AGENT FOR PROTECTION AGAINST ATMOSPHERIC POLLUTANTS AND COMPOSITION FOR PROTECTION AGAINST ATMOSPHERIC POLLUTANTS

Abstract

An agent for protection against atmospheric pollutants containing a tocopherol phosphate ester or a salt thereof as an active ingredient is provided. A composition for protection against atmospheric pollutants containing the above-mentioned agent for protection against atmospheric pollutants and a pharmaceutically acceptable carrier is further provided.

Claims

1. An agent for protection against atmospheric pollutants comprising a tocopherol phosphate ester or a salt thereof as an active ingredient.

2. The agent for protection against atmospheric pollutants according to claim 1, wherein the tocopherol phosphate ester is an α-tocopherol phosphate ester.

3. The agent for protection against atmospheric pollutants according to claim 2, wherein the salt of the tocopherol phosphate ester is a sodium salt of the tocopherol phosphate ester.

4. The agent for protection against atmospheric pollutants according to claim 1, wherein the agent for protection against atmospheric pollutants promotes expression of an NRF2 gene.

5. The agent for protection against atmospheric pollutants according to claim 1, wherein the agent for protection against atmospheric pollutants suppresses production of active oxygen.

6. A composition for protection against atmospheric pollutants comprising: the agent for protection against atmospheric pollutants according to claim 1; and a pharmaceutically acceptable carrier.

7. The composition for protection against atmospheric pollutants according to claim 6, wherein a total content of the tocopherol phosphate ester or the salt thereof is 0.1% to 2% by mass.

8. A method for using the composition for protection against atmospheric pollutants according to claim 6, the method comprising using the composition for protection against atmospheric pollutants by spraying the composition on a body surface.

9. A method for using the composition for protection against atmospheric pollutants according to claim 6, the method comprising using the composition for protection against atmospheric pollutants by applying the composition to a mucous membrane.

10. The agent for protection against atmospheric pollutants according to claim 3, wherein the agent for protection against atmospheric pollutants promotes expression of an NRF2 gene.

11. The agent for protection against atmospheric pollutants according to claim 3, wherein the agent for protection against atmospheric pollutants suppresses production of active oxygen.

12. A composition for protection against atmospheric pollutants comprising: the agent for protection against atmospheric pollutants according to claim 3; and a pharmaceutically acceptable carrier.

13. The composition for protection against atmospheric pollutants according to claim 12, wherein a total content of the tocopherol phosphate ester or the salt thereof is 0.1% to 2% by mass.

14. A method for using the composition for protection against atmospheric pollutants according to claim 12, the method comprising using the composition for protection against atmospheric pollutants by spraying the composition on a body surface.

15. A method for using the composition for protection against atmospheric pollutants according to claim 12, the method comprising using the composition for protection against atmospheric pollutants by applying the composition to a mucous membrane.

Description

EXAMPLES

[0112] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[0113] In the following experimental examples, as the following Na tocopherol phosphate, TPNa (registered trademark) (manufactured by Showa Denko K.K.), which is sodium dl-α-tocopheryl phosphate, was used.

Experimental Example 1

[0114] (Effect of Promoting Expression of NRF2 Gene)

[0115] The effect of Na tocopherol phosphate on promoting the expression of an NRF2 gene in normal human epidermal keratinocytes (NHEK, manufactured by KURABO INDUSTRIES LTD.) was measured under the following conditions.

