KR102203703B1 - Composition for inhibiting cell aging and method using thereof - Google Patents

Abstract

The present invention relates to a composition for inhibiting cell aging for controlling the release of nitrogen monoxide, and a method using the same. The present invention provides the composition for inhibiting cell aging which has no toxic residues, can release nitrogen monoxide for a long time, and has excellent biocompatibility, and thus the composition can maximize an effect of inhibiting cell aging by controlling the release of nitrogen monoxide.

Description

Composition for inhibiting cell aging and method using the same {Composition for inhibiting cell aging and method using thereof}

The present invention relates to a composition for inhibiting cell aging and a method using the same, and more particularly, to a composition for inhibiting cell aging through control of the release of nitrogen monoxide, and a method using the same.

Aging is a phenomenon in which the body functions of living organisms are deteriorated over time due to various causes, resulting in a decrease in vitality. Cell aging is the loss of the ability of cells to divide, and senescent cells secrete a senescence-associated secretory phenotype (SASP), which has been reported to contribute to the onset and progression of various diseases.

Nitric oxide (NO) is a free radical generally produced from L-arginine in vivo by three isomers of nitrogen monoxide synthase. Of these, nNOS and eNOS produce low levels of nitrogen monoxide, and they regulate cell signaling and are responsible for normal physiological functions such as vasodilation, nerve transmission, and wound healing. On the other hand, nitrogen monoxide, which is excessively produced by iNOS, which is mainly present in macrophages, can be converted into toxic substances such as peroxynitrite. It is known that it causes a strong oxidation reaction to damage cells, promotes inflammatory reactions, and is involved in chronic diseases such as cancer and arteriosclerosis.

As such, nitrogen monoxide has a different concentration of nitrogen monoxide required depending on the application site and purpose, so when applying nitrogen monoxide to a living body, it is recommended to apply nitrogen monoxide in an amount suitable for a specific therapeutic purpose within a safe concentration or release time range. very important.

Various types of nitrogen monoxide donors including diazenium dioleate, a functional group that typically releases nitrogen monoxide, have been reported to prevent cell damage by hydrogen peroxide and protect cells. In addition, it has been reported that nitrogen monoxide removes reactive oxygen species and relieves oxidative stress in cells. Through the above results, it has been reported that nitrogen monoxide has an effect of preventing cell aging, but the mechanism is still unclear. Accordingly, there is a need for a method of determining whether nitrogen monoxide has an effect of preventing cell aging due to oxidative stress through an accurate in vitro model. Recently, it has been discovered that cell aging at the cellular level is one of the main causes of individual aging, and controlling cell aging through an in vitro model can provide preventive measures and treatments for various diseases caused by individual aging and senescent cells. There will be a way.

Under this background, it is necessary to develop a material capable of stably controlling the release of nitrogen monoxide in cells and a method using the same in order to suppress cell aging.

An object of the present invention is to provide a composition for inhibiting cell aging including a nitrogen monoxide donor of a new aspect and a method using the same.

In detail, it is an object of the present invention to provide a composition for inhibiting cell aging comprising a nitrogen monoxide donor capable of releasing nitrogen monoxide for a long time without toxic residue.

In detail, it is an object of the present invention to provide a composition for inhibiting cell aging that can easily control the amount of release to provide an amount of nitrogen monoxide suitable for therapeutic purposes in cells.

In detail, an object of the present invention is to provide a method of delivering nitrogen monoxide that is excellent in biocompatibility and can stably provide nitrogen monoxide in cells. In addition, an object thereof is to provide a method for inhibiting cell aging through this.

For the above-described purposes, the present invention provides a composition for inhibiting cell aging, comprising a compound represented by the following Chemical Formulas 1 and 2 as an active ingredient.

[Formula 1]

[Formula 2]

[In Formulas 1 and 2,

Me is independently of each other Li, Na, K, Cs, or Rb.]

