JP2016169164A - Antitumor aqueous solutions and production methods thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide antitumor aqueous solutions exerting an antitumor effect by a relatively simple construction, and to provide production methods thereof.SOLUTION: The invention provides an antitumor aqueous solution containing a culture medium, hydrogen peroxide, and nitrite ion. Preferably, the concentration of hydrogen peroxide is 30 μM to 100 mM and preferably the concentration of nitrite ion is 300 μM to 1M. Though hydrogen peroxide and nitrite ion are preferably generated in a culture medium by irradiating the culture medium with non-equilibrium atmospheric pressure plasma, a plasma generator is not necessarily required.SELECTED DRAWING: None

Description

  The technical field of this specification relates to an antitumor aqueous solution and a method for producing the same.

  Plasma technology is applied in the fields of electricity, chemistry, and materials. In recent years, medical applications have been actively studied. Inside the plasma, ultraviolet rays and radicals are generated in addition to charged particles such as electrons and ions. These have been found to have various effects on living tissues, including sterilization of living tissues.

  For example, Patent Document 1 discloses blood coagulation (see Example 4 of Patent Document 1, paragraphs [0063] to [0068]) and tissue sterilization (Example 5 of Patent Document 1, Paragraphs [0069]-[0074]) and Leishmaniasis (see Example 6 of Patent Document 1, paragraphs [0075]-[0077]) are described as being effective. And it is described that plasma has the effect of killing melanoma cells (malignant melanoma cells) (see Example 7 of Patent Document 1, paragraph [0078]).

  Patent Document 2 discloses a technique for sterilizing bacteria in a liquid by irradiating plasma with a liquid adjusted to have a pH of 4.8 or less (Patent Document 2 paragraph [ 0020] etc.). Further, it is described that a superoxide anion radical, a hydroperoxy radical, or the like may have a bactericidal effect (see paragraphs [0090]-[0099] etc. of Patent Document 2).

Special table 2008-539007 gazette International Publication No. 2009/041049 International Publication No. 2013/128905

  By the way, in the treatment of such cancer, it is generally preferable to 1) kill cancer cells and 2) selectively kill cancer cells so as not to affect normal cells. This is because even if cancer cells can be killed, killing a large number of normal cells for that purpose causes a great physical burden on the patient. Therefore, a treatment technique that selectively kills cancer cells in this way is desired. However, it is not easy to selectively kill cancer cells. In Patent Document 1, the degree of influence on normal cells is not clear.

  Therefore, as described in Patent Document 3, the present inventors have researched and developed a technique related to an antitumor aqueous solution that selectively kills cancer cells (Patent Document 3, paragraphs [0085]-[0087] and FIG. 16 etc.). This antitumor aqueous solution is obtained by irradiating a culture solution with non-equilibrium atmospheric pressure plasma. This anti-tumor aqueous solution can selectively kill cancer cells with little effect on normal cells. In addition, this anti-tumor aqueous solution exhibited an anti-tumor effect not only on cultured cells but also on mice (see paragraphs [0145]-[0152] and FIGS. 45 and 46 of Patent Document 3).

  However, it is difficult to specify what component of this anti-tumor aqueous solution has an anti-tumor effect. This is because many types of active species are generated by irradiation with non-equilibrium atmospheric pressure plasma. Thus, the antitumor aqueous solution irradiated with plasma has many kinds of active species and has a complicated structure.

  The technique of this specification has been made to solve the problems of the conventional techniques described above. That is, the problem is to provide an antitumor aqueous solution that exhibits an antitumor effect with a relatively simple structure, and a method for producing the same.

  The anti-tumor aqueous solution in the first embodiment is an aqueous solution that kills cancer cells. This anti-tumor aqueous solution contains a culture solution, hydrogen peroxide, and nitrite ions.

  This anti-tumor aqueous solution kills cancer cells. Also, its composition is relatively simple. Therefore, a plasma generator is not necessarily required when producing this antitumor aqueous solution.

