Cosmetic composition for skin barrier improvement and skin regeneration, containing rose-derived DNA as active ingredient

Abstract

The present invention relates to a cosmetic composition for skin barrier improvement and skin regeneration, containing rose-derived DNA (polydeoxyribonucleotide (PDRN)) as an active ingredient. The rose-derived DNA minimizes DNA degradation caused by oxidative stress and the like, has a high molecular weight and high yield, and exhibits regeneration, wrinkle reduction, elasticity improvement and skin barrier maintenance and improvement effects superior to those of a conventional salmon-derived DNA or a low molecular weight DNA derived from other plants.

Description

Cosmetic composition for improving skin barrier and regenerating skin, containing rose-derived DNA as an active ingredient

[0001] The present invention relates to a cosmetic composition for improving the skin barrier and regenerating the skin, containing rose-derived DNA (PDRN; polydeoxyribonucleotide) as an active ingredient.

[0002] DNA is a polymer composed of phosphates, bases (adenine, thymine, guanine, and cytosine), and deoxyribose, which are double-bonded in a helical pattern based on complementary base structures. It contains genetic information. DNA activates adenosine receptors in cell membranes, and is therefore used for skin regeneration, scar and wound healing, and arthritis treatment.

[0003] Polydeoxyribonucleotide (PDRN), recently used as a key material in cosmeceuticals and bio-cosmetics, is manufactured from DNA extracted from salmon semen and contains deoxyribonucleotide polymers of 50 to 2,000 base pairs. PDRN is mainly used for the treatment of wounds caused by skin grafts and tissue repair, but it is also used for some tissue regeneration or inflammation treatment where there is no suitable treatment, depending on the medical professional's judgment. It 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. It is also known to stimulate the A2 receptor, a skin regeneration signaling pathway, to promote the secretion of various growth factors, induce capillary formation by VEGF (vascular endothelial growth factor), improve blood circulation, have anti-inflammatory effects, and prevent capillary leakage.

[0004] PDRN, an animal-derived ingredient extracted from salmon semen, has proven its effectiveness. However, it is not suitable for vegan products (which do not contain animal ingredients) or cruelty-free cosmetics (which do not undergo animal testing). Furthermore, animal DNA, such as that from salmon, is obtained by chemically removing the phospholipid cell membrane (using organic solvents such as chloroform or phenol), necessitating further improvements in the extraction method.

[0005] Meanwhile, research is underway to derive DNA from natural sources like seaweed as an alternative to animal-derived sources. However, plants and seaweed contain significant amounts of chlorophyll and other impurities for photosynthesis. Therefore, technologies that efficiently remove these impurities and enable commercialization of high-purity DNA are crucial. Furthermore, existing methods can significantly damage DNA, reducing it to a low molecular weight, thereby losing its inherent functionality. Therefore, diverse research is needed to extract DNA in a high molecular weight with minimal damage and to elucidate its efficacy.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] (Patent Document 1) Republic of Korea Patent No. 10-2524599 (Title of the Invention: Liposomal Composition Containing Vegan PDRN Effective for Elasticity, Nutrition, and Regeneration, Applicant: DermaLoop Co., Ltd., Registration Date: October 21, 2022)

[0009] (Patent Document 2) Republic of Korea Patent No. 10-2249241 (Title of the invention: Polydeoxyribonucleotide derived from seaweed having angiogenesis and cell regeneration effects and method for extracting polynucleotides, Applicant: Han Ji-seong, Registration date: November 20, 2020)

[0010] (Patent Document 3) Republic of Korea Patent No. 10-2237543 (Title of the invention: Method for producing DNA derived from aloe plants, and composition for anti-aging and anti-inflammatory purposes containing DNA derived from aloe plants as an effective ingredient, Applicant: Moachem Co., Ltd., Registration date: February 22, 2019)

[0011] The purpose of the present invention is to provide a cosmetic composition for improving the skin barrier and regenerating the skin, which contains rose-derived DNA (PDRN; polydeoxyribonucleotide) as an active ingredient.

