WO2011071088A1 - Procédé de production d'un superoxyde, procédé d'évaluation de la capacité de piégeage du superoxyde, dispositif de production d'un superoxyde, et dispositif d'évaluation de la capacité de piégeage du superoxyde - Google Patents

Procédé de production d'un superoxyde, procédé d'évaluation de la capacité de piégeage du superoxyde, dispositif de production d'un superoxyde, et dispositif d'évaluation de la capacité de piégeage du superoxyde Download PDF

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WO2011071088A1
WO2011071088A1 PCT/JP2010/072050 JP2010072050W WO2011071088A1 WO 2011071088 A1 WO2011071088 A1 WO 2011071088A1 JP 2010072050 W JP2010072050 W JP 2010072050W WO 2011071088 A1 WO2011071088 A1 WO 2011071088A1
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superoxide
determination
spectrum
spin
electron donor
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PCT/JP2010/072050
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Japanese (ja)
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博匡 藤井
俊志 郡
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北海道公立大学法人 札幌医科大学
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Priority to CA2783422A priority Critical patent/CA2783422A1/fr
Priority to US13/514,876 priority patent/US20120255853A1/en
Priority to JP2011545231A priority patent/JP5723294B2/ja
Publication of WO2011071088A1 publication Critical patent/WO2011071088A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B15/00—Peroxides; Peroxyhydrates; Peroxyacids or salts thereof; Superoxides; Ozonides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
    • B01J19/12—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor employing electromagnetic waves

Definitions

  • the present invention relates to a superoxide production method, a superoxide elimination ability evaluation method, a superoxide production apparatus, and a superoxide elimination ability evaluation apparatus.
  • Superoxide (superoxide, superoxide anion, superoxide anion radical) is a substance in which one electron is added to an oxygen molecule, and is represented by a chemical formula of (.O 2 ⁇ ). That is, superoxide is a kind of radical (free radical, free radical) having unpaired electrons.
  • An unpaired electron is an unpaired electron located in the outermost orbital of a molecule or atom, and radicals generally react or oxidize or reduce other substances in an attempt to eliminate this unpaired electron. It is a high substance.
  • Superoxide is a kind of active oxygen. Active oxygen refers to a derivative of oxygen in which oxygen becomes chemically active and generally exhibits a very unstable and strong oxidizing power. Superoxide is the most frequently generated active oxygen in the living body, and is always produced by enzymes such as xanthine oxidase, NAD (P) H oxidase, and aldehyde oxidase in the energy metabolism system, nucleic acid metabolism system, immune system, and the like. Moreover, the production
  • superoxide generated excessively in the living body is activated oxygen having a stronger oxidizing power than superoxide, such as hydrogen peroxide and hydroxy radical ( ⁇ OH ⁇ ), due to a spontaneous disproportionation reaction.
  • superoxide causes oxidative degeneration of various substances such as nucleic acids, enzymes, and cell membranes. Therefore, superoxide is considered as one of the starting materials for oxidative damage in living organisms, and is considered to cause diseases and aging. Yes. Therefore, research on the effects of superoxide on the living body and the search for substances that suppress the activity of superoxide have been carried out recently, and there is a need for technology that generates superoxide without generating extra radicals. It has been.
  • non-patent document 1 a method of generating hypoxanthine by causing xanthine oxidase to act
  • a method of dissolving potassium superoxide (KO 2 ) in water a method of dissolving potassium superoxide (KO 2 ) in water
  • Patent Document 1 A method of generating an electric current between the anode and the redox polymer-supported cathode (Patent Document 1), a method of generating an aluminum anodic oxide film immersed in water by irradiating ultraviolet light (Patent Document 2), and a mixed gas
  • Patent Document 3 A method of applying a high voltage to a sealed discharge tube (Patent Document 3), a method using a pulse radiolysis method (Non-Patent Document 3), a method of irradiating light to a flavin and an electron donor (Non-Patent Document 4) To 7).
  • JP 2002-273433 A JP 2003-112053 A JP-A-10-152306
  • Non-Patent Document 1 causes a decrease or loss of enzyme activity, stable superoxide generation cannot be ensured.
  • Non-Patent Document 2 super It is difficult to control the amount of oxide generated.