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient comprises a mixture in which the molar ratio of the compound represented by Formula 2 to the compound represented by Formula 1 is 1:0.1 to 1:2.0. Can be.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may be a crystal including the compound represented by Chemical Formulas 1 and 2, and the crystal is a rod-shaped crystal having an aspect ratio of 2 to 20 It can be.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient is a crystal comprising a compound represented by Chemical Formulas 1 and 2, wherein the crystal is a rod-shaped crystal having an aspect ratio of 2 to 20. It may be a conjugated spherical crystal.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the concentration of the active ingredient may be less than 100 μg/ml.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may have a total amount of nitrogen monoxide released from 1 to 5 μ mol/mg.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may have a half-life of 1 to 24 hours based on the total amount of nitrogen monoxide released.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may react with water to release nitrogen monoxide.

The composition for inhibiting cell aging according to an embodiment of the present invention may be to inhibit 　 cell death of vascular endothelial cells.

In addition, in the present invention, there is provided a pharmaceutical composition for treating wounds or regenerating skin tissue, comprising the compounds represented by Chemical Formulas 1 and 2 as an active ingredient.

In addition, in the present invention, there is provided a method of delivering nitrogen monoxide using the release of nitrogen monoxide through the reaction of water with a composition comprising the compound represented by Chemical Formulas 1 and 2 as an active ingredient. Such a method of delivering nitrogen monoxide may be a method of inhibiting cell aging by delivering nitrogen monoxide into cells.

According to the present invention, as a nitrogen monoxide provider that has no toxic residue, can release nitrogen monoxide for a long time, and is excellent in biocompatibility, cell aging containing a compound of a new aspect in which a diazenium dioleate group is bonded to carbon as an active ingredient It is possible to provide a composition for inhibition.

According to the present invention, nitrogen monoxide can be stably provided and controlled to suit the purpose. In addition, it is applied to senescent cells that have lost the ability to produce nitrogen monoxide, and by emphasizing the net function of the cells, the effect of inhibiting cell aging can be maximized. Accordingly, according to the present invention, it is possible to provide new materials and methods for improving and treating various diseases caused by individual aging and senescent cells by controlling cell aging.

1 is a graph showing the results of the effect of delaying cell aging using the composition for inhibiting cell aging according to Example 1 of the present invention,
2 is a graph showing the survival rate of human skin fibroblasts according to the concentration of the composition for inhibiting cell aging according to Example 1 of the present invention,
3 is a graph showing in real time analysis of nitrogen monoxide released through the composition for inhibiting cell aging according to Example 1 of the present invention,
4 is a micrograph of the crystals prepared in Examples 2 to 7 of the present invention,
5 is a graph showing the total amount of nitrogen monoxide released per unit area of the crystals prepared in Examples 2 to 7 of the present invention,
Figure 6 is a graph investigated for 24 hours the effect on the movement of human skin fibroblasts according to the concentration of the composition for inhibiting cell aging according to Example 1 of the present invention.

The composition for inhibiting cell aging according to the present invention and a method using the same will be described later, but unless there are other definitions in the technical and scientific terms used at this time, those of ordinary skill in the art to which this invention belongs are commonly understood. In the following description, a description of known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted.

As used herein, the term "diazeniumdiolate group" has a meaning equivalent to a nonoate group, and may be represented by the general formula RR'N-N(O)=NOR".

As used herein, the term "low level nitrogen monoxide" is not limited as long as it is applied to senescent cells to impart net function of the cell, and specifically, the amount of nitrogen monoxide released in a concentration range that does not cause cytotoxicity It could mean something.

As described above, a low level of nitrogen monoxide can impart antioxidant activity. Accordingly, by providing an appropriate level of nitrogen monoxide to senescent cells, it is possible to provide benefits for cell regeneration as well as preventing cell aging. Low levels of nitrogen monoxide are generally always produced by cells, so little has been discussed regarding aging. On the other hand, nitrogen monoxide has been reported as an entity to be removed because it plays a negative role in promoting aging or exacerbating disease in the event of inflammation or disease. However, prior to such an extreme situation, in general, in a situation in which cells are aging, since the state of the cells is not normal, a vicious cycle in which nitrogen monoxide in the normal range cannot be produced and more and more senescent cells are produced is repeated. Therefore, supplying nitrogen monoxide that does not significantly deviate from the normal range to senescent cells that have lost the production capacity of nitrogen monoxide will activate the cells, and the net function of removing reactive oxygen species will be highlighted, and eventually aging will be prevented.

