KR20240015040A - A Method for manufacturing nucleic acid fragment uniform size - Google Patents

Abstract

This application relates to methods of making nucleic acid fragments and various uses thereof.

Description

{A Method for manufacturing nucleic acid fragment uniform size}

This application relates to methods of making nucleic acid fragments. In particular, the method is to prepare nucleic acid fragments of uniform size by applying a specific high pressure.

This application relates to various uses of nucleic acids prepared by the above method.

Nucleic acid is the most important biomolecule and plays a role in storing and transmitting genetic information in living cells. In particular, fragmented nucleic acids are essential components of cells and are also used for purposes such as treatment and improvement of wounds, cell activation, and wrinkle improvement.

Conventionally, ultrasound is used to fragment nucleic acids; using enzymes; using genetic scissors; Nucleic acids were fragmented using hydrodynamic methods.

However, conventional methods have the following problems.

The method using sonication has the problem that it is not easy to obtain pure nucleic acids because many parts are separated into single-stranded nucleic acids (Biosens Bioelectron. 2004 Nov 15;20(5):945-55.).

Methods using enzymes or specific nucleases (gene scissors, etc.) had the problem of high costs because they required the use of expensive reagents. Additionally, these methods were difficult to use commercially because they were manufactured only in small quantities.

In addition, conventional methods using hydrodynamic methods can obtain fragmented nucleic acids of 1,500 bp or more, but there are still limitations in obtaining low-molecular-size nucleic acid fragments of 500 bp or less (Nanotechnology22 (2011) 494013 (7pp).

In particular, when using the conventional methods described above, the size of the fragmented nucleic acid is 500 bp or more, and the nucleic acid is produced as a high molecular size nucleic acid. When manufactured from polymer nucleic acids in this way, viscosity occurs, making industrial use very difficult. It is already known that the effect varies depending on the size of the fragmented nucleic acid (Mol Med Rep. 2018 Dec;18(6):5166-5172.).

Therefore, when fragmenting nucleic acids, there is a need to produce low-molecular-weight nucleic acids rather than high-molecular-weight nucleic acids.

1. U.S. Patent Publication No. 2011-0070219

One object of the present application is to provide a method for producing nucleic acid fragments. In particular, the method is to prepare nucleic acid fragments of uniform size by applying a specific high pressure.

Another object of the present application is to provide various uses of nucleic acids prepared by the above method.

In order to solve the above-described problems, the present application provides a method for producing nucleic acid fragments. The method may be a method of fragmenting DNA.

The method for fragmenting the DNA is,

i) Prepare first DNA having a size of 500bp to 5000bp; and

ii) applying pressure to the first DNA at a level of 20,000 psi to 30,000 psi for 15 to 30 cycles to obtain a product containing a fragmented second DNA of the first DNA;

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

When providing the pressure, it may be performed at a temperature of 5°C to 25°C.

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

The pressure may be performed by any one or more of a high pressure homogenizer, high pressure disperser, ultra-high pressure homogenizer, and ultra-high pressure disperser. .

The first DNA may be derived from fish. At this time, the fish may be salmon or trout. Additionally, the first DNA may be single-stranded or double-stranded.

The first DNA may have a size of 500bp to 5000bp.

The method for fragmenting the DNA is,

iii) precipitating the obtained product may further include the step of isolating and concentrating the second DNA.

The precipitation may use one or more selected from sodium chloride (NaCl), potassium chloride (KCl), and sodium acetate (CH3COONa). At this time, the sodium chloride may be used in an amount of 15% to 30% compared to the amount of water. At this time, the precipitation may use one or more solvents selected from ethanol, methanol, butanol propanol, pentanol, and isopropyl alcohol.

The second DNA has anti-apoptosis activity, reactive oxygen species (ROS) scavenging, cell proliferation activation, cell migration activation, and anti-inflammatory ( It may have one or more effects selected from anti-inflammatory activation.

According to this application, the following effects occur.

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

Second, according to the present application, various uses of small, uniform nucleic acids prepared by the above method can be provided.

Figure 1 shows the results of electrophoresis by fragmenting the first nucleic acid contained in the first solution by setting the pressure of the high pressure disperser to 10,000 psi and using different cycles. In Figure 1, M is marker, STD is standard (Mastelli, Placentex); 1 performs 1 cycle of high pressure dispersion; 2 performs 2 cycles of high pressure dispersion; 3 performs 5 cycles of high pressure dispersion; 4 indicates that 10 cycles of high pressure dispersion were performed.
Figure 2 shows the results of electrophoresis by fragmenting the first nucleic acid contained in the first solution by setting the pressure of the high pressure disperser to 25,000 psi and using different cycles. In Figure 2, M is a marker, 1 is the first nucleic acid of Experimental Example 1; 2 performs 1 cycle of high pressure dispersion; 3 performs 5 cycles of high pressure dispersion; 4 indicates that 15 cycles of high pressure dispersion were performed.
Figure 3 is a graph showing the results of electrophoresis by fragmenting the first nucleic acid by changing the cycle while setting the pressure of the high pressure disperser to 35,000 psi. At this time, the cycles were 1, 5, and 15, and samples were obtained for each cycle.
Figure 4 shows the results of confirming the cell proliferation (Cell Proliferation) effect of the second nucleic acid obtained for each cycle of Figure 2 using HDF (Human dermal fibroblast) cells using EdU and Ki67. In Figure 4, (-Ve) control is the group treated with serum free, and (+Ve) control is the group treated with 10% FBS. In addition, [Figure 2] Line-2 is a group processed with nucleic acid (hereinafter referred to as nucleic acid-A) with a size ranging from 400bp to 2600bp, and [Figure 2] Line-3 is a group with a size ranging from 150bp to 350bp. is a group treated with nucleic acid (hereinafter referred to as nucleic acid-B), and [Figure 2] Line-4 is a group processed with nucleic acid (hereinafter referred to as nucleic acid-C) having a size ranging from 50bp to 150bp. .
Figure 5 is a graph quantifying the results of Figure 4. In Figure 5, A is an Edu positive cell, and B is a numerical graph for Ki67 positive cells. In FIG. 5, [-]control refers to the (-Ve) control in FIG. 4, and [+]control refers to the (+Ve) control.
Figure 6 shows the results of comparing the mRNA levels of EGF and VEGF in the second nucleic acid obtained for each cycle in Figure 2 using human primary gingival fibroblasts (HGF). In Figure 6, control is the group treated with distilled water, LPS is the group to establish the inflammatory phenotype, LPS+[Figure 2]Line-2 is the group treated with LPS and nucleic acid-A, LPS+[Figure 2]Line- 3 is the group treated with LPS and nucleic acid-B, and LPS+[Figure 2]Line-4 is the group treated with LPS and nucleic acid-C.
Figures 7 to 10 show the results of confirming anti-apoptotic activity by varying the concentration of nucleic acid-C (i.e., PDRN of small molecule size (50bp to 150bp)) using HaCaT cells. At this time, a total of three concentrations (25 μg/ml, 50 μg/ml, and 100 μg/ml) of PDRN of small molecular size (50bp to 150bp) were compared. At this time, 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-acetyl cysteine (N- This is the group treated with Acetylcysteine (NAC). UV-B+25㎍/㎖-PDRN is a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 25㎍/㎖ after UV-B treatment; UV-B+50㎍/㎖-PDRN is a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 50㎍/㎖ after UV-B treatment; UV-B+100㎍/㎖-PDRN is a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 100㎍/㎖ after UV-B treatment.
Figures 7 and 8 show the results of nuclear condensation analysis using Hoechst. Figure 7 is a fluorescence microscope picture of the Hoechst staining results, and Figure 8 is a graph quantifying the results of Figure 7.
Figures 9 and 10 show the results of analysis of sub-G1 hypodiploid cells. Figure 9 shows the results of analysis using a flow cytometer, and Figure 10 is a graph quantifying the results of Figure 9.
Figures 11 and 12 show the results of confirming ROS scavenging by varying the concentration of nucleic acid-C (i.e., PDRN of small molecule size (50bp to 150bp)) using HaCaT cells. Figure 11 shows the results of analyzing active oxygen radical scavenging with 2'-7'dichlorodihydrofluorescein diacetate (DCFH-DA), 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 PDRN of small molecular size (50bp to 150bp) were compared. At this time, 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-acetyl cysteine (N- This is the group treated with Acetylcysteine (NAC). UV-B+25㎍/㎖-PDRN is a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 25㎍/㎖ after UV-B treatment; UV-B+50㎍/㎖-PDRN is a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 50㎍/㎖ after UV-B treatment; UV-B+100㎍/㎖-PDRN is a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 100㎍/㎖ after UV-B treatment.
Figures 13 to 15 show the results of confirming the cell proliferation effect by varying the concentration of nucleic acid-C (i.e., PDRN of small molecule size (50bp to 150bp)) using HDF (Human dermal fibroblast) cells. At this time, a total of three concentrations (25 μg/ml, 50 μg/ml, and 100 μg/ml) of PDRN of small molecular size (50bp to 150bp) were compared. Figure 13 is the MTS (methoxyphenyl tetrazolium salt) analysis result, Figure 14 is the LIVE/DEAD analysis result, and Figure 15 is the DAPI/Phalloidin analysis result.
In Figure 13, [-]control is a group treated with culture medium without FBS; [+]control is the group treated with culture medium containing 10% FBS. In addition, 25㎍/㎖, 50㎍/㎖, and 100㎍/㎖ are groups treated with PDRN of small molecule size (50bp~150bp) at a concentration of 25㎍/㎖, respectively; A group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 50㎍/ml; It means a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 100㎍/㎖.
In Figures 14 and 15, (-Ve)control is a group treated with culture medium without FBS; (+Ve)control is the group treated with culture medium containing 10% FBS. In addition, 25㎍/㎖-PDRN, 50㎍/㎖-PDRN, and 100㎍/㎖-PDRN are groups treated with PDRN of small molecule size (50bp~150bp) at a concentration of 25㎍/㎖; A group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 50㎍/ml; It means a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 100㎍/㎖.
Figures 16 and 17 show the results of confirming the cell migration effect by varying the concentration of nucleic acid-C (i.e., PDRN of small molecule size (50bp to 150bp)) using HDF (Human dermal fibroblast) cells. At this time, a total of three concentrations (25 μg/ml, 50 μg/ml, and 100 μg/ml) of PDRN of small molecular size (50bp to 150bp) were compared. Figure 16 is a micrograph, and Figure 17 is a graph quantifying the results of Figure 16. At this time, (-Ve) control is the group treated with culture medium without FBS; (+Ve)control is the group treated with culture medium containing 10% FBS. In addition, 25㎍/㎖-PDRN, 50㎍/㎖-PDRN, and 100㎍/㎖-PDRN are groups treated with PDRN of small molecule size (50bp~150bp) at a concentration of 25㎍/㎖; A group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 50㎍/ml; It means a group treated with PDRN of small molecule size (50bp~150bp) at a concentration of 100㎍/㎖.
Figures 18 and 19 show the results of confirming anti-inflammatory activity by varying the concentration of nucleic acid-C (i.e., PDRN of low molecular size (50bp to 150bp)) using HDF (Human dermal fibroblast) cells. . At this time, a total of three concentrations (25 μg/ml, 50 μg/ml, and 100 μg/ml) of PDRN of small molecular size (50bp to 150bp) were compared. Figures 18 and 19 show IL-6 (Interleukin-6), IL-1β (interleukin 1-beta), and TNF-α (IL-6) through RT-PCR using human primary gingival fibroblasts (HGF) This is the result of analyzing the mRNA levels of tumor necrosis factor-α), EGF, and VEGF. At this time, control is the group treated with distilled water, and LPS is the group to establish the inflammatory phenotype.

Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by a person skilled in the art to which this application pertains. Although methods and materials similar or equivalent to those described in this application can be used in the practice or testing of this application, 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, the materials, methods and practices are illustrative only and are not intended to be limiting.

Definition of Terms

Fragment and fragmentation

In this application, the term "fragment" means a cut, piece, fragment, or shear product of one component. As used in this application, “nucleic acid fragment” refers to a form having a certain length of materials called nucleic acids, such as RNA, DNA, and PNA, and a representative example is DNA having a certain length. I can hear it.

In one embodiment, full-length DNA extracted from cells, etc. or DNA with a length of 1000 bp or more can be cut to obtain DNA fragments with a shorter length. .

In this application, the term “fragmentation” means splitting one component into the fragments or a process including the same. For example, DNA fragmentation means separating or destroying DNA strands of a certain length or more into pieces of smaller length. In particular, in the present application fragmentation can be achieved using pressure.

base pair (bp)

In this application, the term “base pair (bp)” refers to a unit representing the length or size of nucleic acids such as RNA, DNA, and PNA. The bp can be used interchangeably with nt (nucleotide) and mer. For example, when the nucleic acid is double-stranded, bp can be used, and when the nucleic acid is single-stranded, nt is generally used. However, in this application, bp or nt can be used regardless of whether it is double-stranded or single-stranded. In addition, the mer refers to DNA or RNA itself consisting of a few nucleotides. For example, 24-mer refers to a nucleic acid consisting of 24 bases. In other words, mer indicates length or size regardless of the type of nucleic acid. 1,000bp is 1kb (=kbp=kilo base pair), 1,000,000bp is 1Mb (mega base pair), and 1,000,000,000 bp is 1Gb (giga base pair). Therefore, in this application, bp is used interchangeably with nt and mer as a unit representing the size of a nucleic acid.

About

In this application, the term "about" means 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, for reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. A quantity, level, value, or number that changes by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30%. , means frequency, percentage, dimension, size, quantity, weight or length.

Hereinafter, the invention disclosed in this application will be described in detail.

I. Method for producing nucleic acid fragments of specific size according to specific high pressure

Overview of methods for producing nucleic acid fragments of specific sizes at specific high pressures

One aspect of the present application relates to a method of making nucleic acid fragments. In particular, the method utilizes certain high pressures. Using high pressure at a specific size is intended to fragment nucleic acids into specific size nucleic acids.

For example, a method of producing nucleic acid fragments of a specific size using high pressure,

Preparing a first nucleic acid; and

It may include providing pressure of a specific magnitude to the first nucleic acid.

Using the above method, a product containing a second nucleic acid of a specific size can be obtained.

Below, each step of the method for producing nucleic acid fragments of a specific size using high pressure will be described in detail.

Steps to prepare the first nucleic acid

The method of producing nucleic acid fragments of a specific size using high pressure in the present application,

It includes preparing the first nucleic acid.

first nucleic acid

The first nucleic acid refers to a nucleic acid used as a material used to obtain a final product comprising a second nucleic acid of a specific size. In other words, it refers to nucleic acid as a material before the process of fragmenting nucleic acid.

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

For example, the first nucleic acid is 1000bp (i.e. 1kb), 1500bp, 2000bp, 2500bp, 3000bp, 3200bp, 3400bp, 3600bp, 3800bp, 4000bp, 4500bp, 5000bp, 5500bp, 6000bp, 6500bp, 7000bp, 7500bp, 8000bp, 8500bp , 9000bp, 9500bp, 10000bp, 20000bp, 30000bp, 40000bp, 50000bp, 100000bp, 1000000bp (i.e. 1Mb), 1000000000bp (i.e. 1Gb). For example, the first nucleic acid is a nucleic acid having a size of 2000 bp or more. For another example, the first nucleic acid is a nucleic acid having a size of 3000 bp or more.

In one embodiment, the first nucleic acid may be a nucleic acid having a size of 2000bp to 40000bp. In another embodiment, the first nucleic acid may be a nucleic acid having a size of 2500bp to 20000bp. In a preferred example, the first nucleic acid may be a nucleic acid having a size of 3000bp to 10000bp.

The first nucleic acid is a nucleic acid obtained directly from cells, tissues, etc.; It may be a commercially available nucleic acid, etc.

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

The first nucleic acid may be DNA (Deoxyribonucleic acid), RNA (RiboNucleic Acid), PNA (Peptide nucleic acid), LNA (Locked nucleic acid), and a combination thereof, but is not limited thereto.

For example, the first nucleic acid may be genomic DNA (gDNA) extracted from cells.

The first nucleic acid may be single-stranded or double-stranded. Additionally, the first nucleic acid may be full-length DNA or a DNA fragment.

For example, DNA fragments can be created in a DNA extraction process from cells, etc. In one embodiment, the first nucleic acid may be polydeoxyribonucleotide (PDRN). Preferably, the first nucleic acid may be PDRN with a size of about 300bp to 50,000bp.

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 a freeze-dried or vacuum-dried powder.

How to prepare primary nucleic acid

The method for preparing the first nucleic acid is,

For example, it may include pretreating a sample containing the first nucleic acid.

Pretreatment of the sample containing the first nucleic acid may be performed using a pretreatment method known in the art to obtain the first nucleic acid from the sample containing the first nucleic acid.

The sample containing the first nucleic acid may be, for example, a cell tissue. In one embodiment, the sample containing the first nucleic acid may be fish testis or sperm. In some embodiments, the sample containing the first nucleic acid may be salmon sperm.

Pretreatment of a sample containing the first nucleic acid may include a first process of breaking or removing the cell membrane or cell wall. Through the first process, a product such as nucleic acid present in a sample containing the first nucleic acid can be obtained, and this can be used as the first nucleic acid.

For example, the first process may be performed using enzymes, ultrasound, etc.

In addition, pretreatment of the sample containing the first nucleic acid may optionally further include a second process of separating and/or washing the first nucleic acid from the product obtained according to the first process. The second process may be to remove unnecessary substances other than nucleic acids, such as proteins and histones, or to extract and isolate only the first nucleic acid.

For example, the second process can be performed by centrifugation, precipitation, chromatography, or a kit for protein removal.

Additionally, the step of preparing the first nucleic acid may further include a post-processing step, if necessary. The post-treatment process may be concentration, storage, or drying before applying pressure to the first nucleic acid. Concentrating the nucleic acid may be for mass production of a second nucleic acid obtained from the first nucleic acid. The storage and/or drying may be to minimize external influences on the nucleic acid before applying it to a device that provides pressure. At this time, the external influence may be, for example, destruction of nucleic acids due to the influence of temperature. The drying may be performed by vacuum drying, freeze drying, etc.

Obtaining a second nucleic acid by providing pressure to the first nucleic acid

The method of producing nucleic acid fragments of a specific size using high pressure in the present application,

It includes providing pressure of a specific magnitude to the first nucleic acid obtained above.

By applying pressure of a specific size to the first nucleic acid, a product in which the first nucleic acid is fragmented can be obtained, and the fragmented product includes the second nucleic acid as the final product.

That is, the second nucleic acid is a nucleic acid included in a product containing nucleic acid fragmented to a specific size by applying pressure of a specific size to the first nucleic acid. The size of the second nucleic acid may vary depending on the magnitude of the pressure, but in the present application, it refers to a nucleic acid fragment having a small molecule size of about 50bp to 400bp.

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

Since the second nucleic acid is a fragment (i.e., fragmented) of the first nucleic acid due to pressure, the second nucleic acid is smaller in size and/or length and has an increased number compared to the first nucleic acid.

The second nucleic acid is selected from about 75%, 80%, 85%, 90%, 95%, or 100% of the product of the first nucleic acid (i.e., the product of the fragmented first nucleic acid) to which pressure is applied to the first nucleic acid described above. It may be included (or distributed) as many numbers (%) within the two numerical ranges as possible. For example, the second nucleic acid may be contained in about 80% to 100% of the product of the first fragmented nucleic acid. As another example, the second nucleic acid may be contained in about 85% to 100% of the product of the first fragmented nucleic acid. As another example, the second nucleic acid may be contained in about 90% to 100% of the product of the first fragmented nucleic acid.

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

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 a lyophilized or vacuum-dried powder.

pressure of a certain magnitude

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

The pressure used in the method of the present application can be determined depending on the desired size of the second nucleic acid. Units used for pressure may include pounds per square inch (psi), bar, pascal (Pa), and Torr. Additionally, the units can be converted to one another. For example, 1 psi is approximately 6894.757 Pa, and 1 pa is approximately 0.000145 psi.

For example, the pressures used in the methods of this application are 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, 38000psi, 40000psi, 42000psi, 44000psi, 46000psi , 48000psi, 50000psi, 52000psi, 54000psi, 56000psi, 58000psi, and 60000psi. It may be a pressure within two numerical ranges selected from among.