  • other molecules such as hydroxy radicals and hydrated electrons are generated simultaneously with the generation of superoxide, so that superoxide cannot be selectively generated.
  • the methods disclosed in Patent Document 1 and Patent Document 2 also generate super radicals and other molecules such as hydroxy radicals, hydrogen peroxide, or ozone at the same time. Cannot be generated automatically.
  • Non-Patent Documents 4 to 7 also generate other molecules such as radicals derived from electron donors (electron donor radicals, interfering radicals, TH radicals) simultaneously with superoxide, Again, superoxide cannot be selectively generated. Furthermore, since the method disclosed in Non-Patent Document 3 requires a device for irradiating radiation, it cannot be said to be a simple method. However, the method disclosed in Patent Document 3 also requires a high voltage. At the same time, the inside of the discharge tube must be set to a negative pressure, and it is not a simple method.
  • the present invention has been made in order to solve such problems, and a method and an object for selectively and stably producing superoxide or a radical containing superoxide at a high purity in a simple and stable manner.
  • an apparatus for selectively generating superoxide or a radical containing superoxide with high purity, and producing it simply and stably, and the superoxide of the target sample An object of the present invention is to provide an apparatus for simply evaluating the erasing ability.
  • the inventors of the present invention irradiate light on a determination solution containing a flavin, an electron donor, a spin trap agent, and an aqueous solvent to produce a superoxide spin adduct, an electron donor radical (interfering radical, TH ⁇
  • an electron donor radical interfering radical, TH ⁇
  • a spectrum is obtained by electron spin resonance, and this spectrum and the standard spectrum of a superoxide spin adduct (superoxide standard)
  • the generation of electron donor radicals is obtained by obtaining the concentration of the flavin by determining whether the spectrum is similar to the spectrum and irradiating the raw material solution containing the obtained concentration of flavin, electron donor and aqueous solvent with light.
  • (1) Superoxide production method having the following steps (a), (b), (c), (d), (e), (f) and (g); (A) a determination solution preparation step for preparing a determination solution containing a flavin, an electron donor, a spin trap agent, and an aqueous solvent; (B) a determination spin adduct / radical generation step of irradiating the determination solution with light to generate a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical; (C) a spectrum acquisition step for determination for acquiring a spectrum by detecting the spin adduct of the generated superoxide, the spin adduct of the electron donor radical and / or the electron donor radical by electron spin resonance; (D) a similarity determination step of determining whether a standard spectrum of a superoxide spin adduct is similar to the determination spectrum; (E) a flavin concentration acquisition step of acquiring a similar flavin concentration by the similarity determination; (F) a raw material
  • step (h) When the flavin is riboflavin, it has the following step (h) instead of the steps (a), (b), (c), (d), (e) and (f): 1) Superoxide production method according to (H) A raw material solution preparation step of preparing a raw material solution containing riboflavin, an electron donor and an aqueous solvent so that the concentration C ( ⁇ mol / L) of riboflavin is 0.1 ⁇ C ⁇ 15.
  • the flavin concentration acquisition step is a flavin concentration acquisition step of acquiring a flavin concentration that is similar by the similarity determination so that the purity of the superoxide in the generated radical is 75.6 to 100%. Or the superoxide production method according to (2).
  • a method for evaluating the superoxide scavenging ability of a sample comprising: (a), (b), (c), (d), (e), (i), (j), (k) and The method comprising the step of (l); (A) a determination solution preparation step for preparing a determination solution containing a flavin, an electron donor, a spin trap agent, and an aqueous solvent; (B) a determination spin adduct / radical generation step of irradiating the determination solution with light to generate a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical; (C) a spectrum acquisition step for determination for acquiring a spectrum by detecting the spin adduct of the generated superoxide, the spin adduct of the electron donor radical and / or the electron donor radical by electron spin resonance; (D) a similarity determination step of determining whether a standard spectrum of a superoxide spin adduct is similar to the determination spectrum; (E) a
  • the flavin concentration acquisition step is a flavin concentration acquisition step of acquiring the concentration of flavin that is similar by the similarity determination so that the purity of the superoxide in the generated radical is 75.6 to 100%. ) Or the method according to (8).