[0012] The present invention relates to a cosmetic composition for improving the skin barrier and regenerating the skin, containing PDRN (polydeoxyribonucleotide) extracted from rose as an active ingredient.

[0013] The above PDRN has the effect of restoring protein expression of laminin 5 or collagen type XVII in damaged skin cells, and enhances the expression of filaggrin, thereby improving the skin barrier.

[0014] The above PDRN has the effect of promoting skin cell proliferation and wound healing, and is characterized by enhancing the regenerative effect of skin cells.

[0015] The above rose-derived PDRN has a whitening effect by inhibiting melanin biosynthesis and has ROS (reactive oxygen species) scavenging ability.

[0016] The rose-derived PDRN of the present invention enhances collagen type I or collagen type III protein, thereby restoring skin elasticity and improving wrinkles.

[0017] The rose-derived PDRN of the present invention may be obtained through the following extraction method. Preferably,

[0018] (Step 1) A step of adding a buffer solvent to rose raw materials and crushing them to obtain rose crushed material;

[0019] (Step 2) A step of storing and maturing the above rose powder in a dark room;

[0020] (Step 3) Step of heating the rose powder aged in a dark room;

[0021] (Step 4) Step to remove impurities separated while heating;

[0022] (Step 5) A step of adding salt and alcohol to the reactant from which impurities have been removed to obtain a precipitate containing DNA; and,

[0023] (Step 6) A step of obtaining a precipitate, dissolving it in purified water, and purifying DNA may be included.

[0024] The rose raw material used in Step 1 above can be dried or fresh. Any part of the rose, including the whole plant, roots, aerial parts, stems, leaves, flower buds, and fruit, can also be used.

[0025] The rose of the first step above can be pulverized using a buffer solution (pH 5 to 9). The buffer solution is water, alcohol, or a mixed solvent thereof, and may be a buffer solution to which at least one raw material selected from the group consisting of arginine, vitamin C, Tris-HCl, glycine, diphosphate, phosphate, potassium diphosphate, and potassium phosphate is added.

[0026] Additionally, the buffer solution or the raw material within the buffer solution may be further added prior to the heating in the third step. At this time, the buffer solution may be further added to the rose powder in an amount 1 to 3 times its weight, or the raw material within the buffer solution may be further added in an amount 1 to 3 times its weight, thereby increasing only the concentration by 1 to 3 times.

[0027] The above buffer solution serves to reduce damage during the extraction process of rose DNA.

[0028] Each raw material in the above buffer solution can be included at 4 to 300 mM for rose crushing.

[0029] Additionally, the alcohol used in all steps of the present invention may be a C1 to C4 alcohol, and preferably may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and isobutanol. Additionally, the alcohol may be a mixed solution of 10 to 90 (v / v)% with water, if necessary.

[0030] Additionally, in the second step, the rose powder can be stored in a dark room at room temperature (20-30°C) for 24-96 hours. During this time, chlorophyll synthesis is blocked in each tissue within the rose material, components other than DNA are decomposed by enzymatic action of pectin or cellulose, and the DNA is cut to a size suitable for use in the present invention.

[0031] Through the heating step of the third step, the rose plant cells are softened and DNA is extracted from the cells. The heating step can be performed at 20 to 70°C for 0.5 to 48 hours.

[0032] The impurities in the fourth step may include various cell walls, cytoplasm, proteins, fibers, etc. that make up the rose tissue. Furthermore, this step may involve adjusting the acidity to the isoelectric point of the protein using organic acids such as acetic acid, hydrochloric acid, citric acid, and lactic acid to precipitate the protein impurities, and then performing centrifugation or pressure filtration to remove the impurities. Preferably, this step may also involve removing substances exhibiting viscosity through diatomaceous earth filtration.

