A Method for manufacturing nucleic acid fragment uniform size

Abstract

The present application relates to a method for producing nucleic acid fragments and various uses thereof.

Description

Technology Field

[0001] The present application relates to a method for producing nucleic acid fragments. In particular, the method produces nucleic acid fragments of uniform size by applying a specific high pressure.

[0002] This application relates to various uses of nucleic acids produced by the above method. Background Technology

[0004] Nucleic acids are the most important biomolecules, playing a role in storing and transmitting genetic information within the cells of living organisms. In particular, fragmented nucleic acids are essential components of cells and are utilized for purposes such as wound healing and improvement, cell activation, and wrinkle reduction.

[0005] Conventionally, nucleic acids have been fragmented using ultrasound, enzymes, gene scissors, or hydrodynamic methods.

[0006] However, conventional methods have the following problems.

[0007] The method using sonication has the problem that it is difficult to obtain pure nucleic acid because a large portion is separated into single-stranded nucleic acid (Biosens Bioelectron. 2004 Nov 15;20(5):945-55.).

[0008] Methods using enzymes or specific nucleases (such as gene scissors) had the problem of high costs because they required the use of expensive reagents, and also, since these methods were manufactured only in small quantities, commercial use was difficult.

[0009] In addition, while existing methods using hydrodynamic methods can obtain fragmented nucleic acids of 1,500 bp or more, there have been limitations in obtaining small molecular size nucleic acid fragments of 500 bp or less (Nanotechnology22(2011)494013(7pp).

[0010] In particular, when using the conventional methods described above, fragmented nucleic acids are produced as high-molecular-weight nucleic acids with a size of 500 bp or larger. When produced as high-molecular-weight nucleic acids in this way, viscosity develops, posing a significant problem for industrial use. It is already known that the effectiveness varies depending on the size of the fragmented nucleic acids (Mol Med Rep. 2018 Dec;18(6):5166-5172.).

[0011] Therefore, when fragmenting nucleic acids, production into low-molecular-weight nucleic acids rather than high-molecular-weight nucleic acids is required. Prior art literature

[0013] 1. U.S. Patent Publication No. 2011-0070219 The problem to be solved

[0014] One objective of the present application is to provide a method for producing nucleic acid fragments. In particular, the method produces nucleic acid fragments of uniform size by applying a specific high pressure.

[0015] Another objective of the present application is to provide various uses for the nucleic acid produced by the above method. means of solving the problem

[0017] The present application provides a method for preparing a nucleic acid fragment to solve the aforementioned problem. The method may be a method for fragmenting DNA.

[0018] The method for fragmenting the above DNA is,

[0019] i) preparing a first DNA having a size of 500 bp to 5000 bp; and

[0020] ii) applying pressure of 20,000 psi to 30,000 psi to the first DNA for 15 to 30 cycles to obtain a product comprising fragmented second DNA of the first DNA;

[0021] The second DNA obtained above has a size of 50 to 150 nt and may be included in 70% to 100% of the product.

[0022] When providing the above pressure, it can be performed at a temperature of 5°C to 25°C.

[0023] In the above ii) process, pressure may be provided in 15 cycles. At this time, when providing pressure, one cycle may take 0.5 seconds to 10 seconds.

[0024] The above pressure can be achieved using one or more devices among a high pressure homogenizer, a high pressure disperser, an ultra-high pressure homogenizer, and an ultra-high pressure disperser.

[0025] The first DNA may be of fish origin. In this case, the fish may be salmon or trout. Additionally, the first DNA may be single-stranded or double-stranded.

[0026] The first DNA above may have a size of 500 bp to 5000 bp.

[0027] The method for fragmenting the above DNA is,

[0028] iii) The method may further include a step of precipitating the obtained product to isolate and concentrate the second DNA.

[0029] The above precipitate may use one or more selected from sodium chloride (NaCl), potassium chloride (KCl), and sodium acetate (CH3COONa). In this case, the sodium chloride may be used in an amount of 15% to 30% relative to the amount of water. In this case, the above precipitate may use one or more solvents selected from ethanol, methanol, butanol, propanol, pentanol, and isopropyl alcohol.

[0030] The above second DNA may have one or more effects selected from anti-apoptosis activity, reactive oxygen species (ROS) scavenging, cell proliferation activation, cell migration activation, and anti-inflammatory activation. Effects of the invention

[0032] According to the present application, the following effects occur.

[0033] First, according to the present application, a method for producing nucleic acid fragments of uniform size can be provided. In particular, by the above method, small and uniform nucleic acid fragments of 50 to 150 nt can be obtained by specific high pressure.

[0034] Second, according to the present application, various uses of small and uniform nucleic acids produced by the above method can be provided. Brief explanation of the drawing

[0036] Figure 1 shows the results of electrophoresis performed by fragmenting the first nucleic acid contained in the first solution using a high-pressure disperser at a pressure of 10,000 psi with varying cycles. In Figure 1, M represents the marker and STD represents the standard (Mastelli, Placentex); 1 indicates performing high-pressure dispersion for 1 cycle; 2 indicates performing high-pressure dispersion for 2 cycles; 3 indicates performing high-pressure dispersion for 5 cycles; and 4 indicates performing high-pressure dispersion for 10 cycles. Figure 2 shows the results of electrophoresis performed by fragmenting the first nucleic acid contained in the first solution using a high-pressure disperser at a pressure of 25,000 psi with varying cycles. In Figure 2, M represents the marker and 1 represents the first nucleic acid of Experimental Example 1; 2 indicates performing high-pressure dispersion for 1 cycle; and 3 indicates performing high-pressure dispersion for 5 cycles. 4 refers to the result of performing 15 cycles of high-pressure dispersion. Figure 3 is a graph showing the results of electrophoresis obtained by fragmenting the first nucleic acid with different cycles while setting the pressure of the high-pressure disperser to 30,000 psi. At this time, samples were obtained for each cycle, with cycles of 1, 5, and 15. Figure 4 is the result of confirming the cell proliferation effect of the second nucleic acid obtained for each cycle of Figure 2 using HDF (Human dermal fibroblast) cells with EdU and Ki67. In Figure 4, the (-Ve) control is the group treated with serum-free, and the (+Ve) control is the group treated with 10% FBS. In addition, [Fig. 2] Line-2 is a group treated with nucleic acid having a size range of 400 bp to 2600 bp (hereinafter referred to as nucleic acid-A), [Fig. 2] Line-3 is a group treated with nucleic acid having a size range of 150 bp to 350 bp (hereinafter referred to as nucleic acid-B), and [Fig. 2] Line-4 is a group treated with nucleic acid having a size range of 50 bp to 150 bp (hereinafter referred to as nucleic acid-C). Fig. 5 is a graph quantifying the results of Fig. 4.Figure 5A is a numerical graph for Edu-positive cells, and Figure 5B is a numerical graph for Ki67-positive cells. In Figure 5, [-] control refers to the (-Ve) control in Figure 4, and [+] control refers to the (+Ve) control. Figure 6 is the result of comparing the mRNA levels of EGF and VEGF when human primary gingival fibroblasts (HGF) were treated with the second nucleic acid obtained for each cycle of Figure 2. In Figure 6, the control group was treated with distilled water, LPS was the group to establish an inflammatory phenotype, LPS+[Figure 2] Line-2 was the group treated with LPS and nucleic acid-A, LPS+[Figure 2] Line-3 was the group treated with LPS and nucleic acid-B, and LPS+[Figure 2] Line-4 was the group treated with LPS and nucleic acid-C. Figures 7 to 10 show the results of confirming anti-apoptotic activity using HaCaT cells at different concentrations of nucleic acid-C (i.e., PDRN of low molecular size (50bp–150bp)). At this time, a total of three concentrations of PDRN of low molecular size (50bp–150bp) (25µg / ml, 50µg / ml, and 100µg / ml) were compared. In this case, the control is the group treated with distilled water, UV-B is the group treated with UV-B to induce intracellular oxidative stress, and UV-B-NAC is the group treated with N-acetylcysteine ​​(NAC) after UV-B treatment. UV-B+25 µg / ml-PDRN is the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 25 µg / ml after UV-B treatment; UV-B+50 µg / ml-PDRN is the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 50 µg / ml after UV-B treatment; UV-B+100 µg / ml-PDRN is a group treated with low molecular weight PDRN (50 bp–150 bp) at a concentration of 100 µg / ml after UV-B treatment. Figures 7 and 8 show the results of analyzing nuclear condensation using Hoechst.Figure 7 is a fluorescence microscope image of the results of Hoechst staining, and Figure 8 is a graph quantifying the results of Figure 7. Figures 9 and 10 are the results of analyzing sub-G1 low-diploid cells. Figure 9 is the result of analysis using a flow cytometer, and Figure 10 is a graph quantifying the results of Figure 9. Figures 11 and 12 are the results of confirming reactive oxygen species scavenging (ROS scavenging) using HaCaT cells at different concentrations of nucleic acid-C (i.e., PDRN of low molecular size (50bp–150bp)). Figure 11 is the result of analyzing reactive oxygen species scavenging with DCFH-DA (2'-7'dichlorodihydrofluorescein diacetate), and Figure 12 is a graph quantifying the results of Figure 11. At this time, a total of three concentrations (25 µg / ml, 50 µg / ml, and 100 µg / ml) of low molecular weight (50 bp–150 bp) PDRN were compared. Here, the control group was treated with distilled water, the UV-B group was treated with UV-B to induce intracellular oxidative stress, and the UV-B-NAC group was treated with N-acetylcysteine ​​(NAC) after UV-B treatment. UV-B+25 µg / ml-PDRN is the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 25 µg / ml after UV-B treatment; UV-B+50 µg / ml-PDRN is the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 50 µg / ml after UV-B treatment; UV-B+100 µg / ml-PDRN is a group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 100 µg / ml after UV-B treatment. Figures 13 to 15 show the results of confirming the cell proliferation effect using HDF (Human dermal fibroblast) cells at different concentrations of nucleic acid-C (i.e., low molecular weight (50 bp–150 bp) PDRN). At this time, a total of three concentrations of low molecular weight (50 bp–150 bp) PDRN (25 µg / ml, 50 µg / ml, and 100 µg / ml) were compared.Figure 13 shows the MTS (methoxyphenyl tetrazolium salt) analysis results, Figure 14 shows the LIVE / DEAD analysis results, and Figure 15 shows the DAPI / Phalloidin analysis results. In Figure 13, [-]control refers to the group treated with a culture medium without FBS; [+]control refers to the group treated with a culture medium containing 10% FBS. Additionally, 25 µg / ml, 50 µg / ml, and 100 µg / ml represent the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 25 µg / ml; the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 50 µg / ml; and the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 100 µg / ml, respectively. In Figures 14 and 15, (-Ve)control refers to the group treated with a culture medium without FBS; (+Ve)control is the group treated with a culture medium containing 10% FBS. Additionally, 25 μg / ml-PDRN, 50 μg / ml-PDRN, and 100 μg / ml-PDRN refer to the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 25 μg / ml; the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 50 μg / ml; and the group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 100 μg / ml, respectively. Figures 16 and 17 show the results of confirming the cell migration effect using HDF (Human dermal fibroblast) cells with different concentrations of nucleic acid-C (i.e., low molecular weight (50 bp–150 bp) PDRN). At this time, a total of three concentrations (25 μg / ml, 50 μg / ml, and 100 μg / ml) of PDRN with a low molecular size (50 bp to 150 bp) were compared. Figure 16 is a micrograph, and Figure 17 is a graph quantifying the results of Figure 16. In this case, the (-Ve) control is the group treated with a culture medium without FBS; the (+Ve) control is the group treated with a culture medium containing 10% FBS.In addition, 25 μg / ml-PDRN, 50 μg / ml-PDRN, and 100 μg / ml-PDRN refer to a group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 25 μg / ml; a group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 50 μg / ml; and a group treated with low molecular weight (50 bp–150 bp) PDRN at a concentration of 100 μg / ml, respectively. Figures 18 and 19 show the results of confirming anti-inflammatory activity using human primary gingival fibroblasts (HGF) at different concentrations of nucleic acid-C (i.e., low molecular weight (50 bp–150 bp) PDRN). At this time, a total of three concentrations (25 μg / ml, 50 μg / ml, and 100 μg / ml) of PDRN with a small molecular size (50 bp–150 bp) were compared. Figures 18 and 19 show the results of analyzing the mRNA levels of IL-6 (Interleukin-6), IL-1β (interleukin 1-beta), TNF-α (tumor necrosis factor-α), EGF, and VEGF using RT-PCR with human primary gingival fibroblasts (HGF). In this case, the control group was treated with distilled water, and LPS was the group used to establish an inflammatory phenotype. Specific details for implementing the invention

[0037] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application pertains. Methods and materials similar or identical to those described in this application may be used in the practice or testing of this application, but suitable methods and materials are described below. All publications, patent applications, papers, and other references mentioned in this application are incorporated by reference in their entirety. Additionally, materials, methods, and practices are merely illustrative and are not intended to be limiting.