  In the antitumor aqueous solution in the second aspect, the concentration of hydrogen peroxide is 30 μM or more and 100 mM or less.

  In the anti-tumor aqueous solution in the third aspect, the concentration of nitrite ions is 300 μM or more and 1 M or less.

  The antitumor aqueous solution in the fourth embodiment is one in which hydrogen peroxide and nitrite ions are generated in the culture solution by irradiating the culture solution with non-equilibrium atmospheric pressure plasma.

  The method for producing an anti-tumor aqueous solution in the fifth aspect is a method for producing an anti-tumor aqueous solution that kills cancer cells. In this production method, hydrogen peroxide water and nitrite ion water are added to the culture solution to obtain a mixed solution.

  In the anti-tumor aqueous solution in the sixth aspect, the concentration of hydrogen peroxide in the mixed solution is 30 μM or more and 100 mM or less.

  In the anti-tumor aqueous solution according to the seventh aspect, the concentration of nitrite ions in the mixed solution is 300 μM or more and 1 M or less.

  In the present specification, an antitumor aqueous solution having an antitumor effect with a relatively simple structure and a method for producing the same are provided.

It is a conceptual diagram for demonstrating the structure of the robot arm which scans the plasma irradiation apparatus of embodiment. FIG. FIG. 2A is a cross-sectional view showing the configuration of the first plasma generator, and FIG. B is a figure which shows the shape of an electrode. FIG. FIG. 3A is a cross-sectional view showing the configuration of the second plasma generator, and FIG. B is a partial cross-sectional view in a cross section perpendicular to the longitudinal direction of the plasma region. It is a table | surface which shows the density | concentration of the hydrogen peroxide and nitrite ion which generate | occur | produce in a culture solution when a culture solution is irradiated with plasma. It is a graph which shows the relationship between the irradiation time of a plasma, and the density | concentration of the hydrogen peroxide generated in a culture solution. It is a graph which shows the relationship between the irradiation time of a plasma, and the density | concentration of the nitrite ion which generate | occur | produces in a culture solution. It is a graph which shows the relationship between the culture solution which irradiated plasma for 90 second, the culture solution corresponding to it, and the survival rate of a glioblastoma cell. It is a graph which shows the relationship between the culture solution which irradiated plasma for 180 second, and the culture solution corresponding to it, and the survival rate of a glioblastoma cell. It is a graph which shows the relationship between the plasma irradiation time and the quantity of the additive corresponding to it, and the survival rate of a glioblastoma cell.

  Hereinafter, specific embodiments will be described with reference to the drawings, taking an antitumor aqueous solution and a production method thereof as examples.

(First embodiment)
A first embodiment will be described.

1. 1. Antitumor aqueous solution 1-1. Content of anti-tumor aqueous solution The anti-tumor aqueous solution of the present embodiment is an aqueous solution that kills cancer cells. This anti-tumor aqueous solution contains a culture solution, hydrogen peroxide, and nitrite ions. Here, the culture solution is a commercially available culture solution. For example, DMEM.

DMEM contains the following culture components. The culture components are calcium chloride, ferric nitrate · 9H ₂ O, magnesium sulfate (anhydrous), potassium chloride, sodium bicarbonate, sodium chloride, phosphate-sodium (anhydrous), L-arginine · HCl, L- Cystine · 2HCl, L-glutamine, glycine, L-histidine · HCl · H ₂ O, L-isoleucine, L-leucine, L-lysine · HCl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine · 2Na · 2H ₂ O, L-valine, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid, pyridoxine · HCl, riboflavin, thiamine · HCl, D-glucose, phenol Red Na.

  Hydrogen peroxide and nitrite ions have a synergistic effect in the culture medium. That is, the antitumor effect when both hydrogen peroxide and nitrite ions are present in the culture solution is higher than the antitumor effect when only one of hydrogen peroxide and nitrite ions is present.