Under this background, the present inventors devise a new nitrogen monoxide provider that can replace the conventionally known nitrogen monoxide providers, and develop a method for stably releasing nitrogen monoxide in cells through this, and propose the present invention. Specifically, the nitrogen monoxide donor according to the present invention includes a new compound capable of releasing nitrogen monoxide more stably by bonding a diazenium dioleate group to carbon.

The nitrogen monoxide provider according to the present invention has high solubility in water, and is easily decomposed under conditions of a living body temperature and pH to release nitrogen monoxide. In addition, it can release nitrogen monoxide for a long time and does not produce toxic residues. In addition, the nitrogen monoxide provider according to the present invention has a remarkably improved half-life compared to the conventional nitrogen monoxide provider including an N-diazenium dioleate group, and shows a sustained and long release time. Thus, it is suitable for providing the desired low level of nitrogen monoxide.

In addition, since the solubility in the physiological environment is excellent, it is possible to overcome the limitations due to the dispersibility and aggregation phenomena of the conventional nitrogen monoxide provider introducing nanoparticles. In addition, it is easily removed from the body and does not cause toxicity problems due to accumulation and residue.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition comprising a nitrogen monoxide donor comprising a C-diazenium dioleate group. Specifically, the composition may be a composition for inhibiting cell aging comprising a compound represented by the following Chemical Formulas 1 and 2 as an active ingredient.

[Formula 1]

[Formula 2]

[In Formulas 1 and 2,

Me is independently of each other Li, Na, K, Cs, or Rb.]

As described above, the composition for inhibiting cell aging according to the present invention can release nitrogen monoxide for a long period of time through a nitrogen monoxide donor including a C-diazenium dioleate group having a long half-life. Accordingly, it is possible to easily provide a desired level of nitrogen monoxide in various aspects. For example, it may be appropriately controlled in consideration of the initial emission amount, total emission amount, emission time, etc., and a desired level of nitrogen monoxide may be stably provided. In addition, it is possible to prevent undesired toxicity due to the rapid release of nitrogen monoxide.

In one aspect, the composition for inhibiting cell aging may include a mixture of the compounds represented by Chemical Formulas 1 and 2 as an active ingredient. At this time, the mixing ratio of the mixture may be adjusted according to the starting material and composition used in the synthesis, and the mixing ratio may be adjusted according to the composition added later. In this case, the mixing ratio means a molar ratio. In addition, it goes without saying that the composition for inhibiting cell aging may be used in an appropriate combination of different compounds represented by Formula 1 or different compounds represented by Formula 2.

For example, the active ingredient may include a mixture in which the molar ratio of the compound represented by Formula 2 to the compound represented by Formula 1 is 1:0.1 to 1:2.0. Specifically, the molar ratio of the mixture may be 1:0.5 to 1:1.5, more specifically 1:0.7 to 1:1.3.

For example, the active ingredient is completely soluble in water at room temperature and pressure (25° C., 1 atmosphere), but may not be dissolved in a lower alcohol-based solvent such as methanol or ethanol. At this time, the dissolution may mean exhibiting an average strength (uv absorbance, a.u.) of 0.1 or less in the region of 380 to 600 nm when measuring the absorbance, which means a degree that is transparent and can be evaluated by the naked eye. In addition, this is a measurement result of a UV-vis spectroscopy, and it is evaluated that it becomes opaque as its value increases.

For example, the absorbance (T _{550 nm} ) of the aqueous solution containing the active ingredient (1 mg/ml concentration, in H ₂ O) at room temperature and pressure may have an average strength of 0.1 or less.

For example, the absorbance (T' _550nm ) of the ethanol solution (1 mg/ml concentration, in EtOH) containing the active ingredient at room temperature and pressure may have an average intensity of 0.9 (au) or more.

In one aspect, in the composition for inhibiting cell aging, the active ingredient may be a crystal comprising a compound represented by Chemical Formulas 1 and 2, and the crystal is a membrane having an aspect ratio (L/D) of 2 to 20 It can be a large crystal.

For example, the length (L) of the rod-shaped crystal may be 1 to 60 μm, and the width (D) may be 0.1 to 10 μm. At this time, the length is always greater than the width (L>D).