As an example, the pressure used in the method of the present application may be a pressure ranging from 10,000 psi to 50,000 psi. In another embodiment, the pressure used in the method of the present application may be a pressure ranging from 15,000 psi to 40,000 psi. In another embodiment, the pressure used in the method of the present application may be a pressure ranging from 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 in the range of 20000 psi to 30000 psi.

The present inventor was the first to identify the relationship that a second nucleic acid with a specific size can be obtained by applying pressure of a specific size to the first nucleic acid. In other words, a second nucleic acid having a uniform size can be obtained by applying pressure of a specific size to the first nucleic acid according to the size of the desired second nucleic acid.

As the pressure increases, a second nucleic acid of smaller size 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 fragmenting nucleic acids at high pressure, the smaller the molecule is, the smaller the collision area is when it collides with the disk. In order to make small molecules smaller, increasingly greater pressure is required, but because the collision area becomes smaller, when the pressure exceeds a certain level, the size of the nucleic acid no longer decreases.

Therefore, the method of producing a nucleic acid fragment using high pressure of a specific size of the present application includes providing pressure of a specific size to the first nucleic acid, and at this time, the pressure of the specific size causes the second nucleic acid fragment to have a specific size. Nucleic acids can be obtained.

In one embodiment, by applying a pressure of 10,000 psi to 50,000 psi to the first nucleic acid, a second nucleic acid with 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 with 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 with 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 with a size of 50 bp to 150 bp can be obtained.

Conditions for handling pressure

The pressure applied to the method of the present application can be adjusted according to the size of the first nucleic acid and/or the size of the desired second nucleic acid. At this time, the conditions may be the number of pressure treatments, time, temperature, etc.

The number of pressure treatments may be 1 to 100 times. The term “number of times” should be interpreted as having the same meaning as cycle. For example, 1 cycle means the interval from applying (providing) pressure once to the moment when the applied pressure is completely released or the pressure disappears, and 2 cycles are after the 1 cycle is completed. This refers to the interval from when additional pressure is applied until the moment when the applied pressure disappears.

When the number of pressure treatments is two or more, each treatment session may be treated with pressure of the same magnitude or may be treated with pressure of different magnitudes. For example, the pressure is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. , it can be processed 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, or 30 times. As another example, the pressure may be applied 5 to 30 times. As another example, the pressure may be applied 10 to 30 times. As another example, the pressure may be applied 10 to 20 times. In one embodiment, the pressure may be applied 5 to 20 times.

The pressure processing time may take approximately 0.5 to 10 seconds from the time pressure is applied to the time the pressure disappears for one treatment. For example, when performing one cycle, the pressure processing time may take about 0.5 to 5 seconds. For another example, when performing one cycle, the pressure processing time may take about 1 second to 3 seconds. For example, in one embodiment of the present invention, when applying pressure for 15 cycles, it may take about 7.5 seconds (0.5 seconds x 15 times) to 150 seconds (10 seconds x 15 times). For another specific example, in one embodiment of the present invention, when applying pressure for 15 cycles, it may take about 15 seconds (1 second x 15 times) to 75 seconds (5 seconds x 15 times).

The pressure can be applied at a temperature of about 1°C to 40°C. At this time, the temperature may be a gradually changing temperature rather than a fixed temperature. For example, it may be a gradually lowering 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 gradually lowered by about 1°C to 10°C.

For example, the pressure is approximately 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℃, 30℃, 31℃, It can be processed at a temperature within two numerical ranges selected from 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, and 40℃. As another example, the pressure may be applied at a temperature of about 2°C to 35°C. As another example, the pressure may be applied at a temperature of about 5°C to 35°C. In one embodiment, the pressure may be applied at a temperature of about 5°C to 25°C.

device that provides pressure

Meanwhile, the 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.

For example, the specific device may include a chamber, an inlet, an outlet, a pressure control device, and a flow rate control device. Additionally, the particular device may optionally further include a temperature control device.

Depending on the flow rate, the chamber includes, for example, cavitation nozzle type, impact valve type, V-type, Y-type, Z-type, etc., but is not limited thereto.

The specific device may be a high pressure homogenizer, high pressure disperser, ultra-high pressure homogenizer, ultra-high pressure disperser, etc., but is not limited thereto.

The principle of nucleic acid fragmentation using high pressure is as follows. When nucleic acid is injected into the specific device, high pressure flows into the disk, causing separation and colliding again, or colliding with the wall and receiving high energy, cutting the weak bonds in the DNA molecular chain and fragmenting the nucleic acid.

For example, a method of providing pressure to nucleic acids using a specific device includes:

Including introducing the first nucleic acid into the inlet of the specific device,

At this time, the pressure control device and the flow rate control device within the specific device are each set to specific conditions,

The first nucleic acid introduced into the inlet passes through the chamber, and at this time, the flow rate of the chamber increases and the pressure is released,

Cavitation may occur due to the difference in pressure and flow rate.

additional process

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

For the separation and/or concentration, a precipitation process, a filtration process, etc. may be performed on the product containing the second nucleic acid obtained by applying pressure to the first nucleic acid. The precipitation process can be performed by known methods, such as using salt. For example, the precipitation process may be performed using substances such as sodium chloride (NaCl), potassium chloride (KCl), and sodium acetate (CH3COONa). At this time, the material may be determined depending on the amount of the product containing the second nucleic acid, but the conditions can be changed arbitrarily by the experimenter. The filtration process may be performed by known methods such as reduced pressure filtration, pressure filtration, and natural filtration.

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

Additionally, an organic solvent can be used in the 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 at a ratio of 1:99 to 99:1 relative to the total solution.

Optionally, only the product of the fragmented first nucleic acid or/and the second nucleic acid contained in the product of the fragmented first nucleic acid can be extracted and stored by a known method. This may be for storage until future industrial use. For example, storage may be done using liquid nitrogen, freeze-drying, or using an ultra-low temperature freezer, but is not limited thereto.

Specific examples of the method herein

Hereinafter, an example of a method for producing nucleic acid fragments of a specific size according to the specific high pressure described above will be described, but the method is not limited to the following description.

The method examples described below are merely examples, and those skilled in the art can change the equipment used in the method, pressure treatment conditions, etc. to produce nucleic acid fragments of a specific target size.

Method example (1)

As an example of the present application, the method for producing nucleic acid fragments is

The first nucleic acid having a size of about 500bp to 5000bp prepares a nucleic acid;

providing a pressure of about 15000 psi to 30000 psi 10 to 30 times to the first nucleic acid to obtain a fragmented product of the first nucleic acid;

may include.

The first nucleic acid may be obtained using an ultrasonic grinder.

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 fish testes or sperm.

The first nucleic acid may be in the form of vacuum-dried powder.

The pressure may be provided by a device selected from a high pressure homogenizer, high pressure disperser, ultra-high pressure homogenizer, and ultra-high pressure disperser.

The pressure may be applied to the first nucleic acid for about 5 to 30 minutes in one treatment. Additionally, when providing the pressure, it may be performed at a temperature of about 5°C to 25°C.

The fragmented product of the first nucleic acid may contain about 70% to 100% of the second nucleic acid. At this time, the second nucleic acid may be a nucleic acid having a size of about 50bp to 200bp. As a specific example, the second nucleic acid may be DNA having a size of about 50bp to 150bp.

Method example (2):

As another example of the present application, the method for producing nucleic acid fragments is

The first nucleic acid having a size of about 500bp to 5000bp prepares a nucleic acid;

providing a pressure of about 15000 psi to 30000 psi 10 to 30 times to the first nucleic acid to obtain a fragmented product of the first nucleic acid;

separating a second nucleic acid from the fragmented product of the first nucleic acid;

may include.

The first nucleic acid may be obtained using an ultrasonic grinder.

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 fish testes or sperm.

The first nucleic acid may be in the form of vacuum-dried powder.

The pressure may be provided by a device selected from a high pressure homogenizer, high pressure disperser, ultra-high pressure homogenizer, and ultra-high pressure disperser.

The pressure may be applied to the first nucleic acid for about 5 to 30 minutes in one treatment. Additionally, when providing the pressure, it may be performed at a temperature of about 5°C to 25°C.

The second nucleic acid may be contained in about 70% to 100% of the fragmented product of the first nucleic acid. At this time, the second nucleic acid may be a nucleic acid having a size of about 50bp to 200bp. As a specific example, the second nucleic acid may be PDRN with a size of about 50bp to 150bp.

The second nucleic acid may be in powder form.

Additionally, the method includes a precipitation process to separate the second nucleic acid from the fragmented product of the first nucleic acid. For example, the second nucleic acid can be isolated by treating the fragmented product of the first nucleic acid with sodium chloride. For another example, the fragmented product of the first nucleic acid may be treated with sodium chloride and then treated with ethanol to separate the second nucleic acid.

Separating the second nucleic acid from the fragmented product of the first nucleic acid may optionally further include a filtration process after the precipitation process. For example, the fragmented product of the first nucleic acid can be treated with sodium chloride and then filtered under reduced pressure to separate the second nucleic acid. For another example, the fragmented product of the first nucleic acid may be treated with sodium chloride and ethanol and then filtered under reduced pressure to separate the second nucleic acid.

Features of the method

The method for producing nucleic acid fragments of a specific size according to the specific high pressure of the present application described above may have the characteristics described below, but is not limited thereto.

Feature (1): Nucleic acid fragments of a certain size can be obtained homogeneously at a certain pressure.

In this application, a nucleic acid fragment of a specific size of interest can be manufactured using a pressure of a specific size. In particular, the obtained nucleic acid fragments of a certain size are uniform in size.

Specifically, in the present application, for example, a high pressure homogenizer (HPH) fragments nucleic acids using high pressure to a specific size. In one embodiment, pressure is applied at optimal conditions of about 15,000 psi to 25,000 psi to fragment nucleic acids into small molecules of about 50 bp to 150 bp or less.

Feature (2): Obtaining nucleic acids (DNA fragments) of a smaller size than before.

The method of the present application has the feature of being able to obtain low-molecular-size nucleic acids that are difficult to obtain by methods using enzymes conventionally used for nucleic acid fragmentation and methods using ultrasound. Most conventional methods were able to obtain large nucleic acid fragments of 650 bp or more, but using the method of the present application, small molecule nucleic acid fragments of 50 to 350 bp in size could be obtained.