1-2. Hydrogen peroxide concentration The concentration of hydrogen peroxide in this anti-tumor aqueous solution is 30 μM or more. The concentration of hydrogen peroxide is preferably 30 μM or more and 100 mM or less. Preferably, the concentration of hydrogen peroxide is 40 μM or more and 50 mM or less. More preferably, the concentration of hydrogen peroxide is 50 μM or more and 10 mM or less.

1-3. Nitrite ion concentration The concentration of nitrite ion in this anti-tumor aqueous solution is 300 μM or more. The concentration of nitrite ions is preferably 300 μM or more and 1 M or less. Preferably, the concentration of nitrite ions is 400 μM or more and 500 mM or less. More preferably, it is 500 μM or more and 100 mM or less.

2. Effect of anti-tumor aqueous solution The anti-tumor aqueous solution of the present embodiment naturally has an anti-tumor effect. That is, cancer cells are killed. And the antitumor effect is comparable with the antitumor effect of the antitumor aqueous solution which irradiated the non-equilibrium atmospheric pressure plasma to the culture solution demonstrated in 2nd Embodiment. In addition, as described above, the antitumor effect when both hydrogen peroxide and nitrite ions are present in the culture solution is more than the antitumor effect when only one of hydrogen peroxide and nitrite ions is present. high.

3. 3. Method for producing antitumor aqueous solution 3-1. Culture solution preparation process First, a culture solution is prepared. Here, the culture solution to be prepared is, for example, a commercially available culture solution. Moreover, you may produce a culture solution by adding a predetermined culture component to a pure water.

3-2. Hydrogen peroxide addition process Next, hydrogen peroxide water is added to a culture solution. The hydrogen peroxide solution is, for example, a commercially available hydrogen peroxide solution.

3-3. Nitrite ion addition step Next, nitrite ion water is added to the culture solution. Nitrite ion water is, for example, a sodium nitrite aqueous solution. Or it is potassium nitrite aqueous solution. In addition, any aqueous solution containing nitrite may be used. In this way, a mixed solution is produced by adding hydrogen peroxide water and nitrite ion water to the culture solution. This mixed solution is an antitumor aqueous solution. The order of adding the hydrogen peroxide solution and the nitrite ion water may be reversed.

3-4. Produced anti-tumor aqueous solution The produced anti-tumor aqueous solution contains hydrogen peroxide and nitrite ions. The concentration of hydrogen peroxide in the anti-tumor aqueous solution is 30 μM or more. The concentration of nitrite ions in the antitumor aqueous solution is 300 μM or more.

4). Modification The anti-tumor aqueous solution of this embodiment has a culture solution, hydrogen peroxide, and nitrite ions. For example, DMEM is used as the culture solution. Of course, other culture solutions may be used. For example, RPMI, YPD, NB, etc. are mentioned.

5. Summary of this embodiment As described in detail above, the anti-tumor aqueous solution of this embodiment is an aqueous solution that kills cancer cells. This anti-tumor aqueous solution contains a culture solution, hydrogen peroxide, and nitrite ions. In addition, the presence of hydrogen peroxide and nitrite ions in the culture medium provides a synergistic effect between hydrogen peroxide and nitrite ions.

(Second Embodiment)
A second embodiment will be described. In the second embodiment, hydrogen peroxide and nitrite ions are generated in the culture solution by irradiating the culture solution with non-equilibrium atmospheric pressure plasma.

1. Anti-tumor aqueous solution production apparatus 1-1. Configuration of Antitumor Aqueous Solution Manufacturing Device The antitumor aqueous solution manufacturing device PM of this embodiment includes a plasma irradiation device P1 and an arm robot M1 as shown in FIG. The plasma irradiation apparatus P1 is for generating plasma and irradiating the plasma toward the solution.

