Method for producing Anti-tumor aqueous solution and Anti-tumor aqueous solution

Abstract

To provide an anti-tumor aqueous solution that is produced using a biopolymer as a starting material.SOLUTION: A method for producing an anti-tumor aqueous solution comprises a step of generating a plasma-irradiated aqueous solution by irradiating a chitosan-containing aqueous solution with plasma. In the method for producing the anti-tumor aqueous solution, a gas used as a plasma generation gas for generating the plasma is selected from among Ar gas, a mixed gas of Ar and N2, a mixed gas of Ar, N2, and O2, or a mixed gas of Ar and O2. The plasma-irradiated aqueous solution thus generated is preferably diluted by means of a culture medium. The anti-tumor aqueous solution comprises glyceric acid, ethyl acetate, and tricarballylic acid.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a method for producing an antitumor aqueous solution and an antitumor aqueous solution. [Background technology]

[0002] Plasma technology is applied in the fields of electricity, chemistry, and materials. In recent years, its application to medical treatment has been actively studied. For example, research is being conducted on the production of an antitumor solution that exerts an antitumor effect by irradiating an aqueous solution containing a specific component with plasma.

[0003] Specifically, for example, Patent Document 1 discloses a method for producing an antitumor aqueous solution by irradiating plasma to an aqueous solution in which water is added to a solute containing at least one of disodium hydrogen phosphate, sodium hydrogen carbonate, L-glutamine, L-histidine, and L-tyrosine disodium dihydrate. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 128905 Summary of the Invention [Problem to be solved by the invention]

[0005] In the production of antitumor aqueous solutions using plasma, the selection of starting materials to be added to the aqueous solution before plasma irradiation is important. In Patent Document 1, inorganic compounds such as disodium hydrogen phosphate and sodium hydrogen carbonate are used as starting materials, but from the viewpoint of medical application, it is desirable to use biopolymers with excellent biocompatibility and biodegradability.

[0006] The present invention has been made in view of the above problems, and aims to provide an antitumor aqueous solution using a biopolymer as a raw material. [Means for solving the problem]

[0007] One aspect of the present invention is The method includes a step of irradiating a chitosan-containing aqueous solution containing chitosan with plasma to generate a plasma-irradiated aqueous solution, The plasma generating gas used to generate the plasma is Ar gas, a mixed gas of Ar and N2, a mixed gas of Ar, N2 and O2, or a mixed gas of Ar and O2. The present invention relates to a method for producing an antitumor aqueous solution.

[0008] Another aspect of the present invention is It is an antitumor aqueous solution containing glyceric acid, ethyl acetate, and tricarballylic acid. Effect of the Invention

[0009] The method for producing an antitumor aqueous solution has the above-mentioned configuration. Therefore, according to the method for producing an antitumor aqueous solution, an antitumor aqueous solution can be produced using chitosan, which is a biopolymer, as a raw material.

[0010] The antitumor aqueous solution has the above-mentioned composition. The antitumor aqueous solution can be obtained through the method for producing the antitumor aqueous solution using chitosan, which is a biopolymer, as a raw material.

