Antioxidant enzyme production promotion apparatus

Abstract

To provide a novel antioxidant enzyme production promotion apparatus that utilizes magnetic stimulation by an alternating magnetic field.SOLUTION: An antioxidant enzyme production promotion apparatus that promotes production of antioxidant enzymes by applying magnetic stimulation to cells comprises: an electromagnetic wave generation unit that generates electromagnetic waves of an antioxidant enzyme production promotion frequency in order to apply an alternating magnetic field of the antioxidant enzyme production promotion frequency to the cells; a frequency control unit that controls the antioxidant enzyme production promotion frequency of the electromagnetic waves to be within 100 kHz to 450 MHz so that production of the antioxidant enzymes is promoted by magnetic stimulation with the alternating magnetic field of the antioxidant enzyme production promotion frequency; and an irradiation unit that irradiates the cells with the alternating magnetic field of the antioxidant enzyme production promotion frequency.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an antioxidant enzyme production promoting device that promotes the production of antioxidant enzymes by applying magnetic stimulation to cells. [Background technology]

[0002] Most of the oxygen taken into the body through breathing is consumed by the mitochondria in cells to produce ATP. During this process, some of the oxygen becomes highly oxidizing reactive oxygen species (ROS), which are thought to cause damage to genes, proteins, and other molecules in the body.

[0003] The body's antioxidant system works to eliminate reactive oxygen species. The antioxidant system is comprised of endogenous (primary) antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). (See, for example, OM Ighodaro et al., "First line defense antioxidants - superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defense grid," Alexandria Journal of Medicine 54 (2018)) 287-293 (see Non-Patent Document 1)) and exogenous (secondary) antioxidants. Antioxidants include, for example, water-soluble antioxidants (riboflavin (vitamin B2), niacin, vitamin C, folic acid, etc.), plant-derived water-soluble antioxidants (polyphenols (flavonoids, catechins, tannins, anthocyanins, isoflavones, quercetin, etc.)), other water-soluble antioxidants (maritime pine bark (pycnogenol), grape seeds (procyanidins), etc.), fat-soluble antioxidants (vitamin E, astaxanthin, coenzyme Q10 (CoQ10), etc.), fat-soluble plant pigment carotenoids (α-carotene, β-carotene, γ-carotene, lycopene, xanthophylls, etc.), amphoteric antioxidants (ginkgo leaves, α-lipoic acid, etc.), oxidative stress-related inhibitors (arginase inhibitors, glutathione S-transferase (GST)), and the like. Known anti-inflammatory drugs include nitric oxide synthase (NOS) inhibitors, guanylate cyclase inhibitors, nitric oxide synthase (NOS) inhibitors, Nrf2 (NF-E2-related factor 2) activators, and angiotensin II receptor blockers (ARBs).

[0004] A state in which the oxidative power of reactive oxygen species in the body exceeds the antioxidant power of antioxidant enzymes and antioxidant substances is called a state of oxidative stress, and in recent years, the relationship between oxidative stress and various pathological conditions has become clear. For example, in diabetes, it has been shown that nerve cells in the brain that are in a state of oxidative stress for a long time can be damaged, and the regulation of glucose metabolism can no longer function normally (for example, "Mechanism by which oxidative stress causes diabetes elucidated - Improvement of obesity and diabetes by oxidative stress defense mechanisms" [searched March 6, 2024], Internet<https: / / www.amed.go.jp / news / release_20170222.html> (Non-Patent Document 2; Yoko Yagishita et al., "Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress," Cell Reports 18, 2030-2040, 2017 (Non-Patent Document 3)). Furthermore, for example, lipid peroxides produced by the reaction of lipids with reactive oxygen in the body are said to cause arteriosclerosis and myocardial infarction, and the reaction of nucleic acids with reactive oxygen is said to cause nucleic acid mutations and carcinogenesis. As such, reactive oxygen species are believed to be involved in many lifestyle-related diseases and aging, and diseases that have been suggested to be related to reactive oxygen species generated in the body include arteriosclerosis, myocardial infarction, cancer, Parkinson's disease, Alzheimer's disease, multiple sclerosis, cataracts, bronchial asthma, ulcerative colitis, autoimmune diseases, etc. (See, for example, Nakamura Shigeo, "Chemistry of Reactive Oxygen Species and Antioxidants," Journal of the Japan Medical University Medical Association 2013; 9(3), 164-169 (Non-Patent Document 4); Kunitomo Masaru, "Oxidative Stress and Arteriosclerosis," YAKUGAKU ZASSHI 127(12) 1997-2014(2007) (Non-Patent Document 5)).