Furthermore, the amount of low-molecular-weight nucleic acid fragments of about 50bp to 350bp in size distributed throughout the fragmented nucleic acid increases. In other words, the method of the present application facilitates the preparation of low-molecular-weight nucleic acid fragments and enables mass production.

In particular, nucleic acid fragments of 50 to 150 bp in size obtained by the method disclosed in the present application have the advantage of very high industrial and biological usefulness compared to nucleic acid fragments of 500 to 1,000 bp in size conventionally used in the art.

II. Use of compositions comprising a second nucleic acid

Another aspect of the present application relates to various uses of a second nucleic acid in the above methods.

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

The second nucleic acid has anti-apoptosis activity, reactive oxygen species (ROS) scavenging, cell proliferation activation, cell migration activation, and anti-inflammatory ( It can have effects such as anti-inflammatory activation. Because of this, it can be more useful industrially.

The composition containing the above-described second nucleic acid (hereinafter referred to as the second nucleic acid composition) may have a lower viscosity than the composition containing the first nucleic acid (hereinafter referred to as the first nucleic acid composition). In addition, due to low viscosity, permeability can be high, so it can be administered in high doses when applied to the human body and can be more useful industrially.

For example, the viscosity of the second nucleic acid composition may be about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold. , 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 70 times, 80 times, 90 times, 100 times, 120 times, 150 times, 180 times, 200 The viscosity of the first nucleic acid composition can be lower than the viscosity of the first nucleic acid composition by a multiple within two numerical ranges selected from among 2, 220, 250, 280, 300, 320, 350, 380, 400, 450, and 500 times. there is. 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. As 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. As 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.

As is well known in the art, when the fragmented nucleic acid is manufactured in a size of 500 bp or more, viscosity occurs and it is difficult to apply it industrially. However, the second nucleic acid of the present application is manufactured in a small size of about 50 bp to 150 bp. Therefore, it can be used industrially for a variety of purposes. For example, the use may be cell regeneration, wound treatment and improvement, cell activity, wrinkle improvement, etc.

Hereinafter, various uses of the composition containing the second nucleic acid prepared by the above-described method as an active ingredient will be described.

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

Use (1): Pharmaceutical use

A composition containing the second nucleic acid as an active ingredient prepared by the method of the present application can be used to treat an individual in need of treatment. Examples include wound healing, improvement, cell regeneration, cell activity, and wrinkle improvement.

pharmaceutical composition

For example, a pharmaceutical composition applicable to the above pharmaceutical uses may be disclosed.

The pharmaceutical composition contains the above-mentioned active ingredients.

It may contain a second nucleic acid.

The second nucleic acid is the same as described above.

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

Additional Components of Pharmaceutical Compositions

In addition to the active ingredients, the pharmaceutical composition may optionally further include additional pharmaceutically acceptable components.

In this application, the term "pharmaceutically acceptable" means that it can be used in contact with human and animal tissue without undue toxicity, irritation, allergic reaction or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable benefit/risk ratio. Used herein to refer to materials, compositions and/or dosage forms suitable for use.

The pharmaceutically acceptable additional component may be a physiologically acceptable component that affects the stabilization, dissolution, absorption, and introduction efficiency of the active ingredient of the present application. For example, the pharmaceutically acceptable additional components may be carriers, excipients, diluents, preservatives, etc., but are not limited thereto.

For example, the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium It may be silicate, cellulose, methyl cellulose, distilled water, physiological saline, glycerol, ethanol, HSA (Human serum albumin), etc.

For example, the preservative may be benzoic acid, sodium benzoate, sorbic acid, paraoxybenzoic acid, chlorobutanol, etc.

When formulating the pharmaceutical composition, it may further include fillers, extenders, binders, wetting agents, etc.

Formulation of pharmaceutical compositions

The pharmaceutical composition may be formulated for oral or parenteral use.

For example, when formulated for oral use, it can be manufactured in solid form, liquid form, suspension, granule, capsule, semi-solid form, etc.

For another example, when formulated for parenteral use, it may be manufactured in the form of injections, aerosols, kits, etc. Preferably, it can be formulated as an injection.

In the case of injectable drugs, methods known in the art may be additionally performed to delay the absorption of the drug, if necessary, to prolong the effect of the drug. For example, an injectable depot form can be manufactured by encapsulating the active ingredient using a biodegradable polymer. As another example, an injectable depot form can be prepared by entrapped the active ingredient in liposomes or microemulsions.

In the case of a kit, this means including a storage container, mixing container, mixing tool, etc.

For example, the storage container may be a vial, antioxidant bottle, or plastic container.

For example, the mixing container may be a plastic container, a sterilized bottle, or a syringe container.

For example, mixing tools may be gloves, spoons, sticks, etc.

To apply the above-mentioned pharmaceutical composition for pharmaceutical purposes, that is, for the above-mentioned wound treatment, improvement, cell regeneration, cell activity, wrinkle improvement, etc.,

A method for wound treatment, improvement, cell regeneration, cell activity, and wrinkle improvement including administering a pharmaceutical composition to a subject may be provided.

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

The pharmaceutical composition of the present application may be administered to a subject through various routes.

The administration may be oral administration or parenteral administration. At this time, parenteral administration may be injection administration.

When administered by injection, the administration site may be subcutaneous, intramuscular, intradermal, intravenous, transdermal, etc.

The administration dose of the pharmaceutical composition may be determined in consideration of the administration site, gender, body weight, etc., but is not limited thereto.

In an optional example, the second nucleic acid comprising the pharmaceutical composition of the present application may be administered in the following doses.

For example, when the subject is a 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㎍/㎖, 28㎍/㎖, 29㎍/㎖, 30㎍/㎖, 35㎍/㎖, 40㎍/㎖, 45㎍/㎖, 50㎍/㎖, 55㎍/㎖, 60㎍/㎖, 65㎍/㎖, 70㎍/㎖, 75㎍/㎖, 80㎍/㎖, 85㎍/㎖, 90㎍/㎖, 95㎍/㎖, 100㎍/㎖, 120㎍/㎖, 140㎍/㎖, Administered at a dose within two numerical ranges selected from 160㎍/㎖, 180㎍/㎖, 200㎍/㎖, 220㎍/㎖, 240㎍/㎖, 260㎍/㎖, 280㎍/㎖, and 300㎍/㎖. It can be. At this time, the non-human refers to all mammals excluding humans.

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

For another example, if the subject is a human, the second nucleic acid may be 20 μg/ml, 21 μg/ml, 22 μg/ml, 23 μg/ml, 24 μg/ml, 25 μg/ml, 26 μg/ml, 27㎍/㎖, 28㎍/㎖, 29㎍/㎖, 30㎍/㎖, 35㎍/㎖, 40㎍/㎖, 45㎍/㎖, 50㎍/㎖, 55㎍/㎖, 60㎍/㎖, 65㎍/㎖, 70㎍/㎖, 75㎍/㎖, 80㎍/㎖, 85㎍/㎖, 90㎍/㎖, 95㎍/㎖, 100㎍/㎖, 120㎍/㎖, 140㎍/㎖, Administered at a dose within two numerical ranges selected from 160㎍/㎖, 180㎍/㎖, 200㎍/㎖, 220㎍/㎖, 240㎍/㎖, 260㎍/㎖, 280㎍/㎖, and 300㎍/㎖. It can be.

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

20㎍, 21㎍, 22㎍, 23㎍, 24㎍, 25㎍, 26㎍, 27㎍, 28㎍, 29㎍, 30㎍, 35㎍, 40㎍, 45㎍, 50㎍ per 1mL of the pharmaceutical composition. Μg, 55 µg, 60 μg, 65 µg, 70 µg, 80 µg, 85 µg, 90 µg, 100 µg, 120 µg, 140 µg, 160 µg, 180 µg, 200 µg, 220 µg, The second nucleic acid may be included at a dose within two numerical ranges selected from 240 μg, 260 μg, 280 μg, and 300 μg.

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

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

For example, the administration volume may be 0.1 mL to 10 mL at a time, but is not limited thereto.

Additionally, the number of administrations may be from 1 to 30 times per day, but is not limited thereto. At this time, it may be administered at regular intervals.

The administration interval may be 1 to 30 days, but is not limited thereto. At this time, administration may be continuous administration or discontinuous administration.

Use (2): Cosmetic use

A composition containing the second nucleic acid as an active ingredient prepared by the method of the present application can be used for cosmetic purposes. The cosmetic treatment may include wrinkle improvement, whitening, scar improvement, moisturizing, and elasticity improvement, but is not limited thereto.

Cosmetic composition

As another example, a cosmetic composition applicable to the above cosmetic uses may be disclosed.

The cosmetic composition contains the above-mentioned active ingredients.

It may contain a second nucleic acid.

The second nucleic acid is the same as described above.

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

Additional components of cosmetic compositions

In addition to the active ingredients, the cosmetic composition may optionally further include cosmetically acceptable additional components.

In this application, the term "cosmetically acceptable" means suitable for use in contact with human and animal tissue, without undue toxicity, irritation, allergic reaction or other problems, commensurate with a reasonable benefit/risk ratio, for valid cosmetic purposes. Used herein to refer to materials, compositions and/or dosage forms.

The cosmetically acceptable additional component may be a physiologically acceptable component that affects the stabilization, dissolution, absorption, and introduction efficiency of the active ingredient of the present application. For example, the cosmetically acceptable additional components may be carriers, excipients, diluents, preservatives, etc., but are not limited thereto.

For example, the carriers, excipients and diluents include oil, water, surfactants, humectants, alcohol, chelating agents, 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.

For example, the preservative may be benzoic acid, paraoxybenzoic acid ester, benzoic acid, etc.

Meanwhile, the cosmetically acceptable additional component may optionally further include one or more active ingredients (hereinafter referred to as similar functional ingredients) that exhibit a similar function to the second nucleic acid of the present application. For example, 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.

When adding the above-mentioned similar functional ingredients, skin stability, ease of formulation, stability, etc. due to combined use may be taken into consideration. For example, similar functional ingredients may be kojic acid, arbutin, hydroquinone, vitamin C, retinoic acid, TGF, protein derived from animal placenta, chlorella extract, etc.

When formulating the cosmetic composition, fillers, extenders, binders, wetting agents, etc. may be further included.