[0011] Therefore, according to the above-mentioned method for producing an antitumor aqueous solution and the above-mentioned antitumor aqueous solution, it is possible to provide an antitumor aqueous solution using a biopolymer as a raw material. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing the state in which a chitosan-containing aqueous solution is irradiated with non-equilibrium atmospheric pressure plasma produced by a predetermined plasma generating gas in Experimental Example 1. In FIG. [Diagram 2]FIG. 2 shows photographs of the appearance of (a) an untreated chitosan-containing aqueous solution not irradiated with plasma, (b) an aqueous solution irradiated with Ar plasma, (c) an aqueous solution irradiated with Ar / N2 / O2 plasma, and (d) an aqueous solution irradiated with Ar / O2 plasma, obtained in Experimental Example 1. [Diagram 3] FIG. 3 is a graph showing the cell survival rates of the Ar plasma-irradiated aqueous solution and its diluted solution obtained in Experimental Example 1. [Figure 4] FIG. 4 is a graph showing the cell survival rates of the aqueous solution irradiated with Ar / N 2 plasma and its diluted solution, obtained in Experimental Example 1. [Diagram 5] FIG. 5 is a graph showing the cell survival rates of the aqueous solution irradiated with Ar / N2 / O2 plasma and its diluted solution, obtained in Experimental Example 1. [Figure 6] FIG. 6 is a graph showing the cell survival rates of the aqueous solution irradiated with Ar / O 2 plasma and its diluted solution, obtained in Experimental Example 1. [Figure 7] FIG. 7 shows mass spectra of glyceric acid (GA) detected in the Ar plasma-irradiated aqueous solution, the Ar / N2 plasma-irradiated aqueous solution, the Ar / N2 / O2 plasma-irradiated aqueous solution, and the Ar / O2 plasma-irradiated aqueous solution obtained in Experimental Example 2. [Figure 8] FIG. 8 shows mass spectra of ethyl acetate (EA) detected in the Ar plasma-irradiated aqueous solution, the Ar / N2 plasma-irradiated aqueous solution, the Ar / N2 / O2 plasma-irradiated aqueous solution, and the Ar / O2 plasma-irradiated aqueous solution obtained in Experimental Example 2. [Figure 9] FIG. 9 shows mass spectra of tricarballylic acid (TA) detected in the Ar plasma-irradiated aqueous solution, the Ar / N2 plasma-irradiated aqueous solution, the Ar / N2 / O2 plasma-irradiated aqueous solution, and the Ar / O2 plasma-irradiated aqueous solution obtained in Experimental Example 2. [Figure 10] FIG. 10 is a graph showing the cell viability obtained in Experimental Example 2 using a sample aqueous solution simultaneously containing three compounds, glyceric acid (GA), ethyl acetate (EA) and tricarballylic acid (TA). [Figure 11] FIG. 11 is a graph showing the cell viability obtained in Experimental Example 2 using a sample aqueous solution simultaneously containing two compounds, ethyl acetate (EA) and tricarballylic acid (TA). [Figure 12] FIG. 12 is a graph showing the cell viability obtained in Experimental Example 2 using a sample aqueous solution containing only glyceric acid (GA). [Figure 13] FIG. 13 is a graph showing the cell viability obtained in Experimental Example 2 using a sample aqueous solution containing only ethyl acetate (EA). [Figure 14] FIG. 14 is a graph showing the cell viability obtained in Experimental Example 2 using a sample aqueous solution containing only tricarballylic acid (TA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] (Embodiment 1) A description will be given of a method for producing an antitumor aqueous solution according to embodiment 1. The method for producing an antitumor aqueous solution according to this embodiment includes a step of irradiating a chitosan-containing aqueous solution with plasma to produce a plasma-irradiated aqueous solution.

[0014] The chitosan-containing aqueous solution is an aqueous solution containing chitosan. Chitosan is one of the most abundant biopolymers derived from natural chitin, which is commonly present in arthropods, crustacean shells, insects, and the exoskeleton of fungal cell walls. Chitosan has unique properties such as non-toxicity, biocompatibility, biodegradability, bioactivity, and mucoadhesiveness. Despite its unique physicochemical and biological properties, chitosan has not been widely used in the clinical field due to its low solubility and poor mechanical properties. The method for producing an antitumor aqueous solution of this embodiment uses chitosan, which is rarely used in the clinical field, as a raw material and combines it with plasma treatment technology to produce an antitumor solution with an antitumor effect that can kill cancer cells.

[0015] Specifically, the chitosan-containing solution can be composed of an aqueous solution composed of chitosan, a solvent capable of dissolving chitosan, and water. The chitosan-containing solution can be prepared, for example, by dissolving chitosan in an aqueous solution composed of a solvent capable of dissolving chitosan and water. Examples of the solvent capable of dissolving chitosan include acetic acid (including glacial acetic acid) and dimethyl sulfoxide (DMSO).