  • a superoxide production apparatus comprising the following means (i), (ii), (iii), (iv), (v), (vi) and (vii); (I) a determination solution preparation means for preparing a determination solution containing a flavin, an electron donor, a spin trap agent and an aqueous solvent; (Ii) a determination spin adduct / radical generation means for generating a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical by irradiating the determination solution with light; (Iii) A spectrum acquisition means for determination for acquiring a spectrum by detecting the spin adduct of the generated superoxide, the spin adduct of the electron donor radical and / or the electron donor radical by electron spin resonance, (Iv) Similarity determining means for determining whether or not the standard spectrum of the superoxide spin adduct is similar to the determination spectrum; (V) flavin concentration acquisition means for acquiring the concentration of flavins that are similar by the similarity
  • the flavin concentration acquisition means is a flavin concentration acquisition means for acquiring a flavin concentration that is similar by the similarity determination so that the purity of the superoxide in the generated radical is 75.6 to 100%.
  • the superoxide production apparatus according to (14).
  • the flavin concentration acquisition means is a flavin concentration acquisition means for acquiring the concentration of flavin that is similar by the similarity determination so that the purity of the superoxide in the generated radical is 75.6 to 100%. ) Or (20).
  • (25) Superoxide in vivo production method having the following steps (A), (B), (C), (D), (E), (F) and (G); (A) A raw material administration step of administering an arbitrary amount of flavin, electron donor and spin trap agent into a biological sample or the body of a non-human animal, (B) Superoxide spin adduct, electron donor radical spin adduct and / or a portion where the administered flavin, electron donor and spin trap agent are present in the biological sample or non-human animal body.
  • a determination spin adduct / radical generation step for generating an electron donor radical (C) A spectrum acquisition step for determination, in which the generated superoxide spin adduct, electron donor radical spin adduct and / or electron donor radical is detected by electron spin resonance to acquire a spectrum; (D) a similarity determination step of determining whether the standard spectrum of the superoxide spin adduct is similar to the determination spectrum; (E) Optimum flavin acquisition step of acquiring an optimal amount of flavins similar by the similarity determination, (F) Optimal raw material administration step of administering the obtained optimal amount of flavin and electron donor into the body of the biological sample or non-human animal, (G) A generation step in which superoxide is generated by irradiating light to a site where the administered flavin and electron donor are present in the biological sample or non-human animal body.
  • a determination spin adduct / radical generation step for generating an electron donor radical (C) A spectrum acquisition step for determination, in which the generated superoxide spin adduct, electron donor radical spin adduct and / or electron donor radical is detected by electron spin resonance to acquire a spectrum; (D) a similarity determination step of determining whether the standard spectrum of the superoxide spin adduct is similar to the determination spectrum; (E) Optimum flavin acquisition step of acquiring an optimal amount of flavins similar by the similarity determination, (H) an evaluation specimen administration step of administering an evaluation specimen containing the obtained optimal amount of flavin, electron donor, spin trap agent and superoxide scavenging ability into a biological sample or a non-human animal body; (I) an in vivo spin adduct generation step of generating a spin adduct by irradiating light to the administered evaluation specimen in the biological sample or the non-human animal body; (J) an in vivo spectrum acquisition step of acquiring a spectrum by a
  • the present invention it is possible to selectively generate superoxide or a high-purity superoxide-containing radical in vitro and in vivo and easily and stably produce it. Evaluation and research on the involvement of superoxide can be performed accurately and simply, and the search for treatments / prevention methods for diseases and aging phenomena involving superoxide and the elucidation of the progression mechanism can be promoted.
  • the superoxide scavenging ability of a target sample can be easily evaluated in vitro and in vivo, a search for an antioxidant substance that is truly effective for a living body and various substances can be performed. It is possible to accurately, conveniently and quickly evaluate the antioxidant ability of the.
  • FIG. 10 It is a figure which shows the evaluation solution preparation means 10 in this embodiment. It is a figure which shows the spectrum obtained by performing the measurement by electron spin resonance (ESR) after irradiating visible light to the SOD non-addition water system solution and the SOD addition water system solution.
  • spectrum A shows a spectrum obtained from an SOD-free aqueous solution
  • spectrum B shows a spectrum obtained from an SOD-added aqueous solution.
  • the vertical axis represents the amount of radicals generated
  • the horizontal axis represents the riboflavin concentration.