[0116] The NHEK cells were seeded in a HuMedia KG2 medium manufactured by KURABO INDUSTRIES LTD. at the seeding density of 10000 cells/cm.sup.2, and cultured for 24 hours under the conditions of 37° C. and 5% CO.sub.2. Next, a sample, in which Na tocopherol phosphate or tocopherol acetate was dissolved in an aqueous solution of 0.05% (V/V) ethanol, was added to the culture medium so that the final concentration of Na tocopherol phosphate or tocopherol acetate was 10 μM, and culturing was performed for 24 hours. Furthermore, one obtained by adding 5 μL of the aqueous solution of 0.05% (V/V) ethanol to the culture medium and culturing for 24 hours was also prepared. Thereafter, 0.1 mL of a DMSO solution of atmospheric dust (MST 1648a) was added per 100 mL of the culture medium so that the final concentration of the atmospheric dust in the culture medium was 500 μg/mL, and culturing was further performed for 48 hours. Thereafter, the NHEK cells were recovered to extract total RNA using Nucleospin™ RX (Takara Bio Inc.). From the obtained RNAs, cDNA was synthesized using a PrimeScript (registered trademark) RT Master Mix (Takara Bio Inc.). Using this cDNA as a template, the expression level of the NRF2 gene was quantitatively determined by quantitative real-time PCR using a primer specific to the NRF2 gene (Perfect Real Time Primer, manufactured by Takara Bio Inc.). As an internal standard gene, the expression level of GAPDH was quantitatively determined (primer used: Perfect Real Time Primer, manufactured by Takara Bio Inc.), and the expression level of the NRF2 gene was standardized based on the expression level of GAPDH. One to which atmospheric dust was not added was used as a control.

[0117] Table 1 shows the results. Table 1 shows the expression level of the NRF2 gene in the atmospheric dust-added group as a relative expression level when the expression level of the NRF2 gene in the control to which atmospheric dust was not added was 1. In the Na tocopherol phosphate-added group, the expression of the NRF2 gene was promoted as compared to the 0.05% ethanol-added group and the tocopherol acetate-added group. From these results, it was confirmed that the Na tocopherol phosphate has the effect of restoring the expression of the NRF2 gene suppressed due to atmospheric dust.

TABLE-US-00001 TABLE 1 Relative gene expression level Sample NRF2 Atmospheric 0.05% Ethanol 1.00 dust not added Atmospheric 0.05% Ethanol 0.67 dust added Na tocopherol 0.94 phosphate Tocopherol acetate 0.71

Experimental Example 2

[0118] (Effect of Suppressing ROS Production)

[0119] The effect of Na tocopherol phosphate on suppressing ROS production in normal human epidermal keratinocytes (NHEK, manufactured by KURABO INDUSTRIES LTD.) was measured under the following conditions.

[0120] The NHEK cells were seeded in a HuMedia KG2 medium manufactured by KURABO INDUSTRIES LTD. at the seeding density of 10000 cells/cm.sup.2, and cultured for 24 hours under the conditions of 37° C. and 5% CO.sub.2. Next, a sample, in which Na tocopherol phosphate or tocopherol acetate was dissolved in an aqueous solution of 0.05% (V/V) ethanol, was added to the culture medium so that the final concentration of Na tocopherol phosphate or tocopherol acetate was 10 μM, and culturing was performed for 24 hours under the conditions of 37° C. and 5% CO.sub.2. Furthermore, one obtained by adding 1 μL of the aqueous solution of 0.05% (V/V) ethanol to the culture medium and culturing for 24 hours was also prepared. Thereafter, 0.1 mL of a DMSO solution of atmospheric dust (NIST 1648a) was added per 100 mL of the culture medium so that the final concentration of the atmospheric dust in the culture medium was 500 μg/mL, and culturing was further performed for 48 hours. Thereafter, the ROS production amount was measured using a ROS assay kit (manufactured by OZ BIOSCIENCES). After washing the cells from which the culture medium was removed with a phosphate buffer solution (PBS, manufactured by FUJIFILM Wako Pure Chemical Corporation), 100 μL of dichlorofluorescein diacetate attached to the ROS assay kit was added to each group of the cells, and the cells were left to stand at 37° C. for 30 minutes while being shielded from light. After washing with PBS again, 100 μL of PBS was added, and the fluorescence intensity at an excitation wavelength of 485 nm/an absorption wavelength of 535 nm was measured with a microplate reader i-Control (manufactured by Tecan Group Ltd.). One to which atmospheric dust was not added was used as a control.