[0033] The salt of the fifth step may contain 0.1 to 3.0 M of an ionic component (a mixed component among calcium, iodine, iron, magnesium, selenium, zinc, sodium, phosphorus, sulfur, chlorine, copper, manganese, iron, and guanidine). NaCl can preferably be used as the salt.

[0034] When adding alcohol in the above 5th step, it is recommended to slowly add alcohol at a rate of 0.1 to 10 mL / sec to ensure that the alcohol and the sample containing DNA are well mixed, and it is recommended to maintain the immersion state for 1 to 72 hours.

[0035] The step of purifying DNA in the above 6th step can be performed by continuously repeating the process of separating the precipitate obtained in the 5th step through centrifugation and pressure filtration to obtain only the solid, dissolving the obtained DNA-containing solid in purified water or an ionic buffer solution, centrifuging again, additionally recovering the solid, and repeatedly redissolving the solid to further increase the purity of the DNA.

[0036] The present invention also relates to a cosmetic composition containing rose-derived DNA (PDRN) prepared by the above method.

[0037] Conventionally known DNA extraction methods have a molecular weight of less than 200 bp in an agarose gel, but the rose DNA of the present invention contains DNA having a molecular weight of more than 5,000 bp, approximately 15 to 20 kbp, and approximately 9,750 to 13,000 kDa. DNA with such a high molecular weight and extracted with minimal damage has the effect of inhibiting DNA degradation due to oxidative stress, etc.

[0038] The DNA extracted in the present invention is yellow-green in color, has no specific odor, and has a pH of 5.0 to 7.5.

[0039] The rose-derived PDRN of the present invention inhibits melanin expression by 20-50% when treated at 6-12 ㎍ / ml per unit cell. In addition, the ROS scavenging ability is reduced by 20-40% by the PDRN of 2-12 ㎍ / ml, and the wound healing efficacy is increased by 150-250% when treated at 2-12 ㎍ / ml. The proliferation of skin cells is also increased by 130-150% by the rose-derived PDRN of 6-12 ㎍ / ml, and collagen type I shows the best efficacy by increasing by 130-180% when treated at a concentration of 2-6 ㎍ / ml, and collagen type III by 300-400% at 3 ㎍ / ml and by 500-600% when treated at 6-12 ppm. The expression of laminin is increased by 200-300% with 2 ppm of rose-derived PDRN, 350-500% with 2-12 ppm of collagen type XVII, and 140-160% with 6 ppm of filaggrin.

[0040] The roses used in the present invention can be any of the Western roses, Oriental roses, European roses, variants, hybrids, cultivated hybrids, subspecies, or varieties of the Rosa genus, and can be applied without exception to the colors of the petals being red, white, yellow, blue, pink, orange, pink, purple, black, etc. For example, Rosa persica, Rosa chinensis, Rosa gigantean, Rosa arvensis, Rosa gallica, Rosa moschata, Rosa damascene, Rosa centifolia, Rosa hybrida, Rosa hybrida compassion, Rosa hybrida compassion, Rosa Hybrida Hortorus, Rosa Dr. Huey, and various improved varieties or improved cultivars thereof, and Oriental roses may also be used. About 25,000 species of roses have been developed so far, and 6,000 to 7,000 species currently exist, and more than 200 new cultivars are developed every year.

[0041] In addition, the DNA-containing composition of the present invention can be provided as a cosmetic composition, and the cosmetic composition can be prepared in any formulation commonly manufactured in the art, and can be provided in the form of an essence, a lotion, an emulsion, a pack, a hand cream, a foot cream, a lip balm, a lipstick, an eye shadow, an eyeliner, an eyebrow pencil, a blusher, a highlighter, a general toner, a skin, a cream, a serum, a cosmetic soap, a softening toner, a medicated toner, a body cleanser, a cleansing foam, a cleansing lotion, a gel, a cleansing oil, a cleansing cream, a shampoo, a rinse, a hair treatment, a hair lotion, a cleansing tissue, and a cleansing water.