  As shown in FIG. 1, the arm robot M1 can move the position of the plasma irradiation apparatus P1 in each of the x-axis, y-axis, and z-axis directions. For convenience of explanation, the direction of plasma irradiation is the −z axis direction. Thereby, the distance between the liquid level of a solution and the plasma irradiation apparatus P1 can be adjusted. Moreover, this anti-tumor aqueous solution manufacturing apparatus PM can irradiate plasma only for the time by setting plasma irradiation time beforehand.

  As will be described later, there are two types of plasma irradiation apparatus P1 (first plasma generation apparatus P10 and second plasma generation apparatus P20). Any method may be used.

1-2. First plasma generator FIG. A is a cross-sectional view showing a schematic configuration of the plasma generator P10. Here, the plasma generator P10 is a first plasma generator that ejects plasma in the form of dots. FIG. B is shown in FIG. It is a figure which shows the detail of the shape of the electrodes 2a and 2b of the plasma generator P10 of A.

The plasma generating apparatus P10 includes a casing unit 10, electrodes 2a and 2b, and a voltage application unit 3. The casing 10 is made of a sintered body made of alumina (Al ₂ O ₃ ) as a raw material. And the shape of the housing | casing part 10 is a cylinder shape. The internal diameter of the housing | casing part 10 is 2 mm or more and 3 mm or less. The thickness of the housing | casing part 10 is 0.2 mm or more and 0.3 mm or less. The length of the housing | casing part 10 is 10 cm or more and 30 cm or less. A gas inlet 10 i and a gas outlet 10 o are formed at both ends of the housing 10. The gas inlet 10i is for introducing a gas for generating plasma. The gas outlet 10 o is an irradiation unit for irradiating the outside of the housing unit 10 with plasma. The direction in which the gas moves is the direction of the arrow in the figure.

  The electrodes 2a and 2b are a pair of opposing electrodes arranged to face each other. The lengths of the electrodes 2 a and 2 b in the facing surface direction are smaller than the inner diameter of the housing portion 10. For example, it is about 1 mm. For the electrodes 2a and 2b, FIG. As shown to B, many recessed parts (hollow) H are formed in each of an opposing surface. Therefore, the opposing surfaces of the electrodes 2a and 2b have a fine uneven shape. In addition, the depth of this recessed part H is about 0.5 mm.

  The electrode 2a is disposed inside the housing 10 and in the vicinity of the gas inlet 10i. The electrode 2b is disposed inside the housing portion 10 and in the vicinity of the gas ejection port 10o. Therefore, in the plasma generator P10, gas is introduced from the opposite side of the facing surface of the electrode 2a, and the gas is ejected to the opposite side of the facing surface of the electrode 2b. The distance between the electrodes 2a and 2b is 20 cm or more and 25 cm or less. The distance between the electrodes 2a and 2b may be other distances.

  The voltage application unit 3 is for applying an alternating voltage between the electrodes 2a and 2b. The voltage application unit 3 boosts the voltage to 9 kV using 60 Hz and 100 V, which are commercial AC voltages, and applies a voltage between the electrodes 2 a and 2 b.

  When argon is introduced from the gas inlet 10 i and a voltage is applied between the electrodes 2 a and 2 b by the voltage application unit 3, plasma is generated inside the housing unit 10. FIG. A region where plasma is generated is defined as a plasma generation region P as indicated by the hatched line A in FIG. The plasma generation region P is covered with the casing unit 10.

1-3. Second plasma generator FIG. A is a sectional view showing a schematic configuration of the plasma generator P20. Here, the plasma generator P20 is a second plasma generator that ejects plasma linearly. FIG. B is shown in FIG. It is a fragmentary sectional view in the cross section perpendicular | vertical to the longitudinal direction of the plasma area | region P of the plasma generator P20 of A.

The plasma generating apparatus P20 includes a casing unit 11, electrodes 2a and 2b, and a voltage application unit 3. The casing 11 is made of a sintered body using alumina (Al ₂ O ₃ ) as a raw material. At both ends of the housing portion 11, a gas introduction port 11 i and a large number of gas ejection ports 11 o are formed. The gas inlet 11i is shown in FIG. It has a slit shape with the left-right direction of A as the longitudinal direction. The slit width (the width in the left-right direction in FIG. 3.B) from the gas inlet 11i to just above the plasma region P is 1 mm.