[0039] Definition of Terms

[0040] fragment and fragmentation

[0041] In this application, the term "fragment" refers to a cut, piece, fragment, or sheared product of a component. As used in this application, "nucleic acid fragment" refers to a form having a certain length of substances referred to as nucleic acids, such as RNA, DNA, and PNA; a representative example is DNA having a certain length.

[0042] In one embodiment, whole-length DNA extracted from cells, etc., or DNA having a length of 1000 bp or more can be cut to obtain a DNA fragment having a shorter length.

[0044] In this application, the term "fragmentation" means splitting a component into said fragments or a process including the same. For example, DNA fragmentation means separating or breaking a DNA strand having a length greater than a certain length into fragments having a shorter length. In particular, in this application, fragmentation may be achieved using pressure.

[0046] base pair (bp)

[0047] In this application, the term "base pair (bp)" refers to a unit indicating the length or size of nucleic acids such as RNA, DNA, and PNA. The above-mentioned bp may be used interchangeably with nt (nucleotide) and mer. For example, while it is generally common to use bp when the nucleic acid is double-stranded and nt when it is single-stranded, in this application, either bp or nt may be used regardless of whether the nucleic acid is double-stranded or single-stranded. Furthermore, the above-mentioned mer refers to the DNA or RNA itself composed of several nucleotides. For example, 24-mer refers to a nucleic acid composed of 24 nucleotides. In other words, mer indicates the length or size regardless of the type of nucleic acid. 1,000 bp is 1 kb (= kbp = kilo base pair), 1,000,000 bp is 1 Mb (mega base pair), and 1,000,000,000 bp is 1 Gb (giga base pair). Therefore, in this application, the term bp is used as a unit to denote the size of nucleic acids, interchangeably with nt and mer.

[0049] About

[0050] In this application, the term “about” means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0052] The invention disclosed in this application will be described in detail below.

[0054] I. Method for producing nucleic acid fragments of a specific size under specific high pressure

[0055] Overview of a method for producing nucleic acid fragments of a specific size under specific high pressure

[0056] One aspect of the present application relates to a method for producing nucleic acid fragments. In particular, the method utilizes a specific high pressure. The use of a specific high pressure is intended to fragment into nucleic acids of a specific size.

[0057] For example, a method for manufacturing nucleic acid fragments using high pressure of a specific size is,

[0058] Step of preparing the first nucleic acid; and

[0059] It may include the step of providing pressure of a specific size to the first nucleic acid.

[0060] A product containing a second nucleic acid of a specific size can be obtained by the above method.

[0061] Below, I will explain in detail each step of the method for manufacturing nucleic acid fragments using high pressure of a specific size.

[0063] Step of preparing the first nucleic acid

[0064] The method of producing nucleic acid fragments using high pressure of a specific size according to the present application is,

[0065] It includes the step of preparing the first nucleic acid.

[0067] 1st nucleic acid

[0068] The above-mentioned first nucleic acid refers to a nucleic acid used as a material to obtain a final product containing a second nucleic acid of a specific size. In other words, it refers to a nucleic acid used as a material prior to the process of fragmenting the nucleic acid.

[0069] The first nucleic acid is a nucleic acid that has a size larger than the size of the nucleic acid fragment as the final product.

[0070] For example, the first nucleic acid is 1000 bp (i.e., 1 kb), 1500 bp, 2000 bp, 2500 bp, 3000 bp, 3200 bp, 3400 bp, 3600 bp, 3800 bp, 4000 bp, 4500 bp, 5000 bp, 5500 bp, 6000 bp, 6500 bp, 7000 bp, 7500 bp, 8000 bp, 8500 bp, 9000 bp, 9500 bp, 10000 bp, 20000 bp, 30000 bp, 40000 bp, 50000 bp, 100000 bp, It may be a nucleic acid having a size within two numerical ranges selected from 1,000,000 bp (i.e., 1 Mb) and 1,000,000,000 bp (i.e., 1 Gb). For example, the first nucleic acid is a nucleic acid having a size of 2,000 bp or more. For another example, the first nucleic acid is a nucleic acid having a size of 3,000 bp or more.

[0071] In one embodiment, the first nucleic acid may be a nucleic acid having a size of 2,000 bp to 40,000 bp. In another embodiment, the first nucleic acid may be a nucleic acid having a size of 2,500 bp to 20,000 bp. In a preferred example, the first nucleic acid may be a nucleic acid having a size of 3,000 bp to 10,000 bp.

[0072] The first nucleic acid mentioned above may be a nucleic acid obtained directly from cells, tissues, etc.; a commercially available nucleic acid, etc.

[0073] For example, the first nucleic acid may be a nucleic acid obtained from a prokaryote or a eukaryote. For example, the first nucleic acid may be a nucleic acid obtained from the testes, sperm, eggs, etc. of a fish.

[0074] The first nucleic acid mentioned above may be DNA (Deoxyribonucleic acid), RNA (RiboNucleic Acid), PNA (Peptide nucleic acid), LNA (Locked nucleic acid), and combinations thereof, but is not limited thereto.

[0075] For example, the first nucleic acid may be genomic DNA (gDNA) extracted from a cell.

[0076] The first nucleic acid may be single-stranded or double-stranded. Additionally, the first nucleic acid may be whole-length DNA or a DNA fragment.

[0077] For example, DNA fragments can be produced from a DNA extraction process from cells, etc. In one embodiment, the first nucleic acid may be PDRN (Polydeoxyribonucleotide). Preferably, the first nucleic acid may be PDRN having a size of about 300 bp to 50,000 bp.

[0078] The form of the first nucleic acid may be liquid, powder, semi-solid, etc., but is not limited thereto. For example, the first nucleic acid may be in the form of freeze-dried or vacuum-dried powder.

[0080] Method for preparing the first nucleic acid

[0081] The method for preparing the first nucleic acid above is,

[0082] For example, it may include pre-treating a sample containing the first nucleic acid.

[0083] Pre-treating a sample containing the first nucleic acid can be performed using a pre-treatment method known in the art to obtain the first nucleic acid from a sample containing the first nucleic acid.

[0084] A sample containing the first nucleic acid may be, for example, cell tissue, etc. In one embodiment, the sample containing the first nucleic acid may be fish testes or sperm. In any embodiment, the sample containing the first nucleic acid may be salmon sperm.

[0085] Pre-treating a sample containing the first nucleic acid may include a first process of breaking or removing a cell membrane or cell wall. Through the first process, a product such as nucleic acid present in the sample containing the first nucleic acid can be obtained and used as the first nucleic acid.

[0086] For example, the first process can be carried out using methods such as using enzymes or using ultrasound.

[0087] In addition, pre-treating a sample containing the first nucleic acid may optionally further include a second process for separating or / and washing the first nucleic acid from the product obtained according to the first process. The second process may involve removing unnecessary substances other than nucleic acid, such as proteins and histones, or extracting and separating only the first nucleic acid.

[0088] For example, the second process can be performed using centrifugation, precipitation, chromatography, a kit for protein removal, etc.

[0089] Additionally, the step of preparing the first nucleic acid may further include a post-processing step as needed. The post-processing step may involve concentrating, storing, or drying the first nucleic acid before applying pressure to it. Concentrating the nucleic acid may be for the mass production of a second nucleic acid obtained from the first nucleic acid. The storage and / or drying may be intended to minimize external influences on the nucleic acid before it is applied to a pressure-applying device. In this case, external influences may be, for example, the destruction of the nucleic acid due to the influence of temperature. The drying may be performed by vacuum drying, freeze-drying, etc.

[0091] Step of obtaining a second nucleic acid by applying pressure to the first nucleic acid

[0092] The method of producing nucleic acid fragments using high pressure of a specific size according to the present application is,

[0093] It includes providing a specific amount of pressure to the first nucleic acid obtained above.

[0094] By applying pressure of a specific size to the first nucleic acid, a fragmented product of the first nucleic acid can be obtained, and the fragmented product contains a second nucleic acid as a final product.

[0095] That is, the second nucleic acid is a nucleic acid included in a product comprising nucleic acids fragmented to a specific size by applying a specific amount of pressure to the first nucleic acid. The size of the second nucleic acid may vary depending on the amount of pressure, but in this application, it refers to a nucleic acid fragment having a size of about 50 bp to 400 bp, which is a low molecular size.

[0096] The second nucleic acid is a nucleic acid having a small molecular size. For example, the second nucleic acid may be a nucleic acid having a size within a range of two numerical values ​​selected from about 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, and 400 bp. As an example, the second nucleic acid may be a nucleic acid having a size of about 30 bp to 350 bp. As another example, the second nucleic acid may be a nucleic acid having a size of about 40 bp to 300 bp. As another example, the second nucleic acid may be a nucleic acid having a size of about 45 bp to 200 bp. In one embodiment, the second nucleic acid may be a nucleic acid having a size of about 50 bp to 150 bp.

[0097] Since the second nucleic acid is the first nucleic acid that has been fragmented (i.e., fragmented) by pressure, the second nucleic acid is smaller in size and / or length and has an increased number compared to the first nucleic acid.

[0098] The second nucleic acid may be contained (or distributed) in a numerical percentage (%) within two numerical ranges selected from about 75%, 80%, 85%, 90%, 95%, and 100% within the product of the first nucleic acid to which pressure has been applied to the first nucleic acid described above (i.e., the fragmented product of the first nucleic acid). For example, the second nucleic acid may be contained in about 80% to 100% within the product of the fragmented first nucleic acid. For another example, the second nucleic acid may be contained in about 85% to 100% within the product of the fragmented first nucleic acid. For yet another example, the second nucleic acid may be contained in about 90% to 100% within the product of the fragmented first nucleic acid.

[0099] The size of the second nucleic acid can be determined using known methods. For example, the size of the second nucleic acid can be determined by methods such as electrophoresis, chromatography, and sequencing, but is not limited thereto.

[0100] The form of the second nucleic acid may be liquid, powder, semi-solid, etc., but is not limited thereto. For example, the second nucleic acid may be in the form of freeze-dried or vacuum-dried powder.

[0102] pressure of a specific size

[0103] The method of the present application is characterized by fragmenting the first nucleic acid obtained above by applying pressure of a specific size.

[0104] The pressure used in the method of the present application may be determined according to the desired size of the second nucleic acid. Units used for pressure may include pound per square inch (psi), bar, pascal (Pa), Torr, etc. Additionally, these units may be converted into one another. For example, 1 psi is approximately 6894.757 Pa, and 1 Pa is approximately 0.000145 psi.

[0105] For example, the pressure used in the method of the present application may be a pressure having a size within two numerical ranges selected from 10000 psi, 12000 psi, 14000 psi, 16000 psi, 18000 psi, 20000 psi, 22000 psi, 24000 psi, 26000 psi, 28000 psi, 30000 psi, 32000 psi, 34000 psi, 36000 psi, 38000 psi, 40000 psi, 42000 psi, 44000 psi, 46000 psi, 48000 psi, 50000 psi, 52000 psi, 54000 psi, 56000 psi, 58000 psi, and 60000 psi.

[0106] For example, the pressure used in the method of the present application may be a pressure having a magnitude of 10,000 psi to 50,000 psi. In another embodiment, the pressure used in the method of the present application may be a pressure having a magnitude of 15,000 psi to 40,000 psi. In another embodiment, the pressure used in the method of the present application may be a pressure having a magnitude of 18,000 psi to 38,000 psi. In a preferred example, the pressure used in the method of the present application may be a pressure having a magnitude of 20,000 psi to 30,000 psi.

[0107] The inventors of the present invention have for the first time identified a relationship in which a second nucleic acid having a specific size can be obtained by applying a pressure of a specific size to the first nucleic acid. That is, a second nucleic acid having a uniform size can be obtained by applying a pressure of a specific size to the first nucleic acid according to the size of the desired second nucleic acid.

[0108] As the pressure increases, a smaller second nucleic acid can be obtained. However, when the pressure exceeds a certain level, the fragmentation size of the first nucleic acid no longer decreases. This is because when nucleic acids are fragmented under high pressure, the smaller the molecule, the smaller the collision area when colliding with the disk. Although increasingly greater pressure is required to make small molecules smaller, the size of the nucleic acid no longer decreases once the pressure exceeds a certain level because the collision area becomes smaller.

[0109] Accordingly, the method of preparing a nucleic acid fragment using high pressure of a specific size according to the present application comprises providing pressure of a specific size to the first nucleic acid, and at this time, a second nucleic acid having a specific size can be obtained due to the pressure of a specific size.