[0121] Table 2 shows the results. Table 2 shows the ROS production amount in the atmospheric dust-added group as a relative amount when the fluorescence intensity in the control to which atmospheric dust was not added was 1. In the Na tocopherol phosphate-added group, the ROS production amount was reduced as compared to the 0.05% ethanol-added group and the tocopherol acetate-added group. From these results, it was confirmed that the Na tocopherol phosphate has a high suppression effect on the ROS production induced by atmospheric dust.

TABLE-US-00002 TABLE 2 Relative ROS Sample production amount Atmospheric 0.05% Ethanol 1.00 dust not added Atmospheric 0.05% Ethanol 1.54 dust added Na tocopherol 1.24 phosphate Tocopherol acetate 1.67

Experimental Example 3

[0122] (Effect of Suppressing Proliferation of Cancer Cells)

[0123] The effect of Na tocopherol phosphate on suppressing the proliferation of cancer cells in a cell line derived from Lewis lung carcinoma (LLC, JCRB Cell Bank) was measured under the following conditions.

[0124] The LLC cells were seeded at the seeding density of 50000 cells/cm.sup.2 in a culture medium in which a Ham F10 medium and an L15 medium (both manufactured by Sigma-Aldrich) were mixed at the ratio of 3:7 (volume ratio), and cultured for 24 hours under the conditions of 37° C. and 5% CO.sub.2. Next, a sample, in which Na tocopherol phosphate or tocopherol acetate was dissolved in an aqueous solution of 0.05% (V/V) ethanol, was added to the culture medium so that the final concentration of Na tocopherol phosphate or tocopherol acetate was 10 μM, and culturing was performed for 24 hours. Furthermore, one obtained by adding 1 μL of the aqueous solution of 0.05% (VN) ethanol to the culture medium and culturing for 24 hours was also prepared. Thereafter, 0.1 mL of a DMSO solution of atmospheric dust (NIST 1648a) was added per 100 mL of the culture medium so that the final concentration of the atmospheric dust in the culture medium was 500 μg/mL, and culturing was further performed for 48 hours. Thereafter, the culture medium was replaced with a medium containing 10% (VN) of WST-8 of Nacalai Tesque Inc., and after further culturing for 3 hours, the absorbance at the wavelength of 450 nm was measured with a microplate reader i-Control (manufactured by Tecan Group Ltd.). One to which atmospheric dust was not added was used as a control.

[0125] Table 3 shows the results. Table 3 shows the cell proliferation amount in the atmospheric dust-added group as a relative amount when the absorbance in the control to which atmospheric dust was not added was 1. In the Na tocopherol phosphate-added group, the cell proliferation amount of cancer cells was reduced as compared to the 0.05% ethanol-added group and the tocopherol acetate-added group. From these results, it was confirmed that the Na tocopherol phosphate has a high suppression effect on the cell proliferation of cancer cells accelerating due to atmospheric dust.

TABLE-US-00003 TABLE 3 Relative cell proliferation Sample amount Atmospheric 0.05% Ethanol 1.00 dust not added Atmospheric 0.05% Ethanol 4.25 dust added Na tocopherol 2.33 phosphate Tocopherol acetate 3.98

Experimental Example 4

[0126] (Effect of Suppressing Invasion of Cancer Cells)

[0127] The effect of Na tocopherol phosphate on suppressing the invasion of cells in a cell line derived from Lewis lung carcinoma (LLC, JCRB Cell Bank) was measured under the following conditions. A CytoSelect invasion assay kit manufactured by Cell Biolabs, Inc. was used for a test.