  The gas ejection port 11o is an irradiation unit for irradiating the outside of the casing unit 11 with plasma. The gas ejection port 11o has a cylindrical shape or a slit shape. The gas outlet 11o in the case of a cylindrical shape is formed in a straight line along the longitudinal direction of the plasma region. The inner diameter of the gas ejection port 11o is in the range of 1 mm to 2 mm. In the case of a slit shape, the slit width of the gas ejection port 11o is preferably 1 mm or less. Thereby, a stable plasma is formed. The gas inlet 11i introduces gas in a direction intersecting with a line connecting the electrode 2a and the electrode 2b.

  The electrodes 2a and 2b and the voltage application unit 3 are the same as those in the plasma generator P10 shown in FIG. Similarly, a voltage is applied between the electrodes 2a and 2b using a commercial AC voltage. Thereby, plasma can be ejected in a straight line.

  A plasma generator P20 for ejecting plasma in a straight line is shown in FIG. If arranged in a line in the left-right direction of B, the plasma can be ejected in a plane over a rectangular region.

2. 2. Plasma generated by plasma generator 2-1. The first plasma generator and the second plasma generator The plasma generated by the plasma generators P10 and P20 is non-equilibrium atmospheric pressure plasma. Here, atmospheric pressure plasma refers to plasma having a pressure in the range of 0.5 to 2.0 atmospheres.

  In this embodiment, Ar gas is mainly used as the plasma generating gas. Of course, electrons and Ar ions are generated in the plasma generated by the plasma generators P10 and P20. Ar ions generate ultraviolet rays. Further, since this plasma is released into the atmosphere, it generates radicals derived from molecules in the atmosphere.

The plasma density of this plasma is in the range of 1 × 10 ¹⁴ cm ^{−3 to} 1 × 10 ¹⁷ cm ⁻³ . The plasma density in the plasma generated by the dielectric barrier discharge is about 1 × 10 ¹¹ cm ^{−3 to} 1 × 10 ¹³ cm ⁻³ . Therefore, the plasma density of the plasma generated by the plasma generators P10 and P20 is about three orders of magnitude higher than the plasma density of the plasma generated by the dielectric barrier discharge. Therefore, more Ar ions are generated inside the plasma. Therefore, the amount of radicals and ultraviolet rays is also large. This plasma density is approximately equal to the electron density inside the plasma.

And the plasma temperature at the time of this plasma generation is in the range of about 1000K to 2500K. Moreover, the electron temperature in this plasma is larger than the gas temperature. Moreover, although the electron density is in the range of 1 × 10 ¹⁴ cm ^{−3 to} 1 × 10 ¹⁷ cm ⁻³ , the gas temperature is in the range of about 1000 K to 2500 K. The temperature of this plasma is the temperature in the plasma region P where plasma is generated. Therefore, the plasma temperature at the position of the liquid level can be set to about room temperature by making the conditions of the plasma and the distance from the gas outlet to the liquid level different.

3. Antitumor aqueous solution (plasma culture solution)
The anti-tumor aqueous solution of this embodiment is an aqueous solution obtained by irradiating a culture solution with non-equilibrium atmospheric pressure plasma. This antitumor aqueous solution is one in which hydrogen peroxide (H ₂ O ₂ ) and nitrite ions (NO ₂ ⁻ ) are generated in the culture solution by irradiating the culture solution with non-equilibrium atmospheric pressure plasma. This plasma culture solution has an antitumor effect.

4). 2. Manufacturing method of anti-tumor aqueous solution There are two types of manufacturing methods of the anti-tumor aqueous solution of this embodiment. Therefore, each method will be described.