For example, the aspect ratio of the rod-shaped crystal may be 2.0 to 10.0.

For example, the aspect ratio of the rod-shaped crystal may be 2.5 to 8.5.

In addition, as the length of the rod-shaped crystal increases, the amount of nitrogen monoxide emitted per crystal may increase. Conversely, as the length of the rod-shaped crystal decreases, the amount of nitrogen oxide emitted per crystal may decrease.

In one aspect, in the composition for inhibiting cell aging, the active ingredient is a crystal comprising a compound represented by Chemical Formulas 1 and 2, and the crystal is a spherical shape in which rod-shaped crystals having an aspect ratio of 2 to 20 are mutually conjugated It can be a crystal.

As an example, the average diameter of the spherical crystal may be 25 to 200㎛, specifically 25 to 100㎛, more specifically 25 to 50㎛, most specifically 25 to 35㎛.

The above-described rod-shaped crystals and spherical crystals may be obtained by dispersing and culturing a composition containing the compounds represented by Chemical Formulas 1 and 2 in a human skin fibroblast culture solution to a concentration of 0.1 to 20 mg/ml. . At this time, the shape of the crystal can be easily changed according to the concentration of the composition including the compound represented by Chemical Formulas 1 and 2.

For example, when the concentration of the composition containing the compound represented by Chemical Formulas 1 and 2 is less than 6 mg/ml, only rod-shaped crystals may be formed alone.

For example, when the concentration of the composition containing the compound represented by Formula 1 and Formula 2 is 6 mg/ml or more, spherical crystals are formed, and as the concentration increases, spherical crystals increase.

In addition, the spherical crystal may have a surprisingly improved amount of nitrogen monoxide emission per unit area compared to the rod-shaped crystal. Accordingly, it can be very economical because the desired effect can be exhibited even with a smaller amount of use. In addition, the spherical crystals also have a long half-life and release time, so that a low level of nitrogen monoxide can be stably provided for a long period of time.

In addition, in order to properly control the total emission amount of nitrogen monoxide as well as the emission time according to the half-life, the spherical crystal and the rod-shaped crystal can be mixed and used, as well as any combination with the composition used in the form of a mixture. Do.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the concentration of the active ingredient may be less than 100 μg/ml. As shown in Fig. 2 below, it was confirmed that the composition for inhibiting cell aging according to the present invention did not exhibit toxicity within the above-described concentration range. In addition, it was confirmed that within the above-described concentration range, the cell culture medium only treated the control group, that is, showed a higher cell viability than the negative control group, thereby helping the survival and proliferation of cells. That is, it can be used and applied in various application modes within the above-described concentration range.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may have a total amount of nitrogen monoxide released from 1 to 5 μ mol/mg.

For example, when the mixture of the compounds represented by Formulas 1 and 2 is included as an active ingredient, the total amount of nitrogen monoxide released may be 2.0 to 5.0 μ mol/mg. Specifically, the total amount of daily nitrogen released may be 2.5 to 4.5 μ mol/mg, more specifically 2.8 to 4.3 μ mol/mg.

For example, when the rod-shaped crystal is included as an active ingredient, the total amount of nitrogen monoxide released may be 2.5 to 3.0 μ mol/mg.

For example, when the spherical crystal is included as an active ingredient, the total amount of nitrogen monoxide released may be 2.0 to 3.5 μ mol/mg. The amount of nitrogen monoxide emitted per unit area of the spherical crystals is remarkable compared to the amount of nitrogen monoxide emitted per unit area of the rod-shaped crystals (see FIG. 5).

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may have a half-life of 1 to 24 hours based on the total amount of nitrogen monoxide released. Conventionally, the half-life of a nitrogen monoxide donor containing an N-diazenium dioleate group is very short in moisture, so there was a problem in practical application, but according to the present invention including a composition having a remarkably improved half-life, it is advantageously applied to various fields. I can do it.

For example, the active ingredient may have a half-life of 5 hours or more based on the total amount of nitrogen monoxide released.

For example, the active ingredient may have a half-life of 8 hours or more based on the total amount of nitrogen monoxide released.

For example, when the mixture of the compounds represented by Formulas 1 and 2 is included as an active ingredient, the half-life may be 12 to 24 hours. In addition, the release time of the nitrogen monoxide may be up to 150 minutes (see FIG. 3 below).