Formulation of cosmetic compositions

The cosmetic composition may be formulated for topical use, transdermal use, endothelial use, etc.

The formulation may be in solid, liquid, spray, capsule, granule, semi-solid form, etc.

For example, formulations include solution, external ointment, cream, foam, nourishing lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, shampoo, conditioner, liquid cleanser, bath salt, sunscreen, sun oil, It may be a suspension, emulsion, paste, gel, lotion, powder, cleansing foam, cleansing oil, powder foundation, emulsion foundation, wax foundation, patch, spray, etc., but is not limited thereto.

For example, when the dosage form is an ointment, paste, cream or gel, the carrier, excipient or/and diluent may be animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose water, silicone, bentonite, silica, etc. It can be.

For another example, when the dosage form is a powder or spray, the carrier, excipient or/and diluent may be lactose, talc, calcium silcate, polyamide powder, etc.

For another example, if the formulation is a solution, emulsion, carriers, excipients or/and diluents may be used such as water, ethanol, isopropanol, ethyl carbonate, benzyl benzoate, cottonseed oil, peanut oil, castor oil, glycerol, etc. You can.

For another example, when the dosage form is a suspension, the carrier, excipient or/and diluent may be water, propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, ester, tracant, etc.

For 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.

Meanwhile, an agent that increases percutaneous absorption may be further included. For example, agents that increase percutaneous absorption include dimethyl sulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone (1-dodecylazacycloheptan-2-one), alcohol, urea, ethoxydiglycol, acetone. , propylene glycol, polyethylene glycol, etc.

To apply the above-mentioned cosmetic composition for cosmetic purposes, that is, for the above-mentioned wrinkle improvement, whitening, scar improvement, moisturizing, elasticity promotion, etc.,

A method for improving wrinkles, whitening, improving scars, moisturizing, improving elasticity, etc., including administering a cosmetic composition to a subject, may be provided.

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

The cosmetic composition of the present application may be administered to a subject through various routes.

The administration may be oral administration or parenteral administration. At this time, the administration may be applied to the subject's area or directly injected. The parenteral administration may be injection administration.

When administered by injection, the administration site may be subcutaneous, intramuscular, intradermal, intravenous, transdermal, etc.

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

In an optional example, the second nucleic acid comprising the cosmetic composition of the present application may be administered in the following doses.

For example, when the subject is a 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㎍/㎖, 28㎍/㎖, 29㎍/㎖, 30㎍/㎖, 35㎍/㎖, 40㎍/㎖, 45㎍/㎖, 50㎍/㎖, 55㎍/㎖, 60㎍/㎖, 65㎍/㎖, 70㎍/㎖, 75㎍/㎖, 80㎍/㎖, 85㎍/㎖, 90㎍/㎖, 95㎍/㎖, 100㎍/㎖, 120㎍/㎖, 140㎍/㎖, Administered at a dose within two numerical ranges selected from 160㎍/㎖, 180㎍/㎖, 200㎍/㎖, 220㎍/㎖, 240㎍/㎖, 260㎍/㎖, 280㎍/㎖, and 300㎍/㎖. It can be. At this time, the non-human refers to all mammals excluding humans.

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

For another example, if the subject is a human, the second nucleic acid may be 20 μg/ml, 21 μg/ml, 22 μg/ml, 23 μg/ml, 24 μg/ml, 25 μg/ml, 26 μg/ml, 27㎍/㎖, 28㎍/㎖, 29㎍/㎖, 30㎍/㎖, 35㎍/㎖, 40㎍/㎖, 45㎍/㎖, 50㎍/㎖, 55㎍/㎖, 60㎍/㎖, 65㎍/㎖, 70㎍/㎖, 75㎍/㎖, 80㎍/㎖, 85㎍/㎖, 90㎍/㎖, 95㎍/㎖, 100㎍/㎖, 120㎍/㎖, 140㎍/㎖, Administered at a dose within two numerical ranges selected from 160㎍/㎖, 180㎍/㎖, 200㎍/㎖, 220㎍/㎖, 240㎍/㎖, 260㎍/㎖, 280㎍/㎖, and 300㎍/㎖. It can be.

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

20㎍, 21㎍, 22㎍, 23㎍, 24㎍, 25㎍, 26㎍, 27㎍, 28㎍, 29㎍, 30㎍, 35㎍, 40㎍, 45㎍, 50㎍ per 1mL of the cosmetic composition. Μg, 55 µg, 60 µg, 65 µg, 70 µg, 75 µg, 85 µg, 90 µg, 95 µg, 100 µg, 120 µg, 140 µg, 160 µg, 180 µg, 200 µg, 220 µg, The second nucleic acid may be included at a dose within two numerical ranges selected from 240 μg, 260 μg, 280 μg, and 300 μg.

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

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

For example, the administration volume may be 0.1 mL to 10 mL at a time, but is not limited thereto.

Additionally, the number of administrations may be from 1 to 30 times per day, but is not limited thereto. At this time, it may be administered at regular intervals.

The administration interval may be 1 to 30 days, but is not limited thereto. At this time, administration may be continuous administration or discontinuous administration.

[Form for implementing the application]

Hereinafter, the present application will be described in more detail through examples.

These examples are only for illustrating the present application in more detail, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these examples.

materials and devices

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

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

Human dermal fibroblast (HDF) cells: ATCC- PCS-201-012??

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

Experiment method

● Sample DNA preparation

의 pH 7.0~8.2 범위에서 용해 완충액(lysis buffer)으로 세포 분해 후 초음파를 이용하여 순수한 gDNA(genomic DNA)를 수득하였다.Salmon sperm from which foreign substances have been removed are stored at room temperature (20-25

After cell lysis with lysis buffer in the pH range of 7.0 to 8.2, pure gDNA (genomic DNA) was obtained using ultrasound.

● Electrophoresis

Measurements were made using an agarose gel electrophoresis model WSE1710 from ATTO, Japan, and DNA markers were confirmed using DNA Molecular Weight Marker DNA concentration was measured at 2ug per well on a 2% agarose gel. 10ul of Safe Shine Green (10000X) per 100ml TAE buffer was used in the agarose gel.

● Cell proliferation – using EdU

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

Ki-67 is a protein expressed in the nucleus of cells in all cell cycles except the G1 phase, and is a marker that confirms the proliferation state of cells.

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 through a click reaction.

After treating HDF cells with 100 μg/mL each of nucleic acid-A, nucleic acid-B, and nucleic acid-C, they were exposed to EdU (10 μM), and the procedure was performed according to the kit manual.

The nucleic acid-A, nucleic acid-B and nucleic acid-C refer to:

Nucleic acid-A: Nucleic acid with a size ranging from 400bp to 2600bp (lane 2 in Figure 2),

Nucleic acid-B: Nucleic acid with a size ranging from 150bp to 350bp (lane 3 in Figure 2),

Nucleic acid-C: Nucleic acid with a size ranging from 50bp to 150bp (lane 4 in Figure 2).

Then, for immunofluorescence staining, the cells were fixed with 4% paraformaldehyde (PFA) and cultured for 24 hours at 5% CO ₂ and 37°C before permeabilizing with 0.5% Triton. The cells were then washed with 3% BSA and treated with the reaction cocktail for 30 minutes. After washing with PBS, the cells were blocked with blocking solution (1% BSA in PBS) for 30 minutes, and then incubated with the primary antibody, Ki67 (rabbit anti-Ki67; ab15580, Abcam, 1:100) at 4°C overnight. did. Then, secondary antibody was incubated and stained using DAPI (4,6-diamidino-2-phenylindole) for nuclear staining.

Immunofluorescence staining results were observed using a fluorescence microscope (IX71 and DP72, Olympus, Tokyo, Japan). Additionally, the total number of cells was counted and the percentage of Edu and Ki67 positive cells was calculated.

● RT-PCR analysis

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

In addition, the anti-inflammatory properties of PDRN of small molecule size (50bp to 150bp) were analyzed by RT-PCR. During anti-inflammatory analysis, mRNA expression levels of EGF, VEGF, tumor necrosis factor-α (TNF-α), interleukin 1-beta (IL-1β), and interleukin-6 (IL-6) were confirmed.

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

Cultures were grown at 37°C and 5% CO ₂ conditions, and the culture medium was replaced every 2 days. HGF cells were seeded at 4Х 10 ⁵ cells/Ml in a 6-well plate and cultured overnight.

Afterwards, the cell culture medium was replaced with medium containing 500 ng/mL LPS (lipopolysaccharide).

After culturing with LPS for 4 hours, the cells were washed, treated with 100 μg/mL each of nucleic acid-A, nucleic acid-B, and nucleic acid-C, and cultured 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).

QuantiTect SYBR Green PCR kit (Qiagen, USA) was used to analyze gene-specific mRNA expression. Primers used in the analysis are listed in Table 1.

The result analysis of EGF and VEGF was calculated using the Livak 2 ^-△△CT method, and β-actin was used as a control.

Gene Forward primer Reverse primer β-actin 5'-AGAGCTACGAGCTGCCTGAC-3' (SEQ ID NO: 1) 5'-AGCACTGTGTTGGCGTACAG-3' (SEQ ID NO: 2) IL-6 5'-TTCGGTCCAGTTGCCTTCTC-3' (SEQ ID NO: 3) 5'-CAGCTCTGGCTTGTTCCTCA-3' (SEQ ID NO: 4) IL-1β 5'-GATCACTGAACTGCACGC-3' (SEQ ID NO: 5) 5'-CATCAGCACCTCCAAGC-3' (SEQ ID NO: 6) EGF 5'-AGAGGGAGAGGATGCCACAT-3' (SEQ ID NO: 7) 5'-GGTTGCATTGACCCATCTGC-3' (SEQ ID NO: 8) VEGF 5'-AGGCCAGCACATAGGAGAGA-3' (SEQ ID NO: 9) 5'-ACGCGAGTCTGTGTTTTTGC-3' (SEQ ID NO: 10) TNF-α 5'-CAGAGGGCCTGTACCTCATC-3' (SEQ ID NO: 11) 5'-GGAAGACCCCTCCCAGATAG-3' (SEQ ID NO: 12)

● Nuclear condensation analysis

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

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

● Flow cytometry

To analyze apoptosis of PDRN of small molecular size (50bp to 150bp), sub-G1 hypodiploid cells were analyzed.