4-1. Method for producing antitumor aqueous solution (first method)
4-1-1. Aqueous solution preparation step (first method)
First, the first method will be described. A commercially available culture solution is prepared as an aqueous solution. Alternatively, a culture solution in which a culture component is added to pure water is prepared.

4-1-2. Plasma irradiation process (first method)
Next, the culture solution is irradiated with atmospheric pressure plasma generated in the plasma generation region by the plasma generators P10 and P20 described above. The distance between the liquid level and the plasma jet outlet when the plasma is irradiated is, for example, 1 cm. Further, this distance may be changed within a range of 0.5 cm to 3 cm. Table 1 shows the plasma conditions. As a result, hydrogen peroxide and nitrite ions are generated in the culture solution.

[Table 1]
Conditions Numerical range Liquid surface-jet distance 0.5 cm or more 3 cm or less Plasma density 1 × 10 ¹⁴ cm ⁻³ or more 1 × 10 ¹⁷ cm ⁻³ or less Plasma temperature 1000 K or more 2500 K or less Oxygen radical density 2 × 10 ¹⁴ cm ⁻³ or more 1.6 × 10 ¹⁵ cm ^-3 or less

4-2. Method for producing antitumor aqueous solution (second method)
4-2-1. Aqueous solution preparation process (second method)
Here, among the culture components, disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium hydrogen carbonate (NaHCO ₃ ), L-glutamine (L-Glutamine), L-histidine (L-histidine), An aqueous solution is prepared by adding a solute containing at least one of L-tyrosine disodium dihydrate (L-tyrosine · 2Na · 2H ₂ O) to water.

4-2-2. Plasma irradiation process (second method)
Next, the aqueous solution is irradiated with the atmospheric pressure plasma generated in the plasma generation region by the above-described plasma generators P10 and P20. Various conditions for plasma irradiation are the same as in the first method. As a result, hydrogen peroxide and nitrite ions are generated in the aqueous solution.

4-2-3. Culture component addition step (second method)
Next, a culture component is added to the aqueous solution irradiated with atmospheric pressure plasma. Thereby, an antitumor aqueous solution in which hydrogen peroxide and nitrite ions are present in the culture solution is produced.

5. Summary of this embodiment As described in detail above, the anti-tumor aqueous solution of this embodiment is an aqueous solution that kills cancer cells. This anti-tumor aqueous solution contains a culture solution, hydrogen peroxide, and nitrite ions. In addition, the presence of hydrogen peroxide and nitrite ions in the culture medium provides a synergistic effect between hydrogen peroxide and nitrite ions.

1. Experiment A (hydrogen peroxide and nitrite ions generated by plasma irradiation)
FIG. 4 is a table showing the concentrations of hydrogen peroxide and nitrite ions generated in the culture solution when the culture solution is irradiated with plasma. As the nitrite ion water, an aqueous sodium nitrite solution (manufactured by Wako Pure Chemical Industries, manufacturer code 192-12565) was used. As shown in FIG. 4, when the culture solution is irradiated with plasma for 30 seconds, the concentration of hydrogen peroxide (H ₂ O ₂ ) is 11 μM, and the concentration of nitrite ion (NO ₂ ⁻ ) is 104 μM. Met. When the culture solution was irradiated with plasma for 60 seconds, the concentration of hydrogen peroxide (H ₂ O ₂ ) was 21 μM, and the concentration of nitrite ions (NO ₂ ⁻ ) was 207 μM. When the culture solution was irradiated with plasma for 180 seconds, the concentration of hydrogen peroxide (H ₂ O ₂ ) was 64 μM, and the concentration of nitrite ions (NO ₂ ⁻ ) was 621 μM. When the culture solution was irradiated with plasma for 300 seconds, the concentration of hydrogen peroxide (H ₂ O ₂ ) was 106 μM, and the concentration of nitrite ions (NO ₂ ⁻ ) was 1036 μM.