[0005] Reactive oxygen species are constantly produced in the body due to breathing, ultraviolet light, inflammatory responses, etc., but they are usually eliminated by the antioxidant mechanisms mentioned above, preventing oxidative stress. However, when reactive oxygen species are produced in excess due to aging, ultraviolet light, smoking, stress, etc., when antioxidant enzymes are not produced easily, or when antioxidant intake is low, the balance between reactive oxygen species and the antioxidant mechanism is disrupted, resulting in oxidative stress.

[0006] In living organisms under oxidative stress, it is believed that if antioxidant enzymes and antioxidant substances can be used to increase antioxidant power and counteract reactive oxygen species, this could potentially contribute to the prevention of diseases related to oxidative stress.

[0007] Conventional methods for removing reactive oxygen species or enhancing antioxidant capacity include the intake of antioxidants such as vitamin C, foods, drinks, and supplements. There are also prescription drugs such as glutathione S-transferase (GST) inhibitors that act as oxidative stress-related inhibitors (see "Oxidative stress-related inhibitors" [searched March 6, 2024], Internet <https: / / www.merckmillipore.com / JP / ja / life-science-research / inhibitors-biochemicals / calbiochem-inhibitors / oxidative-stress / a6ib.qB..WwAAAFBRwhlvxeu,nav> (See Non-Patent Document 6). Among antioxidants, the antioxidant activity of coenzyme Q10, for example, has attracted attention for its ability to eliminate lipid radicals in cell membranes and mitochondrial membranes as a fat-soluble antioxidant, preventing oxidative damage (see Yoshiaki Tanaka et al., "Oxidative Stress and Antioxidant Therapy," Journal of the Japanese Society for Parenteral and Enteral Nutrition 3(11):3-12:2016 (Non-Patent Document 7)). Coenzyme Q10 is also sold as a pharmaceutical under the generic name ubidecarenone, and oral administration of 10 mg three times daily after meals in adults is considered effective for the treatment of mild to moderate congestive heart failure symptoms during basic therapy (see package insert for metabolic cardiac stimulant ubidecarenone preparations "Neuquinon® Tablets 5 mg, Neuquinon® Tablets 10 mg, Neuquinon® Sugar-Coated Tablets 5 mg" (Non-Patent Document 8)).

