WO2018194207A1

WO2018194207A1 - Plasma-treated water having anticancer activity, and preparation method therefor

Info

Publication number: WO2018194207A1
Application number: PCT/KR2017/004629
Authority: WO
Inventors: 최은하; 구세광; 송창현; 판카지아트리; 한인; 박지훈
Original assignee: 광운대학교 산학협력단
Priority date: 2017-04-21
Filing date: 2017-05-01
Publication date: 2018-10-25

Abstract

The purpose of the present invention is to evaluate whether plasma-treated water has anticancer activity and the toxicity thereof when the plasma-treated water is prepared and is orally administered. Therefore, the purpose of the present invention is to provide a method for preparing plasma-treated water, and a method for orally administering the same. The present invention prepares plasma-treated water by emitting plasma at distilled water by using a plasma generation device, measures hydrogen peroxide (H₂O₂), nitrogen oxide (NO), nitrite ions (NO₂ ^-) and nitrate ions (NO₃ ^-), which are the active species generated in the plasma-treated water, checks for safety by orally administering the same to rodents and testing for safety, and then orally administers the plasma-treated water to rodents having tumor cells transplanted therein, thereby carrying out anticancer therapy exhibiting anticancer activity.

Description

Plasma treated water having anticancer activity and method for producing same

The present invention relates to plasma treated water having anticancer activity, and more particularly, to the manufacture of plasma treated water and a technique for applying the same to anticancer treatment.

Conventionally, plasma technology, which has played a key role in the semiconductor manufacturing field, has recently been studied to expand its application field. The application of bio-plasma technology has been expanded in various ways such as sterilization, air purification, skin beauty, teeth whitening, hair growth promotion, skin disease treatment, agricultural food production and storage technology.

Plasma has been studied to be effective in treating wounds, blood clots, sterilization and oral diseases, including various ions, electrons, free radicals, neutral particles, and photons. It is becoming. Low dose plasma irradiation promotes cell differentiation, while high dose plasma irradiation has been studied to result in apoptosis of cells [Kalghatgi et al., 2010: Vandamme et al., 2012]. It is expected to make a difference.

Republic of Korea Patent Publication No. 10-2004-0003340 describes a method of treating cancer cells with ultraviolet rays emitted during plasma discharge.

However, as a method of treating tumors using plasma, plasma irradiation treatment directly on tumor cells requires limitations such as surgery. Problems include the inability to treat all tumor points and the difficulty of undergoing multiple surgeries for ongoing treatment. Under such circumstances, a method of orally administering the plasma treated water to the patient may be considered. So far, oral administration of plasma-treated water and its toxicity have not been evaluated.

Therefore, an object of the present invention is to evaluate whether the anti-cancer effect and toxicity when the plasma treated water is prepared orally.

Accordingly, it is intended to provide a method for preparing plasma treated water and a method for oral administration.

The present invention is made in the plasma to be treated by irradiating plasma in distilled water by using a plasma generating apparatus, the plasma processing can be the generated active species, hydrogen peroxide (H _₂ O _{_2),} nitric oxide (NO), nitrite ion (NO ₂ ^- ), nitrate ion (NO ₃ ^-), the measurement and, after this, to rodents, confirming the safety by testing for safety and oral administration, by oral administration, the number of the plasma processing to a rodent transplanted with tumor cells to anti-cancer therapy represent the antitumor activity Was carried out.

That is, the present invention investigated a plasma discharge for the distilled water (助射) and hydrogen peroxide (H _₂ O _2), nitric oxide (NO), nitrite ion (NO ₂ ^{^-) -} one of the nitrate ion (NO ₃₎ It provides a method for producing oral plasma treatment water, characterized in that the plasma treated water containing the active species is used as an anticancer treatment water for oral administration.

In the above, the plasma treated water is diluted with sterile distilled water provides a method for producing orally administered plasma treated water, characterized in that used as chemotherapy water for oral administration.

In the above, the plasma treated water provides a method for producing oral plasma treated water, characterized in that the concentration of 0.5ml / kg to 2ml / kg.

In the above, there is provided a method for producing orally administered plasma treated water, characterized in that to increase the plasma treatment time to increase the concentration of the active species contained in the plasma treated water.

In the above, in the process of irradiating the plasma to the distilled water, by placing the refrigerant around the vessel containing the distilled water to prepare the plasma treated water for oral administration, characterized in that to sustain the life of the active species generated by the plasma irradiation Provide a method.