For example, when the rod-shaped crystal is included as an active ingredient, the half-life may be 10 to 20 hours.

For example, when the spherical crystal is included as an active ingredient, the half-life may be 13 to 185 hours.

In the composition for inhibiting cell aging according to an embodiment of the present invention, the active ingredient may react with water to release nitrogen monoxide. In this case, the active ingredient is decomposed when the pH is lowered, and effective release of nitrogen monoxide can be controlled under conditions of biological temperature and pH.

For example, the pH condition may be in the range of 2.0 to 6.0.

The present invention evaluated anti-aging, that is, antioxidant activity based on the effect of preventing cell aging in vitro. In addition, the present invention provides a composition comprising a nitrogen monoxide donor comprising a novel C-diazenium dioleate group effective in improving and treating various diseases caused by aging and senescent cells from the results thereof, and a use thereof. .

For example, for the effect of preventing cell aging, a senescent cell model was prepared by oxidation of free radicals, the composition for inhibiting cell aging according to the present invention was treated, and the results were compared through various evaluation methods. Examples of the evaluation method include, but are not limited to, identification of senescent cells through cell viability, identification of senescent cells through beta-galactosidase staining, and identification of senescent cells through hTERT analysis.

For example, the composition for inhibiting cell aging may be a composition for inhibiting apoptosis of vascular endothelial cells (HUVEC, human umbilical vein endothelial cells).

For example, the composition for inhibiting cell aging may be formulated as a composition for external application for skin. The composition for external application for skin may be a cosmetic composition or a pharmaceutical composition depending on the purpose.

For example, the composition for inhibiting cell aging may be a pharmaceutical composition for wound treatment or skin tissue regeneration. In this case, the wound may be any one or more selected from burns, lacerations, epidermal wounds, ulcers, trauma, surgical operations, childbirth, chronic wounds, and damage caused by dermatitis. In addition, the chronic wound may be a pressure sore or a pressure sore, and the dermatitis may be impetigo, interstitial, folliculitis, or eczema.

For example, the composition for inhibiting cell senescence may be used as an index for evaluating the degree of inhibition of cell senescence in vitro.

In addition, the present invention provides a method of delivering nitrogen monoxide using the release of nitrogen monoxide through the reaction of water with a composition comprising the compound represented by Chemical Formulas 1 and 2 as an active ingredient. Such a method of delivering nitrogen monoxide may be a method of inhibiting cell aging by delivering a therapeutic amount of nitrogen monoxide in a cell. At this time, the therapeutic amount may be a low level capable of providing nitrogen monoxide in a normal range to senescent cells or abnormal cells, or a low level of nitrogen monoxide released so that the normal cells can exert the normal function of cells, and intracellular toxicity. May be a concentration level that does not cause

In addition, the present invention may be used for the purpose of additional pharmacological effects in addition to the beneficial effects of nitrogen monoxide as described above.

For example, as in the pharmaceutical composition for wound treatment or skin tissue regeneration, in combination with at least one or more substances selected from antioxidants, anti-inflammatory agents, wound healing promoting substances, antibacterial agents, etc., in addition to the therapeutic effect of the drug It is possible to impart a therapeutic effect of nitrogen monoxide, thereby providing a more desirable therapeutic effect.

In the following, for a detailed understanding of the present invention, a composition for inhibiting cell aging according to the present invention and a method using the same will be described, and the following examples are for illustrative purposes of the present invention and are intended to limit the protection scope of the present invention no.

(Assessment Methods)

1. Confirmation of aging delay effect

1-1. Identification of senescent cells through cell viability

Cell viability of senescent cells was analyzed using the composition prepared in Example. First, in order to evaluate the effect of inhibiting cell aging, senescent cells were prepared by inducing cell aging due to oxidation of free radicals using hydrogen peroxide. Specifically, after treating the vascular endothelial cells at a concentration of 100, 400, 800 μM hydrogen peroxide for 24 hours, the medium was replaced and cultured for 3 days. The vascular endothelial cells cultured for 3 days were treated with a CCK reagent and incubated for 4 hours, and then UV absorbance was measured at 450 nm. As the concentration of hydrogen peroxide increased, the viability of cells decreased, and even when treated with 100 μM of hydrogen peroxide, it was confirmed that a majority of cells were killed compared to the control group (sample not treated with hydrogen peroxide). When cells are aged, the cell cycle is delayed or stopped, resulting in slow cell proliferation or cell death. Therefore, the low viability of the cells treated with hydrogen peroxide may be one of the results that can prove that the cells are senescent.