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

● Reactive oxygen species (ROS scavenging) analysis

To evaluate the oxygen scavenging ability of PDRN of small molecular size (50bp to 150bp), the following experiment was performed.

Briefly, HaCaT cells were seeded at 4x10 ⁵ cells/well in a 6-well plate and cultured for 16 hours. Afterwards, the cells were treated with PDRN at three concentrations (25 μg/ml, 50 μg/ml, and 100 μg/ml) and exposed to UV-B (30 mJ/cm2). Exposing cells to UV-B radiation induces intracellular oxidative stress.

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

● Cell proliferation - MTS, LIVE/DEAD, DAPI/Phalloidin

To confirm cell proliferation of PDRN of small molecular size (50bp to 150bp), the following experiment was performed.

Cell proliferation was confirmed through MTS (methoxyphenyl tetrazolium salt) assay and staining of live/dead cells.

Human dermal fibroblast (HDF) cells were cultured in low serum (PCS-201-041, Manassas, USA) fibroblast growth basic medium (PCS-201-030, Manassas, USA).”

Briefly, HDF cells were seeded in a cell culture plate at 5 x 10 ⁴ cells/well and cultured for 24 hours at 37°C and 5% CO ₂ conditions. Afterwards, the culture medium was removed from each well plate and the cells were starved in medium without FBS for an additional 18 h.

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

MTS analysis used Promega's CellTiter 96 Aqueous One Solution, and LIVE/DEAD evaluation was also performed through staining. Additionally, the morphology of PDRN-treated HDF cells was confirmed using DAPI/Phalloidin-TRITC staining.

● Cell migration

To confirm cell migration of PDRN of small molecular size (50bp to 150bp), the following experiment was performed using fibroblasts.

Briefly, a total of 2Х10 ⁵ cells/mL of cells 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.

HDF cells spread and adhered to form a monolayer - the culture insert was removed and a gap (500 μm) between cell layers was formed.

Afterwards, 1 mL of FBS-free medium was filled, and PDRN at three concentrations (25 ㎍/㎖, 50 ㎍/㎖, 100 ㎍/㎖) was treated with serum-free culture medium. Culture medium without FBS and culture medium containing 10% FBS were used as negative and positive controls, respectively.

This was done by taking images of the cell free gap at 0 and 24 h using an inverted light microscope (Leica® DMi8, Germany) to estimate the time taken to fill the cell free gap by cell migration. The experiment was performed three times, and the ratio of initial scratches (Ao) and healed scratches (At) was calculated using ImageJ software (ImageJ, ver. 1.6, USA).

The percentage of open area (A) was quantified as percentage of open area A (%) = [Ao - At]/AoХ100.

The first nucleic acid described below is described as gDNA (genomic DNA), but as described in the detailed description of the invention, the first nucleic acid is only one of the materials used to prepare a nucleic acid fragment to obtain a final product. It is not limited to the following experimental examples.

Experimental Example 1: Preparation of first nucleic acid

10 g of salmon sperm and 20 ml of 0.9% sodium chloride solution were added to a 500 ml flask and mixed for 5 minutes at room temperature. Afterwards, 250ml of lysis buffer (purified water: SDS:EDTA:Tris prepared in a ratio of 530:10.6:3.2) was added. This was stirred at room temperature for 10 minutes and then decomposed by ultrasonic waves for 10 to 30 minutes using an ultrasonic grinder (Q700, QSONICA USA).

Afterwards, 250 ml of 6M sodium chloride aqueous solution was added and mixed, placed in a centrifuge, and centrifuged at 4,000 to 10,000 rpm for 30 minutes to 1 hour to separate the supernatant. The supernatant was filtered using an 8um filter, 500ml of ethanol (>99%) cooled to 0~4℃ was added, stirred slowly, and then centrifuged (4000rpm, 30 minutes). Afterwards, after vacuum drying at 30~50℃ for 9 hours, 4.3~4.7g of gDNA (genomic DNA), the white primary nucleic acid, was obtained.

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

Experimental Example 2: Preparation of second nucleic acid

40 g of the first nucleic acid in powder form, prepared by repeating Experiment 1, was added to 800 ml of purified water and stirred at room temperature for 6 hours to prepare a first solution containing the first nucleic acid.

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 1 cycle.

To produce the second nucleic acid, experiments were conducted to optimize the conditions of the high pressure disperser in Examples 2-1 and 2-2. Afterwards, 165 g of sodium chloride was added to the high-pressure dispersion solution, stirred for 20 minutes, and 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.

Experimental Example 2-1: Optimization of pressure conditions of high pressure disperser for production of secondary nucleic acid

To determine the pressure conditions of the high-pressure disperser to obtain DNA fragments of small size (50bp to 150bp), fragmentation of the first nucleic acid contained in the first solution was performed at 10,000 psi and 1 cycle to 10 cycles. Looking at Figure 1, despite high-pressure dispersion of the first nucleic acid at 10,000 psi for 10 cycles, the nucleic acid was fragmented to a size of 200bp to 350bp, as seen in lane 4 of Figure 1. In particular, the STD (Mastelli, Placentex) in Figure 1 is a commercially available nucleic acid with a size of 50bp to 200bp, and 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 STD size.

In other words, it was confirmed that a pressure condition of at least 10,000 psi or more is necessary to obtain nucleic acid fragments of 50bp to 150bp, the small size desired in this technology. In other words, it was found that small-sized nucleic acid fragments could not be obtained at temperatures below 10,000 psi even if dispersion was repeated several times.

Experimental Example 2-2: Optimization of cycle conditions of high pressure disperser for production of secondary nucleic acid

Through the experiment of Example 2-1, it was confirmed that conditions of 10,000 psi or more were necessary to obtain small-sized nucleic acid fragments, and it was attempted to confirm that small-sized DNA fragments could be obtained under the high pressure disperser pressure conditions of 20,000 to 30,000 psi.

First, in order to fragment the first nucleic acid of Experimental Example 1, the pressure of the high pressure disperser was set to 25000 psi, and samples were collected for each cycle and electrophoresis was performed (Figure 2).

Looking at Figure 2, it can be seen that if only 1 cycle is applied, the size of the second nucleic acid is distributed in the range of 400bp to 2600bp, and if 5 cycles are applied, the size of the second nucleic acid is distributed in the range of 150bp to 350bp (Figure 2 lanes 2 and 3). It can be seen that at least 15 cycles must be applied to obtain a DNA fragment of 50bp to 150bp in size, which is the second nucleic acid (lane 4 in Figure 2). In other words, it can be confirmed that in order to obtain a second nucleic acid having a size of 50bp to 150bp, which is similar to the STD (Mastelli, Placentex) of Figure 1, a pressure of 25000 psi must be applied for at least 15 cycles.

Additionally, the pressure of the high pressure disperser was set to 30000 psi, and the same experiment was repeated to confirm the results (Figure 3). Specifically, the pressure of the high pressure disperser was set to 30,000 psi, samples were collected for each cycle (1 cycle, 5 cycle, and 15 cycle), electrophoresis was performed, and a graph quantifying the results is shown in FIG. 3. That is, Figure 3 shows the relationship between the average size of DNA fragments and the number of cycles under 30 kpsi.

The size of the DNA fragment for each cycle in Figure 3 had the following distribution range: 800bp to 2800bp for 1 cycle, 150bp to 800bp for 5 cycles, and 50bp to 150bp for 15 cycles.

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

Combining the results of Figures 1, 2, and 3, it was found that fragmentation of nucleic acids with a size of 3,000 bp or more was difficult to obtain small sized (50 to 150 bp) DNA fragments under 10,000 psi pressure conditions. Furthermore, it was confirmed that small-sized DNA fragments (50bp to 150bp) could be obtained under pressure conditions of 20,000 psi to 30,000 psi. In order to obtain such small-sized DNA fragments, high-pressure dispersion is performed for at least 15 cycles under specific pressure conditions (20,000 psi to 30,000 psi) to obtain nucleic acids with a size of 50 bp to 150 bp, similar to STD (Mastelli, Placentex), a commercially available nucleic acid. I knew I could do it.

Meanwhile, it was found that the size of the second nucleic acid obtained was uniform (50bp to 150bp) even if the cycle was repeated a certain number of times at a pressure above a certain size. In other words, it was confirmed that the size of the DNA fragment no longer decreased even when pressure above a certain level was repeatedly applied.

This suggests that when nucleic acids are fragmented at high pressure, the nucleic acids are fragmented and become smaller, reducing the area in which they collide with the disk, so the size of the nucleic acids does not decrease even if the pressure and cycle exceed a certain level.

Experimental Example 3: Viscosity measurement

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

In a beaker, prepare 100 ml of the first nucleic acid (3000bp or more in size) of Example 1 and the second nucleic acid of 50bp to 150bp in size (i.e., Lane 4 in Figure 2) obtained in Example 2 at a concentration of 2% in purified water. After stirring at room temperature for 30 minutes, the mixture was allowed to stand for about 2 minutes using a CAS digital viscometer (CL-1) from ATTO, Japan, and the measurement was repeated three times to calculate the average value.

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).

first nucleic acid Second nucleic acid (Lane4) 1 time 5486.11cp 19.21cp Episode 2 5491.87cp 19.19cp 3rd time 5497.71cp 19.15cp average 5491.90cp 19.18cp

This indicates that the small-sized second nucleic acid of 50bp-150bp obtained by the method of the present invention has a sufficiently low viscosity to be industrially useful.

Based on Experimental Examples 1 to 3, it was confirmed that nucleic acids were fragmented by applying high pressure, suggesting that it is important to optimize specific pressures and cycles to obtain nucleic acid fragments of specific sizes, which are features of the present invention.

Experimental Example 4: Confirmation of effect by size of fragmented first nucleic acid

In Experimental Examples 1 to 3, the following experiment was conducted to determine whether the effects of nucleic acid fragments of various sizes obtained by subjecting the first nucleic acid to high pressure were different.

The cell proliferation effect and the mRNA levels of EGF and VEGF were compared for each of the three sizes of nucleic acids obtained in Figure 2.

