KR101917255B1 - Inhibiting agent against disinfectant - Google Patents

Abstract

The present invention relates to a substance which inhibits fungicide toxicity. More particularly, the present invention provides a composition for preventing or treating pulmonary disease caused by fungicide toxicity and a composition for inhibiting DNA damage. The composition for preventing or treating pulmonary disease caused by fungicide toxicity according to the present invention contains at least one selected from vitamin C, berberine, curcumin and resveratrol as an active ingredient to exhibit a remarkable effect on inhibiting cell damage in A549 cell, which is a lung mucosal cell. In addition, the composition containing the active ingredient exhibits a remarkable effect in inhibiting DNA damage induced by chloromethylisothiazolinone.

Description

Inhibiting agent against disinfectant < RTI ID = 0.0 >

The present invention relates to a substance which inhibits fungicide toxicity, and more particularly, to a composition for preventing or treating pulmonary disease caused by fungicide toxicity and a composition for inhibiting DNA damage.

Since the introduction of the disinfectant for humidifiers in 1997, there have been incidents of unknown lung diseases and deaths. In 2011, based on the results of veterinary surveys and animal inhalation tests, the cause of the disinfectants for humidifiers was the Ministry of Health and Welfare The management headquarters confirmed and announced.

Among the components contained in the disinfectant for humidifiers, polyhexamethylene guanidine (PHMG), oligo (2-) ethoxy ethoxyethyl guanidine chloride (PGH), chloromethyl isothiazolyl The sterilization preservative such as Chloromethylisothiazolinone (CMIT) is hazardous. When humidifier spray liquid is injected into the air, humans inhale toxic substance of the disinfectant. When overdosed, lung injury such as pulmonary fibrosis appears and chronic obstructive pulmonary disease, , And interstitial lung disease.

Among them, CMIT is mixed with Methyl (Methylisothiazolinone) and used as a disinfectant in the form of CMIT / MIT. It is easy to dissolve in water and has high volatility. It gives strong stimulation to eyes and skin when exposed to certain concentration. In Korea and Europe, only 15 ppm is used for washing products. In Japan, it is allowed to dilute to 0.1% for washing products. CMIT emits free radicals to induce bacterial cell damage and ultimately leads to cell death.

Therefore, research for protecting the human body from the toxicity contained in the sterilizing agent for humidifier is urgently required, but there is no research on it yet.

In the present invention, the extent of damage to the lymphocyte of the rat by the harmful substance of the humectant disinfectant is checked by a comet assay which can confirm the damage level of the cell at the single cell level, A representative humicidal disinfectant toxic substance, CMIT, was treated by dividing the lymphocyte of the rat into several concentrations, and the degree of damage to each single cell of each harmful substance was determined and quantified. Among them, it is intended to provide a composition for preventing or treating pulmonary disease caused by bactericide toxicity and a composition for inhibiting DNA damage by using Phytochemical which is a chemical substance present in plants.

 In Vitro pulmonary toxicity of humidifier disinfectants in BEAS-2B cells - 2012.5, 145page

It is an object of the present invention to inhibit DNA damage by a bactericide containing toxic components such as CMIT (Chloromethylisothiazolinone), including at least one selected from vitamin C, berberine, curcumin and resveratrol, Which is capable of preventing or treating cancer.

According to a representative aspect of the present invention, there is provided a composition for preventing or treating pulmonary disease caused by toxic fungicide comprising at least one selected from vitamin C, berberine, curcumin and resveratrol as an active ingredient.

It is preferable that the composition for inhibiting and preventing pulmonary disease inhibits cytotoxicity in lung mucosal cells.

The lung mucosal cells are preferably human lung cancer cells A549 cells.

The lung disease is preferably at least one selected from chronic obstructive pulmonary disease, pneumonia, lung abscess, pulmonary tuberculosis, interstitial lung disease, lung tumor and lung cancer.

The bactericide preferably contains CMIT (Chloromethylisothiazolinone) as an active ingredient.