[0110] In one embodiment, by applying a pressure of 10,000 psi to 50,000 psi to the first nucleic acid, a second nucleic acid having a size of 30 bp to 350 bp can be obtained. In another embodiment, by applying a pressure of 15,000 psi to 40,000 psi to the first nucleic acid, a second nucleic acid having a size of 40 bp to 300 bp can be obtained. In another embodiment, by applying a pressure of 18,000 psi to 38,000 psi to the first nucleic acid, a second nucleic acid having a size of 45 bp to 200 bp can be obtained. In a preferred example, by applying a pressure of 20,000 psi to 30,000 psi to the first nucleic acid, a second nucleic acid having a size of 50 bp to 150 bp can be obtained.

[0112] Conditions for handling pressure

[0113] The pressure applied to the method of the present application may be adjusted according to the size of the first nucleic acid and / or the size of the desired second nucleic acid. In this case, the conditions may include the number of times the pressure is applied, time, temperature, etc.

[0114] The number of times pressure is processed may be from 1 to 100. The term "number of times" is interpreted as having the same meaning as "cycle." For example, 1 cycle refers to the interval from when pressure is applied (provided) once until the moment the applied pressure is completely released or the pressure is eliminated, and 2 cycles refer to the interval from when additional pressure is applied after the completion of the 1 cycle until the moment the applied pressure is eliminated again.

[0115] If the number of times pressure is processed is two or more, it may be processed with pressure of the same magnitude or with pressure of a different magnitude in each processing cycle. For example, pressure may be processed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times. As another example, pressure may be processed 5 to 30 times. As another example, pressure may be processed 10 to 30 times. As yet another example, pressure may be processed 10 to 20 times. In one specific example, the pressure can be applied 5 to 20 times.

[0116] The time required to process the pressure during one cycle may be approximately 0.5 seconds to 10 seconds from the time the pressure is applied until the time the pressure is released. For example, the time required to process the pressure during one cycle may be approximately 0.5 seconds to 5 seconds. As another example, the time required to process the pressure during one cycle may be approximately 1 second to 3 seconds. In one embodiment, in one embodiment of the present invention, when the pressure is processed for 15 cycles, it may take approximately 7.5 seconds (0.5 seconds x 15 times) to 150 seconds (10 seconds x 15 times). In another embodiment, in one embodiment of the present invention, when the pressure is processed for 15 cycles, it may take approximately 15 seconds (1 second x 15 times) to 75 seconds (5 seconds x 15 times).

[0117] The pressure can be processed at a temperature of about 1°C to 40°C. In this case, the temperature may be a gradually changing temperature rather than a fixed temperature. For example, it may be a gradually decreasing temperature or a gradually increasing temperature. In one embodiment, the temperature may be a temperature that gradually increases by about 1°C to 10°C. In another embodiment, the temperature may be a temperature that gradually decreases by about 1°C to 10°C.

[0118] For example, pressure can be processed at a temperature within a range of two selected numerical values ​​from about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C. As another example, pressure can be processed at a temperature of about 2°C to 35°C. As another example, the pressure can be processed at a temperature of about 5°C to 35°C. In one embodiment, the pressure can be processed at a temperature of about 5°C to 25°C.

[0120] device that provides pressure

[0121] Meanwhile, the above pressure may be provided using a specific device. The specific device is not otherwise limited as long as it is a device capable of providing pressure.

[0122] For example, the specific device may include a chamber, an inlet, an outlet, a pressure regulator, and a flow rate regulator. Additionally, the specific device may optionally further include a temperature regulator.

[0123] The above chamber may be, for example, a cavitation nozzle type, impact valve type, V-type, Y-type, Z-type, etc. depending on the flow rate, but is not limited thereto.

[0124] The above specific device may be a high pressure homogenizer, a high pressure disperser, an ultra-high pressure homogenizer, and an ultra-high pressure disperser, but is not limited thereto.

[0125] The principle of nucleic acid fragmentation using high pressure is as follows. When nucleic acids are introduced into the aforementioned specific device, high pressure is applied within the disk; after separation, the nucleic acids collide again or collide with the walls, receiving high energy that breaks the weak bonds of the DNA molecular chains, thereby fragmenting the nucleic acids.

[0126] For example, a method of applying pressure to nucleic acids using a specific device is,

[0127] Including introducing a first nucleic acid into the inlet of the specific device;

[0128] At this time, the pressure regulating device and the flow rate regulating device within the aforementioned specific device are each set to specific conditions, and

[0129] The first nucleic acid introduced into the above inlet passes through the chamber, and at this time, the chamber increases the flow rate and releases pressure, and

[0130] Cavitation may occur due to the difference in pressure and flow velocity mentioned above.

[0132] Additional processes

[0133] In the above method, if necessary, a process of separating or / and concentrating only the second nucleic acid may be additionally performed.

[0134] For the above separation or / and concentration, a precipitation process, a filtration process, etc., may be performed on a product containing a second nucleic acid obtained by applying pressure to the first nucleic acid. The precipitation process may be performed by known methods, such as using salts. For example, the precipitation process may be performed using substances such as sodium chloride (NaCl), potassium chloride (KCl), or sodium acetate (CH3COONa). At this time, the substance may be determined according to the amount of the product containing the second nucleic acid, but the conditions may be changed at the experimenter's discretion. The filtration process may be performed by known methods such as vacuum filtration, pressurized filtration, or natural filtration.

[0135] For example, when using sodium chloride, the amount of sodium chloride can be 15% to 30% of the amount of water.

[0136] In addition, an organic solvent may be used in the above precipitation process. For example, the organic solvent may be one or more selected from ethanol, methanol, butanol, propanol, pentanol, and isopropyl alcohol, but is not limited thereto. For example, the solvent may be used in a ratio of 1:99 to 99:1 relative to the total solution.

[0137] Optionally, only the product of the fragmented first nucleic acid or / and the second nucleic acid contained within the product of the fragmented first nucleic acid may be extracted and stored by a known method. This may be for storage until industrial use. For example, storage may be, but is not limited to, methods using liquid nitrogen, freeze-drying, or ultra-low temperature freezers.

[0139] Specific examples of the method of this institution

[0140] Examples of methods for producing nucleic acid fragments of a specific size under the aforementioned specific high pressure will be described below, but are not limited to those described below.

[0141] The method examples described below are merely examples, and a person skilled in the art may change the apparatus used in the method, the pressure treatment conditions, etc., to produce a nucleic acid fragment of a specific size intended.

[0143] Method example (1)

[0144] As an example of the present application, a method for manufacturing a nucleic acid fragment

[0145] A first nucleic acid having a size of approximately 500 bp to 5000 bp is prepared as a nucleic acid;

[0146] A fragmented product of the first nucleic acid is obtained by applying a pressure of approximately 15,000 psi to 30,000 psi to the first nucleic acid 10 to 30 times;

[0147] It may include.

[0148] The above first nucleic acid may be obtained using an ultrasonic grinder.

[0149] At this time, the first nucleic acid may be DNA, RNA, or a mixture thereof. In one embodiment, the first nucleic acid may be DNA obtained from the testes or sperm of a fish.

[0150] The above first nucleic acid may be in the form of a vacuum-dried powder.

[0151] The above pressure may be provided by a device selected from a high pressure homogenizer, a high pressure disperser, an ultra-high pressure homogenizer, and an ultra-high pressure disperser.

[0152] The above pressure may be applied to the first nucleic acid for about 5 to 30 minutes during a single treatment. Additionally, when applying the above pressure, the process may be performed at a temperature of about 5°C to 25°C.

[0153] The second nucleic acid may be contained in an amount of about 70% to 100% within the fragmented product of the first nucleic acid. In this case, the second nucleic acid may be a nucleic acid having a size of about 50 bp to 200 bp. As a specific example, the second nucleic acid may be DNA having a size of about 50 bp to 150 bp.

[0155] Method example (2):

[0156] As another example of the present application, a method for manufacturing a nucleic acid fragment

[0157] A first nucleic acid having a size of approximately 500 bp to 5000 bp is prepared as a nucleic acid;

[0158] A fragmented product of the first nucleic acid is obtained by applying a pressure of approximately 15,000 psi to 30,000 psi to the first nucleic acid 10 to 30 times;

[0159] Separating the second nucleic acid from the fragmented product of the first nucleic acid;

[0160] It may include.

[0161] The above first nucleic acid may be obtained using an ultrasonic grinder.

[0162] At this time, the first nucleic acid or / and the second nucleic acid may be DNA, RNA, or a mixture thereof. As a specific example, the first nucleic acid may be DNA obtained from the testes or sperm of a fish.

[0163] The above first nucleic acid may be in the form of a vacuum-dried powder.

[0164] The above pressure may be provided by a device selected from a high pressure homogenizer, a high pressure disperser, an ultra-high pressure homogenizer, and an ultra-high pressure disperser.

[0165] The above pressure may be applied to the first nucleic acid for about 5 to 30 minutes during a single treatment. Additionally, when applying the above pressure, the process may be performed at a temperature of about 5°C to 25°C.

[0166] The second nucleic acid may be contained in an amount of about 70% to 100% within the fragmented product of the first nucleic acid. In this case, the second nucleic acid may be a nucleic acid having a size of about 50 bp to 200 bp. As a specific example, the second nucleic acid may be PDRN having a size of about 50 bp to 150 bp.

[0167] The above second nucleic acid may be in powder form.

[0168] In addition, the above method includes a precipitation process for separating the second nucleic acid from the fragmented product of the first nucleic acid. For example, the second nucleic acid can be separated by treating the fragmented product of the first nucleic acid with sodium chloride. As another example, the second nucleic acid can be separated by treating the fragmented product of the first nucleic acid with sodium chloride and then treating it with ethanol.

[0169] Separating the second nucleic acid from the fragmented product of the first nucleic acid may optionally include a filtration process after the precipitation process. For example, the second nucleic acid can be separated by treating the fragmented product of the first nucleic acid with sodium chloride and then filtering under reduced pressure. As another example, the second nucleic acid can be separated by treating the fragmented product of the first nucleic acid with sodium chloride and ethanol and then filtering under reduced pressure.

[0171] Characteristics of the method

[0172] The method for producing a nucleic acid fragment of a specific size according to a specific high pressure according to the above-described application may have the features described below, but is not limited thereto.

[0173] Feature (1): Homogeneous nucleic acid fragments of a specific size can be obtained at a specific pressure

[0174] The present application can produce nucleic acid fragments of a desired specific size using pressure of a specific size. In particular, the size of the obtained nucleic acid fragments of a specific size is uniform.

[0175] Specifically, the present application fragments nucleic acids using high pressure of a specific size, for example, with a high pressure homogenizer (HPH). In one embodiment, pressure is applied at an optimal condition of about 15,000 psi to 25,000 psi to fragment the nucleic acids into low molecular sizes of about 50 bp to 150 bp or less.

[0177] Feature (2): Obtaining nucleic acid (DNA fragment) smaller than conventional ones

[0178] The method of the present application is characterized by being able to obtain small molecular size nucleic acids that are difficult to obtain using conventional methods for nucleic acid fragmentation, such as methods using enzymes or ultrasound. While most conventional methods could obtain large nucleic acid fragments of 650 bp or larger, the method of the present application can obtain small molecular size nucleic acid fragments ranging from 50 bp to 350 bp.

[0179] Furthermore, the amount of small molecular nucleic acid fragments distributed throughout the fragmented nucleic acid, ranging in size from about 50 bp to 350 bp, also increases. In other words, the method of the present application makes it easy to manufacture small molecular nucleic acid fragments and enables mass production.

[0180] In particular, the nucleic acid fragment of size 50 to 150 bp obtained by the method disclosed in this application has the advantage of being highly industrial and biologically useful compared to the nucleic acid fragment of size 500 to 1000 bp that is conventionally used in the field.

[0182] II. Uses of compositions containing secondary nucleic acid

[0183] Another aspect of the present application relates to various uses of the second nucleic acid by the above method.

[0184] As described above, the second nucleic acid of the present application is a nucleic acid having a specific size fragmented under a specific pressure. In particular, the size of the second nucleic acid may be about 50 bp to 150 bp. The specific size of the second nucleic acid is characterized by being smaller than that of a nucleic acid fragmented by a conventional nucleic acid fragmentation method.

[0185] The above-mentioned second nucleic acid may have effects such as anti-apoptosis activity, scavenging of reactive oxygen species (ROS), activation of cell proliferation, activation of cell migration, and anti-inflammatory activity. As a result, it can be utilized more effectively in industry.

[0186] The composition containing the aforementioned second nucleic acid (hereinafter referred to as the second nucleic acid composition) may have lower viscosity than the composition containing the first nucleic acid (hereinafter referred to as the first nucleic acid composition). In addition, due to the lower viscosity, the permeability may be high, allowing for high-dose administration when applied to the human body and making it more industrially useful.