The three sizes of nucleic acids used here are as follows:

Lane 2 in Figure 2: Nucleic acid with a size ranging from 400bp to 2600bp (hereinafter referred to as nucleic acid-A),

Lane 3 in Figure 2: Nucleic acid with a size ranging from 150bp to 350bp (hereinafter referred to as nucleic acid-B),

Lane 4 of Figure 2: Nucleic acid having a size ranging from 50bp to 150bp (hereinafter referred to as nucleic acid-C).

Experimental Example 4-1: Confirmation of cell proliferation

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 subjecting the first nucleic acid to high pressure in Experimental Examples 1 to 3, are different, EdU Cell Proliferation Kit was used with HDF cells. Cell proliferation rates were compared using . In addition, the expression of Ki67, a cell proliferation marker, was compared with HDF cells (Figures 4 and 5).

In Figure 4, (-Ve) control is the group treated with serum free, and (+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. .

In FIG. 5, [-]control refers to the (-Ve) control in FIG. 4, and [+]control refers to the (+Ve) control.

EdU results

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 larger number of EdU positive cells than the (-Ve) control. Looking at Figure 5, which quantifies the results of Figure 4, the percentage of EdU positive cells was 28.8 ± 2.2% in the [-] control group and 84.8 ± 4.2% in 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, the group treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a higher proportion of EdU-positive cells than the [-]control group, and 50bp to 150bp of nucleic acid-A, nucleic acid-B, and nucleic acid-C. It was confirmed that nucleic acid-C, a nucleic acid with a range of sizes, had the highest proportion of EdU positive cells. The ratio of EdU positive cells in nucleic acid-C was found to be closest to the results of the positive control group, [+]control group.

Ki67 results

Looking at the Ki67 expression results in Figure 4, it can be seen that the group treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a larger number of Ki67-expressing cells compared to the (-Ve) control. Looking at Figure 5, which quantifies the results of Figure 4, the percentage of Ki67 positive cells was 29.9 ± 3.1% in the [-] control group and 81.78 ± 2.89% in 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, the group treated with nucleic acid-A, nucleic acid-B, and nucleic acid-C had a higher proportion of Ki67 positive cells than the [-]control group, and the proportion of Ki67 positive cells among nucleic acid-A, nucleic acid-B, and nucleic acid-C was 50bp to 150bp. It was confirmed that nucleic acid-C, a nucleic acid with a range of sizes, had the highest percentage of Ki67 positive cells. The ratio of Ki67 positive cells in nucleic acid-C was found to be closest to the results of the positive control group, [+]control group.

Through the results of Experimental Example 4-1, it was confirmed that the cell proliferation effect was different depending on the size of the first fragmented nucleic acid, and specifically, the smaller the nucleic acid fragment, the higher the cell proliferation effect. In particular, it was confirmed that nucleic acid-C, a PDRN with a small molecule size of 50bp to 150bp, had the best cell proliferation effect.

Experimental Example 4-2: Confirmation of mRNA levels of EGF (Epidermal Growth Factor) and VEGF (vascular endothelial growth factor)

In Experimental Examples 1 to 3, HGF cells were used to determine whether the growth factor expression levels of nucleic acid fragments of various sizes, nucleic acid-A, nucleic acid-B, and nucleic acid-C, obtained by subjecting the first nucleic acid to high pressure, were different. The mRNA levels of EGF and VEGF were compared through PCR (Figure 6).

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.

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

Looking at the results of EGF mRNA expression in Figure 6, it can be seen that when treated with LPS, the mRNA expression of EGF is reduced by almost half compared to the control. In contrast, the group treated with LPS and nucleic acid-A; Group treated with LPS and nucleic acid-B; In the group treated with LPS and nucleic acid-C, it was confirmed that the mRNA expression of EGF increased compared to the LPS-treated group. Among them, it was confirmed that the group treated with LPS and nucleic acid-C had EGF mRNA expression at almost the same level as the control group. Furthermore, the group treated with LPS and nucleic acid-A; It can be seen that the mRNA expression of EGF in the group treated with LPS and nucleic acid-C was significantly higher than that in the group treated with LPS and nucleic acid-B.

Looking at the results of VEGF mRNA expression in Figure 6, it can be seen that when treated with LPS, the mRNA expression of VEGF is reduced by almost half compared to the control. In contrast, the group treated with LPS and nucleic acid-A; Group treated with LPS and nucleic acid-B; In the group treated with LPS and nucleic acid-C, it was confirmed that the mRNA expression of VEGF increased compared to the LPS-treated group. Among them, the group treated with LPS and nucleic acid-C was confirmed to have VEGF mRNA expression at almost the same level as the control group. Furthermore, the group treated with LPS and nucleic acid-A; It can be seen that the mRNA expression of EGF in the group treated with LPS and nucleic acid-C was significantly higher than that in the group treated with LPS and nucleic acid-B.

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

To summarize Experimental Example 4, the 50bp ~ 50bp produced by the method of the present application has a high cell proliferation ability, promotes skin regeneration, elasticity, cell production, and has a high mRNA expression level of EGF and VEGF involved in wound healing and regeneration. This suggests that PDRN, which has a small molecule size of 150bp, can be used for industrial purposes such as skin regeneration, elasticity, promoting cell production, wound healing, and regeneration.

Experimental Example 5: Confirmation of the effect of PDRN of small molecule size (50bp to 150bp) by concentration

Through Experimental Example 4, the effect of the fragmented first nucleic acid by size was confirmed, and as a result, the cell proliferation ability and increased expression of growth factors of PDRN, a small molecule-sized second nucleic acid of 50bp to 150bp, were confirmed.

Therefore, we compared the effects depending on the concentration of PDRN with a small molecule size of 50bp to 150bp.

At this time, a total of 3 concentrations (25 ㎍/㎖, 50 ㎍/㎖, 100 ㎍/㎖) of PDRN of small molecule size (50bp~150bp) used were compared.

Experimental Example 5-1: Confirmation of anti-apoptotic activity

The apoptosis-inhibiting properties of PDRN of small molecular size (50bp to 150bp) were confirmed by nuclear condensation analysis and sub-G1 hypodiploid cell analysis.

Nuclear condensation analysis

Figure 7 shows 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 the number of Apoptotic bodies indicated by arrows increases in UV-B treated HaCaT cells compared to the control group, and that the Apoptotic bodies decrease upon NAC treatment. In addition, it can be seen that the group treated with low-molecular-size PDRN showed a decrease in Apoptotic bodies compared to the group treated with UV-B. Specifically, compared to the group treated with 25㎍/㎖ of small-molecule PDRN and the group treated with 50㎍/㎖ of small-molecule PDRN, the number of apoptotic bodies was increased in the group treated with 100㎍/㎖ of small-molecule PDRN. It can be seen that it decreases. In other words, it can be seen that the higher the concentration of small-molecular-sized PDRN, the smaller the number of Apoptotic bodies.

This can be seen more clearly by looking at the graph of Figure 8, which quantifies the results of Figure 7. In HaCaT cells treated with UV-B, the apoptotic cell index increased by about 9 times or more compared to the negative control, and in NAC treatment, apoptotic cells The index decreased significantly. In addition, treatment with PDRN of small molecular size at different concentrations decreased the index of apoptotic cells compared to UV-B treatment. Specifically, compared to UV-B treatment, the group treated with 50㎍/㎖ small molecule PDRN decreased by more than 2 times, and the group treated with 100㎍/㎖ small molecule PDRN decreased by more than 3 times. .

Sub-G1 hypodiploid cell analysis

Figure 9 shows the results of sub-G1 hypodiploid cell analysis using flow cytometry. Looking at Figure 9, it can be seen that the percentage of sub-G1 cell content (i.e., the content of apoptotic cells) was high in UV-B treated HaCaT cells and decreased in the NAC treated group. In addition, it can be seen that the percentage of sub-G1 cell content decreased in the groups treated with low-molecular-size PDRN compared to the group treated with UV-B. Specifically, the percentage of sub-G1 cell content in the group treated with 50 μg/ml small molecule PDRN was lower than that of the group treated with 25 μg/ml small molecule PDRN, and the percentage of sub-G1 cell content in the group treated with 50 μg/ml small molecule PDRN was lower. It can be seen that the percentage of sub-G1 cell content in the group treated with 100㎍/ml was lower than that of the other group.

This can be seen more clearly by looking at the graph of Figure 10, which quantifies the results of Figure 9. In UV-B treated HaCaT cells, the sub-G1 cell content increased by about 40 times or more compared to the negative control group, and in the NAC treated group, the sub-G1 cell content increased by more than 40 times. -G1 cell content decreased. In addition, treatment with PDRN of small molecular size at different concentrations decreased the sub-G1 cell content compared to UV-B treatment. Specifically, compared to UV-B treatment, the group treated with 50㎍/㎖ small molecule PDRN decreased by more than 2 times, and the group treated with 100㎍/㎖ small molecule PDRN decreased by more than 3 times. .

Experimental Example 5-2: Confirmation of ROS scavenging

The intracellular ROS scavenging ability of PDRN of small molecular size (50bp to 150bp) was analyzed.

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 in the group treated with UV-B, which induced intracellular oxidative stress, the number of cells expressing the DCFH-DA probe (i.e., cells in which ROS was not removed) increased compared to the control group, and decreased upon NAC treatment. You can. On the other hand, it can be seen that the cells expressing the DCFH-DA probe decreased in the group treated with low-molecular-size PDRN compared to the group treated with UV-B.

This can be seen more clearly by looking at the graph of Figure 12, which quantifies the results of Figure 11. In UV-B treated HaCaT cells, the intensity of cells expressing the DCFH-DA probe increased by about 3 times or more compared to the control group, and NAC treated It decreased in the military. In addition, treatment with PDRN of small molecular size at different concentrations decreased the intensity of cells expressing the DCFH-DA probe compared to UV-B treatment. In particular, the group treated with 100 μg/ml of PDRN of small molecular size showed significantly lower fluorescence intensity than that treated with UV-B. In other words, a concentration of 100 μg/ml of PDRN of small molecular size means excellent ROS scavenging activity.

Through the results of Experimental Example 5-2, it was confirmed that the intracellular ROS scavenging ability increased as the concentration of PDRN of small molecule size (50bp to 150bp) increased, suggesting that it effectively alleviates intracellular oxidative stress.

Experimental Example 5-3: Confirmation of cell proliferation

Cell proliferation of PDRN of small molecular size (50bp to 150bp) was confirmed by MTS analysis, LIVE/DEAD analysis, and DAPI/Phalloidin analysis.