[0042] More specifically, when the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivatives, 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 particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane-butane or dimethyl ether may be additionally included. When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, and examples thereof include 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. When 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, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, and the like can be used as a carrier component. When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.The cosmetic composition of the present invention may additionally contain excipients including fluorescent substances, fungicides, hydrotropism inducers, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc. The amounts of the above components may be selected according to the formulation or intended use within a range that does not impair the inherent effects of the cosmetic composition. The amount of the above components may be, for example, 0.1 to 10 wt%, preferably 0.1 to 6 wt%, based on the total weight of the composition, but is not limited thereto.

[0043] The present invention relates to a cosmetic composition for improving skin barrier and regenerating skin, containing rose-derived DNA (PDRN; polydeoxyribonucleotide) as an active ingredient. The rose-derived DNA is characterized by minimizing DNA degradation caused by oxidative stress and the like, and possessing high molecular weight and high yield. Compared to existing salmon-derived DNA or low-molecular-weight DNA derived from other plants, it exhibits superior regeneration, wrinkle improvement, elasticity improvement, and skin barrier maintenance and improvement effects.

[0044] In addition, the rose-derived DNA of the present invention maintains its unique functionality by maintaining a DNA structure without loss of molecular weight, and can be provided as various biomaterials utilizing the same.

[0045] Figure 1 shows the results of confirming the quantitative and quality status of the PDRN composition extracted from roses in Example 1 of the present invention by electrophoresis, and it can be confirmed that it has an average size of 20 kb.

[0046] Figure 2 is a graph showing the results of confirming the cytotoxicity of PDRN extracted from roses at different concentrations.

[0047] Figure 3 is a graph showing the effect of rose-derived PDRN on the biosynthesis of melanin per unit cell.

[0048] Figure 4 is a graph showing the results of confirming the ROS (reactive oxygen species) scavenging ability of rose-derived PDRN and salmon-derived PDRN.

[0049] Figure 5a is a photograph showing the results comparing the wound healing efficacy of rose-derived PDRN with that of FBS, and Figure 5b is a graph showing the results in numerical form.

[0050] Figure 6 is a graph showing the results comparing the proliferation efficacy of rose-derived PDRN and salmon-derived PDRN with that of FBS.

[0051] Figure 7 is a photograph (top) of the Western blot results confirming that rose-derived PDRN has the effect of increasing the protein expression of collagen type I (COL-I, Figure 7a) and collagen type III (COL-III, Figure 7b) related to skin elasticity or wrinkle improvement, and a graph (bottom) showing the numerical value of the results.

[0052] Figure 8 is a photograph of the Western blot results and a graph showing the numerical results that confirmed that the expression of laminin 5 (Laminin5, Figure 8a) and collagen type XVII (COL-XVII, Figure 8b) proteins was restored by treatment with rose-derived PDRN in the cell basement membrane damaged by UVB treatment.

[0053] Figure 9a is a photograph showing the results of fluorescent immunostaining on skin cells, confirming that rose-derived PDRN and salmon-derived PDRN enhance the expression of filaggrin, a skin barrier-related factor, and Figure 9b is a graph showing the results in numerical form.

[0054] Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the contents introduced herein are provided to ensure thoroughness and completeness, and to sufficiently convey the spirit of the present invention to those skilled in the art.

[0055] <Preparation of raw materials>

[0056] Salmon PDRN (DNA) was purchased commercially and was confirmed to have a typical size of approximately 500–1500 bp.

[0057] To obtain rose PDRN (DNA), first, the whole rose plant in its raw state was mixed in a buffer solution (aqueous solution, pH 8.0) containing 20 mM glycine, 20 mM Tris-HCl, 75 mM diphosphate, 24 mM phosphate, 20 mM arginine, and 10 mM vitamin C, and ground using a conventional natural product / food grinder. At this time, purified water for preparing the buffer solution was added in an amount 1 to 5 times the weight of the rose raw material. After that, the ground rose material was stored in a dark room maintained at room temperature of 25℃ and relative humidity of 50% for 3 days.