[0008] As mentioned above, to obtain sustained antioxidant effects from antioxidants, regular intake of foods, beverages, supplements, and pharmaceuticals is necessary. For example, coenzyme Q10 reaches its peak blood concentration six hours after oral administration and declines to approximately three-fifths of the peak concentration ten hours after administration (see Non-Patent Document 8, cited above). Furthermore, antioxidants often require oral intake, making them difficult to obtain for those who have difficulty eating due to certain illnesses. Water-soluble vitamins, for example, cannot be synthesized in the body and therefore must be continuously replenished through daily meals. Furthermore, they may be decomposed or denatured during digestion, which limits the sustainability of their effects due to factors such as intake efficiency (see Akio Miyazawa et al., "Absorption and Metabolism of Natural Antioxidants," Chemistry and Biology, Vol. 38, No. 2, 2000, pp. 104-114 (Non-Patent Document 9) and Non-Patent Document 7, cited above). Therefore, there is a need for a method to remove reactive oxygen species or enhance antioxidant capacity and suppress oxidative stress in a more convenient manner than regular intake of exogenous antioxidants. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] OM Ighodaro et al., “First line defense antioxidants-superoxide dismutase (SOD), catalase(CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defense grid”, Alexandria Journal of Medicine 54 (2018) 287-293 [Non-patent document 2] "The mechanism by which oxidative stress causes diabetes elucidated - Improvement of obesity and diabetes by oxidative stress defense mechanisms" [Retrieved March 6, 2024], Internet<https: / / www.amed.go.jp / news / release_20170222.html> [Non-patent document 3] Yoko Yagishita et al., “Nrf2 Improves Leptin and Insulin Resistance Provoked by Hypothalamic Oxidative Stress”, Cell Reports 18, 2030-2040, 2017 [Non-patent document 4] Shigeo Nakamura, "Chemistry of reactive oxygen species and antioxidants," Journal of the Japan Medical University Medical Association 2013; 9(3), 164-169 [Non-Patent Document 5] Kunitomo, Masaru, "Oxidative stress and arteriosclerosis", YAKUGAKU ZASSHI 127(12) 1997-2014(2007) [Non-patent document 6] "Oxidative stress-related inhibitors" [searched March 6, 2024], Internet <https: / / www.merckmillipore.com / JP / ja / life-science-research / inhibitors-biochemicals / calbiochem-inhibitors / oxidative-stress / a6ib.qB..WwAAAFBRwhlvxeu,nav> [Non-Patent Document 7] Yoshiaki Tanaka et al., "Oxidative Stress and Antioxidant Therapy," Journal of the Japanese Society for Parenteral and Enteral Nutrition 3(11):3-12:2016 [Non-patent document 8] Metabolic cardiac stimulant ubidecarenone preparation "Neuquinon® Tablets 5 mg, Neuquinon® Tablets 10 mg, Neuquinon® Sugar-Coated Tablets 5 mg" package insert [Non-Patent Document 9] Akio Miyazawa et al., "Absorption and Metabolism of Natural Antioxidants," Chemistry and Biology Vol.38, No. 2, 2000, 104-114 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made to solve the above problems, and its purpose is to provide a novel antioxidant enzyme production promoting device that utilizes magnetic stimulation by an alternating magnetic field. [Means for solving the problem]

[0011] The present invention is a device for promoting the production of antioxidant enzymes by applying magnetic stimulation to cells (antioxidant enzyme production promotion device), an electromagnetic wave generating unit that generates electromagnetic waves of a frequency for promoting antioxidant enzyme production so that an alternating magnetic field of the frequency for promoting antioxidant enzyme production acts on the cells; a frequency control unit that controls the frequency of the electromagnetic waves for promoting antioxidant enzyme production to 100 kHz to 450 MHz so that production of the antioxidant enzyme is promoted by magnetic stimulation using an alternating magnetic field of the frequency for promoting antioxidant enzyme production; The present invention is characterized by comprising an irradiation unit for irradiating cells with an alternating magnetic field of the frequency for promoting production of antioxidant enzymes.

[0012] In the antioxidant enzyme production promoting device of the present invention, the antioxidant enzyme is preferably superoxide dismutase (SOD).

[0013] In the antioxidant enzyme production promoting device of the present invention, the frequency for promoting antioxidant enzyme production is preferably within the range of 50 MHz to 300 MHz. [Effects of the Invention]

[0014] According to the present invention, by irradiating the human body with an alternating magnetic field of a frequency for promoting the production of antioxidant enzymes, the alternating magnetic field penetrates the body and reaches cells, promoting the production of antioxidant enzymes. This makes it possible to remove reactive oxygen species or enhance antioxidant power, suppress oxidative stress, and contribute to the prevention of diseases related to oxidative stress more easily than the conventional method of regularly ingesting exogenous antioxidants. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the results of the relative value of SOD amount in the antioxidant capacity evaluation test in Experimental Example 1. [Figure 2] 1 is a schematic diagram showing a specific example of an antioxidant enzyme production promoting device of the present invention. [Figure 3] 1A and 1B are schematic diagrams showing an irradiation unit in an antioxidant enzyme production promoting device according to one embodiment, in which (a) is a top view, (b) is a rear view, and (c) is an AA cross-sectional view. [Figure 4] 1A and 1B are printed wiring diagrams showing the coil arrangement of an antioxidant enzyme production promoting device according to one embodiment, in which FIG. 1A is a top view and FIG. 1B is a bottom view. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to an apparatus for promoting the production of antioxidant enzymes by applying magnetic stimulation to cells, the apparatus comprising: an electromagnetic wave generating unit that generates electromagnetic waves of a frequency for promoting the production of antioxidant enzymes in order to apply an alternating magnetic field of the frequency for promoting the production of antioxidant enzymes to the cells; a frequency control unit that controls the frequency of the electromagnetic waves for promoting the production of antioxidant enzymes to 100 kHz to 450 MHz so that the production of antioxidant enzymes is promoted by the magnetic stimulation caused by the alternating magnetic field of the frequency for promoting the production of antioxidant enzymes; and an irradiation unit that irradiates the alternating magnetic field of the frequency for promoting the production of antioxidant enzymes to the cells.