It provides a plasma-treated water for oral administration prepared by the above production method.

In addition, the present invention,

Hollow needle electrodes made of metal;

A cylindrical dielectric disposed outside the needle electrode and surrounding the needle electrode; And

And a cylindrical external electrode disposed outside the cylindrical dielectric.

The lower end of the cylindrical external electrode is gradually narrowed in diameter to form a nozzle,

Provided is a plasma treated water production system for producing plasma treated water for oral administration by applying electric power to the needle electrode and the external electrode to discharge the plasma to irradiate the plasma to distilled water.

In the above, further comprising a cover for covering the bowl containing distilled water to prevent evaporation of distilled water, wherein the cover portion can cover the periphery of the opening in which the nozzle is fitted and extending from the opening to cover the bowl of distilled water Provided is a plasma treated water production system comprising a surface.

In the above, there is provided a plasma treatment water production system further comprises a refrigerant disposed around the vessel to maintain the life of the active species.

According to the present invention, the plasma treated water showed safety for rodents, so the maximum tolerated dose was determined to be 20 ml / kg or more, and the tumor treatment by oral administration showed concentration-dependent anticancer activity of the plasma treated water, and tumor-related cachexia The phenomenon was also significantly reduced.

1 is a photograph showing a process of manufacturing plasma treated water according to the present invention.

2 is a graph of a result of measuring the concentration of hydrogen peroxide (H ₂ O ₂ ) contained in the prepared plasma treated water.

3 is a graph of a result of measuring the concentration of nitrogen oxide (NO) contained in the prepared plasma treated water.

4 is a graph illustrating a result of concentration measurement of nitrite ions (NO ₂ ⁻ ) contained in the prepared plasma treated water.

FIG. 5 is a graph illustrating a result of concentration measurement of nitrate ions (NO ₃ ⁻ ) contained in the prepared plasma treated water.

6 is a view showing the structure of the plasma jet and the applied voltage waveform of the plasma treated water of the present invention.

FIG. 7 shows the plasma jet optical emission spectroscopy (OES) spectrum of FIG. 6.

Hereinafter, with reference to the accompanying drawings will be described in detail a preferred embodiment of the present invention.

1 is a photograph showing a process of manufacturing a plasma treated water according to the present invention.

Plasma treated water is produced by directly irradiating plasma with distilled water. Plasma discharge is disposed at a point spaced from the surface of the distilled water, and the plasma is irradiated to the distilled water, it is desirable to provide a means for preventing the evaporation of distilled water. For example, the cover part which can fully cover the opening part of a bowl containing distilled water can be provided in a plasma discharge part, or the plasma discharge part itself can be made into the surface discharge structure which can cover the opening part of a bowl containing distilled water. Dishes containing distilled water are placed around a cool refrigerant such as ice to sustain the life of the active species produced by the plasma treatment. Plasma treatment time can be adjusted according to the amount of distilled water, the longer the treatment time increases the content of active species. The approximate treatment time can be from 10 minutes to 30 minutes for 5 ml distilled water. As will be described later, the anticancer activity is proportional to the concentration of the plasma treated water, that is, the concentration of the active species, so that the treatment time is a major variable.

In addition, the type and amount of active species can be controlled by controlling the applied voltage and processing time. Numerical values, including the amount of distilled water, given in the examples of producing the actual plasma treated water may be modified to be exemplary.

In the present embodiment, the plasma jet of FIG. 6 is used, and a detailed description thereof will be described later.

In this embodiment, the plasma treated water was prepared by treating 5 ml of distilled water with a plasma jet. The plasma treatment time was 20 minutes and 30 minutes, and the distance from the nozzle of the plasma jet to the distilled water surface was fixed at 5 mm. In order to prevent evaporation of distilled water that may occur during the plasma treatment, a square acrylic plate 6 cm in width and length was installed in the plasma jet nozzle. That is, an opening was formed slightly above the end of the plasma jet nozzle in the acrylic plate to sandwich the nozzle, and the outer wall surface forming the nozzle was attached to the periphery of the acrylic plate opening. Attachment can be carried out using means such as welding, glue gun, or the like. The acrylic plate may be composed of another dielectric plate, or may be a metal plate of an insulator only in contact with the nozzle. Acrylic plates can help to prevent the evaporation of distilled water and at the same time keep the active species remaining.