[0058] The rose powder was taken out from the darkroom and further raw materials were added to the buffer solution to make the initial volume of glycine 20 mM, Tris-HCl 20 mM, diphosphate 75 mM, phosphate 24 mM, arginine 20 mM, and vitamin C 10 mM.

[0059] In this state, when the rose tissue and DNA were separated by heating at 60℃ for 1 to 24 hours, the separated tissue impurities were removed.

[0060] In addition, after most of the impurities were removed, 99.9% ethanol was added to the remaining solution so that the NaCl concentration was 1M, and so that the rose-derived DNA in the solution could be precipitated, and so that the ethanol concentration of the solution was 50 (v / v)%.

[0061] By adding an aqueous solution, the obtained precipitate was dissolved in purified water, precipitated again with a 50 (v / v)% ethanol aqueous solution, centrifuged, and the process of rehydrating only the precipitate in purified water was repeated 2 to 4 times to remove the ethanol component in the precipitate and purify only the DNA component, thereby obtaining only high-purity rose PDRN.

[0062] <Example 1. Quality and Quantity Confirmation of PDRN>

[0063] The extracted rose PDRN was quantified by electrophoresing 10 μl of DNA at a concentration of 200 μg / ml on an acrylamide gel and measuring it with NanoDrop.

[0064] As a result, as shown in Fig. 1, it was confirmed that the PDRN was in good condition and had an average size of 20 kbp and a molecular weight of 13,000 kDa. Through repeated experiments, rose PDRN with a molecular weight of 15 to 20 kbp and 9,750 to 13,000 kDa was extracted.

[0065] This result was confirmed in various ways by changing the rose flower color, the rose raw materials such as commercially available Western roses, Oriental roses, European rose varieties and hybrids, but it was similar even when the experiment was repeated.

[0066] Salmon PDRN was identified as 500–1500 bp.

[0067] Additionally, PDRN was extracted from roses using three methods, including the conventional DNA phenol-chloroform extraction method, the ion bridge extraction method, and the room temperature ethanol immersion method, rather than the raw material preparation method of the present invention. Although it was the same rose-derived DNA, most of the DNA was found to be around 200 bp in size and the degree of DNA damage was high, so it was not used in subsequent experiments.

[0068] <Example 2. Confirmation of cytotoxicity>

[0069] Cytotoxicity tests on fibroblasts were conducted by treating rose PDRN with various concentrations. The experimental method is as follows.

[0070] Cells were seeded in a 24-well plate and cultured for 24 hours. The medium of the cultured cells was replaced with serum-free medium, and the samples were treated at various concentrations and cultured for 24 hours. After the culture was completed, the medium was replaced with a 10-fold dilution of a 2.5 mg / ml MTT solution and the reaction was performed. The supernatant was removed, and 1 ml of DMSO was added to dissolve the produced MTT-formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader. The control group used here was DMSO (Dimethyl Sulfoxide) as a dissolution solvent. Cytotoxicity was expressed as a percentage of absorbance compared to the control group, and the results are shown in Figure 2.

[0071] As a result, it was confirmed that the rose PDRN extracted in the present invention is a very safe substance with no cytotoxicity up to a high concentration of about 60㎍ / ㎖.

[0072] <Example 3. Evaluation of whitening efficacy through inhibition of melanin secretion>

[0073] B16-F1 was inoculated into 6-well plates and cultured for 24 hours. The medium was then replaced with fresh DMEM containing α-melanocyte stimulating hormone (α-MSH) and 10% FBS. Each rose PDRN sample was then treated with different concentrations and cultured for 72 hours. After 72 hours, the medium containing melanin was transferred to a 96-well plate, and the absorbance was measured at 450 nm.