[0016] The chitosan concentration in the chitosan-containing aqueous solution may be preferably 1% by mass or less, more preferably 0.8% by mass or less, and even more preferably 0.6% by mass or less, from the viewpoints of viscosity suppression, solubility, etc. The concentration of the solvent capable of dissolving chitosan may be appropriately adjusted depending on the desired chitosan concentration.

[0017] Specifically, the plasma irradiated to the chitosan-containing aqueous solution is non-equilibrium atmospheric pressure plasma. In the method for producing an antitumor aqueous solution of this embodiment, in order to obtain an antitumor aqueous solution having an antitumor effect, the type of plasma generating gas used for generating the plasma is important. Specifically, the plasma generating gas used for generating the plasma is Ar gas, a mixed gas of Ar and N2, a mixed gas of Ar, N2 and O2, or a mixed gas of Ar and O2. From the viewpoint of effectively obtaining an antitumor effect of killing cancer cells with almost no effect on normal cells, the plasma generating gas is preferably a mixed gas of Ar and N2 or a mixed gas of Ar, N2 and O2.

[0018] In addition, the plasma irradiation device for irradiating the chitosan-containing solution with plasma, the plasma irradiation conditions other than the plasma generating gas, etc. will be exemplified in the experimental examples described later, but other known techniques described in JP 2016-169164 A, Japanese Patent No. 6099277, Japanese Patent No. 6381111, etc. may also be appropriately referred to. Furthermore, the techniques of each of these publications may be incorporated into the present disclosure as necessary.

[0019] In the method for producing an antitumor aqueous solution of this embodiment, the plasma-irradiated aqueous solution produced by irradiating a chitosan-containing aqueous solution with plasma can be diluted with a culture medium. According to this configuration, the produced plasma-irradiated aqueous solution can be used as a stock solution for the antitumor aqueous solution, and the stock solution can be diluted to obtain the antitumor aqueous solution. Also, according to this configuration, by adjusting the dilution rate, it becomes easier to obtain an antitumor effect that kills cancer cells with almost no effect on normal cells. In other words, according to this configuration, an antitumor aqueous solution having selectivity for killing only cancer cells can be relatively easily prepared.

[0020] In addition, in the method for producing an antitumor aqueous solution of this embodiment, it is also possible to use the plasma-irradiated aqueous solution produced by irradiating a chitosan-containing aqueous solution with plasma as it is without diluting it with culture medium.

[0021] When the generated plasma-irradiated aqueous solution is diluted with a culture solution, the culture solution used for dilution may be water to which culture components have been added. The culture components are components contained in the culture solution for culturing cells. Specific examples of the culture solution include DMEM and RPMI. Details of the components of DMEM are shown in the experimental examples. The pH of the culture solution may be selected from the range of 5.5 to 8.5, and may preferably be approximately 7.

[0022] The dilution ratio when the generated plasma-irradiated aqueous solution is diluted with a culture solution can be selected, for example, from the range of 1 to 64, preferably 1 to 50, more preferably 1 to 40, and even more preferably 1 to 32. The dilution ratio is preferably determined according to the type of plasma-generating gas from the viewpoint of the selectivity of the antitumor aqueous solution to cancer cells.

[0023] Specifically, when the plasma generating gas is Ar gas, it is preferable to dilute the plasma irradiated aqueous solution at a dilution ratio of 32 times or more. When the plasma generating gas is a mixed gas of Ar and N2, it is preferable to dilute the plasma irradiated aqueous solution at a dilution ratio of 4 times or more, more preferably 4 times or more and 32 times or less. When the plasma generating gas is a mixed gas of Ar, N2 and O2, it is preferable to dilute the plasma irradiated aqueous solution at a dilution ratio of 8 times or more. When the plasma generating gas is a mixed gas of Ar and O2, it is preferable to dilute the plasma irradiated aqueous solution at a dilution ratio of 32 times or more. The dilution ratios can be arbitrarily combined within the above-mentioned range.

[0024] The method for producing an antitumor aqueous solution of this embodiment has the above-mentioned configuration, and therefore, according to the method for producing an antitumor aqueous solution, a novel antitumor aqueous solution can be produced using chitosan, which is a biopolymer, as a raw material.