According to another representative aspect of the present invention, there is provided a composition for inhibiting DNA damage caused by bactericide toxicity comprising at least one selected from vitamin C, berberine, curcumin and resveratrol as an active ingredient.

The active ingredient is preferably contained at a concentration of 1 to 10 占 퐂 / ml.

The composition preferably has an activity to inhibit DNA damage due to cytotoxicity induced by chloromethyl isothiazolinone in vitro .

According to another representative aspect of the present invention, there is provided a method of treating a cell, comprising: (A) treating a cell with a positive control or a sample;

(B) treating the cells treated with the positive control or the sample with a bactericide;

(C) electrophoresing the cells treated with the bactericide; And

(D) staining the cells after the electrophoresis and confirming them by fluorescence microscopy,

The cells are A549 cells which are lymphocytes or lung mucosal cells collected from rats,

Wherein the sample is at least one selected from vitamin C, berberine, curcumin and resveratrol.

The positive control group was N-acetylcysteine,

The bactericide preferably contains CMIT (Chloromethylisothiazolinone) as an active ingredient.

The composition for preventing or treating pulmonary diseases according to the present invention comprises at least one selected from the group consisting of vitamin C, berberine, curcumin and resveratrol as an active ingredient to inhibit cell damage in A549 cell, a lung mucosal cell And exhibits remarkable effects.

In addition, the composition containing the active ingredient exhibits a remarkable effect in inhibiting DNA damage induced by chloromethyl isothiazolinone.

FIG. 1 is a graph showing the results of a comet assay of an experimental group treated with control (CON) and CMIT (1, 3, 5, and 10 μg / ml) as a control group without CMIT treatment.
FIG. 2 is a graph showing a result of a comet assay of an experimental group treated with vitamin C at different concentrations (1, 3, 5 / / ml).
FIG. 3 is a graph showing the results of a comet assay of an experimental group treated with berberine at different concentrations (1, 3, 5, and 7 / / ml).
4 is a graph showing the results of a comet assay of an experimental group treated with curcumin at different concentrations (3, 5, and 7 / / ml).
5 is a graph showing the results of a comet assay of an experimental group treated with resveratrol at different concentrations (1, 3, and 5 / / ml).
Figure 6 shows the cell survival rate of A549 cells after 24 hours of control (CON) and control (CMIT) treatment groups treated with CMIT (10, 20, 30, 40, 50 ㎍ / viability,%).
FIG. 7 is a graph showing the cell survival rate of an experimental group treated with N-acetylcysteine at a concentration (3, 7 / / ml).
FIG. 8 is a graph showing the results of measurement of cell viability of an experimental group treated with resveratrol at different concentrations (3, 5, and 7 / / ml).

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a composition for preventing or treating pulmonary disease caused by bactericide toxicity comprising at least one selected from vitamin C, berberine, curcumin and resveratrol as an active ingredient.

The vitamin C has an antioxidative activity to remove free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) C radicals have a relatively long half-life of 5 to 10 seconds compared to other antioxidants and thus serve as stable antioxidants.

Berberine is an isoquinoline alkaloid obtained from the roots of Coptis japonica root, Phellodendron amurense bark, Berberis vulgaris, and the alkaloid is a relatively small amount It is a substance that exhibits a remarkable pharmacological action in humans or animals.

Curcumin is a kind of alkaloids contained in Indian turmeric, and has antioxidant, antiinflammatory, anti-cancer and neuroprotective effect, and polyphenol possessed by curcumin has a role of inducing antioxidative action.

Resveratrol is a substance contained in a large amount in red wine. It is composed of polyphenols and has a strong antioxidant ability, and has an antimutagenic and anti-inflammatory effect.

The bactericide contains CMIT (Chloromethylisothiazolinone) as an active ingredient.

The pulmonary disease refers to a pulmonary disease caused by bactericide toxicity including the active ingredient, and specifically includes chronic obstructive pulmonary disease, pneumonia, lung abscess, pulmonary tuberculosis, interstitial lung disease, lung tumor and lung cancer.