When the additional experiment was repeated, the same trend was found, and it was confirmed that the survival rate of cells differed depending on the state of the cells or the number of cells. Therefore, based on the result of this experiment, the concentration of hydrogen peroxide is fixed to 100 μM and the treatment time is adjusted from 8 to 24 hours depending on the state of the cells, thereby minimizing cell death and efficiently producing senescent cells.

The senescent cells prepared by the above method were treated with each of the compositions for inhibiting cell aging of Examples 1 to 7 below, and the survival rate of the cells was confirmed.

1-2. Identification of senescent cells through betagalactosidase staining

As cells age, since the number and size of lysosomes generally increase, the pH inside the cells decreases to about 4 to 4.5, and the activity of beta galactosidase increases due to a low pH environment. Therefore, only senescent cells can be stained by using these characteristics of senescent cells. This is the principle that X-gal is hydrolyzed by activated beta-galactosidase and turns blue, resulting in staining. As expected from the cell viability, as the concentration of hydrogen peroxide increased, a larger number of stained cells were observed. Therefore, more senescent cells were produced in proportion to the concentration of hydrogen peroxide, and the association with the cell viability was confirmed. Thus, the senescent cells prepared by the above method were treated with each of the compositions for inhibiting cell aging of Examples 1 to 7 below, and the cells were observed by staining beta galactosidase.

1-3. Identification of senescent cells through hTERT analysis

Among the characteristics of senescent cells, there is a phenomenon in which the telomere portion of DNA is shortened. This is because the activity of telomerase, an enzyme that prolongs telomeres, decreases as cells age. Therefore, senescent cells can be more accurately identified through a comparison of the activity of telomerase. In order to confirm the activity of telomerase, telomerase reverse transcriptase (hTERT), one of the catalytic subunits of telomerase, was quantitatively analyzed. Specifically, 100 μM of hydrogen peroxide was treated for 24 hours to prepare aged cells and untreated cells in 100 pie dishes, respectively, to remove all, and then wash once with phosphate buffered saline (PBS) to prepare a pellet. After that, the cells were lysed to extract all RNA, and cDNA was synthesized using PCR. The synthesized cDNA was quantified by amplifying only the hTERT portion by performing RT-PCR based on SyGreen. In addition, the senescent cells prepared by the above method were treated with each of the compositions for inhibiting cell aging of Examples 1 to 7 below, and hTERT analysis was performed.

2. Evaluation of cell migration effects

In order to confirm the effect of the composition prepared in the examples on migration of human skin fibroblasts, cell migration evaluation was performed. Specifically, human skin fibroblasts were seeded at a rate of 60,000 per well in a 12 well plate, and cultured in an incubator for 24 hours. After all the cell culture solution was removed, it was replaced with a culture solution containing no serum. After 12 hours, a vertical wound was formed in the middle of the well using a ruler and a 200 μl pipette tip. All of the cell culture solution was removed, and 0.5 ml of the cell culture solution containing serum was added and washed. 1 ml of the composition solution of the above example prepared by filtering the culture solution containing no serum was added to each well. Using a microscope, the top, middle, and bottom of the wound were photographed and the shooting points were marked. After incubating the well plate for 24 hours, the indicated location was photographed under a microscope to confirm the degree of cell migration before and after 24 hours. The results according to the concentration of the composition and the negative control group treated with only the cell culture solution were compared by calculating the cell area using ImageJ.