[0128] The LLC cells were seeded in a chamber plate for invasion tests attached to the above-mentioned invasion assay kit so that the concentration was 100000 cells/mL using a culture medium in which a Ham F10 medium and an L15 medium (both manufactured by Sigma-Aldrich) were mixed at the ratio of 3:7 (volume ratio), and cultured for 24 hours under the conditions of 37° C. and 5% CO.sub.2. Next, a sample, in which Na tocopherol phosphate or tocopherol acetate was dissolved in an aqueous solution of 0.05% (V/V) ethanol, was added to the culture medium so that the final concentration of Na tocopherol phosphate or tocopherol acetate was 10 μM, and culturing was further performed for 24 hours under the conditions of 37° C. and 5% CO.sub.2. Furthermore, one obtained by adding 5 μL of the aqueous solution of 0.05% (V/V) ethanol to the culture medium and culturing for 24 hours was also prepared. Thereafter, 0.1 mL of a DMSO solution of atmospheric dust (NIST 1648a) was added per 100 mL of the culture medium so that the final concentration of the atmospheric dust in the culture medium was 500 μg/mL, and culturing was further performed for 6 hours. Next, the medium containing atmospheric dust was removed and replaced with a new medium, 0.1 mL of a DMSO solution of atmospheric dust (NIST 1648a) was added per 100 mL of the culture medium so that the final concentration of the atmospheric dust in the culture medium was 500 μg/mL, and culturing was further performed for 18 hours. Thereafter, the cells were stained using the above-mentioned invasion assay kit, and the fluorescence intensity at an excitation wavelength of 480 nm/an absorption wavelength of 570 nm was measured with a microplate reader i-Control (manufactured by Tecan Group Ltd.). One to which atmospheric dust was not added was used as a control.

[0129] Table 4 shows the results. Table 4 shows the cell invasion in the atmospheric dust-added group as a relative amount when the fluorescence intensity in the control to which atmospheric dust was not added was 1. In the Na tocopherol phosphate-added group, the cell invasion of cancer cells was reduced as compared to the 0.05% ethanol-added group and the tocopherol acetate-added group. From these results, it was confirmed that the Na tocopherol phosphate suppresses the cell invasion of cancer cells accelerating due to atmospheric dust.

TABLE-US-00004 TABLE 4 Relative cell Sample invasion Atmospheric 0.05% Ethanol 1.00 dust not added Atmospheric 0.05% Ethanol 2.53 dust added Na tocopherol 1.17 phosphate Tocopherol acetate 2.08

Prescription Examples

[0130] Prescription examples of the composition for protection against atmospheric pollutants are described below. As Na tocopherol phosphate in the following prescription examples, TPNa (registered trademark) (manufactured by Showa Denko K.K.), which is sodium dl-α-tocopheryl phosphate, is the exemplary example.

Prescription Example 1

[0131] Table 5 shows prescription examples of a spreading agent (spray).

TABLE-US-00005 TABLE 5 Prescription Component (mass %) Na tocopherol phosphate 0.2 Carboxy vinyl polymer 1 Ethyl alcohol 30 Phenoxyethanol 0.2 Water 68.6

Prescription Example 2

[0132] Table 6 shows prescription examples of a spreading agent (aerosol).

TABLE-US-00006 TABLE 6 Prescription Component (mass %) Na tocopherol phosphate 0.2 Ethyl alcohol 35 Water 10 Nitrogen gas (propellant) 54.8

Prescription Example 3

[0133] Table 7 shows prescription examples of a spreading agent (spray for skin).

TABLE-US-00007 TABLE 7 Prescription Component (mass %) Na tocopherol phosphate 0.2 Carboxy vinyl polymer 1 Ethyl alcohol 25 Titanium oxide 0.5 Sodium hyaluronate 0.5 Phenoxyethanol 0.2 Water 72.6

Prescription Example 4

[0134] Table 8 shows prescription examples of a nasal drop.

TABLE-US-00008 TABLE 8 Prescription Component (mass %) Na tocopherol phosphate 0.2 Carboxy vinyl polymer 0.5 Sodium chloride 1 Benzalkonium chloride 0.2 Ethanol 0.1 Edetic acid 0.2 Menthol Minute amounts Sorbitan sesquioleate 0.2 Water 97.1

INDUSTRIAL APPLICABILITY

[0135] According to the present invention, an agent for protection against atmospheric pollutants which can protect cells from harmful actions due to atmospheric pollutants, and a composition for protection against atmospheric pollutants which contains the above-mentioned agent for protection against atmospheric pollutants are provided.