[0025] (Embodiment 2) The antitumor aqueous solution of the second embodiment will be described.

[0026] The antitumor aqueous solution of this embodiment contains glyceric acid (hereinafter, sometimes referred to as GA), ethyl acetate (hereinafter, sometimes referred to as EA), and tricarballylic acid (hereinafter, sometimes referred to as TA).

[0027] It is important that the antitumor aqueous solution of this embodiment simultaneously contains glyceric acid, ethyl acetate, and tricarballylic acid. In other words, an aqueous solution containing three compounds, glyceric acid, ethyl acetate, and tricarballylic acid, in combination has an antitumor effect that can kill cancer cells without affecting normal cells, and can function as an antitumor aqueous solution. If one or two of glyceric acid, ethyl acetate, and tricarballylic acid are missing, the antitumor effect cannot be obtained.

[0028] The antitumor aqueous solution of this embodiment may contain components derived from the chitosan-containing solution or culture medium (including decomposition products caused by plasma irradiation) to the extent that the antitumor effect is not impaired.

[0029] In the antitumor aqueous solution of this embodiment, the total concentration of GA, EA and TA can be, for example, from 0.04 mM to 40 mM, preferably from the viewpoint of cell viability of normal cells, from 0.2 mM to 40 mM, more preferably from 0.4 mM to 40 mM, even more preferably from 2 mM to 40 mM, and even more preferably from the viewpoint of the selectivity of the antitumor aqueous solution for cancer cells, from 4 mM to 40 mM.

[0030] In the antitumor aqueous solution of this embodiment, the GA concentration can be, for example, 0.01 mM or more and 10 mM or less, the EA concentration can be, for example, 0.02 mM or more and 20 mM or less, and the TA concentration can be, for example, 0.01 mM or more and 10 mM or less.

[0031] The antitumor aqueous solution of this embodiment can be obtained through the method for producing an antitumor aqueous solution of this embodiment using chitosan, which is a biopolymer, as a raw material.

[0032] For other configurations and effects, the explanation of the method for producing the antitumor aqueous solution in embodiment 1 can be referred to as appropriate.

[0033] The method for producing the antitumor aqueous solution and the antitumor aqueous solution of this embodiment will be described in more detail below using experimental examples.

[0034] (Experimental Example 1) 1.1 Preparation of plasma-irradiated aqueous solution A chitosan-containing aqueous solution was prepared by mixing 0.5% by mass of chitosan (Sigma Aldrich, low molecular weight, poly(D-glucosamine), deacetylated chitin) with a 1% by mass aqueous solution of acetic acid.

[0035] Next, as shown in Figure 1, 8 mL of chitosan-containing aqueous solution was irradiated with non-equilibrium atmospheric pressure plasma by a specific plasma generating gas using a beta-plasma irradiation device (manufactured by FUJI Co., Ltd.) to obtain four types of plasma-irradiated aqueous solutions. The plasma irradiation conditions were as follows. Plasma irradiation width: 0.3mm x 20mm Plasma generation gas and flow rate Ar gas 1slm A mixture of Ar gas and N2 gas (hereinafter referred to as Ar / N2) Ar / N20.9slm / 0.1slm Mixture of Ar, N2 and O2 (hereinafter referred to as Ar / N2 / O2) Ar / N2 / O20.8slm / 0.1slm / 0.1slm A mixture of Ar gas and O2 gas (hereinafter referred to as Ar / O2) Ar / O20.9slm / 0.1slm Applied voltage: 9kV Frequency: 9kHz Plasma irradiation distance: 5mm Plasma exposure time: 5 minutes

[0036] In the above, the plasma-irradiated aqueous solution prepared by using Ar gas as the plasma generating gas is called Ar plasma-irradiated aqueous solution. Similarly, the plasma-irradiated aqueous solution prepared by using a mixed gas of Ar gas and N2 gas as the plasma generating gas is called Ar / N2 plasma-irradiated aqueous solution. The plasma-irradiated aqueous solution prepared by using a mixed gas of Ar, N2, and O2 as the plasma generating gas is called Ar / N2 / O2 plasma-irradiated aqueous solution. The plasma-irradiated aqueous solution prepared by using a mixed gas of Ar gas and O2 gas as the plasma generating gas is called Ar / O2 plasma-irradiated aqueous solution.