[0074] The final concentration of α-MSH used in the experiment was 100 nM, the positive control group, Arbutin, was 100 μg / ml, and each rose PDRN was treated at different concentrations, as shown in Figure 3.

[0075] As shown in Figure 3, it can be seen that the melanin secretion inhibitory effect of rose PDRN is very excellent.

[0076] <Example 4. Confirmation of ROS (Reactive Oxygen Species) Scavenging Ability>

[0077] To evaluate the antioxidant efficacy of rose PDRN, the amount of ROS production was measured in HaCaT cells (human keratinocyte cells).

[0078] For this, HaCaT cells were seeded in a 96-well plate at 3x10 4 Cells were seeded at 1 / well and cultured for 24 hours. After replacing each well with serum-free medium, each complex extract (final treatment concentration 300 ppm) was treated and cultured for 24 hours. At this time, vitamin E, known as a powerful antioxidant, was treated as a control group. After culture, DCFDA (D6883, Sigma-Aldrich, Germany) was treated for dark reaction to stain for ROS, and 1 mM H2O2 was treated for an additional 15 minutes to induce ROS (reactive oxygen species) production. Fluorescence was measured at a wavelength of 485 / 528 (excitation / emission) nm after all sample treatments. The measurement results were calculated as the amount of ROS production / cell viability x 100 (%) and are shown in Fig. 4.

[0079] Referring to Figure 4, it is observed that after inducing ROS generation using H2O2, the ROS scavenging ability is significantly reduced due to treatment with rose PDRN. In comparison, salmon PDRN was found to have a low or very minimal effect in reducing ROS activity.

[0080] <Example 5. Wound healing efficacy - scratch assay>

[0081] Human dermal fibroblasts were cultured in a 24-well plate (corning) for 1 day (37°C, CO25%), scraped with a Scar™ Scratcher (SPL), and washed with HBSS (Hank's Balanced Salt Solution, Gibco). The medium was replaced with serum-free medium (IMDM; Iscove's Modified Dulbecco's Medium, Welgene), and the samples were treated at various concentrations and cultured in an incubator at 37°C, CO25% for 26 hours.

[0082] Cell photographs were taken using a microscope (Leica) immediately after scratching with a scratcher and after 26 hours of culture, and are shown in Fig. 5a. The wound closure value was calculated using ImageJ and is shown in Fig. 5b.

[0083] As shown in the results of Figure 5, it was confirmed that the cell regeneration effect increases depending on the concentration of rose PDRN extracted in the present invention.

[0084] <Example 6. Evaluation of cell proliferation efficacy>

[0085] To confirm the cell proliferation and cell regeneration efficacy of the rose PDRN of the present invention, 1 × 10 HDF (Human Dermal fibroblast) was seeded in a 12-well plate. 5 Cells were dispensed per well and stabilized in a humidified incubator at 37°C and 5% CO2 for 18 hours. After replacing the cell medium with IMDM containing 0.25% FBS, samples were treated with rose PDRN and salmon PDRN at different concentrations and reacted for 48 hours.

[0086] After the reaction, the medium was removed, and the mixture was reacted in a 0.24 mg / ml MTT solution for 4 hr. Then, formazan was dissolved in DMSO and the absorbance was measured at 590 nm. The results are shown in Figure 6.

[0087] <Example 7. Evaluation of skin elasticity enhancement efficacy>

[0088] To evaluate the efficacy of improving skin elasticity, the efficacy of increasing the expression of collagen type I (COL-I) and collagen type III (COL-III), which are fundamental for anti-aging, in HDF (Human dermal fibroblast) cells was analyzed. HDF cells were seeded at 1 x 10 in a 60 mm plate. 6 The cells / well were cultured in IMDM medium supplemented with 10% FBS for 24 hours at 37°C in a 5% CO2 incubator. After removing the medium from the plate, the plate was washed once with PBS (phosphate-buffered saline), replaced with serum-free medium, and each extract was treated at a final concentration of 300 ppm and cultured for 24 hours. Ascorbic acid (100 μg / ml), which helps collagen synthesis enzyme activity, was treated as a positive control. After the culture was completed, the culture medium was removed and washed once with PBS (phosphate-buffered saline) to remove the medium components. The cells were lysed using Proprep (Invitron) buffer, and proteins were extracted and quantified from the cells, and Western blotting was performed.