(Example 1)

A 40 ml solution was prepared by adding methanol-based sodium methoxide having a concentration of 0.5M to 30 ml of ethanol inside the vial. After sufficiently mixing the solution for 1 minute with a stirrer, it was injected into the reactor and the reactor was sealed. Argon gas having a pressure of 10 atm was injected into the reactor for 10 minutes, and then the process of discharging it was rapidly repeated three times to remove all gases in the reactor and the solution. Thereafter, nitrogen monoxide gas of 10 atmospheres was injected into the reactor and the reaction was performed at room temperature for 3 days. After 3 days, the process of discharging nitrogen monoxide, injecting and discharging argon gas of 10 atm, was repeated 3 times to remove unreacted nitrogen monoxide gas. After confirming that a white solid suspension was formed in the solution, the solution was transferred to a conical tube, and then centrifuged at 10,000 rpm for 10 minutes to remove the supernatant. After dispersing by adding 40 ml of cold ethanol to the remaining white pellet, the process of washing by centrifugation under the same conditions was repeated twice. After removing ethanol as much as possible from the washed white solid, a vacuum pump was connected, dried within 30 minutes, vacuum-packed, and frozen to obtain a product in the form of a salt.

The product is a mixture of two substances, sodium methane-1,1-bis-(diazene-N-oxide-N ' -hydroxylate)), a portion containing a diazeniumdiolate group bonded to carbon and a mineral portion of sodium formate.

The physical properties of the product obtained by the above method were analyzed through the methods described in Application Nos. 10-2018-0136820 and 10-2018-0136820. In addition, the results are shown in Table 1 below.

(Examples 2 to 7)

The product obtained in Example 1 was dispersed in a human skin fibroblast culture solution containing serum so that the concentration of the product was 15 mg/ml, and then filtered through a 0.45 μm filter. Cell culture solutions of various concentrations (2, 4, 6, 8, 10, 15 mg/ml) were prepared through dilution. The cell culture solution was cultured after 1 ml of the cell culture solution was injected into each well of a 24-well plate. After 24 hours, each well was observed under a microscope to confirm that crystals of various sizes and shapes were formed.

Example 2 is a case where the concentration of the product obtained in Example 1 is 2 mg/ml, and Example 3 is a case where the concentration of the product obtained in Example 1 is 4 mg/ml, and Example 4 is a case where the concentration of the product obtained in Example 1 is 6 mg/ml, Example 5 is a case where the concentration of the product obtained in Example 1 is 8 mg/ml, and Example 6 Is the case where the concentration of the product obtained in Example 1 is 10 mg/ml, and Example 7 corresponds to the case where the concentration of the product obtained in Example 1 is 15 mg/ml.

As a result, it was confirmed that the shape and size of the crystals were different depending on the concentration of the product obtained in Example 1 in the culture medium (see FIG. 4 below). At low concentration, short rod-shaped crystals were formed, and as the concentration increased, the length of the crystal became longer, and at high concentration, the rod crystals were conjugated to each other to form a three-dimensional spherical crystal. The crystals were not dispersed in water and existed in a stable state, allowing further analysis.

The physical properties of each crystal obtained by the above method were analyzed through the methods described in Application Nos. 10-2018-0136820 and 10-2018-0136820. In addition, the results are shown in Table 1 below.

(Table 1)

Based on the nitrogen monoxide emission graph, the total emission amount, half-life, maximum emission amount, and total emission time of nitrogen monoxide were analyzed. As shown in Table 1, the product prepared in the Example of the present invention, that is, the composition for inhibiting cell aging, was found to have a maximum total amount of nitrogen monoxide released of 4.0 μ mol/mg. Such a value may be slightly higher than the 0.30 μ mol/mg of Comparative Example 1, but it is possible to gradually release nitrogen monoxide as the total release time increases significantly, so that more stable nitrogen monoxide release behavior can be realized It has an advantage in terms of

In addition, Figure 5 below is a comparative comparison of the amount of nitrogen monoxide emitted per unit area of each crystal by calculating the surface area according to the size and shape of each crystal obtained from Examples 2 to 7. As a result, it was confirmed that the amount of emission per unit area increased rapidly as the size of the rod-shaped crystals formed from the composition at a concentration of 2 to 6 mg/mL increased, or as a spherical crystal formed from the composition at a concentration of 6 mg/mL or higher was formed. .