[0017] The antioxidant enzyme production promoting device of the present invention is a device that promotes the production of antioxidant enzymes by applying magnetic stimulation to cells. As demonstrated in the experimental examples described below, the inventors have found that magnetic stimulation using an alternating magnetic field can significantly promote the production of antioxidant enzymes in cells.

[0018] In the present invention, since cells generally have the ability to produce antioxidant enzymes, there are no particular limitations on the cells to which magnetic stimulation is applied.

[0019] In the present invention, the antioxidant enzyme is not particularly limited as long as it is one generally known as an antioxidant enzyme, and examples thereof include superoxide dismutase (SOD), catalase, glutathione peroxidase, etc. Among these, SOD is preferred because it is a major antioxidant enzyme and is often used as an indicator of oxidative stress markers.

[0020] Here, Figure 1 is a graph showing the results of Experimental Example 1, which will be described later. The vertical axis of Figure 1 represents the relative ratio of SOD amount, and the horizontal axis represents frequency (10 kHz, 100 kHz, 500 kHz, 1 MHz, 5 MHz, 10 MHz, 50 MHz, 100 MHz, 150 MHz, 200 MHz, 250 MHz, 300 MHz, 350 MHz, 400 MHz, 450 MHz, and 500 MHz). Figure 1 shows that production of SOD, an antioxidant enzyme, is significantly increased within the frequency range of 100 kHz to 450 MHz (the "frequency for promoting antioxidant enzyme production" in the present invention), preferably within the range of 50 MHz to 300 MHz.

[0021] According to the present invention, by irradiating the human body with an alternating magnetic field of a frequency for promoting antioxidant enzyme production, the alternating magnetic field penetrates the body and reaches cells, promoting the production of antioxidant enzymes within the cells. This makes it possible to remove reactive oxygen species or enhance antioxidant power and suppress oxidative stress more easily than the conventional method of regularly ingesting exogenous antioxidants, thereby contributing to the prevention of diseases related to oxidative stress. As described above, since cells normally have the ability to produce antioxidant enzymes, the alternating magnetic field may be irradiated to the entire body or to a local area, and there are no particular restrictions on the area to be irradiated.

[0022] The antioxidant enzyme production promoting device of the present invention comprises an electromagnetic wave generating unit that generates electromagnetic waves of a frequency for promoting antioxidant enzyme production in order to apply an alternating magnetic field of the frequency for promoting antioxidant enzyme production to the cells, a frequency control unit that controls the frequency for promoting antioxidant enzyme production of the electromagnetic waves so that the production of the antioxidant enzymes is promoted by magnetic stimulation from the alternating magnetic field of the frequency for promoting antioxidant enzyme production, and an irradiation unit that irradiates the cells with the alternating magnetic field of the frequency for promoting antioxidant enzyme production.

[0023] The electromagnetic wave generating unit includes, for example, a signal generating unit, a signal amplifying unit, and a signal wave output unit having an antenna for generating electromagnetic waves. The signal generating unit generates a signal of 100 kHz or more. The signal generated by the signal generating unit is supplied to the signal amplifying unit, where it is amplified and output to the antenna for generating electromagnetic waves. The antenna for generating electromagnetic waves is, for example, a coil formed by printed wiring on a printed circuit board, and generates electromagnetic waves in response to the signal from the signal amplifying unit. The electromagnetic waves include an alternating magnetic field and an alternating electric field.

