiting the expression of EDNRb (Endothelin receptor B) induced by PGE2 stimulation, thereby inducing an anti-redness effect while causing vasoconstriction.

[0090] HUVEC was inoculated into 6-well slides, the medium was changed to serum, and then the PDRN sample was treated and cultured for 24 hours at 37°C in a 5% CO2 incubator. Subsequently, the sample and prostaglandin E2 (PGE2) 0.1 μM were treated and cultured again. The cells were then treated with QIAzol TM RNA was collected using Lysis Reagent (Qiagen) and extracted according to the manufacturer's method. After quantifying the extracted RNA, cDNA was synthesized and EDNRb gene expression was confirmed by Real-time PCR (Applied Biosystems, 7500 FAST Real-Time PCR System).

[0091] [Table 13]

[0092] Referring to Figure 12 and Table 13, it was found that stimulation of vascular cells with PGE2 increased the expression of EDNRb (Endothelin receptor B), but when treated with camellia PDRN at a concentration of 5-10 μg / mL, EDNRb expression was maintained at almost the same level as in the normal group. Furthermore, when the MTT assay was performed on the aforementioned HUVEC, no cytotoxicity was observed up to 10 μg / mL of camellia PDRN, as before.

[0093] These results suggest that camellia PDRN can improve erythema reactions and redness of the skin caused by UVB, or accelerate recovery from such skin damage, and may even prevent or block skin damage before it occurs. It may also mean that applying camellia PDRN can prevent UVB-induced photoaging reactions of the skin.

Claims

1. An anti-aging and antioxidant composition containing PDRN (polydeoxyribonucleotide) extracted from camellia.

2. The anti-aging and antioxidant composition according to claim 1, characterized in that the PDRN has anti-glycation efficacy.

3. The anti-aging and antioxidant composition according to claim 1, characterized in that the PDRN suppresses the generation of ROS (Reactive Oxygen Species).

4. The anti-aging and antioxidant composition according to claim 1, characterized in that the PDRN has the efficacy of inducing lysosome biosynthesis via autophagy and removing intracellular waste products.

5. The anti-aging and antioxidant composition according to claim 1, characterized in that the PDRN has the efficacy of enhancing mitochondrial vitality and promoting the regeneration of damaged cells.

6. The anti-aging and antioxidant composition according to claim 1, characterized in that the PDRN has the efficacy of reducing CPD (Cyclobutane pyrimidine dimer) generated by DNA damage.

7. The anti-aging and antioxidant composition according to claim 1, characterized in that the PDRN has the effect of inhibiting skin pigmentation, whitening effect by inhibiting melanin production, skin erythema inhibiting effect, or wrinkle improvement effect.

8. A cosmetic composition for anti-aging and antioxidant purposes, characterized by comprising the composition described in any one of claims 1 to 7.

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