  FIG. 5 is a graph showing the relationship between the plasma irradiation time and the concentration of hydrogen peroxide generated in the culture solution. The horizontal axis in FIG. 5 represents the plasma irradiation time. The vertical axis in FIG. 5 is the concentration of hydrogen peroxide. As shown in FIG. 5, the concentration of hydrogen peroxide is proportional to the plasma irradiation time. Moreover, when the culture solution contains FBS, the concentration of hydrogen peroxide slightly decreases. That is, FBS captures a portion of the hydrogen peroxide.

  FIG. 6 is a graph showing the relationship between the plasma irradiation time and the concentration of nitrite ions generated in the culture solution. The horizontal axis in FIG. 6 is the plasma irradiation time. The vertical axis in FIG. 6 is the concentration of nitrite ions. As shown in FIG. 6, the concentration of nitrite ions is proportional to the plasma irradiation time. The presence or absence of FBS in the culture does not affect the concentration of nitrite ions.

2. Experiment B (Type of anti-tumor aqueous solution and anti-tumor effect)
2-1. Experimental Method In the experiment, five types of culture solutions shown in Table 2 were prepared. Here, two types of culture broth (b) were produced: one that was irradiated with plasma for 90 seconds and one that was irradiated for 180 seconds. Based on FIG. 4, the culture solution (c)-(e) was added with hydrogen peroxide and nitrite ions corresponding to the case where the plasma was irradiated for 90 seconds and the case where it was irradiated for 180 seconds. In other words, two types of culture solution (c) were prepared: a culture solution containing 32 μM hydrogen peroxide corresponding to 90 seconds and a culture solution containing 64 μM hydrogen peroxide corresponding to 180 seconds. Similarly, two types of culture solutions (d) and (e) were prepared.

  Then, glioblastoma cells were prepared as cancer cells for examining the antitumor effect. Specifically, 5000 glioblastoma cells were cultured in the culture solution. Next, the culture solution was replaced with the five types of culture solutions shown in Table 2. And each sample was cultured for 24 hours using five types of culture solutions. Thereafter, viable glioblastoma cells were counted by MTS assay.

[Table 2]
Type of culture solution Properties of culture solution Culture solution (a) Culture solution (no plasma irradiation, no addition)
Culture solution (b) Culture solution (90 seconds or 180 seconds plasma irradiation)
Culture solution (c) Culture solution (hydrogen peroxide added)
Culture solution (d) Culture solution (hydrogen peroxide and nitrite ion added)
Culture solution (e) Culture solution (Nitrite ion added)

2-2. Experimental Results FIG. 7 is a graph showing the relationship between the culture solution irradiated with plasma for 90 seconds and the corresponding culture solution and the viability of glioblastoma cells. As shown in FIG. 7, the normal culture solution (a) which was not irradiated with plasma and not added with hydrogen peroxide or the like did not show an antitumor effect. The culture solution (b) irradiated with plasma for 90 seconds showed an antitumor effect. Of 5000 glioblastoma cells, 50% were killed. The culture solution (c) to which hydrogen peroxide was added did not show an antitumor effect. The culture solution (d) to which both hydrogen peroxide and nitrite ions were added showed an antitumor effect. Of 5000 glioblastoma cells, 50% were killed. That is, the culture solution (d) showed the same antitumor effect as the culture solution (b) irradiated with plasma for 90 seconds. The culture solution (e) to which nitrite ions were added did not show an antitumor effect.

  As described above, the culture solution (c) to which only hydrogen peroxide was added and the culture solution (e) to which only nitrite ions were added showed no antitumor effect. However, the culture solution (d) to which both hydrogen peroxide and nitrite ions were added showed an antitumor effect. That is, it became clear that hydrogen peroxide and nitrite ions show a synergistic effect in the culture solution. It was revealed that when the culture solution was added with hydrogen peroxide of 30 μM or more and nitrite ion of 300 μM or more, the culture solution showed an antitumor effect.