[0024] The frequency control unit is configured to control the operation of the signal wave output unit and control the frequency of the electromagnetic waves for promoting antioxidant enzyme production. The frequency control unit is configured as a circuit using, for example, a graphics processing unit (GPU) or the like, and is displayed on an image display unit such as a liquid crystal display (LCD) of a touch panel display, so that a user of the antioxidant enzyme production promoting device can control it by operating the image display unit. For example, the frequency control unit is realized so that it can detect the position where the user's finger touches the touch panel of the touch panel display from changes in static electricity at that position, and control the signal wave generation, output, etc. of the signal wave output unit, as well as the alternating magnetic field generated by the irradiation unit, in accordance with the user's instructions, in response to a signal indicating an instruction input using an operation button displayed on the LCD or the like corresponding to the touch position.

[0025] The irradiation unit is configured to irradiate cells with an alternating magnetic field of the frequency for promoting antioxidant enzyme production, which is generated from the electromagnetic wave generating antenna. The irradiation unit is, for example, a flexible plate-like body incorporating the electromagnetic wave generating antenna, and is preferably realized so that it can be manually deformed to fit the area that the user of the antioxidant enzyme production promoting device wants to irradiate.

[0026] Specific examples of the antioxidant enzyme production promoting device of the present invention will be described below with reference to the drawings. Note that the drawings are schematic and may differ from the actual device. Furthermore, the following description exemplifies a method for embodying the technical concept of the present invention, and does not limit the configuration to the one described below. The technical concept of the present invention can be modified in various ways within the technical scope described in the claims.

[0027] Here, Fig. 2 is a schematic diagram showing a specific example of the antioxidant enzyme production promoting device of the present invention. The antioxidant enzyme production promoting device shown in Fig. 2 comprises a device main body 1 and a probe 11 (corresponding to the above-mentioned irradiation unit) connected to the device main body 1 via a signal cable 21, and the device main body 1 also comprises a power cable (not shown) that is detachably inserted and attached.

[0028] 2 has a resin casing 2, a touch panel display 3 (corresponding to the touch panel display of the frequency control unit described above) housed diagonally upward in the front part of the casing 2 and exposed from an opening in the front of the casing 2, a signal wave output unit housed in the upper part of the rear of the casing 2, and a power supply unit housed in the lower part of the rear of the casing 2. An alarm stop button and a power switch button are provided on the left and right sides below the opening in the front of the casing 2 of the device main body 1. Further below these buttons, multiple sockets for plugging in signal cables 21 are provided side by side to enable connection of multiple probes 11 to the device main body 1.

[0029] 3A shows a top view, a back view, and an AA cross-sectional view of a probe 11 of an antioxidant enzyme production promoting device according to this embodiment. In this embodiment, the probe 11 includes a roughly quadrilateral exterior 12 made of a flexible material and a truncated pyramidal circuit section 13 for protecting electrical circuits, etc. A signal cable 21 is led out from the back of the circuit section.

[0030] As shown in Figure 3(c), the probe 11 contains a flexible thin plate 14, which is a printed circuit board on which a coil and an electric circuit are arranged, in an exterior 12 made of a soft resin such as an elastomer or rubber material. The probe 11 can be manufactured by resin molding such as injection molding or RIM molding. Since resin molding involves self-heating, it is preferable to apply a resin mold to the circuit part 13 in advance in order to protect the electric circuit, etc.

[0031] FIG. 4 is a printed wiring diagram showing the coil arrangement of the antioxidant enzyme production promoting device of this embodiment, showing a top view (a) and a bottom view (b). A circuit section, a high-frequency output coil, a high-frequency detection coil, a low-frequency output coil, etc. are arranged on a printed circuit board. In this embodiment, the high-frequency output coil is arranged as an annular disk on the top surface of the printed circuit board, and the high-frequency detection coil is arranged as an annular ring around it. A spiral-shaped low-frequency output coil is arranged inside the high-frequency output coil. The high-frequency output coil and the low-frequency output coil are also arranged on the bottom surface of the printed circuit board, and are configured symmetrically with respect to each other so that the high-frequency current and the low-frequency current flow in the same direction on both the top and bottom surfaces. This allows the centers of the magnetic fields generated by the coils on both the top and bottom surfaces to be aligned. The high-frequency output coil, the high-frequency detection coil, and the low-frequency output coil may be changed from annular to rectangular.