[0187] For example, the viscosity of the second nucleic acid composition may be lower than the viscosity of the first nucleic acid composition by a multiple within a range of two numbers selected from approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 150, 180, 200, 220, 250, 280, 300, 320, 350, 380, 400, 450, and 500. That is, because the size of the second nucleic acid included in the second nucleic acid composition is smaller than the size of the first nucleic acid included in the first nucleic acid composition, the second nucleic acid composition may have lower viscosity than the first nucleic acid composition. For example, the viscosity of the second nucleic acid composition may be about 2 to 300 times lower than the viscosity of the first nucleic acid composition. For another example, the viscosity of the second nucleic acid composition may be about 3 to 250 times lower than the viscosity of the first nucleic acid composition. For yet another example, the viscosity of the second nucleic acid composition may be about 3 to 150 times lower than the viscosity of the first nucleic acid composition. In one embodiment, the viscosity of the second nucleic acid composition may be about 20 to 300 times lower than the viscosity of the first nucleic acid composition.

[0188] As is well known in the art, when fragmented nucleic acids are manufactured with a size of 500 bp or larger, viscosity is generated, making industrial application difficult; however, since the second nucleic acid of the present application is manufactured with a small size of about 50 bp to 150 bp, it can be utilized for various industrial applications. For example, such applications may include cell regeneration, wound healing and improvement, cell activation, wrinkle improvement, etc.

[0189] Hereinafter, various uses of a composition containing a second nucleic acid prepared by the above-described method as an active ingredient are described.

[0190] In particular, among the various uses, pharmaceutical and cosmetic uses are described, but are not limited thereto.

[0191] Use (1): Pharmaceutical use

[0192] A composition comprising a second nucleic acid prepared by the method of the present application as an active ingredient can be utilized to treat an individual in need of treatment. Examples include wound healing, improvement, cell regeneration, cell activation, and wrinkle improvement.

[0194] Pharmaceutical composition

[0195] For example, a pharmaceutical composition applicable to the above pharmaceutical use may be disclosed.

[0196] The above pharmaceutical composition is the aforementioned active ingredient

[0197] It may include a second nucleic acid.

[0198] The second nucleic acid above is the same as described above.

[0199] To briefly describe, for example, the first nucleic acid may be DNA having a size of about 500 bp to 5000 bp. For example, the second nucleic acid may be DNA having a size of about 50 bp to 150 bp. In one embodiment, the second nucleic acid may be PDRN having a size of about 50 bp to 150 bp. In addition, the pharmaceutical composition containing the second nucleic acid may have a viscosity about 50 to 300 times lower than that of the pharmaceutical composition containing the first nucleic acid.

[0201] Additional components of a pharmaceutical composition

[0202] In addition to the active ingredient, the above pharmaceutical composition may optionally further include additional pharmaceutically acceptable components.

[0203] In this application, the term “pharmaceuticalally acceptable” is used herein to refer to materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the scope of reasonable medical judgment and corresponding to a reasonable benefit / risk ratio.

[0204] The above-mentioned pharmaceutically acceptable additional components may be physiologically acceptable and act on the stabilization, dissolution, absorption, and introduction efficiency of the active ingredient of the present application. For example, the above-mentioned pharmaceutically acceptable additional components may be, but are not limited to, carriers, excipients, diluents, preservatives, etc.

[0205] For example, the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, distilled water, physiological saline, glycerol, ethanol, HSA (Human serum albumin), etc.

[0206] For example, the above preservative may be benzoic acid, sodium benzoate, sorbic acid, parahydroxybenzoic acid, chlorobutanol, etc.

[0207] When formulating the above pharmaceutical composition, it may further include fillers, extenders, binders, humectants, etc.

[0209] Formulation of pharmaceutical compositions

[0210] The above pharmaceutical composition may be formulated for oral or parenteral use.

[0211] For example, when formulated for oral use, it can be manufactured in the form of a solid, liquid, suspension, granule, capsule, semi-solid, etc.

[0212] As another example, when formulated for parenteral use, it can be manufactured in the form of an injectable, aerosol, or kit. Preferably, it can be formulated as an injectable.

[0213] In the case of injectable formulations, additional methods known in the art may be performed as needed to prolong the effect of the drug or to slow down drug absorption. For example, an injectable depot form may be prepared by encapsulating the active ingredient using a biodegradable polymer. As another example, an injectable depot form may be prepared by capturing the active ingredient in a liposome or microemulsion.

[0214] In the case of a kit, it means including storage containers, mixing containers, mixing tools, etc.

[0215] For example, the storage container can be a vial, an antioxidant bottle, or a plastic container.

[0216] For example, the mixing container may be a plastic container, a sterile bottle, a syringe, etc.

[0217] For example, mixing tools can be gloves, spoons, sticks, etc.

[0219] To apply the aforementioned pharmaceutical composition for pharmaceutical purposes, that is, for the aforementioned wound healing, improvement, cell regeneration, cell activation, and wrinkle improvement, etc.

[0220] A method for wound healing, improvement, cell regeneration, cell activity, and wrinkle improvement may be provided, comprising administering a pharmaceutical composition to a target.

[0221] The above subject may be a mammal. For example, the mammal may be a mouse, rat, rabbit, dog, cow, cat, monkey, human, etc.

[0222] The pharmaceutical composition of the present application may be administered to a subject via various routes.

[0223] The above administration may be oral or parenteral. In this case, parenteral administration may be by injection.

[0224] When administering the above injection, the injection site may be subcutaneous, muscular, intradermal, intravenous, transdermal, etc.

[0225] The administration dose of the above pharmaceutical composition may be determined by considering the site of administration, gender, body weight, etc., but is not limited thereto.

[0226] As an optional example, a second nucleic acid comprising the pharmaceutical composition of the present application may be administered in the following doses.

[0227] For example, if the subject is non-human, the second nucleic acid is 20 µg / ml, 21 µg / ml, 22 µg / ml, 23 µg / ml, 24 µg / ml, 25 µg / ml, 26 µg / ml, 27 µg / ml, 28 µg / ml, 29 µg / ml, 30 µg / ml, 35 µg / ml, 40 µg / ml, 45 µg / ml, 50 µg / ml, 55 µg / ml, 60 µg / ml, 65 µg / ml, 70 µg / ml, 75 µg / ml, 80 µg / ml, 85 µg / ml, 90 µg / ml, 95 µg / ml, 100 µg / ml, 120 µg / ml, 140 µg / ml, 160 µg / ml, 180 µg / ml, 200 µg / ml, 220 µg / ml, It may be administered at a dose within a range of two selected numerical values ​​from 240 µg / ml, 260 µg / ml, 280 µg / ml, and 300 µg / ml. In this case, the above non-human refers to all mammals excluding humans.

[0228] In one embodiment, the second nucleic acid may be administered to a non-human at a dose of 20 µg / ml to 200 µg / ml. Preferably, the second nucleic acid may be administered to a non-human at a dose of 20 µg / ml to 120 µg / ml. Most preferably, the second nucleic acid may be administered to a non-human at a dose selected from 25 µg / ml, 50 µg / ml, or 100 µg / ml.

[0229] As another example, when the subject is human, the second nucleic acid is 20 µg / ml, 21 µg / ml, 22 µg / ml, 23 µg / ml, 24 µg / ml, 25 µg / ml, 26 µg / ml, 27 µg / ml, 28 µg / ml, 29 µg / ml, 30 µg / ml, 35 µg / ml, 40 µg / ml, 45 µg / ml, 50 µg / ml, 55 µg / ml, 60 µg / ml, 65 µg / ml, 70 µg / ml, 75 µg / ml, 80 µg / ml, 85 µg / ml, 90 µg / ml, 95 µg / ml, 100 µg / ml, 120 µg / ml, 140 µg / ml, 160 µg / ml, 180 µg / ml, 200 µg / ml, 220 µg / ml, It can be administered at a dose within a range of two selected values ​​from 240 µg / ml, 260 µg / ml, 280 µg / ml, and 300 µg / ml.

[0230] In one embodiment, the second nucleic acid may be administered to a human at a dose of 20 µg / ml to 200 µg / ml. Preferably, the second nucleic acid may be administered to a human at a dose of 20 µg / ml to 120 µg / ml. Most preferably, the second nucleic acid may be administered to a human at a dose selected from 25 µg / ml, 50 µg / ml, or 100 µg / ml.

[0231] The above pharmaceutical composition may contain a second nucleic acid in a dose within a range of two numerical values ​​selected from 20 µg, 21 µg, 22 µg, 23 µg, 24 µg, 25 µg, 26 µg, 27 µg, 28 µg, 29 µg, 30 µg, 35 µg, 40 µg, 45 µg, 50 µg, 55 µg, 60 µg, 65 µg, 70 µg, 75 µg, 80 µg, 85 µg, 90 µg, 95 µg, 100 µg, 120 µg, 140 µg, 160 µg, 180 µg, 200 µg, 220 µg, 240 µg, 260 µg, 280 µg, and 300 µg per 1 mL.

[0232] For example, the pharmaceutical composition of the present application may contain 20 µg to 200 µg of a second nucleic acid per 1 mL. Preferably, the pharmaceutical composition may contain 20 µg to 120 µg of a second nucleic acid per 1 mL. Most preferably, the pharmaceutical composition may contain the second nucleic acid in a dose selected from 25 µg, 50 µg, or 100 µg per 1 mL.

[0234] The administration volume of the above pharmaceutical composition can be selected at an appropriate dose considering the administration method, target, etc.

[0235] For example, the administration volume may be 0.1 mL to 10 mL per dose, but is not limited thereto.

[0236] In addition, the number of administrations may be from 1 to 30 times per day, but is not limited thereto. At this time, administration may also be performed at intervals of a certain period.

[0237] The above administration interval may be 1 to 30 days, but is not limited thereto. At this time, administration may be continuous or non-continuous.

[0239] Use (2): Cosmetic use

[0240] A composition comprising a second nucleic acid prepared by the method of the present application as an active ingredient can be utilized for cosmetic purposes. The cosmetic uses may include, but are not limited to, wrinkle improvement, whitening, scar improvement, moisturization, and elasticity enhancement.

[0242] Cosmetic composition

[0243] As another example, a cosmetic composition applicable to the above cosmetic use may be disclosed.

[0244] The above cosmetic composition is the aforementioned active ingredient

[0245] It may include a second nucleic acid.

[0246] The second nucleic acid above is the same as described above.

[0247] To briefly describe, for example, the first nucleic acid may be DNA having a size of about 500 bp to 5000 bp. For example, the second nucleic acid may be DNA having a size of about 50 bp to 150 bp. In one embodiment, the second nucleic acid may be PDRN having a size of about 50 bp to 150 bp. In addition, the cosmetic composition containing the second nucleic acid may have a viscosity about 50 to 300 times lower than that of the cosmetic composition containing the first nucleic acid.

[0249] Additional components of a cosmetic composition

[0250] In addition to the active ingredient, the above cosmetic composition may optionally include additional cosmetically acceptable components.

[0251] In this application, the term “cosmetically acceptable” is used herein to refer to materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems, corresponding to a reasonable effect / risk ratio, under reasonable cosmetic purposes.

[0252] The above-mentioned cosmetically acceptable additional components may be physiologically acceptable and act on the stabilization, dissolution, absorption, and introduction efficiency of the active ingredient of the present application. For example, the above-mentioned cosmetically acceptable additional components may be, but are not limited to, carriers, excipients, diluents, preservatives, etc.

[0253] For example, the carrier, excipient, and diluent may be oil, water, surfactant, humectant, alcohol, chelating agent, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan ester, ether sulfate, sulfate, betaine, glucoside, maltoside, nonoxynol, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, etc.

[0254] For example, the above preservative may be benzoic acid, para-hydroxybenzoic acid ester, benzoic acid, etc.

[0255] Meanwhile, the above-mentioned cosmetically acceptable additional components may optionally include one or more active ingredients (hereinafter referred to as similar functional ingredients) that exhibit a function similar to the second nucleic acid of the present application. For example, the similar functional ingredients may be known skin whitening, elasticity enhancement, wrinkle improvement, moisturizing ingredients, etc. Including additional similar functional ingredients may further increase the skin whitening, elasticity enhancement, wrinkle improvement, and moisturizing effects of the cosmetic composition of the present application.

[0256] When adding the above-mentioned similar functional ingredients, skin safety, ease of formulation, and stability due to combined use may be considered. Any example of a similar functional ingredient may be kojic acid, arbutin, hydroquinone, vitamin C, retinoic acid, TGF, protein derived from animal placenta, Chlorella extract, etc.

[0258] When formulating the above cosmetic composition, it may further include fillers, extenders, binders, humectants, etc.

[0260] Formulation of cosmetic compositions

[0261] The above cosmetic composition can be formulated for topical, transdermal, endodermal use, etc.

[0262] The above formulation may be in the form of a solid, liquid, spray, capsule, granule, semi-solid, etc.

[0263] For example, the formulation may be a solution, topical ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, shampoo, rinse, liquid cleanser, bath additive, sun cream, sun oil, suspension, emulsion, paste, gel, lotion, powder, cleansing foam, cleansing oil, powder foundation, emulsion foundation, wax foundation, patch, spray, etc., but is not limited thereto.