  • the vertical axis represents signal intensity
  • the horizontal axis represents visible light irradiation time or time after stopping visible light irradiation.
  • production inhibition rate by SOD in the case where superoxide is generated by light irradiation of riboflavin and in the case where superoxide is generated by xanthine oxidase.
  • the vertical axis represents the radical generation inhibition rate
  • the horizontal axis represents the added SOD concentration.
  • the superoxide production method includes the following steps (a), (b), (c), (d), (e), (f) and (g);
  • A a determination solution preparation step for preparing a determination solution containing a flavin, an electron donor, a spin trap agent, and an aqueous solvent
  • B a determination spin adduct / radical generation step of irradiating the determination solution with light to generate a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical
  • D a similarity determination step of determining whether a standard spectrum of a superoxide spin adduct is similar to the determination spectrum
  • E a flavin concentration acquisition step of acquiring a similar flavin concentration by the similarity determination;
  • the “radical containing high purity and superoxide” is a set of radicals composed of superoxide and an electron donor radical (interfering radical, TH ⁇ radical), and superoxide occupies the set.
  • a radical whose ratio (purity of superoxide) is sufficiently large compared to the ratio occupied by radicals other than superoxide, and its purity is preferably 70% or more and less than 100%, preferably 75.6% or more and less than 100%. Is more preferably 83.3% or more and less than 100%.
  • the purity of superoxide in a radical containing superoxide can be calculated according to a conventional method. For example, first, a radical containing superoxide is generated in an aqueous solution and measured by electron spin resonance. A spectrum similar to the oxide standard spectrum is obtained, and the amount of superoxide generated is measured based on this spectrum. Next, radicals are generated after adding superoxide dismutase (SOD), an enzyme that specifically eliminates superoxide, to an aqueous solution having the same composition as when the amount of superoxide generated is measured. A spectrum is obtained by measurement, and the amount of electron donor radical (interfering radical, TH.radical) generated is measured based on this spectrum. Subsequently, the purity of the superoxide can be calculated from the measured superoxide generation amount and the amount of electron donor radical (interfering radical, TH ⁇ radical) generation by the following equation 1.
  • SOD superoxide dismutase
  • ESR electron spin resonance
  • a sample is placed in a magnetic field, microwaves are irradiated to resonate unpaired electrons of radicals contained in the sample, and the absorption energy generated when the sample is resonated is measured.
  • the absorption energy is measured while changing the magnetic field, and a series of absorption energy changes are acquired as a spectrum. Since the shape of the spectrum is determined depending on the type of radical, the radical contained in the sample can be identified by observing the shape of the acquired spectrum.
  • the standard spectrum of a superoxide spin adduct is a superoxide spin adduct, a high-purity radical containing a superoxide, or a high-purity radical containing a superoxide.
  • This refers to a spectrum obtained by performing measurement by electron spin resonance or a spectrum assumed to be obtained by measurement by electron spin resonance for a mixture of a spin adduct and a radical.
  • the superoxide standard spectrum can be obtained, for example, by computer simulation, and the one described in the previous report (MASATO K. et al., Free Radical Research, Vol. 40, No. 11, pp. 1166-1172, 2006) is used.
  • it can be obtained by generating a high-purity radical containing superoxide with xanthine oxidase and performing measurement by electron spin resonance.
  • the determination solution can be prepared by dissolving a flavin, an electron donor, and a spin trap agent in an aqueous solvent. It may contain substances.
  • Flavin is a group of derivatives having a substituent at the 10-position of dimethylisoalloxazine. As shown in the following formulas 2 and 3, when flavin is irradiated with light in the presence of an electron donor, it is excited to a higher energy level, takes one electron from the electron donor, and becomes a flavin radical. At the same time, since the electron donor deprived of one electron has an unpaired electron, it becomes an electron donor radical (interfering radical, TH.radical) (Formula 2). Subsequently, the flavin radical adds one electron to the oxygen molecule and generates superoxide (Formula 3).
  • the above reaction can be used to produce superoxide or a radical containing superoxide with high purity.
  • the flavin that can be used in the present invention include riboflavin, flavin mononucleotide (FMN), isoalloxazine, alloxazine, lumichrome, lumiflavin, flavin-adenine dinucleotide (FAD), galactoflavin, D-araboflavin, lysoflavin and their Arbitrary combinations and the like can be mentioned, but riboflavin, FMN, or a mixture thereof can be preferably used.