[0032] There is no particular limitation on the shape of the probe 11, but by making it an approximately quadrilateral, particularly an approximately rectangular flat plate as in the example shown in Figure 3, it has the advantage of being easy to attach to the target object and to fix with tape.

[0033] The flexible thin plate 14 is a film-like printed circuit board, and preferably has a base layer made of a flexible insulating film and, if necessary, an adhesive layer. The base layer can be made of polyimide resin, polyester resin, polyamide paper-based epoxy resin, glass cloth-based epoxy resin, glass-based BT resin, or the like. Each coil can be a conductor such as metal foil printed or etched onto the printed circuit board 14.

[0034] In the antioxidant enzyme production promoting device of the present invention, the power supply unit is configured as a circuit, for example, with a CPU and two AC-DC converters (not shown), and a predetermined DC power supply is obtained from 100V commercial AC power supplied via a power cable (not shown) by the two AC-DC converters, and these DC power supplies are connected in series to obtain a charging voltage for the battery, which is then charged. The predetermined DC power supply is stepped down from these DC power supplies by a switching power supply and a linear regulator, and supplied as DC power of the required voltage to the signal wave output unit and screen control unit of the device main body 1 and the operation state detection unit of the probe 11. During normal use of the antioxidant enzyme production promoting device without a power cable attached, the power supply unit steps down the DC power supply from the battery to the signal wave output unit and screen control unit of the device main body 1 and the operation state detection unit of the probe 11 to the required DC voltages by the switching power supply and linear regulator, thereby making the antioxidant enzyme production promoting device portable for use.

[0035] The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited to these examples.

[0036] <Experimental Example 1> (cell culture) PC12 cells (CRL-1721), a rat-derived neuronal cell line, were obtained from the ATCC Cell Bank and cultured in RPMI containing 10% horse serum and 5% fetal bovine serum.

[0037] (H2O2 treatment and magnetic stimulation) 2 × 10 PC12 cells in a 6 cm dish 5 Two days after seeding, the cells were divided into two groups: H2O2 treatment group H2O2 treatment-magnetic stimulation group was allocated to.

[0038] The cells were treated with hydrogen peroxide (Fujifilm Wako Pure Chemical Industries, Ltd., 081-04215) at a concentration of 200 μM as an oxidative stress stimulus, and then cultured for 3 hours.

[0039] Magnetic stimulation was performed using a function generator (81160A, Keysight Technologies, Inc.) and a coil (WT505090-20K2-A10-G, TDK Corporation). For the H2O2-treated group, magnetic stimulation was initiated 3 hours before hydrogen peroxide treatment, for a total of 6 hours. Magnetic stimulation was performed at frequencies of 10 kHz, 100 kHz, 500 kHz, 1 MHz, 5 MHz, 10 MHz, 50 MHz, 100 MHz, 150 MHz, 200 MHz, 250 MHz, 300 MHz, 350 MHz, 400 MHz, 450 MHz, and 500 MHz. The waveform was a square or sine wave with a 50% duty cycle, and the output voltage (v(max)) was 2.5–3.0 Vpp.

[0040] (Cell extraction) After magnetic stimulation, the cells were extracted using the following procedure. (1) The cells were collected in a 1.5 mL tube and washed twice with 1 mL of PBS. (2) After removing the PBS, the cells were homogenized using a pestle to disrupt the cell membrane. (3) 200 μL of fresh PBS was added and mixed well. (4) Centrifuged at 10,000 × g for 15 minutes at 0 to 4°C. (5) The supernatant was used as the measurement sample.

[0041] (Protein concentration measurement) The amount of protein in the measurement sample was quantified using a BCA Protein Assay Kit (manufactured by Takara Bio Inc.). (1) A dilution series of the BSA standard solution was prepared as shown in Table 1. PBS was used to dilute the BSA standard solution and samples.

[0042] [Table 1]

[0043] (2) 25 μL of each diluted solution of the prepared BSA standard solution and sample were dispensed into each well of the microplate. Each concentration was measured in duplicate (n=2) or more. (3) 200 μL of working solution was added and immediately mixed. (4) The mixture was reacted at 37°C for 30 minutes, and then returned to room temperature. (5) The absorbance at 562 nm was measured using a spectrophotometer. For the measurement, the absorbance at 562 nm was measured using a plate reader. PBS was used for zero point calibration. (6) For the dilutions of the BSA standard solution, the absorbance of each standard solution was subtracted from the blank value, and the average value was calculated to create a standard curve. For the samples, the average absorbance minus the blank value was used to calculate the sample concentration against the standard curve of the BSA standard solution.