The effect of delaying aging of the composition for inhibiting cell aging according to the present invention was confirmed. Specifically, as a result of treating the example that releases nitrogen monoxide to senescent cells prepared using hydrogen peroxide, it was confirmed that the cell viability compared to the untreated control group was significantly increased through all evaluation methods. In addition, as shown in Figure 1 below, when treated with Example 1, which is a composition for inhibiting cell aging according to the present invention, it was confirmed that significant cell proliferation of 10% or more occurred compared to the control group. That is, it can be seen that the nitrogen monoxide released from the composition for inhibiting cell aging according to the present invention inhibits or delays the aging of cells aged by hydrogen peroxide, thereby inhibiting cell death and promoting proliferation.

In addition, as shown in Figure 2 below, in the case of Example 1, which is a composition for inhibiting cell aging according to the present invention, toxicity was confirmed when the concentration of the composition was 100 µg/ml or more, whereas when the concentration was less than the above concentration, the negative control It was confirmed that it shows a high cell viability compared to that it helps the survival and proliferation of cells.

In addition, as shown in FIG. 6 below, when Example 1, which is a composition for inhibiting cell aging according to the present invention, was treated, it can be seen that more cells migrated toward the wound compared to the control group. Particularly, referring to the photograph when the concentration is 1 μg/ml, it can be easily confirmed that the wound is more closed due to the movement of cells even with the naked eye. From these results, it can be inferred that the composition for inhibiting cell aging according to the present invention has an effect on the treatment or regeneration of cell damage caused by wounds.

In addition, the following Figure 4 is a photograph taken with a microscope of the shape and size of the product prepared in the Example of the present invention, that is, the crystal formed according to the concentration of Example 1. The length of the rod-shaped crystals formed at low concentration (2, 4 mg/mL) increases as the concentration increases, and from 6 mg/mL, the middle portions of the rod-shaped crystals are conjugated to each other to form spherical crystals. have. In addition, it can be seen that at a concentration of 8 mg/ml or more, most of the crystals are converted into spherical crystals, and the size thereof increases unevenly. Specifically, at a concentration of 2 to 6 mg/ml, there were rod-shaped crystals, and their length increased from 26 μm to 53 μm. In addition, spherical crystals were generated from a concentration of 6 mg/ml, and when only the size of uniform spherical crystals were analyzed, it was confirmed that they showed similar sizes regardless of the concentration.

Although the embodiments of the present invention have been described in detail as described above, those of ordinary skill in the art to which the present invention pertains, without departing from the spirit and scope of the present invention as defined in the appended claims. The invention may be implemented by various modifications. Therefore, changes to the embodiments of the present invention will not be able to depart from the technology of the present invention.

Claims (11)

It contains as an active ingredient a crystal comprising a compound represented by the following formulas 1 and 2,
The crystal is a spherical crystal in which rod-shaped crystals having an aspect ratio of 2 to 20 are bonded to each other, an antioxidant cosmetic composition:
[Formula 1]

[Formula 2]

In Chemical Formulas 1 and 2,
Me is independently of each other Na or K. The method of claim 1,
The active ingredient,
A cosmetic composition for antioxidant, wherein the molar ratio of the compound represented by Formula 2 to the compound represented by Formula 1 is 1:0.1 to 1:2.0. delete delete The method of claim 1,
The concentration of the active ingredient is,
Less than 100㎍ / ㎖, antioxidant cosmetic composition. The method of claim 1,
The active ingredient,
The total amount of nitrogen monoxide released is 1 to 5 μ mol/mg, an antioxidant cosmetic composition. The method of claim 6,
The active ingredient is based on the total amount of nitrogen monoxide released,
It will have a half-life of 1 to 24 hours, an antioxidant cosmetic composition. The method of claim 1,
The active ingredient,
It reacts with water to release nitrogen monoxide, an antioxidant cosmetic composition. The method of claim 1,
The composition,
To inhibit apoptosis of vascular endothelial cells, an antioxidant cosmetic composition. It contains as an active ingredient a crystal comprising a compound represented by the following formulas 1 and 2,
The crystal is a spherical crystal in which rod-shaped crystals having an aspect ratio of 2 to 20 are conjugated to each other, a pharmaceutical composition for wound treatment or skin tissue regeneration:
[Formula 1]

[Formula 2]

In Chemical Formulas 1 and 2,
Me is independently of each other Na or K. delete

Images