[0089] As confirmed in each result in Figure 7, it was confirmed that the expression of collagen type I (COL-I, Figure 7a) and collagen type III (COL-III, Figure 7b) increased under the influence of rose PDRN.

[0090] <Example 8. Confirmation of the Skin Barrier Improvement Efficacy of Rose PDRN - I>

[0091] A431 (keratinocyte) cells were prepared under the same conditions as in Example 7, but before treatment with rose PDRN, 50 mJ / cm of UVB 2was investigated for 30 minutes. After that, rose PDRN was treated to the cells, and 24 hours later, the expression of Laminin 5 and collagen type XVII (COL-XVII) proteins, which are factors related to skin barrier improvement, was confirmed through Western blot.

[0092] The results for this are shown in Fig. 8. When the Western blot result bands were confirmed by numerical substitution, it was found that the expression of Laminin 5 (Laminin5, Fig. 8a) and collagen type XVII (COL-XVII, Fig. 8b) proteins was significantly reduced in cells damaged by UVB treatment, but it was confirmed that most of them were restored to normal levels by treatment with rose-derived PDRN.

[0093] <Example 9. Confirmation of the Skin Barrier Improvement Efficacy of Rose PDRN - II>

[0094] Using rose PDRN and salmon PDRN, we directly examined the protein expression level of filaggrin, a skin barrier factor, within cells. To this end, normal human kiratinocyte cells were fixed with 4% paraformaldehyde solution, permeablized with 0.2% Triton X-100 solution, and immunostained sequentially with anti-Filagrin antibody and secondary fluorescent antibody, and images were obtained using a fluorescence microscope.

[0095] As a result, when examining the fluorescent staining photograph (Fig. 9a) and the graph showing the digitized image (Fig. 9b), it can be confirmed that the protein expression amount of filaggrin is higher when rose PDRN is treated, even though the treatment concentration of salmon PDRN is twice as high.

Claims

1. A cosmetic composition for improving the skin barrier and regenerating the skin, characterized by containing PDRN (polydeoxyribonucleotide) extracted from rose as an active ingredient.

2. In paragraph 1, The above PDRN is a cosmetic composition for improving the skin barrier and regenerating the skin, characterized in that it has the effect of restoring the protein expression of laminin 5 or collagen type XVII in damaged skin cells.

3. In paragraph 1, The above PDRN is a cosmetic composition for improving the skin barrier and regenerating the skin, characterized in that it enhances the expression of filaggrin.

4. In paragraph 1, The above PDRN is a cosmetic composition for improving the skin barrier and regenerating the skin, characterized by having a whitening effect.

5. In paragraph 1, A cosmetic composition for improving the skin barrier and regenerating the skin, characterized in that the above PDRN has the effect of inhibiting melanin biosynthesis.

6. In paragraph 1, The above PDRN is a cosmetic composition for improving the skin barrier and regenerating the skin, characterized in that it has ROS (reactive oxygen species) scavenging ability.

7. In paragraph 1, The above PDRN is a cosmetic composition for improving the skin barrier and regenerating the skin, characterized by having skin cell proliferation effects and wound healing effects.

8. In paragraph 1, The above PDRN is a cosmetic composition for improving skin barrier and skin regeneration, characterized by its effect of improving skin elasticity and wrinkles.

9. In paragraph 1, The above PDRN is a cosmetic composition for improving the skin barrier and regenerating the skin, characterized in that it has a protein enhancing effect of collagen type I or collagen type III.

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