[0037] Figure 2 shows the appearance of an untreated chitosan-containing solution that was not irradiated with plasma, an aqueous solution irradiated with Ar plasma, an aqueous solution irradiated with Ar / N2 / O2 plasma, and an aqueous solution irradiated with Ar / O2 plasma. As shown in Figure 2, the color of the liquid changed from transparent (slightly translucent) to yellow by irradiating the chitosan-containing solution with plasma. The aqueous solution irradiated with Ar / O2 plasma exhibited the most intense yellow color through the oxidation process by Ar / O2 plasma. From these results, it is inferred that the structure of chitosan changes and new compounds are generated by irradiating the chitosan-containing solution with plasma.

[0038] In addition, the same tendency as above was obtained when a chitosan-containing aqueous solution prepared by mixing 1% by mass of chitosan was used. When the chitosan content was 1% by mass, the viscosity of the chitosan-containing aqueous solution increased. Therefore, in the following, from the viewpoint of handling, etc., the antitumor effect was confirmed for each of the above plasma-irradiated aqueous solutions using a chitosan-containing aqueous solution containing 0.5% by mass of chitosan.

[0039] 1.2 Preparation of normal and cancer cells MCF-10A cells, which are human mammary epithelial cells, were prepared as normal cells, and MCF-7 cells, which are human breast cancer cells, were prepared as cancer cells.

[0040] 1.3 Normal and cancer cell cultures MCF-10A cells were cultured in a 96-well plate to prepare a normal cell culture medium. MCF-7 cells were cultured in a 96-well plate to prepare a cancer cell culture medium. The culture medium used was a mixture of DMEM, serum (FBS), and antibiotics (penicillin and streptomycin). The number of cells cultured was 5,000 per sample. The culture period for each cell was 24 hours.

[0041] The components of the DMEM are calcium chloride, ferric nitrate·9H2O, magnesium sulfate (anhydrous), potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate (anhydrous), L-arginine·HCl, L-cystine·2HCl, L-glutamine, glycine, L-histidine·HCl·H2O, L-isoleucine, L-leucine, L-lysine·HCl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine·2Na·2H2O, L-valine, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid, pyridoxine·HCl, riboflavin, thiamine·HCl, D-glucose, and phenol red·Na.

[0042] 1.4 Confirmation of antitumor effects of various aqueous solutions As antitumor aqueous solutions, we prepared each plasma-irradiated aqueous solution (Treat) as it was (not diluted with DMEM), and each plasma-irradiated aqueous solution (Treat) was diluted with DMEM at a stepwise dilution rate from 1 to 32 times to prepare each plasma-irradiated aqueous solution (1x, 2x, 4x, 8x, 16x, 32x). In this experimental example, an untreated chitosan-containing solution (Untreat) that was not irradiated with plasma was used as the reference aqueous solution. In addition, a normal cell culture medium containing 5,000 cells cultured in DMEM and a cancer cell culture medium were used as controls (Ctrl).

[0043] The culture medium in the 96-well plate in which MCF-10A cells and MCF-7 cells were cultured was replaced with the above-mentioned untreated chitosan-containing solution (Untreat), each plasma-irradiated aqueous solution as prepared (Treat), or each plasma-irradiated aqueous solution diluted 1 to 32 times with DMEM (1x, 2x, 4x, 8x, 16x, 32x), and cultured for 2 hours. After this 2-hour culture, the above-mentioned various aqueous solutions were removed from the 96-well plate and replaced with only DMEM, and then cultured for 24 hours. Then, for each sample, the surviving MCF-10A cells or MCF-7 cells were counted by MTS assay to determine the cell viability.