In particular, the composition for preventing or treating pulmonary disease due to bactericidal toxicity is characterized by inhibiting cytotoxicity in lung mucosal cells, wherein the lung mucosal cells are A549 cells, which are human lung cancer cells.

According to another aspect of the present invention, there is provided a composition for inhibiting DNA damage caused by bactericide toxicity, which comprises at least one selected from vitamin C, berberine, curcumin and resveratrol as an active ingredient.

It is preferable that the active ingredient is contained at a concentration of 1 to 10 占 퐂 / ml. If the concentration range is less than 1 占 퐂 / ml, the effect of inhibiting DNA damage due to bactericide toxicity is insignificant. Which is undesirable. More preferably, the active ingredient has a concentration of 1 to 7 占 퐂 / ml, and the experimental error is the least within the above range, and it is confirmed that it is effective in suppressing DNA damage.

In particular, the composition has an activity of inhibiting DNA damage due to cytotoxicity induced by chloromethylisothiazolinone (CMIT) in vitro .

Formulations of the compositions according to the present invention may be selected from the group consisting of granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops or solutions.

The preferred dosage of the composition according to the present invention varies depending on the condition and body weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a daily dose of 0.0001 to 2000 mg / kg, preferably 0.001 to 1000 mg / kg. It is, however, within the knowledge of the art to gradually increase the dose until the desired effect is achieved, starting with the dose of the compound at a level lower than that required for achieving the desired therapeutic effect, , Body shape, and body weight. The composition may be further processed before being formulated into a pharmaceutically acceptable pharmaceutical formulation and may be preferably ground or ground into smaller particles. Also, the composition will vary depending on the condition and the patient being treated, but it can be determined non-ingeniously.

The concentration of one or more active ingredients selected from the group consisting of vitamin C, berberine, curcumin and resveratrol may be in the range of 1 nM to 1 M, or 1 μM to 1 mM, or 0.1 mM to 0.2 mM, or 0.05 μM to 5 μM But are not limited thereto. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, the composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. Preferably intravenously, intraarterially, intraperitoneally, intramuscularly, intraarterially, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, , Transdermal, non-intranasal, inhalation, topical, rectal, oral, intra-ocular, or intradermal routes.

According to another aspect of the present invention, there is provided a method for producing a cell, comprising: (A) treating a cell with a positive control or a sample; (B) treating the cells treated with the positive control or the sample with a bactericide; (C) electrophoresing the cells treated with the bactericide; And (D) staining the cells after the electrophoresis and confirming them by fluorescence microscope, wherein the cells are A549 cells which are lymphocytes or lung mucosal cells collected from mice, and the samples are vitamin C, berberine, curcumin and The present invention provides a method for confirming inhibition of DNA damage due to bactericide toxicity.

The step (A) preferably treats the cells with a positive control or sample capable of inhibiting the toxicity of the bactericidal agent prior to treating the bactericidal agent. Specifically, in order to maintain the environment of the cells, a buffer solution such as PBS (phosphate buffered saline) was used as all the solvents, and the cells suspended in the buffer solution were pretreated by centrifugation. The positive control group is preferably N-acetylcysteine.

In the step (B), the bactericide may be treated with a bactericide to the cells of the positive control or the treated sample, and the bactericide preferably contains CMIT (Chloromethylisothiazolinone) as an active ingredient.

The step (C) is a step of electrophoresis of the cells treated with the bactericide. Since the DNA with negative charge moves to the anode during electrophoresis, the larger the damaged cell, the more the DNA is cut into smaller fragments and moves quickly to the anode. The result is a tail shape due to the movement of DNA fragments, And to compare the mean value of the tail value, the olive tail moment, to determine the degree of damage.

The step (D) is a step of staining the cells after the electrophoresis and confirming the cells by a fluorescence microscope. The average value of the olive tail moment, which is the above-mentioned significant tail value, is confirmed. The dyeing may be performed using a dye such as ethidium bromide, but is not limited thereto.