  FIG. 8 is a graph showing the relationship between the culture solution irradiated with plasma for 180 seconds and the corresponding culture solution and the viability of glioblastoma cells. As shown in FIG. 8, the normal culture solution (a) did not show an antitumor effect. The culture solution (b) irradiated with plasma for 180 seconds showed a very strong antitumor effect. Of the 5000 glioblastoma cells, about 98% were killed. The culture solution (c) to which hydrogen peroxide was added showed an antitumor effect. Of the 5000 glioblastoma cells, about 80% were killed. The culture solution (d) to which both hydrogen peroxide and nitrite ions were added showed a strong antitumor effect. Of the 5000 glioblastoma cells, about 90% were killed. That is, the culture solution (d) showed an antitumor effect to a degree close to that of the culture solution (b) irradiated with plasma for 180 seconds. The culture solution (e) to which nitrite ions were added did not show an antitumor effect.

  Therefore, it was also clarified that hydrogen peroxide and nitrite ions showed a synergistic effect in the culture solution. Further, it was revealed that the addition of about 621 μM of hydrogen peroxide to the culture solution shows an antitumor effect. For this reason, the concentration of hydrogen peroxide alone has an antitumor effect at 500 μM or more.

  FIG. 9 is a graph showing the relationship between plasma irradiation time and the amount of additive corresponding thereto and the viability of glioblastoma cells. The horizontal axis in FIG. 9 is the plasma irradiation time. Further, the amount of addition of hydrogen peroxide or nitrite ions generated in the culture medium when plasma is irradiated is converted to the plasma irradiation time. As shown in FIG. 9, when only nitrite ions were added alone to the culture solution, the antitumor effect was not shown regardless of the amount added. The culture solution (b) irradiated with plasma showed the strongest antitumor effect. The culture solution (d) to which both hydrogen peroxide and nitrite ions were added showed the strongest antitumor effect next to the culture solution (b). The culture solution (c) to which only hydrogen peroxide was added showed the strongest antitumor effect next to the culture solution (d).

  In FIG. 9, when the plasma irradiation time was 240 seconds or longer, no difference was observed in the culture solutions (b), (c), and (d). However, if the number of glioblastoma cells is increased, the difference between these cultures may appear.

P1 ... Plasma irradiation device M1 ... Robot arm PM ... Anti-tumor aqueous solution production device P10, P20 ... Plasma generator 10, 11 ... Case 10i, 11i ... Gas inlet 10o, 11o ... Gas outlet 2a, 2b ... Electrode P ... Plasma area H ... Recess (hollow)

Claims (7)

In an anti-tumor aqueous solution that kills cancer cells,
A culture solution;
Hydrogen peroxide,
Nitrite ions,
An antitumor aqueous solution, comprising: In the anti-tumor aqueous solution according to claim 1,
The concentration of hydrogen peroxide is
An antitumor aqueous solution, which is 30 μM or more and 100 mM or less. In the anti-tumor aqueous solution according to claim 1 or 2,
The concentration of nitrite ions is
An antitumor aqueous solution, which is 300 μM or more and 1 M or less. In the anti-tumor aqueous solution according to any one of claims 1 to 3,
An antitumor aqueous solution, wherein hydrogen peroxide and nitrite ions are generated in a culture solution by irradiating the culture solution with non-equilibrium atmospheric pressure plasma. In a method for producing an antitumor aqueous solution for killing cancer cells,
A method for producing an antitumor aqueous solution, which comprises adding a hydrogen peroxide solution and nitrite ion water to a culture solution to form a mixed solution. In the manufacturing method of the anti-tumor aqueous solution of Claim 5,
The concentration of hydrogen peroxide in the mixed solution,
The manufacturing method of the anti-tumor aqueous solution characterized by setting it as 30 to 100 mM. In the manufacturing method of the anti-tumor aqueous solution of Claim 5 or Claim 6,
The concentration of nitrite ions in the mixed solution,
300 μM or more and 1 M or less, A method for producing an antitumor aqueous solution.