[0044] The experimental results are shown in Figures 3 to 6. In Figures 3 to 6, the horizontal axis indicates the type of sample used, and the vertical axis indicates the cell viability. The p-value in the figure indicates a value related to a statistically significant difference. As shown in Figures 3 to 6, the Ar plasma irradiated aqueous solution, the Ar / N2 plasma irradiated aqueous solution, the Ar / N2 / O2 plasma irradiated aqueous solution, the Ar / O2 plasma irradiated aqueous solution, and their diluted solutions can all kill cancer cells and have an antitumor effect. Therefore, according to the present disclosure, it can be seen that a novel antitumor aqueous solution can be obtained by using chitosan, a biopolymer, as a raw material and subjecting it to plasma irradiation treatment.

[0045] In addition, since glacial acetic acid is used to solubilize chitosan in the MTS assay, it is considered that the chitosan-containing solution that was not treated with plasma irradiation (Untreat) completely killed all types of cells.

[0046] Here, in the treatment of cancer, it is preferable to kill cancer cells while not affecting normal cells. In other words, it is preferable to kill only cancer cells. Even if it is possible to kill cancer cells, killing a large number of normal cells would be a great physical burden on the patient. Therefore, we will consider below whether Ar plasma irradiated aqueous solution, Ar / N2 plasma irradiated aqueous solution, Ar / N2 / O2 plasma irradiated aqueous solution, and Ar / O2 plasma irradiated aqueous solution can kill cancer cells with almost no effect on normal cells.

[0047] As shown in Figure 3, the Ar plasma irradiated aqueous solution showed good selectivity to kill only cancer cells in samples diluted 32 times with culture medium. Also, as shown in Figure 4, the Ar / N2 plasma irradiated aqueous solution showed good selectivity to kill only cancer cells in a wide range of diluted samples (samples diluted 4 times, 8 times, 16 times, and 32 times with culture medium). In Figure 4, the significant difference was p<0.01 in the 8-fold diluted sample and the 16-fold diluted sample, and the significant difference was p<0.05 in the 4-fold diluted sample and the 32-fold diluted sample. Also, as shown in Figure 5, the Ar / N2 / O2 plasma irradiated aqueous solution showed good selectivity to kill only cancer cells in a relatively wide range of diluted samples (samples diluted 8 times, 16 times, and 32 times with culture medium). In Figure 5, the significant difference was p<0.05 in the 8-fold diluted sample and the 16-fold diluted sample, and the significant difference was p<0.1 in the 32-fold diluted sample. Furthermore, as shown in FIG. 6, the Ar / O2 plasma-irradiated aqueous solution exhibited selectivity for killing only cancer cells in a sample diluted 32-fold with culture medium.

[0048] In addition, according to Figures 3 to 6, when the plasma generating gas contains N2, it is possible to protect normal cells and kill cancer cells at the same time with a relatively low dilution rate. This shows that the nitrogen component in the plasma generating gas plays an important role in the survival rate of normal cells. From the above, it was confirmed that by using a mixed gas of Ar and N2, or a mixed gas of Ar, N2, and O2 as the plasma generating gas, it is possible to effectively obtain an antitumor effect that kills cancer cells with almost no effect on normal cells.

[0049] (Experimental Example 2) This experimental example is an experiment to identify anticancer substances that exhibit antitumor effects for the Ar plasma-irradiated aqueous solution, Ar / N2 plasma-irradiated aqueous solution, Ar / N2 / O2 plasma-irradiated aqueous solution, and Ar / O2 plasma-irradiated aqueous solution, which are the antitumor aqueous solutions prepared in Experimental Example 1. In this experimental example, an untreated chitosan-containing solution (Untreat) that was not irradiated with plasma was used as a control (Ctrl).

[0050] 2.1 Mass spectrometry of various aqueous solutions Mass spectrometry was performed on the above various aqueous solutions. The results are shown in Figures 7 to 9. As shown in Figures 7 to 9, it was identified that each plasma-irradiated aqueous solution obtained by irradiating a chitosan-containing solution with Ar plasma, Ar / N2 plasma, Ar / N2 / O2 plasma, or Ar / O2 plasma contained at least glyceric acid, ethyl acetate, and tricarballylic acid. Therefore, in the following, the possibility of these compounds generated in each plasma-irradiated aqueous solution as anticancer substances was investigated.