Through the above-described method of confirming the inhibition of DNA damage due to the bactericide toxicity, the DNA damage-inhibiting effect on the above-mentioned composition can be confirmed.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

In addition, the experimental results presented below only show representative experimental results of the embodiments and the comparative examples, and the respective effects of various embodiments of the present invention which are not explicitly described below will be specifically described in the corresponding part.

(Example)

1. Cell culture

Experimental Example The cells used were lymphocytes obtained from SD rats (7 weeks old, male). For the evaluation of toxicity of CMIT, 200 μl of blood was used for each group and 400 μl of each group was used for evaluating the protection effect of phytochemicals. To maintain the cell environment, 1XPBS (phosphate buffered saline) was used for all the solvents. For toxicity assessment, 800 μl of 1XPBS was taken up with 200 μl of blood suspension, placed on a 200 μl histopaque, and centrifuged at 1450 rpm for 5 minutes at a temperature of 20 ° C. Then 200 μl of the buffy coat was taken and suspended in 1 ml of 1 × PBS and suspended. After centrifugation at 1450 rpm at 20 ° C for 5 minutes, the supernatant was removed and 1 ml of toxic material was taken out to release the pellet. The reaction was carried out in ice for 5 minutes and then centrifuged at 1450 rpm for 5 minutes at a temperature of 20 ° C. The supernatant was taken, pelleted with 1 ml of 1XPBS and centrifuged for 5 minutes. After pouring the supernatant again, the pellet was dissolved in 75 μl of 0.7% normal melting point agrose (NMA) and dropped on a slipped 1% low melting point agarose (LMA). Covered with a cover glass and slowly set in a 4 ° C refrigerator for 30 minutes. The cover glass was removed, and 100 μl of 0.7% NMA was dropped on the slide, covered with a cover glass, and slowly cooled in a 4 ° C refrigerator for 30 minutes. Cell lysis buffer was prepared with 2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1% Triton X100, 10% DMSO (Dimethylsuloxide, pH 10) and the slides were immersed therein and allowed to react in a dark room for 2 hours . In the case of the evaluation of the protective action, the experimental method is totally similar to the toxicity evaluation, and the pellet is dissolved with 1 ml of the concentration-specific protecting substance before the toxic substance treatment step, and the reaction is carried out for 30 minutes in a 37 ° C water bath.

Cell-mounted slides were immersed in an electrophoresis buffer (0.2 M Na ₂ EDTA, 10 N NaOH) for 20 minutes in a dark room and then subjected to electrophoresis for 20 minutes at a voltage of 25 V and a current of 300 mA. After the electrophoresis, the slides were washed three times for 5 minutes with Neutralization buffer (0.5 M Tris, pH 7.5), dehydrated in 100% ethanol for 5 minutes, and then dried at room temperature for 10 minutes. Slides were stained with Ethidium Bromide 50 μM and observed under a fluorescence microscope. The Kinetic Komet 5.5 program read 100 single cells per slide and the average of the olive tail moments was obtained.

2. Culture of A549 cells

Human lung cancer cell A549 cells were purchased from Korean Cell Line Bank (KCLB). Cells were subcultured using RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin. Cell culture dishes were 100 mm and 150 mm, cultured in a 5% CO ₂ incubator at 37 ° C, and subcultured every 2 to 3 days. In the subculture, trypsin-EDTA was treated with 0.25%, 3 ml for 100 mm dish, and 8 ml for 150 mm dish. The A549 cells used were distributed to passage 36 and MTT assays were carried out by passage 45.

(Test Example 1: Comet assay analysis)

Since the DNA with negative charge moves to the anode during electrophoresis, the larger the damaged cell, the more the DNA is cut into small fragments and moves to the anode quickly. As a result, the shape of the tail is revealed by the movement of the DNA fragments and the degree of damage is measured by comparing the mean value of the olive tail moment, which is the significant tail value. All cells were counted 100 per experimental group, and the difference between the groups was compared as the average of the respective olive tail moments. For control, 100 cells were measured on two slides for accuracy and a total of 200 averages were obtained.