MTS Analysis

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

LIVE/DEAD analysis

Figure 14 shows the results of LIVE/DEAD analysis observed by staining the cells analyzed in Figure 13. Looking at Figure 14, it can be seen that the number of living HDF cells (green fluorescence) increased in all groups treated with low-molecular-size 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 small molecule PDRN compared to the group treated with 25 μg/ml small molecule PDRN. In addition, the number of living HDF cells (green fluorescence) increased in the group treated with 100 μg/ml small molecule PDRN compared to the group treated with 50 μg/ml small molecule PDRN. This result, like the MTS result, once again confirms that the cell survival rate increases as the concentration of small molecule-sized PDRN increases. Meanwhile, it can be confirmed that there were no dead HDF cells in any of the groups treated with low-molecular-sized PDRN.

DAPI/Phalloidin analysis

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

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-size PDRN compared to the (-VE) control. In particular, the number of Phalloidin-expressing cells increased in the group treated with 50 μg/ml small molecule PDRN compared to the group treated with 25 μg/ml small molecule PDRN. In addition, the number of Phalloidin-expressing cells increased in the group treated with 100 μg/ml small molecule PDRN compared to the group treated with 50 μg/ml small molecule PDRN. These results are similar to the MTS results and LIVE/DEAD results, confirming that the cell proliferation rate increases as the concentration of small molecule-sized PDRN increases.

Experimental Example 5-4: Confirmation of cell migration activity

The cell migration activity of PDRN of small molecular size (50bp to 150bp) was analyzed (Figures 16 and 17).

Figure 16 shows the results of microscopic observation of cell migration. Looking at Figure 16, it can be seen that the open area was reduced in all groups treated with PDRN of a small molecule size compared to the (-VE) control in the results at 24 hours compared to 0 hours. In particular, compared to the group treated with 25 ㎍/㎖ small molecule PDRN, the open area was reduced in the group treated with 50 ㎍/㎖ small molecule PDRN, and the group treated with 50 ㎍/㎖ small molecule PDRN. Compared to , the open area was further reduced in the group treated with 100㎍/㎖ of small molecule PDRN.

This can be seen more clearly by looking at the graph of FIG. 17, which quantifies the results of FIG. 16. Compared to the (-VE) control, all groups treated with PDRN of a small molecule size had a higher open area (%) in the results at 24 hours compared to 0 hours. It can be seen that has been reduced. Specifically, the group treated with 25 ㎍/㎖ of small molecule PDRN decreased by about 30-40% at 24 hours compared to 0 hours. And in the group treated with 50㎍/㎖ of small molecule PDRN, there was a decrease of about 40-50% at 24 hours compared to 0 hours. In addition, the group treated with 100㎍/㎖ of small molecule PDRN decreased by about 50-60% at 24 hours compared to 0 hours. This result is similar to the previous results, confirming that the cell migration activity of small molecule-sized PDRN increases in a concentration-dependent manner.

Experimental Example 5-5: Confirmation of anti-inflammatory activity

The anti-inflammatory activity of PDRN of small molecule size (50bp to 150bp) can be confirmed through RT-PCR using EGF, VEGF, tumor necrosis factor-α (TNF-α), interleukin 1-beta (IL-1β), and IL-6. The mRNA levels of (Interleukin-6) were compared (Figure 6).

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

TNF-α mediates inflammatory responses, regulates cellular immune responses, and plays a role in eliminating intracellular infections. It is mainly secreted by stimulated macrophages, but is also secreted by fibroblasts, T cells, B cells, endothelial cells, and epithelial cells, playing a major role in acute inflammatory responses.

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

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

Looking at the mRNA expression results of IL-6 in Figure 18, it can be seen that treatment with LPS significantly increases the mRNA expression of IL-6 compared to the control. However, compared to the group treated with LPS, it was confirmed that the mRNA expression of IL-6 was decreased in all groups treated with LPS and PDRN of small molecule size (50bp to 150bp) at three concentrations. Specifically, the group treated with LPS and small molecule-sized PDRN at 50㎍/㎖ decreased by more than 50% compared to the LPS-treated group, and the group treated with LPS and small molecule-sized PDRN at 100㎍/㎖ decreased by more than 50%. Compared to the LPS-treated group, it decreased by more than 60%. In particular, the mRNA expression of IL-6 was decreased in a concentration-dependent manner in the groups treated with LPS and three concentrations of PDRN of small molecule size (50bp to 150bp).

Looking at the mRNA expression results of IL-1β in Figure 18, it can be seen that treatment with LPS significantly increases the mRNA expression of IL-1β compared to the control. However, compared to the group treated with LPS, it can be seen that the mRNA expression of IL-1β was decreased in all groups treated with LPS and PDRN of small molecule size (50bp to 150bp) at three concentrations. Specifically, the group treated with LPS and small molecule-sized PDRN at 50㎍/㎖ decreased by more than about 30% compared to the LPS-treated group, and the group treated with LPS and small molecule-sized PDRN at 100㎍/㎖ decreased by more than 30%. Compared to the LPS-treated group, it decreased by more than 70%. In particular, the mRNA expression of IL-1β was reduced in a concentration-dependent manner in the groups treated with LPS and three concentrations of PDRN of small molecule size (50bp to 150bp).

Looking at the mRNA expression results of TNF-α in Figure 18, it can be seen that treatment with LPS significantly increases the mRNA expression of TNF-α compared to the control. However, compared to the group treated with LPS, it can be seen that the mRNA expression of TNF-α decreased in all groups treated with LPS and PDRN of small molecule size (50bp to 150bp) at three concentrations. Specifically, the group treated with LPS and small molecule-sized PDRN at 50㎍/㎖ decreased by more than 40% compared to the LPS-treated group, and the group treated with LPS and small molecule-sized PDRN at 100㎍/㎖ decreased by more than 40%. Compared to the LPS-treated group, it decreased by more than 50%. In particular, the mRNA expression of TNF-α was decreased in a concentration-dependent manner in the groups treated with LPS and three concentrations of PDRN of small molecule size (50bp to 150bp).

Figure 19 shows the results of confirming the mRNA levels of growth factors EGF and VEGF. Looking at the EGF mRNA expression results in Figure 19, it can be seen that when LPS is treated, the EGF mRNA expression is greatly reduced compared to the control. However, compared to the group treated with LPS, it can be seen that the mRNA expression of EGF increased in all groups treated with LPS and PDRN of small molecule size (50bp to 150bp) at three concentrations. Specifically, the group treated with LPS and small molecule-sized PDRN at 50㎍/㎖ increased by more than twice that of the LPS-treated group, and the group treated with LPS and small molecule-sized PDRN at 100㎍/㎖ increased. Compared to the LPS-treated group, it increased by more than three times. In particular, the groups treated with LPS and three concentrations of PDRN of small molecule size (50bp to 150bp) showed an increase in EGF mRNA expression in a concentration-dependent manner.

Looking at the mRNA expression results of VEGF in Figure 19, it can be seen that when treated with LPS, the mRNA expression of VEGF is greatly reduced compared to the control. However, compared to the group treated with LPS, it can be seen that the mRNA expression of VEGF increased in all groups treated with LPS and PDRN of small molecule size (50bp to 150bp) at three concentrations. Specifically, the group treated with LPS and small molecule-sized PDRN at 50㎍/㎖ increased by more than twice that of the LPS-treated group, and the group treated with LPS and small molecule-sized PDRN at 100㎍/㎖ increased. Compared to the LPS-treated group, it increased by more than three times. In particular, in groups treated with LPS and three concentrations of PDRN of small molecule size (50bp to 150bp), the mRNA expression of VEGF increased in a concentration-dependent manner.

Through the above-mentioned experimental examples, the nucleic acid fragment production method of the present application is

i) providing a certain pressure to the nucleic acid; ii) The technical significance is that the specific pressure is provided to the nucleic acid to obtain low-molecular-weight nucleic acid fragments.

Furthermore, PDRN of small molecule size (50bp to 150bp) prepared by the nucleic acid fragment production method of the present application has low viscosity, high cell proliferation rate, and has effects such as growth factor activation.

In addition, PDRN of small molecular size (50bp to 150bp) has concentration-dependent effects such as activation of anti-apoptosis, oxygen scavenging ability, cell proliferation ability, activation of cell migration, and activation of anti-inflammation, so it is suitable for industrial application. I think it will be very easy.

Claims (13)

Methods of fragmenting DNA, including:
i) Prepare first DNA having a size of 500bp to 5000bp; and
ii) applying pressure to the first DNA at a level of 20,000 psi to 30,000 psi for 15 to 30 cycles to obtain a product containing a fragmented second DNA of the first DNA;
The obtained second DNA has a size of 50 to 150 nt and is characterized in that it is contained in 70% to 100% of the product.
According to clause 1,
When providing the pressure, the method is characterized in that it is carried out at a temperature of 5 ℃ to 25 ℃.
According to clause 1,
In the process ii), the pressure is provided in 15 cycles.
According to clause 3,
When providing pressure, one cycle is characterized in that it takes 0.5 to 10 seconds.
According to clause 1,
The pressure is characterized in that it is performed by one or more devices among a high pressure homogenizer, high pressure disperser, ultra-high pressure homogenizer, and ultra-high pressure disperser. How to do it.
According to clause 1,
A method wherein the first DNA is derived from fish.
According to clause 6,
A method wherein the fish is salmon or trout.
According to clause 1,
A method wherein the first DNA is single-stranded or double-stranded.
According to clause 1,
iii) a method characterized in that it further comprises the step of separating and concentrating the second DNA by precipitating the obtained product.
According to clause 9,
The precipitation is a method characterized by using at least one selected from sodium chloride (NaCl), potassium chloride (KCl), and sodium acetate (CH3COONa).
According to clause 10,
A method characterized in that the sodium chloride is used in an amount of 15% to 30% compared to the amount of water.
According to clause 10,
The precipitation is characterized in that using one or more solvents selected from ethanol, methanol, butanol propanol, pentanol, and isopropyl alcohol.
According to clause 1,
The second DNA has anti-apoptosis activity, reactive oxygen species (ROS) scavenging, cell proliferation activation, cell migration activation, and anti-inflammatory ( A method characterized by having one or more effects selected from anti-inflammatory activation.