  • the electron donor used in the present invention may be any substance having a low redox potential that gives electrons to the excited flavin, and examples thereof include oxygen-containing compounds, nitrogen-containing compounds, phosphorus-containing compounds, and sulfur-containing compounds. Nitrogen-containing compounds are preferred. Examples of such nitrogen-containing compounds include ethylenediaminetetraacetic acid (EDTA), methionine, tetramethylethylenediamine (TMD, TMED), and EDTA can be preferably used.
  • EDTA ethylenediaminetetraacetic acid
  • TMD tetramethylethylenediamine
  • TMED tetramethylethylenediamine
  • a spin trap agent is a reagent that forms a stable spin adduct by covalent bonding (adduct) with an unstable radical that changes to another substance in a short time. Since superoxide is also an unstable radical, it is difficult to detect it directly by electron spin resonance, but it can be detected by generating a spin adduct.
  • the spin trapping agent used in the present invention is not particularly limited as long as it generates at least a superoxide and a spin adduct.
  • CYPMPO 5,5-Dimethyl-2-oxo-2 ⁇ 5- [1,3,2] dioxphosphinan- 2-yl) -2-methyl-3,4-dihydro-2H-pyrrole 1-oxide
  • DMPO 5,5-Dimethyl-1-pyrroline N-oxide
  • DEPMPO 5-Diethylphosphoryl 5-methyl-1- pyrroline N-oxide
  • M 3 PO 2,5,5-triethyl-1-pyrroline N-Oxide
  • T PO 3,3,5,5-Tetramethyl-1-pyrroline N-Oxide
  • the aqueous solvent used in the present invention is not particularly limited as long as it has a function of keeping the pH of the solution containing the aqueous solvent constant.
  • phosphate buffer, acetate buffer, citrate buffer, borate buffer , Tartrate buffer solution, Tris buffer solution and the like, and phosphate buffer solution can be preferably used.
  • the light source that can be used in the present invention include a xenon lamp, a fluorescent lamp, a halogen lamp, a krypton lamp, a sodium lamp, a mercury lamp, and a metal halide lamp.
  • the type of light (wavelength), light intensity, and irradiation time used in the present invention are not particularly limited, and the type of light source, the positional relationship between the light source and the target to be irradiated, the amount of target to be irradiated, and the target to be irradiated.
  • the concentration can be appropriately set according to conditions such as the necessary amount of superoxide generated as a result of irradiation.
  • the spin trapping agent in the determination solution forms a spin adduct with an electron donor radical (interfering radical, TH radical)
  • the spin adduct of the electron donor radical (interfering radical, TH radical) Produces.
  • high homology means at least 70% homology, preferably 80% or more homology, more preferably 85% homology, more preferably 90% or more homology, still more preferably Refers to homology of 95% or more.
  • step (e): flavin concentration acquisition step the flavin concentration that is similar by similarity determination can be acquired so that the purity of the superoxide in the generated radical is in the range of 75.6 to 100%.
  • radicals are irradiated by irradiating light to a raw material solution containing riboflavin, an electron donor and an aqueous solvent so that the riboflavin concentration C ( ⁇ mol / L) is 0.1 ⁇ C ⁇ 15.
  • the produced radical can be produced so that the purity of the superoxide is 75.6% to 100%. Furthermore, when a radical was generated by irradiating light to a raw material solution containing riboflavin, an electron donor and an aqueous solvent so that the concentration C ( ⁇ mol / L) of riboflavin was 0.1 ⁇ C ⁇ 10, it was generated. The radicals can be produced so that the purity of superoxide is 83.3% to 100%.
  • the superoxide scavenging ability evaluation method is a method for evaluating the superoxide scavenging ability of a sample, and includes the following (a), (b), (c), (d), (e), Having steps (i), (j), (k) and (l);
  • A a determination solution preparation step for preparing a determination solution containing a flavin, an electron donor, a spin trap agent, and an aqueous solvent;
  • B a determination spin adduct / radical generation step of irradiating the determination solution with light to generate a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical;
  • C a spectrum acquisition step for determination for acquiring a spectrum by detecting the spin adduct of the generated superoxide, the spin adduct of the electron donor radical and / or the electron donor radical by electron spin resonance;
  • D a similarity determination step of determining whether a standard spectrum of a superoxide spin
  • the evaluation solution can be prepared by dissolving a flavin, an electron donor, a spin trap agent, and a sample for evaluating superoxide elimination ability in an aqueous solvent.