1. Evaluation of Toxicity of Hazardous Ingredients by Means of Comet Assay

Control and CMIT, which were not treated with CMIT, were treated with lymphocytes at concentrations of 1, 3, 5, and 10 ㎍ / ml. When the concentration of CMIT was 1 ㎍ / ml, the olive tail moment value was similar to that of the control, but the longer the tail length was, the higher the CMIT concentration was. Duncan 's post - analysis showed that the CMIT - treated group contained 1 ㎍ / ml and 3 ㎍ / ml a group, but 5 ㎍ / ml and 10 ㎍ / ml belong to groups b and c, respectively. Thus, DNA damage is induced when CMIT is treated to cells above 5 / / ml (Fig. 1).

2. Through comet assay phytochemical Evaluation of the protective action of

As a result of the CMIT protective action of vitamin C, the olive tailing torque, which is remarkably lowered when the vitamin C was treated at 1, 3 and 5 ㎍ / ml, was confirmed. Duncan showed that the group treated with 5 ㎍ / ml of vitamin C showed a significant level of DNA damage to the normal cells not treated with CMIT while belonging to the same group as control. Therefore, vitamin C can inhibit cell damage as a protective substance against CMIT (Fig. 2).

Berberine was analyzed for the protective effect against CMIT of cells treated at the concentrations of 1, 3, 5, and 7 / / ml. Referring to FIG. 3, the higher the concentration of berberine, the lower the olive tail moment Able to know. This means that there is less oxidative damage to the cells by berberine. Duncan 's post - mortem analysis showed that the treatment with 7 ㎍ / ml berberine was in the same group a as the control and protected the cells from damage.

Referring to FIG. 4, as the concentration of curcumin increased, the olive tail moment value decreased. This suggests that curcumin induced a protective action against oxidative damage to cells, thereby preventing cell damage caused by a strong oxidative damaging substance called CMIT. Duncan showed that curcumin 7 μg / ml inhibited DNA damage at the same level as the control.

Also, referring to FIG. 5, resveratol also showed protection against DNA oxidative damage induced by CMIT. In the experimental group treated with 1, 3, and 5 ㎍ / ml of resveratol, the values were similar to the olive tail moment of the control.

(Test Example 2: MTT assay assay)

A549 cells were plated at 2 × 10 ⁴ cells / well in a 96-well plate at 200 μl each. The cells were incubated in an incubator at 37 ° C and 5% CO ₂ for 22 hours. After the medium was sucked in, the material was treated. In the case of the plate for the inhibition of CMIT, 100 .mu.l of medium was added to the control group not treated with CMIT and 100 .mu.l of CMIT at different concentrations of 10, 20, 30, 40, 50. In the case of the plate for the protection effect against CMIT, only 100 ㎕ of the medium was added to the control group. In the experimental group, the treatment volume of CMIT was 50 ㎍ / ml and the treatment concentration of the protective substance was 3 ㎍ / ml and 7 ㎍ / 100 [mu] l of material was treated. Twenty-four hours later, 50 μl of 2 mg / ml MTT reagent was added to all wells and incubated in an incubator for 3 hours. Then, 500 μl of DMSO was added to all wells, and the mixture was stirred at room temperature for 30 minutes while being wrapped in foil. After stirring, cell viability was obtained by measuring the absorbance with an ELISA reader set at 570 nm, center.

The MTT reagent is yellow soluble MTT tetrazolium, which is reduced from living cells to a water-insoluble formazan crystal. This formazan melts in DMSO and becomes bluish purple. The more living cells there are, the more formazan is produced, and the darker it becomes. MTT formazan has a maximum wavelength at 540 nm, and the absorbance measured at this wavelength reflects the concentration of cells that are metabolically active. The results of the MTT assay described below are repeated six times and the measured values are averaged.