[0044] (Antioxidant capacity evaluation) Antioxidant activity was evaluated using SOD Assay Kit-WST (manufactured by Dojindo Laboratories, Inc.) according to the product protocol. (1) A 96-well plate was used, and 20 μL of sample solution (sample, blank2) diluted 1, 1 / 2, 1 / 4, or 1 / 8 times or pure water (blank1, blank3) was placed in each well. (2) 200 μL of WST working solution was added to each well and mixed well using a plate mixer. (3) 20 μL of dilution buffer was added to each of blank 2 and blank 3 wells. (4) 20 μL of enzyme working solution was added to each of the wells containing the sample solution and the blank 1 well. (5) Incubated at 37°C for 20 minutes. (6) The absorbance at 450 nm was measured using a microplate reader (Perkin Elmer). The SOD inhibitory activity (inhibition rate %) was calculated using the following formula: SOD inhibitory activity (inhibition rate%) = [(A blank1-A blank3)-(A sample-A blank2)] / (A blank1-A blank3)×100 was calculated by

[0045] The unit definition in the WST method was "the amount of SOD contained in 20 μL of sample solution that shows 50% inhibition of WST reduction is 1 unit (U)."

[0046] Unit (U) Rules: (1) The dilution rate at which the inhibition rate reached 50% (IC50) was determined from the inhibition curve. (2) The point showing 50% inhibition (IC50) is 1 unit (U), so the SOD unit of the original sample was calculated by multiplying this by the dilution factor, and then converted to volume to calculate the concentration (U / mL). (3) The amount of protein (mg / mL) in the sample was quantified using a BCA Protein Assay Kit (manufactured by Takara Bio Inc.) and corrected to determine the final amount of SOD (U / mg). (4) The relative ratio of the SOD amount in each H2O2 treatment-magnetic stimulation group was calculated, assuming the SOD amount in the control H2O2 treatment group to be 1, and the frequency that promotes the production of antioxidant enzymes was confirmed.

[0047] Figure 1 shows the relative SOD levels at each frequency in the H2O2 treatment-magnetic stimulation group. In Figure 1, the vertical axis represents the relative ratio of SOD levels, and the horizontal axis represents frequency (10 kHz, 100 kHz, 500 kHz, 1 MHz, 5 MHz, 10 MHz, 50 MHz, 100 MHz, 150 MHz, 200 MHz, 250 MHz, 300 MHz, 350 MHz, 400 MHz, 450 MHz, and 500 MHz). Figure 1 shows that SOD production is significantly promoted within the frequency range of 100 kHz to 450 MHz.

[0048] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0049] 1 Main body of antioxidant enzyme production promotion device, 2 Casing, 3 Display (touch panel display), 11 Probe, 12 Exterior, 13 Circuit section, 14 Flexible thin plate (printed circuit board), 21 Signal cable

Claims

1. A device for promoting the production of antioxidant enzymes by applying magnetic stimulation to cells, an electromagnetic wave generating unit that generates electromagnetic waves of a frequency for promoting antioxidant enzyme production so that an alternating magnetic field of the frequency for promoting antioxidant enzyme production acts on the cells; a frequency control unit that controls the frequency of the electromagnetic waves for promoting antioxidant enzyme production to 100 kHz to 450 MHz so that production of the antioxidant enzyme is promoted by magnetic stimulation using an alternating magnetic field of the frequency for promoting antioxidant enzyme production; an irradiation unit for irradiating cells with an alternating magnetic field having a frequency for promoting antioxidant enzyme production;

2. 2. The antioxidant enzyme production promoting device according to claim 1, wherein the antioxidant enzyme is superoxide dismutase.

3. 3. The antioxidant enzyme production promoting device according to claim 1, wherein the frequency for promoting antioxidant enzyme production is within the range of 50 MHz to 300 MHz.

Images