  • the concentration of flavin in the evaluation solution is adjusted to be the concentration obtained in step (e).
  • the evaluation solution may contain other substances as long as the characteristics are not impaired.
  • Examples of the sample for evaluating the superoxide scavenging ability in the present invention include compounds such as foods, plant extracts, cosmetic compositions, and pharmaceutical compositions.
  • the sample to be evaluated is evaluated as “having an erasing ability” by erasing the superoxide before the superoxide changes into a spin adduct.
  • the evaluation of the superoxide scavenging ability of a sample was conducted when the signal intensity of the spectrum for evaluation was smaller than the signal intensity of the superoxide standard spectrum. It has an erasing ability.
  • the sample is evaluated as “not superoxide-erasing ability”, assuming that the superoxide is not erased. Is done.
  • the signal intensity in the evaluation spectrum is the same as the signal intensity in the superoxide standard spectrum.
  • the signal intensity of the evaluation spectrum is almost the same as the signal intensity of the superoxide standard spectrum.
  • An evaluation solution preparation step of preparing an evaluation solution including
  • the superoxide production apparatus comprises the following means (i), (ii), (iii), (iv), (v), (vi) and (vii); (I) a determination solution preparation means for preparing a determination solution containing a flavin, an electron donor, a spin trap agent and an aqueous solvent; (Ii) a determination spin adduct / radical generation means for generating a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical by irradiating the determination solution with light; (Iii) A spectrum acquisition means for determination for acquiring a spectrum by detecting the spin adduct of the generated superoxide, the spin adduct of the electron donor radical and / or the electron donor radical by electron spin resonance, (Iv) Similarity determining means for determining whether or not the standard spectrum of the superoxide spin adduct is similar to the determination spectrum; (V) flavin concentration acquisition means
  • FIG. 1 is a conceptual diagram illustrating the basic configuration of a superoxide production apparatus 1 according to this embodiment.
  • the superoxide production apparatus 1 mainly includes a determination solution preparation means 2, a determination spin adduct / radical generation means 3, a determination spectrum acquisition means 4, a similarity determination means 5, and a flavin.
  • the concentration acquisition means 6, the raw material solution preparation means 7, and the generation means 8 are configured.
  • the determination solution preparation means 2 only needs to have a configuration and a function capable of appropriately preparing a determination solution containing a flavin, an electron donor, a spin trap agent, and an aqueous solvent.
  • a determination injection tube 22 inserted into the determination solution storage unit 23 is formed, and a determination having an injection amount adjusting function according to the number of substances to be injected.
  • a configuration including a plurality of containers 21 for use can be exemplified.
  • an appropriate amount of flavin is supplied from each determination injection tube 22 of the determination container 21 having an injection amount adjusting function, each of which contains a flavin, an electron donor, a spin trap agent, and an aqueous solvent.
  • the electron donor, the spin trap agent, and the aqueous solvent can be injected into the determination solution storage unit 23, and as a result, the determination solution can be prepared.
  • the determination solution preparation unit 2 is configured to be controllable by a control signal output from a control signal output unit 62 described later.
  • maintains a liquid can be mentioned, for example.
  • the material constituting the container is preferably a material having high transparency, water resistance, corrosion resistance, and chemical resistance. Examples of such a material include glass and plastic.
  • the determination spin adduct / radical generation means 3 only needs to have a configuration and a function capable of appropriately irradiating the determination solution with light.
  • a configuration comprising a light source.
  • Mercury lamps and metal halide lamps are examples of such a configuration comprising a light source.
  • the spectrum acquisition means 4 for determination includes a spin adduct of superoxide obtained by irradiating the determination solution with light, a spin adduct of electron donor radicals (interfering radicals, TH radicals) and / or an electron donor radical. It is only necessary to have a configuration or function capable of detecting (interfering radicals, TH ⁇ radicals) by electron spin resonance and acquiring the spectrum. Examples of such a configuration include an electromagnet 41, a microwave oscillator 42, a crystal diode detector 43, an amplifier 44, and a recorder 45 as shown in FIG.