Referring to FIG. 6, MTT assay was performed by setting the control group treated with CMIT and control group CMIT to 10, 20, 30, 40, and 50 ㎍ / ml, respectively. As a result, cell viability was as high as CMIT concentration of 40 ㎍ / ml, but cell viability of CMIT at 50 ㎍ / ml was 78%. Therefore, cytotoxicity is shown when CMIT concentration is 50 ㎍ / ml against lung cells.

Evaluation of phytochemical protection by MTT assay

In order to confirm the action of the protective substance against CMIT at the cellular level, the active oxygen scavenging ability was previously measured by N-acetylcysteine (NAC), which is known as a powerful antioxidant. NAC is a molecule with an acetyl group attached to cysteine. It acts as a powerful antioxidant in the body itself, plays a role in the regeneration of glutathione, or acts directly as a precursor to glutathione. NAC was treated with 3, 7 ㎍ / ml in each well, and CMIT 50 ㎍ / ml was simultaneously treated to confirm the protective action.

Referring to FIG. 7, the cell viability of the group treated with CMIT alone was less than 80%, and the cell survival rate was increased as the NAC treatment concentration was increased. Duncan 's post - mortem analysis showed that 7 μg / ml of NAC was co - treated with CMIT and belonged to the same group b as control, thus protecting the cells against cytotoxicity. The significance level was less than 0.05.

Vitamin C, berberine, curcumin and vesberatol were simultaneously treated at a concentration of 3, 5, and 7 / / ml in 50 ㎍ / ml of CMIT, respectively. The results are shown in FIGS. 8 to 11.

Referring to FIGS. 8 to 10, it can be seen that vitamin C, berberine and curcumin exhibit a high cell survival rate similar to that of the control at a concentration of 7 ㎍ / ml.

11, in the CMIT-treated group, the cell survival rate was about 75% as compared with the control group. However, in the group treated with 7 ㎍ / ml of resveratol, the cell survival rate was similar to that of control, Duncan's post-test analysis was the only group b that belonged to the control group. The significance level was less than 0.05.

Accordingly, the composition for preventing or treating pulmonary disease caused by bactericide toxicity according to the present invention comprises at least one selected from vitamin C, berberine, curcumin and resveratrol as an active ingredient, Lt; / RTI >

In addition, the composition containing the active ingredient exhibits a remarkable effect in inhibiting DNA damage induced by chloromethyl isothiazolinone.

Claims (10)

A composition for preventing or treating pulmonary disease caused by toxic fungicide containing resveratrol as an active ingredient,
Wherein the bactericide contains CMIT (Chloromethylisothiazolinone) as an active ingredient,
The composition for preventing or treating lung disease suppresses cytotoxicity in lung mucosal cells,
Wherein the pulmonary disease is for preventing or treating at least one selected from chronic obstructive pulmonary disease, pneumonia, lung abscess, pulmonary tuberculosis, interstitial lung disease, lung tumor and lung cancer.
delete The method according to claim 1,
Wherein said lung mucosal cell is A549 cell which is a human lung cancer cell.
delete delete A composition for suppressing DNA damage due to toxic fungicide containing resveratrol as an active ingredient,
Wherein the bactericide contains CMIT (Chloromethylisothiazolinone) as an active ingredient,
Wherein the composition has an activity of inhibiting DNA damage due to cytotoxicity induced by chloromethyl isothiazolinone in vitro in a composition for inhibiting DNA damage due to bactericidal toxicity.
The method according to claim 6,
Wherein the effective ingredient is contained at a concentration of 1 to 10 占 퐂 / ml.
delete (A) treating a cell with a positive control or sample;
(B) treating the cells treated with the positive control or the sample with a bactericide;
(C) electrophoresing the cells treated with the bactericide; And
(D) staining the cells after the electrophoresis and confirming them by fluorescence microscopy,
The cells are lymphocytes obtained from mice,
The sample is resveratrol,
Wherein the bactericide comprises CMIT (Chloromethylisothiazolinone) as an active ingredient.
10. The method of claim 9,
Wherein the positive control is N-acetylcysteine.

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