[0264] For example, if the formulation is an ointment, paste, cream, or gel, the carrier, excipient, or / and diluent may be animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose, silicone, bentonite, silica, etc.

[0265] For other examples, if the formulation is a powder or spray, the carrier, excipient, or / and diluent may be lactose, talc, calcium silicate, polyamide powder, etc.

[0266] As another example, when the formulation is a solution or emulsion, the carrier, excipient, or / and diluent may be water, ethanol, isopropanol, ethyl carbonate, benzyl benzoate, cottonseed oil, peanut oil, castor oil, glycerol, etc.

[0267] As another example, when the formulation is a suspension, the carrier, excipient, or / and diluent may be water, propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, ester, tracan, etc.

[0268] As another example, when the formulation is soap, the carrier, excipient, or / and diluent may be fatty acid hemiester salt, isethionate, vegetable oil, glycerol, sugar, lanolin, etc.

[0269] Meanwhile, it may further include agents that increase percutaneous absorption. For example, agents that increase percutaneous absorption may be dimethyl sulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone (1-dodecyl azacycloheptan-2-one), alcohol, urea, ethoxydiglycol, acetone, propylene glycol, polyethylene glycol, etc.

[0271] To apply the aforementioned cosmetic composition for cosmetic purposes, that is, for the aforementioned wrinkle improvement, whitening, scar improvement, moisturization, elasticity enhancement, etc.,

[0272] A method for improving wrinkles, whitening, scars, moisturizing, and enhancing elasticity may be provided, comprising administering a cosmetic composition to a subject.

[0273] The above subject may be a mammal. For example, the mammal may be a mouse, rat, rabbit, dog, cow, cat, monkey, human, etc.

[0274] The cosmetic composition of the present application may be administered to a subject via various routes.

[0275] The above administration may be oral or parenteral. In this case, the administration may be applied to the target area or directly injected. The above parenteral administration may be an injection.

[0276] When administering the above injection, the injection site may be subcutaneous, muscular, intradermal, intravenous, transdermal, etc.

[0277] The administration dose of the above cosmetic composition may be determined by considering the site of administration, gender, body weight, etc., but is not limited thereto.

[0278] As an arbitrary example, the second nucleic acid comprising the cosmetic composition of the present application may be administered in the following doses.

[0279] For example, if the subject is non-human, the second nucleic acid is 20 µg / ml, 21 µg / ml, 22 µg / ml, 23 µg / ml, 24 µg / ml, 25 µg / ml, 26 µg / ml, 27 µg / ml, 28 µg / ml, 29 µg / ml, 30 µg / ml, 35 µg / ml, 40 µg / ml, 45 µg / ml, 50 µg / ml, 55 µg / ml, 60 µg / ml, 65 µg / ml, 70 µg / ml, 75 µg / ml, 80 µg / ml, 85 µg / ml, 90 µg / ml, 95 µg / ml, 100 µg / ml, 120 µg / ml, 140 µg / ml, 160 µg / ml, 180 µg / ml, 200 µg / ml, 220 µg / ml, It may be administered at a dose within a range of two selected numerical values ​​from 240 µg / ml, 260 µg / ml, 280 µg / ml, and 300 µg / ml. In this case, the above non-human refers to all mammals excluding humans.

[0280] In one embodiment, the second nucleic acid may be administered to a non-human at a dose of 20 µg / ml to 200 µg / ml. Preferably, the second nucleic acid may be administered to a non-human at a dose of 20 µg / ml to 120 µg / ml. Most preferably, the second nucleic acid may be administered to a non-human at a dose selected from 25 µg / ml, 50 µg / ml, or 100 µg / ml.

[0281] As another example, when the subject is human, the second nucleic acid is 20 µg / ml, 21 µg / ml, 22 µg / ml, 23 µg / ml, 24 µg / ml, 25 µg / ml, 26 µg / ml, 27 µg / ml, 28 µg / ml, 29 µg / ml, 30 µg / ml, 35 µg / ml, 40 µg / ml, 45 µg / ml, 50 µg / ml, 55 µg / ml, 60 µg / ml, 65 µg / ml, 70 µg / ml, 75 µg / ml, 80 µg / ml, 85 µg / ml, 90 µg / ml, 95 µg / ml, 100 µg / ml, 120 µg / ml, 140 µg / ml, 160 µg / ml, 180 µg / ml, 200 µg / ml, 220 µg / ml, It can be administered at a dose within a range of two selected values ​​from 240 µg / ml, 260 µg / ml, 280 µg / ml, and 300 µg / ml.

[0282] In one embodiment, the second nucleic acid may be administered to a human at a dose of 20 µg / ml to 200 µg / ml. Preferably, the second nucleic acid may be administered to a human at a dose of 20 µg / ml to 120 µg / ml. Most preferably, the second nucleic acid may be administered to a human at a dose selected from 25 µg / ml, 50 µg / ml, or 100 µg / ml.

[0284] The above cosmetic composition may contain a second nucleic acid in a dosage within a range of two numerical values ​​selected from 20 µg, 21 µg, 22 µg, 23 µg, 24 µg, 25 µg, 26 µg, 27 µg, 28 µg, 29 µg, 30 µg, 35 µg, 40 µg, 45 µg, 50 µg, 55 µg, 60 µg, 65 µg, 70 µg, 75 µg, 80 µg, 85 µg, 90 µg, 95 µg, 100 µg, 120 µg, 140 µg, 160 µg, 180 µg, 200 µg, 220 µg, 240 µg, 260 µg, 280 µg, and 300 µg per 1 mL.

[0285] For example, the cosmetic composition of the present application may contain 20 µg to 200 µg of a second nucleic acid per 1 mL. Preferably, the cosmetic composition may contain 20 µg to 120 µg of a second nucleic acid per 1 mL. Most preferably, the cosmetic composition may contain the second nucleic acid in an amount selected from 25 µg, 50 µg, or 100 µg per 1 mL.

[0287] The administration volume of the above cosmetic composition can be selected at an appropriate dose considering the administration method, target, etc.

[0288] For example, the administration volume may be 0.1 mL to 10 mL per dose, but is not limited thereto.

[0289] In addition, the number of administrations may be from 1 to 30 times per day, but is not limited thereto. At this time, administration may also be performed at intervals of a certain period.

[0290] The above administration interval may be 1 to 30 days, but is not limited thereto. At this time, administration may be continuous or non-continuous.

[0292] [Form for implementing the application]

[0293] The present application will be explained in more detail below through examples.

[0294] These examples are provided solely to illustrate the present application more specifically, and it will be obvious to those skilled in the art that the scope of the present application is not limited by these examples.

[0296] Materials and devices

[0297] High-pressure disperser: Microfludizer M-110EH-30 from Microfludics International Corp. USA

[0298] Salmon sperm: Obtained from mature male salmon during the spawning season.

[0299] HDF (Human dermal fibroblast) cells: ATCC-PCS-201-012

[0300] Human primary gingival fibroblasts (HGF): ATCC-PCS-PCS201-018

[0302] Experimental method

[0303] ● Preparation of DNA samples

[0304] Salmon sperm from which foreign substances have been removed is placed at room temperature (20~25 Pure gDNA (genomic DNA) was obtained by using ultrasound after cell lysis with a lysis buffer in the pH range of 7.0 to 8.2.

[0306] ● Electrophoresis

[0307] Measurements were performed using the WSE1710 model agarose gel electrophoresis from ATTO, Japan, and DNA markers were identified using the DNA Molecular Weight Marker XIII 50-base pair Ladder (Lot No. 55309920) from Roche Diagnostics GmbH, Germany. Measurements were taken on a 2% agarose gel at a DNA concentration of 2 µg per well. 10 µl of Safe Shine Green (10000X) was used per 100 ml of TAE buffer on the agarose gel.

[0309] ● Cell proliferation - Using EdU

[0310] Cell proliferation was evaluated using the EdU Cell Proliferation Kit (EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488; C10337, Invitrogen) and by immunofluorescence staining with Ki67 (abchem, ab 15580).

[0311] Ki-67 is a protein expressed in the cell nucleus during all phases of the cell cycle except the G1 phase, and it is a marker used to confirm the cell's proliferation status.

[0312] EdU (5-ethynyl-2'-deoxyuridine) is a thymidine analog used to detect cell proliferation. EdU is incorporated during DNA synthesis and labeled with Alexa Fluor 488 via a click reaction.

[0313] After treating HDF cells with nucleic acid-A, nucleic acid-B, and nucleic acid-C at 100 μg / mL each, they were exposed to EdU (10 μM), and the process was carried out according to the kit manual.

[0314] The above nucleic acid-A, nucleic acid-B, and nucleic acid-C refer to the following:

[0315] Nucleic acid-A: Nucleic acid having a size in the range of 400 bp to 2600 bp (lane 2 of Fig. 2),

[0316] Nucleic acid-B: Nucleic acid having a size in the range of 150 bp to 350 bp (lane 3 of Fig. 2),

[0317] Nucleic acid-C: Nucleic acid having a size in the range of 50 bp to 150 bp (lane 4 of Fig. 2).

[0319] Subsequently, for immunofluorescence staining, cells were fixed with 4% paraformaldehyde (PFA) and cultured for 24 hours under 5% CO2 at 37°C before permeation with 0.5% Triton. Afterward, the cells were washed with 3% BSA and treated with the reaction cocktail for 30 minutes. Then, after washing with PBS, the cells were blocked with a blocking solution (1% BSA in PBS) for 30 minutes, followed by incubation with the primary antibody Ki67 (rabbit anti-Ki67; ab15580, Abcam, 1:100) overnight at 4°C. Then, the secondary antibody was cultured, and the nuclei were stained using DAPI (4,6-diamidino-2-phenylindole).

[0320] Immunofluorescence staining results were observed using a fluorescence microscope (IX71 and DP72, Olympus, Tokyo, Japan). In addition, the total number of cells was counted, and the percentages of Edu and Ki67-positive cells were calculated.

[0322] ● RT-PCR analysis

[0323] The mRNA expression levels of EGF (Epidermal Growth Factor) and VEGF (vascular endothelial growth factor) were confirmed by RT-PCR.

[0324] In addition, the anti-inflammatory activity of small molecule size (50bp–150bp) PDRN was analyzed using RT-PCR. For the anti-inflammatory analysis, mRNA expression levels of EGF, VEGF, TNF-α (tumor necrosis factor-α), IL-1β (interleukin 1-beta), IL-6 (Interleukin-6) were checked.

[0325] To this end, human primary gingival fibroblasts (hereinafter referred to as HGF) were cultured in DMEM (Dulbecco's modified Eagles medium; Hyclone, GE Healthcare Life Sciences, USA) containing heat-inactivated 10% FBS (FBS, Hyclone, Fisher Scientific, USA) and antibiotic-antimycotic (Gibco, USA).

[0326] Cultures were performed at 37°C under 5% CO2 conditions, and the culture medium was replaced every 2 days. HGF cells were placed in 4 x 10 well plates. 5 We seeded at cells / mL and cultured overnight.

[0327] Afterwards, the cell culture medium was replaced with a medium containing 500 ng / mL of LPS (lipopolysaccharide).

[0328] After culturing with LPS for 4 hours, the cells were washed and treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C at 100 µg / mL each, followed by incubation for 24 hours. Then, RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen, USA), and cDNA was synthesized using reverse transcriptase (Toyobo, Japan).

[0329] The QuantiTect SYBR Green PCR Kit (Qiagen, USA) was used for gene-specific mRNA expression analysis. The primers used for the analysis are listed in Table 1.

[0330] Analysis of EGF and VEGF results is Livak 2 -△△CT Calculated using the method, and β-actin was used as a control.

[0331] Gene Forward primer Reverse primer β-actin 5'-AGAGCTACGAGCTGCCTGAC-3'(Sequence No. 1) 5'-AGCACTGTGTTGGCGTACAG-3'(Sequence No. 2) IL-6 5'-TTCGGTCCAGTTGCCTTCTC-3'(Sequence No. 3) 5'-CAGCTCTGGCTTGTTCCTCA-3'(Sequence No. 4) IL-1β 5'-GATCACTGAACTGCACGC-3'(Sequence No. 5) 5'-CATCAGCACCTCCAAGC-3'(Sequence No. 6) EGF 5'-AGAGGGAGAGGATGCCACAT-3'(Sequence No. 7) 5'-GGTTGCATTGACCCATCTGC-3'(Sequence No. 8) VEGF 5'-AGGCCAGCACATAGGAGAGA-3'(Sequence No. 9) 5'-ACGCGAGTCTGTGTTTTTGC-3'(Sequence No. 10) TNF-α 5'-CAGAGGGCCTGTACCTCATC-3'(Sequence No. 11) 5'-GGAAGACCCCTCCCAGATAG-3'(Sequence No. 12)

[0333] ● Nuclear Condensation Analysis

[0334] Nuclear condensation analysis of small molecular size (50 bp–150 bp) PDRN was performed using the DNA-specific fluorescent dye Hoechst 33342 (Sigma-Aldrich, USA).