In addition, a 6 well plate (SPL Life Sciences co., Ltd) with a diameter of 4 cm containing distilled water was treated with plasma while it was placed on ice in order to maintain the life of active species in distilled water generated during plasma treatment.

The active species produced in the plasma treated water thus prepared were investigated and their contents were measured.

That is, hydrogen peroxide (H ₂ O ₂ ), nitrogen oxides (NO), nitrite ions (NO ₂ ⁻ ), and nitrate ions (NO ₃ ⁻ ), which are active species generated in plasma treated water by plasma, were measured.

Hydrogen peroxide (H ₂ O ₂ ) produced 0.14 mM after 20 minutes of plasma treatment, 0.15 mM after 30 minutes treatment. Titanium sulfate colorimetric method was used to measure hydrogen peroxide. When hydrogen peroxide is applied to an aqueous solution of titanium sulfate (Ti (SO ₄ ) ₂ ), a yellow solution of titanium peroxide can be obtained. The absorption wavelength band is 407 nm and the intensity of yellow is proportional to the concentration of hydrogen peroxide.

Nitrtic oxide (NO) was measured as 1.2 ± 0.5 and 1.9 ± 0.7 after 20 and 30 minutes of plasma treatment, respectively. DAF-FM (4-amino-5-methylamino-2 ', 7'-difluorescein) was used to measure nitric oxide. Fluorescent benzotriazole was reacted with nitric oxide as a reagent used to detect nitric oxide at low concentrations. To form a fluorescent state. The fluorescence excitation wavelength is 495 nm and the emission wavelength is 515 nm.

Nitrite ions (NO ₂ ⁻ ) generated 0.11 mM after 20 minutes of plasma treatment, and 0.18 mM after 30 minutes of treatment. Grease reagent was used for nitrite ion measurement. The nitrite ions in the solution generated after plasma treatment react with the grease reagent to give pink color, and the more nitrite ions are produced, the more pink. The amount of nitrite ions was quantified by measuring the absorbance at the maximum absorbance at the wavelength of 540 nm.

Nitrate ions (NO ₃ ⁻ ) were 0.64 ± 0.18 mM and 1.12 mM after 20 and 30 minutes of plasma treatment, respectively. In order to measure the nitrate ions in the solution was used Acorn Ion 6 meter of OAKTON Instruments. Acorn Ion 6 meter is used by connecting Ion selective electrodes (ISE) which can measure various ions. It contains 15ml of reference electrolyte, so the ion concentration can be measured.

The detailed configuration of the plasma jet is as follows.

The hollow needle electrode 100 made of metal is used as an internal electrode, and the dielectric cylinder 200 is disposed outside the needle electrode 100. In this example, a crystal cylinder was used. The cylindrical external electrode 300 made of stainless steel is disposed outside the dielectric cylinder 200. The lower end of the external electrode 300 gradually narrows the diameter to form the nozzle 310. The gap from the end of the needle electrode 100 where the plasma is discharged and the flame starts to the lower end of the nozzle 310 of the external electrode 300 is called a gap gap, and the gap gap may be several mm (for example, 1 to 5 mm). In addition, the cross-sectional area of the discharge flame can be adjusted by adjusting the opening of the lower end of the nozzle. In this example, the gap spacing was 2 mm. When a voltage is applied across the plasma jet electrode as described above, the discharge flame is exposed from the nozzle opening and directly irradiated with distilled water. The waveform is shown on the right side of FIG.

FIG. 7 shows the plasma jet optical emission spectroscopy (OES) spectrum of FIG. 6. This supports the content for the active species described above.

The plasma-treated water thus prepared was orally administered to rodents and subjected to oral toxicity test (based on Food and Drug Safety Notice No. 2015-082 "Toxicity Test Standard for Drugs, etc.").

Results of death, clinical symptoms, and body weights of 5ml / kg and 10ml / kg samples diluted in sterile distilled water at the azeotropic ratio of 2 ml for a maximum dose of 20 ml / kg for rodent administration, and for 2 weeks in a single dose for 2 weeks. , Organ weight, gross necropsy, and histopathologic findings were not recognized and were considered safe drugs.