  • the determination solution storage unit 23 is placed in the magnetic field formed by the electromagnet 41, and the microwave oscillator 42 irradiates microwaves to resonate unpaired electrons of the spin adduct contained in the determination solution.
  • the generated absorption energy is detected by the crystal diode detector 43, and the detected signal is amplified by the amplifier 44 and then recorded by the recorder 45, whereby the determination spectrum can be obtained.
  • a configuration conforming to a commercially available ESR measurement device such as JES-RE1X (JEOL Ltd.) may be adopted.
  • the similarity determination means 5 only needs to have a configuration and a function capable of determining whether or not the acquired determination spectrum is similar to the superoxide standard spectrum.
  • a configuration including an input unit 51 for inputting spectrum data and a display unit 52 for displaying the input spectrum can be given.
  • the data of the superoxide standard spectrum and the determination spectrum are input to the input unit 51, and these spectra are displayed on the display unit 52 to determine whether or not they are similar by comparing their shapes. be able to.
  • the flavin concentration acquisition unit 6 can acquire the concentration of flavin related to the determination solution. What is necessary is just to have a structure and a function. That is, according to the flavin concentration acquisition means 6, when the similarity determination means 5 determines that the superoxide standard spectrum and the determination spectrum are not similar, the determination solution preparation means 2 again changes the flavin concentration. Thereafter, the determination process by the determination spin adduct / radical generation means 3 ⁇ the determination spectrum acquisition means 4 ⁇ the similarity determination means 5 is repeated until a determination spectrum similar to the superoxide standard spectrum is obtained. Therefore, the desired flavin concentration can be finally obtained.
  • the flavin concentration acquisition means 6 can acquire the flavin concentration that is similar by similarity determination so that the purity of the superoxide in the generated radical is in the range of 75.6 to 100%.
  • the similarity determination means 5 and the flavin concentration acquisition means 6 described above may be configured by, for example, a personal computer. Specifically, as shown in FIG. 2, a storage unit R for storing the similarity determination program and the flavin concentration acquisition process described above, various data necessary for the similarity determination, and the like, and the storage unit R And arithmetic processing means C that acquires various data from the spectrum acquisition means 4 for determination and performs arithmetic processing.
  • a storage unit R for storing the similarity determination program and the flavin concentration acquisition process described above, various data necessary for the similarity determination, and the like
  • arithmetic processing means C that acquires various data from the spectrum acquisition means 4 for determination and performs arithmetic processing.
  • the storage means R is composed of ROM (ReadReOnlymMemory), RAM (Random Access Memory), HDD (Hard Disk Drive), flash memory, and the like, and stores various data, and the arithmetic processing means C performs arithmetic processing. It functions as a working area when performing.
  • the storage unit R has a similarity determination program installed therein.
  • the arithmetic processing means C makes a computer function as each component mentioned later by executing the similarity determination program.
  • the usage form of the similarity determination program is not limited to the above configuration, but may be stored in a recording medium such as a CD-ROM, and directly started and executed from this recording medium.
  • the storage means R stores standard spectrum data that serves as a reference for similarity determination.
  • this standard spectrum data is a database of data obtained by computer simulation or previous reports, for example.
  • the arithmetic processing means C is composed of a CPU (Central Processing Unit) and the like, and by executing a similarity determination program installed in the storage means R, as shown in FIG.
  • the unit 53, the standard spectrum data acquisition unit 54, the spectrum comparison determination unit 55, the flavin concentration acquisition unit 61, and the control signal output unit 62 are configured to function.
  • each of these components will be described in more detail.
  • the determination spectrum data acquisition unit 53 acquires the determination spectrum acquired by the determination spectrum acquisition means 4 as data.
  • the determination spectrum recorded in the recorder 45 may be converted into digital data and input from the input unit 51 including a predetermined interface.
  • the standard spectrum data acquisition unit 54 reads out and acquires standard spectrum data stored in the storage means R.
  • the spectrum comparison / determination unit 55 compares the determination spectrum with the standard spectrum to determine whether or not they are similar. Specifically, the spectrum comparison determination unit 55 acquires the determination spectrum data acquired by the determination spectrum data acquisition unit 53 and the standard spectrum data acquired by the standard spectrum data acquisition unit 54, compares the two, It is determined whether or not both are similar based on a predetermined similarity condition. Then, the determination result is output to the control signal output unit 62.