[0335] HaCaT cells were treated with three concentrations of PDRN (25 µg / ml, 50 µg / ml, and 100 µg / ml), exposed to UV-B (30 mJ / cm2), and cultured for 24 hours. 1 mM N-acetylcysteine ​​(NAC) was used as a positive control. After 24 hours of incubation, 1.5 μL of Hoechst 33342 (10 mg / mL stock solution) was added to each well and incubated at 37°C for 10 minutes. Subsequently, the stained cells were observed using a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera to determine the degree of nuclear condensation.

[0337] ● Flow cytometry

[0338] To analyze the apoptosis of small molecule size (50bp–150bp) PDRN, sub-G1 hypoploid cells were analyzed.

[0339] To assess apoptotic sub-G1 hypodiploid cells, HaCaT cells were pretreated with three concentrations of PDRN (25 µg / ml, 50 µg / ml, and 100 µg / ml), exposed to UV-B (30 mJ / cm2), harvested, and washed twice with ice water. They were washed twice with cold PBS and fixed with 75% ethanol at 4°C for 30 minutes. Then, the cells were incubated with PI and RNase A (1:1000) in the dark at room temperature for 30 minutes. Flow cytometry was performed using a FACSCalibur (Becton-Dickinson, San Jose, CA, USA).

[0341] ● Reactive Oxygen Species (ROS) Scavenging Analysis

[0342] To evaluate the reactive oxygen species scavenging activity of low molecular size (50bp–150bp) PDRN, the following experiment was conducted.

[0343] To briefly explain, 4 x 10 HaCaT cells in a 6-well plate 5 Cells were seeded into cells / well and cultured for 16 hours. Subsequently, cells were treated with three concentrations of PDRN (25 µg / ml, 50 µg / ml, and 100 µg / ml) and exposed to UV-B (30 mJ / cm2). Exposure of cells to UV-B radiation induces intracellular oxidative stress.

[0344] After 30 minutes, 25 μM of DCFH-DA (2'-7'dichlorodihydrofluorescein diacetate) solution was added for 10 minutes, and cell fluorescence was captured using a fluorescence microscope (IX71 and DP72, Olympus, Tokyo, Japan). Fluorescence intensity was quantified using ImageJ software (ImageJ, ver. 1.6, Bethesda, MD, USA).

[0346] ● Cell proliferation - MTS, LIVE / DEAD, DAPI / Phalloidin

[0347] To confirm cell proliferation of low molecular weight (50bp–150bp) PDRN, the following experiment was conducted.

[0348] Cell proliferation was confirmed through the MTS (methoxyphenyl tetrazolium salt) assay and staining of living and dead cells.

[0349] Human dermal fibroblast (HDF) cells were cultured in low serum (PCS-201-041, Manassas, USA) basal fibroblast growth medium (PCS-201-030, Manassas, USA).

[0350] Briefly, 5 x 10 HDF cells in a cell culture plate 4 Cells were seeded at 1 cell / well and cultured for 24 hours at 37°C under 5% CO2 conditions. Afterward, the culture medium was removed from each well plate, and the cells were starved for an additional 18 hours in FBS-free medium.

[0351] Then, cells were treated with PDRN at three concentrations (25 µg / ml, 50 µg / ml, and 100 µg / ml). Culture medium without FBS and culture medium containing 10% FBS were used as the negative control and positive control, respectively.

[0352] MTS analysis was performed using Promega’s CellTiter 96 Aqueous One Solution, and live / dead evaluations were also conducted through staining. In addition, the morphology of PDRN-treated HDF cells was confirmed using DAPI / Phalloidin-TRITC staining.

[0354] ● Cell migration

[0355] To confirm the cell migration of small molecule size (50bp–150bp) PDRN, experiments were conducted using fibroblasts as follows.

[0356] To explain briefly, a total of 2 x 10 cells 5 Cells / mL were seeded on each side of the culture insert (Culture-Insert 2 Well in μ-Dish 35 mm, Ibidi, Germany) and cultured at 37°C for 24 hours.

[0357] As HDF cells spread and attached, they formed a monolayer - the culture insert was removed and a gap (500 μm) was formed between the cell layers.

[0358] Afterward, 1 mL of FBS-free medium was added, and then three concentrations of PDRN (25 µg / mL, 50 µg / mL, and 100 µg / mL) were added to the serum-free culture medium. The FBS-free culture medium and the culture medium containing 10% FBS were used as the negative control and positive control, respectively.

[0359] This involved estimating the time required to fill the cell free gap by cell migration by capturing images of the cell free gap at 0 and 24 hours using an inverted optical microscope (Leica® DMi8, Germany). The experiment was performed three times, and the ratio of initial scratch (Ao) to healed scratch (At) was calculated using ImageJ software (ImageJ, ver. 1.6, USA).

[0360] The percentage of the open area (A) was quantified as Percentage of Open Area A (%) = [Ao - At] / AoX100.

[0362] The first nucleic acid described below is described as gDNA (genomic DNA), but as stated in the detailed description of the invention, the first nucleic acid is merely one of the materials used to produce a nucleic acid fragment to be obtained as a final product and is not limited to the experimental examples below.

[0364] Experimental Example 1: Preparation of the first nucleic acid

[0365] 10 g of salmon sperm and 20 ml of 0.9% sodium chloride aqueous solution were placed in a 500 ml flask and mixed at room temperature for 5 minutes. Subsequently, 250 ml of lysis buffer (prepared with purified water: SDS: EDTA: Tris in a ratio of 530:10.6:3.2) was added. After stirring at room temperature for 10 minutes, the mixture was sonicated using an ultrasonic analyzer (Q700, QSONICA USA) for 10 to 30 minutes.

[0366] Subsequently, 250 ml of 6 M sodium chloride aqueous solution was added and mixed. The mixture was then placed in a centrifuge and centrifuged at 4,000–10,000 rpm for 30 minutes to 1 hour to separate the supernatant. The supernatant was filtered using an 8 µm filter, 500 ml of ethanol (>99%) cooled to 0–4°C was added, and the mixture was slowly stirred before centrifugation (4,000 rpm, 30 minutes). Afterward, the mixture was vacuum dried at 30–50°C for 9 hours to obtain 4.3–4.7 g of gDNA (genomic DNA), a white primary nucleic acid.

[0367] Looking at Figure 2, it can be confirmed that the size of the first nucleic acid obtained in Experimental Example 1 is distributed in a range of 3000 bp or more.

[0369] Experimental Example 2: Preparation of Secondary Nucleic Acid

[0370] 40g of powdered first nucleic acid prepared by repeating Experimental Example 1 was added to 800ml of purified water and stirred at room temperature for 6 hours to prepare a first solution containing first nucleic acid.

[0371] The entire line of the high-pressure disperser was filled with purified water, the cooling water temperature was set to 10°C, the pressure was adjusted, and then high-pressure dispersion of the first solution was performed. When the first solution (800 ml) completely exited the high-pressure disperser, it was set to one cycle.

[0372] Experiments were conducted in Examples 2-1 and 2-2 to optimize the conditions of the high-pressure disperser for the preparation of the second nucleic acid. Subsequently, 165 g of sodium chloride was added to the solution after high-pressure dispersion and stirred for 20 minutes, then 800 ml of cooled ethanol was added and precipitated. The precipitated solid was filtered under reduced pressure to obtain 38.4–39.2 g of white powder.

[0374] Experimental Example 2-1: Optimization of Pressure Conditions of a High-Pressure Disperser for the Preparation of Secondary Nucleic Acid

[0375] To determine the pressure conditions of a high-pressure disperser for obtaining DNA fragments of small size (50 bp to 150 bp), fragmentation was performed on the first nucleic acid contained in the first solution at 10,000 psi for 1 to 10 cycles. As shown in Figure 1, even though the first nucleic acid was high-pressure dispersed at 10,000 psi for 10 cycles, lane 4 of Figure 1 shows that the nucleic acid fragments were fragmented to a size of 200 bp to 350 bp. In particular, the STD (Mastelli, Placentex) in Figure 1 is a commercially available nucleic acid with a size of 50 bp to 200 bp, so it can be seen that even if the first nucleic acid is high-pressure dispersed at 10,000 psi for 10 cycles, it does not reach the size of the STD.

[0376] In other words, it was confirmed that in order to obtain nucleic acid fragments of a small size of 50bp to 150bp, which is the target of this technology, a pressure condition of at least 10,000psi is required. That is to say, it was found that at 10,000psi or less, small nucleic acid fragments cannot be obtained even if dispersion is repeated several times.

[0378] Experimental Example 2-2: Optimization of Cycle Conditions for High-Pressure Disperser for Secondary Nucleic Acid Production

[0379] Through the experiment of Example 2-1, it was confirmed that conditions of 10,000 psi or higher are required to obtain small nucleic acid fragments, and it was intended to confirm that small DNA fragments can be obtained at high-pressure disperser pressure conditions of 20,000 to 30,000 psi.

[0380] First, to fragment the first nucleic acid of Experimental Example 1, the pressure of the high-pressure disperser was set to 25,000 psi, and samples were collected for each cycle and electrophoresis was performed (Fig. 2).

[0381] Looking at Figure 2, it can be seen that when only 1 cycle is applied, the size of the second nucleic acid is distributed in the range of 400 bp to 2600 bp, and when 5 cycles are applied, the size of the second nucleic acid is distributed in the range of 150 bp to 350 bp (lanes 2 and 3 of Figure 2). It can be confirmed that at least 15 cycles must be applied to obtain DNA fragments of the second nucleic acid with a size of 50 bp to 150 bp (lane 4 of Figure 2). That is, it can be confirmed that a pressure of 25,000 psi must be applied for at least 15 cycles to obtain a second nucleic acid with a size of 50 bp to 150 bp, which is similar in size to the STD of Figure 1.

[0382] Additionally, the pressure of the high-pressure disperser was set to 30,000 psi, and the same experiment was repeated to verify the results (Fig. 3). Specifically, samples were collected for each cycle (1 cycle, 5 cycles, and 15 cycles) with the pressure of the high-pressure disperser set to 30,000 psi, and electrophoresis was performed. The quantified graph is shown in Fig. 3. That is, Fig. 3 illustrates the relationship between the average size of DNA fragments and the number of cycles under 30 kpsi.

[0383] The DNA fragment sizes for each cycle in Figure 3 had a distribution range as follows: 800 bp to 2800 bp for cycle 1, 150 bp to 800 bp for cycle 5, and 50 bp to 150 bp for cycle 15.

[0384] Even when the pressure of the high-pressure disperser was set to 30,000 psi, at least 15 cycles were applied, similar to Figure 2 where it was set to 25,000 psi, to obtain a second nucleic acid, which is a DNA fragment of 50 bp to 150 bp in size.

[0385] By combining the results of Figures 1, 2, and 3, it was found that when fragmenting nucleic acids with a size of 3000 bp or larger, it is difficult to obtain DNA fragments of small size (50 to 150 bp) under pressure conditions of 10,000 psi. Furthermore, it was confirmed that DNA fragments of small size (50 bp to 150 bp) can be obtained under pressure conditions of 20,000 psi to 30,000 psi. It was found that in order to obtain such small DNA fragments, if high-pressure dispersion is performed for at least 15 cycles under specific pressure conditions (20,000 psi to 30,000 psi), nucleic acids with a size of 50 bp to 150 bp, similar to commercially available STD nucleic acids, can be obtained.

[0386] Meanwhile, it was found that even when the cycle was repeated more than a certain number of times with pressure exceeding a certain level, the size of the obtained secondary nucleic acid appeared uniformly (50bp to 150bp). In other words, it was confirmed that the size of the DNA fragment did not decrease further even when pressure exceeding a certain level was repeatedly applied.

[0387] This suggests that when nucleic acids are fragmented under high pressure, the size of the nucleic acids does not decrease even beyond a certain pressure and cycle, because the fragmented nucleic acids become smaller and the surface area for collision with the disk is reduced.

[0389] Experimental Example 3: Viscosity Measurement

[0390] The physical properties regarding the viscosity of the second nucleic acid obtained in Example 2 were confirmed as follows.

[0391] In a beaker, 100 ml each of the first nucleic acid of Example 1 (size of 3000 bp or larger) and the second nucleic acid of Example 2 (size of 50 bp to 150 bp, i.e., Lane 4 of Fig. 2) obtained in Example 2 were prepared at a concentration of 2% in purified water, stirred at room temperature for 30 minutes, then allowed to stand for about 2 minutes using a CAS digital viscometer (CL-1) from ATTO, Japan, and the measurements were repeated 3 times to calculate the average value.