[0051] 2.2 Investigation of the possibility of compounds produced in plasma-irradiated aqueous solutions as anticancer substances 2.2.1 Confirmation of the antitumor effects of glyceric acid (GA), ethyl acetate (EA) and tricarballylic acid (TA) Glyceric acid (GA), ethyl acetate (EA) and tricarballylic acid (TA) were prepared in a lactate aqueous solution so as to have a predetermined mM ratio, and thus a sample aqueous solution containing GA, EA and TA in a predetermined mM ratio was obtained. In this experimental example, the total concentration of GA, EA and TA was set to 0.04 mM or more and 40 mM or less. In addition, a normal cell culture medium containing 5000 cells cultured in DMEM and a cancer cell culture medium were used as controls (Ctrl).

[0052] Next, in the same manner as in Experimental Example 1, the culture medium in the 96-well plate in which MCF-10A cells and MCF-7 cells were cultured was replaced with the above-prepared sample aqueous solution, and the cells were cultured for 2 hours. After the 2-hour culture, the above-mentioned various aqueous solutions were removed from the 96-well plate, and the cells were replaced with only DMEM, and then cultured for 24 hours. Then, for each sample, the number of surviving MCF-10A cells or MCF-7 cells was counted by MTS assay, and the cell survival rate was calculated.

[0053] The experimental results are shown in FIG. 10. In FIG. 10, the horizontal axis represents the mM ratio of glyceric acid (GA):ethyl acetate (EA):tricarballylic acid (TA), and the vertical axis represents the cell viability. As shown in FIG. 10, the cell viability of normal cells increased up to about 150% at lower GA, EA, and TA concentrations (0.05:0.1:0.05 mM to 0.1:0.2:0.1 mM). When the GA, EA, and TA concentrations were increased (GA:EA:TA=1:2:1 mM and 5:10:5 mM), good selectivity for killing only cancer cells was observed.

[0054] 2.2.2 Confirmation of the antitumor effects of ethyl acetate (EA) and tricarballylic acid (TA) The cell viability of MCF-10A cells and MCF-7 cells was determined in the same manner as in "2.2.1" above, except that glyceric acid (GA) was not used, and instead ethyl acetate (EA) and tricarballylic acid (TA) were prepared in a lactate aqueous solution so as to have a predetermined mM ratio, thereby obtaining a sample aqueous solution containing EA and tricarballylic acid (TA) in a predetermined mM ratio.

[0055] The experimental results are shown in FIG. 11. In FIG. 11, the horizontal axis represents the mM ratio of ethyl acetate (EA):tricarballylic acid (TA), and the vertical axis represents the cell viability. As shown in FIG. 11, the sample aqueous solution of EA:TA=1:1 mM showed slight selectivity in killing cancer cells. However, this result cannot be said to have a sufficient antitumor effect of killing cancer cells with almost no effect on normal cells, compared to the sample aqueous solution containing the three compounds GA, EA, and TA simultaneously.

[0056] 2.2.3 Confirmation of the antitumor effect of glyceric acid (GA) The cell viability of MCF-10A cells and MCF-7 cells was determined in the same manner as in "2.2.1" above, except that ethyl acetate (EA) and tricarballylic acid (TA) were not used, and glyceric acid (GA) was prepared at a specified content (mM) in a lactate aqueous solution to obtain sample aqueous solutions containing different concentrations of GA.

[0057] The experimental results are shown in Figure 12. In Figure 12, the horizontal axis represents mM of glyceric acid (GA), and the vertical axis represents cell viability. As shown in Figure 12, in the sample aqueous solution containing only GA, the cell viability increased, and no antitumor effect of killing cancer cells was observed.