In addition, after the experiment, an additional plasma treated water solution or a solution diluted 2 and 4 times with sterile distilled water was orally administered to rodents once daily for 28 days at a dose of 20 ml / kg, and in the control group, only 20 ml of sterile distilled water having the same capacity was used. The dose was repeated at a dose of / kg. As a result of the experiment, the maximum contents of both the male and female were not recognized because of death, clinical symptoms, body weight, organ weight, blood and blood biochemical changes, macroscopic and histopathological changes associated with the treatment of plasma treated water up to the rodent dose limit of 20 ml / kg. It was judged to be more than 20ml / kg, very safe substance and no special target organ was recognized.

Next, to test the anticancer effect of the plasma treated water on human lung cancer, the plasma treated water 2, 1 or 15 days after implantation of the human squamous cell carcinoma non-small cell lung cancer cell line NCI-H520 cell line in rodents. 0.5 ml / kg was orally administered for 35 days, and then all animals were sacrificed to weigh body weight, tumor volume, tumor weight, immune organ weight, blood interferon (IFN) -γ content, NK cell activity, spleen TNF-α, IL- Changes in the 1β and IL-10 contents were observed along with histopathological changes in tumors and lymphocytes, and anticancer and immune activity effects were observed. Other effects on tumor-related cachexia were also observed. In other words, changes in the ovarian fat weight and blood IL-6 were observed.

Experimental results showed that plasma treated water 2, 1 or 0.5ml / kg oral administration showed dose-dependent anticancer activity through immunological stress and oxidative stress on NCI-H520 tumor cells, in particular, plasma The treated water showed an anticancer effect comparable to the representative oral anticancer drug gefitinib 120 mg / kg in the NCI-H520 cell transplant nude mouse model. Plasma treated water also significantly reduced tumor-related cachexia. For reference, gefitinib 120mg / kg has no effect on tumor-related cachexia.

In the experimental example, hydrogen peroxide (H ₂ O ₂ ) and nitrate ions (NO ₃ ⁻ ) contained in the plasma treated water were ₂ uM and 40 ppm, respectively, and these were considered to act as oxidative stress on tumor cells with rapid self-dividing.

As salpin, the plasma treated water of the present invention can be an effective oral administration agent for anticancer treatment of human and animal.

On the other hand, the specific numerical values given in the above embodiments and experimental examples are illustrative and can be modified as necessary, and those skilled in the art to which the present invention pertains can change the technical spirit or essential features of the present invention without changing the technical spirit or essential characteristics thereof. It will be appreciated that it may be implemented in a form. Therefore, the embodiments described above are to be understood as illustrative and not restrictive in all aspects. The scope of the present invention is shown by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

The present invention can be used in the industry of manufacturing anticancer drugs for humans or animals.

Claims

The plasma discharged to distilled water is irradiated to include one or more active species of hydrogen peroxide (H 2 O 2 ), nitric oxide (NO), nitrite ion (NO 2 − ), and nitrate ion (NO 3 − ). A method of producing plasma treated water for oral administration, characterized in that to prepare a plasma treated water and use as chemotherapy water for oral administration.
The method of claim 1, wherein the plasma treated water is diluted with sterile distilled water and used as an anticancer treatment water for oral administration.
The method of claim 2, wherein the plasma treated water has a concentration of 0.5ml / kg to 2ml / kg.
The method of claim 1, wherein the plasma treatment time is increased to increase the concentration of the active species contained in the plasma treated water.
The method of claim 1, wherein in the process of irradiating the plasma to distilled water, by placing a refrigerant around the vessel containing the distilled water to maintain the life of the active species generated by plasma irradiation, oral plasma treatment water Manufacturing method.
Plasma treated water for oral administration prepared by the method of any one of claims 1 to 5.
Hollow needle electrodes made of metal;

A cylindrical dielectric disposed outside the needle electrode and surrounding the needle electrode; And

And a cylindrical external electrode disposed outside the cylindrical dielectric.

The lower end of the cylindrical external electrode is gradually narrowed in diameter to form a nozzle,

Plasma treated water production system for producing plasma-treated water for oral administration by applying electric power to the needle electrode and the external electrode to discharge the plasma to irradiate the plasma to distilled water.
The method of claim 7, further comprising a cover for covering the bowl containing the distilled water to prevent evaporation of the distilled water, wherein the cover portion can cover the periphery of the opening in which the nozzle is fitted and extending from the opening to cover the bowl containing distilled water. Plasma treatment water production system comprising a cover surface.
10. The system of claim 8, further comprising a refrigerant disposed around the vessel to maintain the lifetime of the active species.