[0392] At the measured concentration (5 mg / mL), the viscosity of the first nucleic acid was measured to be 5491.90 cp (centi Poise), and the concentration of the second nucleic acid was measured to be 19.18 cp (centi Poise) (Table 2).

[0393] 1st nucleic acid Secondary nucleic acid (Lane4) 1 time 5486.11cp 19.21cp 2nd time 5491.87cp 19.19cp 3 times 5497.71cp 19.15cp average 5491.90cp 19.18cp

[0394] This indicates that the second nucleic acid of small size, 50bp-150bp, obtained by the method of the present invention has a viscosity low enough to be industrially useful.

[0396] Based on Experimental Examples 1 to 3, it was confirmed that nucleic acids are fragmented by applying high pressure, suggesting that it is important to optimize specific pressure and cycles to obtain nucleic acid fragments of a specific size, which is a feature of the present invention.

[0398] Experimental Example 4: Confirmation of Size-Based Effects of Fragmented I-Nucleic Acid

[0399] In order to determine whether the effects of nucleic acid fragments of various sizes obtained by treating the first nucleic acid with high pressure in Experimental Examples 1 to 3 were different, the following experiment was conducted.

[0400] For each of the three sizes of nucleic acids obtained in Figure 2, the cell proliferation effect and the confirmation of mRNA levels of EGF and VEGF were compared.

[0401] The three sizes of nucleic acids used at this time are as follows:

[0402] Lane 2 of Fig. 2: Nucleic acid having a size in the range of 400 bp to 2600 bp (hereinafter referred to as Nucleic Acid-A),

[0403] Lane 3 of Fig. 2: Nucleic acid having a size in the range of 150bp to 350bp (hereinafter referred to as nucleic acid-B),

[0404] Lane 4 of FIG. 2: Nucleic acid having a size in the range of 50 bp to 150 bp (hereinafter referred to as nucleic acid-C).

[0406] Experimental Example 4-1: Confirmation of cell proliferation

[0407] In order to determine whether the cell proliferation effects of nucleic acid-A, nucleic acid-B, and nucleic acid-C, which are nucleic acid fragments of various sizes obtained by treating the first nucleic acid with high pressure in Experimental Examples 1 to 3, were different, the cell proliferation rates were compared using the EdU Cell Proliferation Kit with HDF cells. In addition, the expression of Ki67, a cell proliferation marker, was also compared in HDF cells (Figs. 4 and 5).

[0408] In Figure 4, the (-Ve) control is the group treated with serum free, and the (+Ve) control is the group treated with 10% FBS. [Figure 2] Line-2 is the group treated with nucleic acid-A, [Figure 2] Line-3 is the group treated with nucleic acid-B, and [Figure 2] Line-4 is the group treated with nucleic acid-C.

[0409] In Fig. 5, [-]control refers to the (-Ve) control of Fig. 4, and [+]control refers to the (+Ve) control.

[0410] EdU results

[0411] Looking at the EdU results in Figure 4, it can be seen that the groups treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a higher number of EdU-positive cells compared to the (-Ve) control. Looking at Figure 5, which quantifies the results of Figure 4, the proportion of EdU-positive cells was 28.8±2.2% for the [-] control group and 84.8±4.2% for the [+] control group. Additionally, the group treated with nucleic acid-A was 34.7±4.2%, the group treated with nucleic acid-B was 44.9±3.7%, and the group treated with nucleic acid-C was 58.9±2.8%. Through this, it was confirmed that the groups treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a higher proportion of EdU-positive cells compared to the [-] control group, and that among nucleic acid-A, nucleic acid-B, and nucleic acid-C, the proportion of EdU-positive cells was highest in nucleic acid-C, which has a size range of 50bp to 150bp. It was found that the proportion of EdU-positive cells in nucleic acid-C was closest to the results of the positive control group [+]control.

[0413] Ki67 results

[0414] Looking at the Ki67 expression results in Figure 4, it can be seen that the groups treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a higher number of Ki67-expressing cells compared to the (-Ve) control. Looking at Figure 5, which quantifies the results of Figure 4, the proportion of Ki67-positive cells was 29.9±3.1% for the [-] control group and 81.78±2.89% for the [+] control group. Additionally, the group treated with nucleic acid-A was 34.7±3.4%, the group treated with nucleic acid-B was 52.6±3.6%, and the group treated with nucleic acid-C was 58.3±4.7%. Through this, it was confirmed that the groups treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a higher proportion of Ki67-positive cells compared to the [-]control group, and that among nucleic acid-A, nucleic acid-B, and nucleic acid-C, the proportion of Ki67-positive cells was highest in nucleic acid-C, a nucleic acid with a size range of 50bp to 150bp. It was found that the proportion of Ki67-positive cells in nucleic acid-C was closest to the results of the positive control group, the [+]control group.

[0416] Through the results of Experimental Example 4-1, it was confirmed that the cell proliferation effect varied depending on the size of the fragmented first nucleic acid, and specifically, that the smaller the nucleic acid fragment, the higher the cell proliferation effect. In particular, it was confirmed that the cell proliferation effect of nucleic acid-C, a low-molecular-weight PDRN of 50 bp to 150 bp, was the best.

[0418] Experimental Example 4-2: Confirmation of mRNA levels of EGF (Epidermal Growth Factor) and VEGF (Vascular Endothelial Growth Factor)

[0419] In order to determine whether the growth factor expression levels of nucleic acid-A, nucleic acid-B, and nucleic acid-C, which are nucleic acid fragments of various sizes obtained by treating the first nucleic acid with high pressure in Experimental Examples 1 to 3, were different, the mRNA levels of EGF and VEGF were compared by RT-PCR using HGF cells (Fig. 6).

[0420] EGF (Epidermal Growth Factor) and VEGF (vascular endothelial growth factor) are known to be involved in skin regeneration, elasticity, promoting cell production, wound healing, and regeneration.

[0421] In Fig. 6, control is the group treated with distilled water, LPS is the group to establish an inflammatory phenotype, LPS+[Fig. 2] Line-2 is the group treated with LPS and nucleic acid-A, LPS+[Fig. 2] Line-3 is the group treated with LPS and nucleic acid-B, and LPS+[Fig. 2] Line-4 is the group treated with LPS and nucleic acid-C.

[0422] Looking at the results of EGF mRNA expression in Figure 6, it can be seen that treatment with LPS reduces EGF mRNA expression by more than half compared to the control group. In contrast, in the groups treated with LPS and nucleic acid-A; LPS and nucleic acid-B; and LPS and nucleic acid-C, it was observed that EGF mRNA expression increased compared to the LPS-treated group. Among them, the group treated with LPS and nucleic acid-C showed EGF mRNA expression at a level almost identical to that of the control group. Furthermore, it can be seen that the EGF mRNA expression in the group treated with LPS and nucleic acid-C is exceptionally high compared to the groups treated with LPS and nucleic acid-A and LPS and nucleic acid-B.

[0423] Looking at the VEGF mRNA expression results in Figure 6, it can be seen that treatment with LPS reduces VEGF mRNA expression by more than half compared to the control group. In contrast, in the groups treated with LPS and nucleic acid-A; LPS and nucleic acid-B; and LPS and nucleic acid-C, VEGF mRNA expression was found to increase compared to the LPS-treated group. Among them, the group treated with LPS and nucleic acid-C showed VEGF mRNA expression at a level almost identical to that of the control group. Furthermore, it can be seen that the EGF mRNA expression in the group treated with LPS and nucleic acid-C is exceptionally high compared to the groups treated with LPS and nucleic acid-A and LPS and nucleic acid-B.

[0424] Through the results of Experimental Example 4-2, it was confirmed that the mRNA expression levels of EGF and VEGF varied depending on the size of the fragmented first nucleic acid, and specifically, that the mRNA expression levels of EGF and VEGF were higher for smaller nucleic acid fragments. In particular, it was confirmed that the mRNA expression levels of EGF and VEGF were best for nucleic acid-C, a PDRN with a small molecular size of 50 bp to 150 bp.

[0426] To summarize Experimental Example 4, the high cell proliferation ability and the high mRNA expression levels of EGF and VEGF involved in skin regeneration, elasticity, promotion of cell production, wound healing, and regeneration suggest that the low molecular weight PDRN of 50bp to 150bp prepared by the method of the present application can be utilized for industrial purposes such as skin regeneration, elasticity, promotion of cell production, wound healing, and regeneration.

[0428] Experimental Example 5: Confirmation of effects of low molecular weight (50bp–150bp) PDRN at different concentrations

[0429] Through Experimental Example 4, the effects of fragmented first nucleic acid by size were confirmed, and as a result, it was found that PDRN, a low-molecular-weight second nucleic acid of 50bp to 150bp, increased cell proliferation ability and growth factor expression.

[0430] So, we compared the effects according to the concentration of PDRN with a small molecular size of 50bp to 150bp.

[0431] At this time, a total of three concentrations of low molecular weight (50bp–150bp) PDRN were compared (25µg / ml, 50µg / ml, 100µg / ml).

[0433] Experimental Example 5-1: Confirmation of anti-apoptotic activity

[0434] The apoptosis-inhibiting properties of small molecular weight PDRN (50bp–150bp) were confirmed by nuclear condensation analysis and sub-G1 low-diploid cell analysis.

[0435] Nuclear condensation analysis

[0436] Figure 7 illustrates the results of row condensation analysis. Apoptotic body formation was detected using Hoechst 33342 staining (20 μM). Looking at Figure 7, it can be seen that UV-B treated HaCaT cells show an increase in apoptotic bodies (indicated by the arrow) compared to the control group, while apoptotic bodies decrease upon NAC treatment. Additionally, it can be observed that the groups treated with low molecular weight PDRN show a decrease in apoptotic bodies compared to the UV-B treated group. Specifically, compared to the groups treated with low molecular weight PDRN at 25 μg / ml and 50 μg / ml, apoptotic bodies are reduced in the group treated with 100 μg / ml of low molecular weight PDRN. In other words, it can be confirmed that apoptotic bodies decrease as the concentration of low molecular weight PDRN increases.

[0437] This can be confirmed more clearly by looking at the graph in Figure 8, which quantifies the results of Figure 7. UV-B treated HaCaT cells showed an apoptosis index that increased by more than 9 times compared to the negative control group, while NAC treatment showed a significant decrease in the apoptosis index. In addition, treatment with different concentrations of low molecular weight PDRN resulted in a decrease in the apoptosis index compared to UV-B treatment. Specifically, compared to UV-B treatment, the group treated with 50 μg / ml of low molecular weight PDRN showed a decrease of more than 2 times, and the group treated with 100 μg / ml of low molecular weight PDRN showed a decrease of more than 3 times.

[0439] sub-G1 hypoploid cell analysis

[0440] Figure 9 illustrates the results of sub-G1 low-diploid cell analysis using a flow cytometer. Looking at Figure 9, it can be seen that the percentage of sub-G1 cells (i.e., the content of apoptotic cells) is high in UV-B-treated HaCaT cells and decreases in the NAC-treated group. Additionally, it can be seen that the percentage of sub-G1 cells is lower in the groups treated with low molecular weight PDRN compared to the UV-B-treated group. Specifically, it can be seen that the percentage of sub-G1 cells in the group treated with 50 μg / ml of low molecular weight PDRN is lower than that of the group treated with 25 μg / ml, and the percentage of sub-G1 cells in the group treated with 100 μg / ml of low molecular weight PDRN is lower than that of the group treated with 50 μg / ml.

[0441] This can be confirmed more clearly by looking at the graph in Figure 10, which quantifies the results of Figure 9. Compared to the negative control group, the sub-G1 cell content of UV-B-treated HaCaT cells increased by more than 40 times, while the sub-G1 cell content decreased in the NAC-treated group. In addition, treatment with different concentrations of low molecular weight PDRN resulted in a decrease in sub-G1 cell content compared to UV-B treatment. Specifically, compared to UV-B treatment, the group treated with 50 μg / ml of low molecular weight PDRN showed a decrease of more than 2 times, and the group treated with 100 μg / ml of low molecular weight PDRN showed a decrease of more than 3 times.

[0443] Experimental Example 5-2: Confirmation of ROS Scavenging

[0444] The intracellular ROS scavenging ability of small molecular size PDRN (50bp–150bp) was analyzed.

[0445] Figure 11 shows the results of observing the intracellular ROS scavenging ability of PDRN using a fluorescence microscope. Looking at Figure 11, it can be seen that the group treated with UV-B, which induces intracellular oxidative stress, showed an increase in cells expressing the DCFH-DA probe (i.e., cells with unremoved ROS) compared to the control group, while this decreased upon NAC treatment. In contrast, it can be confirmed that the groups treated with small molecule PDRN showed a decrease in cells expressing the DCFH-DA probe compared to the group treated with UV-B.