[0058] 2.2.4 Confirmation of the antitumor effect of ethyl acetate (EA) The cell viability of MCF-10A cells and MCF-7 cells was determined in the same manner as in "2.2.1" above, except that glyceric acid (GA) and tricarballylic acid (TA) were not used, and ethyl acetate (EA) was prepared at a specified content (mM) in a lactate aqueous solution to obtain sample aqueous solutions containing EA at different concentrations.

[0059] The experimental results are shown in Figure 13. In Figure 13, the horizontal axis represents mM of ethyl acetate (EA), and the vertical axis represents cell viability. As shown in Figure 13, the sample aqueous solution containing only EA does not have a cytotoxic effect that kills cancer cells, and no antitumor effect was observed.

[0060] 2.2.5 Confirmation of the antitumor effect of tricarballylic acid (TA) The cell viability of MCF-10A cells and MCF-7 cells was determined in the same manner as in "2.2.1" above, except that glyceric acid (GA) and ethyl acetate (EA) were not used, and tricarballylic acid (TA) was prepared at a specified content (mM) in a lactate aqueous solution to obtain sample aqueous solutions containing different concentrations of TA.

[0061] The experimental results are shown in FIG. 14. In FIG. 14, the horizontal axis represents mM of tricarballylic acid (TA), and the vertical axis represents cell viability. As shown in FIG. 14, the sample aqueous solution containing only TA completely killed both normal cells and cancer cells at a TA concentration of more than 0.5 mM. However, at a TA concentration of 0.5 mM or less, the cell viability of cancer cells actually increased. Therefore, it was determined that the sample aqueous solution containing only TA had no antitumor effect.

[0062] According to the above results, it was confirmed that an aqueous solution simultaneously containing glyceric acid, ethyl acetate and tricarballylic acid, i.e., an aqueous solution using the three compounds glyceric acid, ethyl acetate and tricarballylic acid in combination, has an antitumor effect that can kill cancer cells without affecting normal cells, and can function as an antitumor aqueous solution. It was also confirmed that this antitumor aqueous solution can be obtained by the above-mentioned method for producing an antitumor aqueous solution, which is to irradiate a chitosan-containing aqueous solution with Ar plasma, Ar / N2 plasma, Ar / N2 / O2 plasma or Ar / O2 plasma.

[0063] The present invention is not limited to the above-mentioned embodiment and the above-mentioned experimental example, and various modifications are possible within the scope of the gist of the present invention. Furthermore, the configurations shown in the above-mentioned embodiment and the above-mentioned experimental example can be arbitrarily combined with each other. Furthermore, the claims described in the claims at the time of filing can be arbitrarily combined with each other.

Claims

1. The method includes a step of irradiating a chitosan-containing aqueous solution containing chitosan with plasma to generate a plasma-irradiated aqueous solution, The plasma generating gas used to generate the plasma is Ar gas, Ar and N ２ A mixture of Ar and N ２ and O ２ or a mixture of Ar and O ２ It is a mixed gas of A method for producing an antitumor aqueous solution.

2. The plasma-irradiated aqueous solution thus produced is diluted with a culture medium. A method for producing the antitumor aqueous solution according to claim 1.

3. When the plasma generating gas is Ar gas, the plasma irradiation aqueous solution is diluted at a dilution ratio of 32 times or more; The plasma generating gas is Ar and N ２ When the mixed gas is a gas containing 1,000 to 1,000 times the plasma irradiation aqueous solution, the plasma irradiation aqueous solution is diluted at a dilution ratio of 4 times or more. The plasma generating gas is Ar and N ２ and O ２ When the mixed gas is a gas containing 1,000 to 1,000 times the plasma irradiation aqueous solution is diluted at a dilution rate of 8 times or more. The plasma generating gas is Ar and O ２ When the mixed gas is a gas containing 1,000,000,000, the plasma irradiation aqueous solution is diluted at a dilution ratio of 32 times or more. A method for producing the antitumor aqueous solution according to claim 2.

4. The chitosan concentration in the chitosan-containing aqueous solution is 1% by mass or less. A method for producing the antitumor aqueous solution according to any one of claims 1 to 3.

5. An antitumor aqueous solution comprising glyceric acid, ethyl acetate, and tricarballylic acid.

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