[0446] This can be confirmed more clearly by looking at the graph in Figure 12, which quantifies the results of Figure 11. Compared to the control group, UV-B-treated HaCaT cells showed an intensity of DCFH-DA probe expression that increased by more than three times, whereas in the NAC-treated group, it decreased. Additionally, treatment with different concentrations of small molecular weight PDRN resulted in a decrease in the intensity of DCFH-DA probe expression compared to UV-B treatment. In particular, the group treated with 100 μg / ml of small molecular weight PDRN exhibited significantly lower fluorescence intensity than the UV-B treatment. In other words, a concentration of 100 μg / ml of small molecular weight PDRN signifies excellent ROS scavenging activity.

[0447] Through the results of Experimental Example 5-2, it was confirmed that the intracellular ROS scavenging ability increased as the concentration of low molecular weight PDRN (50bp to 150bp) increased, which suggests that it effectively alleviates intracellular oxidative stress.

[0449] Experimental Example 5-3: Confirmation of cell proliferation

[0450] Cell proliferation of small molecule PDRN (50bp–150bp) was confirmed by MTS analysis, LIVE / DEAD analysis, and DAPI / Phalloidin analysis.

[0451] MTS analysis

[0452] Figure 13 shows the results of the MTS analysis. Looking at Figure 14, it can be seen that compared to the [-]control, the groups treated with 25 μg / ml, 50 μg / ml, and 100 μg / ml of low molecular weight PDRN all showed an increase. Specifically, the group treated with 50 μg / ml of low molecular weight PDRN showed 110.8±5.4%, and the group treated with 50 μg / ml of low molecular weight PDRN showed 118.6±2.2%. As a result, it can be seen that cell proliferation is higher as the concentration of low molecular weight PDRN increases. In other words, cell proliferation increased in a concentration-dependent manner with low molecular weight PDRN.

[0454] LIVE / DEAD Analysis

[0455] Figure 14 illustrates the LIVE / DEAD analysis results obtained by staining and observing the cells analyzed in Figure 13. Looking at Figure 14, it can be observed that the number of living HDF cells (green fluorescence) increased in all groups treated with low molecular weight PDRN compared to the (-VE) control. In particular, the number of living HDF cells (green fluorescence) increased in the group treated with 50 µg / ml of low molecular weight PDRN compared to the group treated with 25 µg / ml. Additionally, the number of living HDF cells (green fluorescence) increased in the group treated with 100 µg / ml of low molecular weight PDRN compared to the group treated with 50 µg / ml. Similar to the MTS results, this once again confirms that cell viability increases with increasing concentrations of low molecular weight PDRN. Meanwhile, it can be confirmed that there were no dead HDF cells in any of the groups treated with low molecular weight PDRN.

[0456] DAPI / Phalloidin Analysis

[0457] Figure 15 shows the results of the DAPI / Phalloidin analysis. Phalloidin is a marker that can confirm cell proliferation through actin filaments.

[0458] Looking at Figure 15, it can be seen that the number of Phalloidin-expressing cells (red fluorescence) increased in all groups treated with low molecular weight PDRN compared to the (-VE) control. In particular, the number of Phalloidin-expressing cells increased in the group treated with 50 μg / ml of low molecular weight PDRN compared to the group treated with 25 μg / ml of low molecular weight PDRN. Additionally, the number of Phalloidin-expressing cells increased in the group treated with 100 μg / ml of low molecular weight PDRN compared to the group treated with 50 μg / ml of low molecular weight PDRN. These results also confirm that, similar to the MTS and LIVE / DEAD results, the cell proliferation rate increases as the concentration of low molecular weight PDRN increases.

[0460] Experimental Example 5-4: Confirmation of cell migration activity

[0461] The cell migration activity of small molecule PDRN (50 bp–150 bp) was analyzed (Figs. 16 and 17).

[0462] Figure 16 illustrates the results of microscopic observation of cell migration. Looking at Figure 16, it can be seen that the open area decreased at 24 hours compared to 0 hours in all groups treated with low molecular weight PDRN compared to the (-VE) control. In particular, the open area decreased in the group treated with 50 μg / ml of low molecular weight PDRN compared to the group treated with 25 μg / ml of low molecular weight PDRN, and the open area decreased even more in the group treated with 100 μg / ml of low molecular weight PDRN compared to the group treated with 50 μg / ml of low molecular weight PDRN.

[0463] This can be confirmed more clearly by looking at the graph in Figure 17, which quantifies the results of Figure 16. Compared to the (-VE) control, all groups treated with low molecular weight PDRN showed a decrease in open area (%) at 24 hours compared to 0 hours. Specifically, the group treated with 25 μg / ml of low molecular weight PDRN showed a decrease of approximately 30–40% at 24 hours compared to 0 hours. The group treated with 50 μg / ml of low molecular weight PDRN showed a decrease of approximately 40–50% at 24 hours compared to 0 hours. Additionally, the group treated with 100 μg / ml of low molecular weight PDRN showed a decrease of approximately 50–60% at 24 hours compared to 0 hours. These results also confirm that, similar to previous findings, the cell migration activity of low molecular weight PDRN increases in a concentration-dependent manner.

[0465] Experimental Example 5-5: Confirmation of anti-inflammatory activity

[0466] The anti-inflammatory activity of small molecule PDRN (50bp–150bp) was confirmed by comparing the mRNA levels of EGF, VEGF, TNF-α (tumor necrosis factor-α), IL-1β (interleukin 1-beta), and IL-6 (Interleukin-6) via RT-PCR (Figs. 18 to 19).

[0467] IL-6 is an interleukin that acts as a pro-inflammatory cytokine and an anti-inflammatory myokine. It is secreted by helper T cells, macrophages, mast cells, neutrophils, epithelial cells, fibroblasts, and others.

[0468] TNF-α mediates inflammatory responses, regulates cellular immune responses, and plays a role in eliminating intracellular infections. While primarily secreted by stimulated macrophages, it is also secreted by fibroblasts, T cells, B cells, endothelial cells, and epithelial cells, playing a major role in acute inflammatory responses.

[0469] IL-1β is an immune cytokine produced in response to inflammatory factors, infections, or microbial toxins. It is primarily secreted by macrophages, lymphocytes, vascular endothelial cells, neutrophils, fibroblasts, and monocytes.

[0470] Figure 18 shows the results of confirming the mRNA levels of the cytokines IL-6, IL-1β, and TNF-α.

[0471] As shown in the results of IL-6 mRNA expression in Figure 18, it can be confirmed that treatment with LPS significantly increases IL-6 mRNA expression compared to the control. However, compared to the group treated with LPS, it can be confirmed that IL-6 mRNA expression decreased in all groups treated with LPS and three concentrations of low-molecular-weight PDRN (50bp–150bp). Specifically, the group treated with LPS and low-molecular-weight PDRN at 50µg / ml showed a decrease of more than 50% compared to the group treated with LPS, and the group treated with LPS and low-molecular-weight PDRN at 100µg / ml showed a decrease of more than 60% compared to the group treated with LPS. In particular, IL-6 mRNA expression decreased in a concentration-dependent manner in the groups treated with LPS and three concentrations of low-molecular-weight PDRN (50bp–150bp).

[0472] As shown in the results of IL-1β mRNA expression in Figure 18, it can be confirmed that treatment with LPS significantly increases IL-1β mRNA expression compared to the control. However, compared to the group treated with LPS, it can be confirmed that IL-1β mRNA expression decreased in all groups treated with LPS and three concentrations of low-molecular-weight PDRN (50bp–150bp). Specifically, the group treated with LPS and low-molecular-weight PDRN at 50µg / ml showed a decrease of more than 30% compared to the group treated with LPS, and the group treated with LPS and low-molecular-weight PDRN at 100µg / ml showed a decrease of more than 70% compared to the group treated with LPS. In particular, IL-1β mRNA expression decreased in a concentration-dependent manner in the groups treated with LPS and three concentrations of low-molecular-weight PDRN (50bp–150bp).

[0473] As shown in the TNF-α mRNA expression results in Figure 18, it can be confirmed that treatment with LPS significantly increases TNF-α mRNA expression compared to the control. However, compared to the group treated with LPS, it can be confirmed that TNF-α mRNA expression decreased in all groups treated with LPS and three concentrations of low molecular weight PDRN (50bp–150bp). Specifically, the group treated with LPS and low molecular weight PDRN at 50µg / ml showed a decrease of more than 40% compared to the group treated with LPS, and the group treated with LPS and low molecular weight PDRN at 100µg / ml showed a decrease of more than 50% compared to the group treated with LPS. In particular, the groups treated with LPS and low molecular weight PDRN at three concentrations showed a concentration-dependent decrease in TNF-α mRNA expression.

[0474] Figure 19 shows the results of confirming the mRNA levels of the growth factors EGF and VEGF. Looking at the EGF mRNA expression results in Figure 19, it can be seen that EGF mRNA expression decreases significantly compared to the control when treated with LPS. However, compared to the group treated with LPS, it can be seen that EGF mRNA expression increased in all groups treated with LPS and low molecular weight PDRN at three concentrations. Specifically, the group treated with LPS and low molecular weight PDRN at 50 μg / ml showed an increase of more than twofold compared to the group treated with LPS, and the group treated with LPS and low molecular weight PDRN at 100 μg / ml showed an increase of more than threefold compared to the group treated with LPS. In particular, EGF mRNA expression increased in a concentration-dependent manner in the groups treated with LPS and low molecular weight PDRN at three concentrations.

[0475] As shown in the VEGF mRNA expression results in Figure 19, it can be confirmed that treatment with LPS significantly reduces VEGF mRNA expression compared to the control group. However, compared to the group treated with LPS, it can be confirmed that VEGF mRNA expression increased in all groups treated with LPS and three concentrations of low-molecular-weight PDRN (50bp–150bp). Specifically, the group treated with LPS and low-molecular-weight PDRN at 50µg / ml showed an increase of more than twofold compared to the group treated with LPS, and the group treated with LPS and low-molecular-weight PDRN at 100µg / ml showed an increase of more than threefold compared to the group treated with LPS. In particular, VEGF mRNA expression increased in a concentration-dependent manner in the groups treated with LPS and three concentrations of low-molecular-weight PDRN (50bp–150bp).

[0477] Through the aforementioned experimental examples, the nucleic acid fragment preparation method of the present application is

[0478] i) the fact that a specific pressure is applied to the nucleic acid; and ii) the fact that the specific pressure is applied to the nucleic acid to obtain a small molecule nucleic acid fragment; are technically significant.

[0479] Furthermore, the low molecular size (50bp to 150bp) PDRN produced by the nucleic acid fragment preparation method of the present application has low viscosity and effects such as high cell proliferation rate and growth factor activation.

[0480] Furthermore, since low molecular weight PDRN (50bp–150bp) exhibits concentration-dependent effects such as anti-apoptotic activation, reactive oxygen species scavenging, cell proliferation, activation of cell migration, and anti-inflammatory activation, it is considered to be very suitable for industrial application.

Claims

Claim 1 A method for fragmenting DNA comprising: i) preparing a first DNA having a size of 500 bp to 5000 bp by using an enzyme or ultrasound; and ii) applying pressure of 25000 psi to 30000 psi to the first DNA for 15 to 30 cycles to obtain a product comprising a second DNA fragmented from the first DNA; wherein the obtained second DNA has a size of 50 to 150 nt and is contained in the product as 70% to 100%. Claim 2 A method according to claim 1, characterized in that the first DNA is prepared by a method using ultrasound. Claim 3 A method according to claim 1, characterized in that the second DNA having a size of 50 to 150 nt has one or more effects selected from anti-apoptosis activity, reactive oxygen species (ROS) scavenging, cell proliferation activation, cell migration activation, and anti-inflammatory activation. Claim 4 A method according to claim 1, characterized by performing the above pressure at a temperature of 5°C to 25°C. Claim 5 A method according to claim 1, characterized in that, when providing the pressure, one cycle takes 0.5 to 10 seconds. Claim 6 A method according to claim 1, characterized in that the pressure is provided by one or more devices among a high pressure homogenizer, a high pressure disperser, an ultra-high pressure homogenizer, and an ultra-high pressure disperser. Claim 7 A method according to claim 1, characterized in that the first DNA is of fish origin. Claim 8 A method according to claim 7, characterized in that the fish is salmon or trout. Claim 9 A method according to claim 1, characterized in that the first DNA is a single strand or a double strand. Claim 10 A method according to claim 1, further comprising the step of precipitating the obtained product to isolate and concentrate the second DNA. Claim 11 A method according to claim 10, characterized in that the above-mentioned precipitate uses one or more selected from sodium chloride (NaCl), potassium chloride (KCl), and sodium acetate (CH3COONa). Claim 12 A method according to claim 11, characterized in that the sodium chloride is used in an amount of 15% to 30% relative to the amount of water. Claim 13 In claim 11, the method is characterized by using one or more solvents selected from ethanol, methanol, butanol, propanol, pentanol, and isopropyl alcohol for the precipitation.

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