  • the flavin concentration acquisition unit 61 acquires the flavin concentration of the determination solution prepared by the determination solution preparation means 2.
  • the flavin concentration acquisition part 61 acquires the flavin density
  • concentration measuring device as data, when it determines with the spectrum comparison determination part 55 being similar.
  • the control signal output unit 62 outputs a control signal to the determination solution preparation means 2 and the raw material solution preparation means 7.
  • the control signal output unit 62 in the present embodiment receives a determination result that is not similar from the spectrum comparison determination unit 55, the control signal output unit 62 changes the flavin concentration and re-adjusts the determination solution preparation unit 2.
  • the control signal is transmitted.
  • the control signal indicating that the raw material solution is prepared with the flavin concentration acquired by the flavin concentration acquisition unit 61 is transmitted to the raw material solution preparation means 7.
  • the raw material solution preparation means 7 only needs to have a configuration and a function capable of appropriately preparing a raw material solution containing a flavin, an electron donor, and an aqueous solvent.
  • a raw material injection tube 72 that can be inserted into the raw material solution storage portion 73 is provided, and the raw material for the raw material is provided with an injection amount adjusting function according to the number of substances to be injected.
  • a configuration including a plurality of containers 71 can be given.
  • an appropriate amount of flavin, electron donor, and aqueous system are supplied from each raw material injection pipe 72 of the raw material container 71 having an injection amount adjustment function, each containing flavin, an electron donor, and an aqueous solvent.
  • a solvent is injected into the raw material solution storage unit 73 to prepare a raw material solution.
  • the structure similar to the solution storage part 23 for determination mentioned above can be mentioned, for example.
  • the raw material solution preparation means 7 is configured to be controllable by the control signal output from the control signal output unit 62 described above.
  • the generation means 8 only needs to have a configuration and a function capable of appropriately irradiating light to the raw material solution.
  • Examples of such a configuration include the above-described determination spin adduct / radical.
  • a configuration similar to that of the generation unit 3 can be given.
  • the determination solution preparation means 2 may be configured to also serve as the raw material solution preparation means 7, and the determination spin adduct / radical generation means 3 may also be configured to serve as the generation means 8. It is good also as a separate structure.
  • the present invention also provides a superoxide scavenging ability evaluation apparatus.
  • the superoxide scavenging ability evaluation apparatus is an apparatus for evaluating the superoxide scavenging ability of a sample, and includes the following (i), (ii), (iii), (iv), (v), (ix), Having means (x), (xi) and (xii);
  • a determination spin adduct / radical generation means for generating a superoxide spin adduct, an electron donor radical spin adduct and / or an electron donor radical by irradiating the determination solution with light;
  • a spectrum acquisition means for determination for acquiring a spectrum by detecting the spin adduct of the generated superoxide, the spin adduct of the electron donor radical and / or the electron donor radical by electron spin resonance
  • FIG. 7 is a conceptual diagram illustrating the basic configuration of the superoxide scavenging ability evaluation apparatus 9 of the present embodiment.
  • the superoxide scavenging ability evaluation device 9 mainly includes a determination solution preparation means 2, a determination spin adduct / radical generation means 3, a determination spectrum acquisition means 4, and a similarity determination means 5.
  • the same or corresponding components as those of the superoxide production device 1 described above are denoted by the same reference numerals, and the description thereof is omitted.
  • the evaluation solution preparation means 10 only needs to have a configuration and a function capable of appropriately preparing an evaluation solution containing a flavin, an electron donor, a spin trap agent, a sample for evaluating superoxide elimination ability, and an aqueous solvent. .
  • a configuration for example, as shown in FIG. 9, an evaluation container having an evaluation injection tube 102 inserted into the evaluation solution storage unit 103 and having an injection amount adjusting function according to the number of substances to be injected is provided.
  • a configuration including a plurality of 101 can be given.
  • each evaluation injection tube 102 of the evaluation container 101 having an injection amount adjusting function, each containing a flavin, an electron donor, a spin trap agent, a sample for evaluating superoxide elimination ability, and an aqueous solvent.
  • an appropriate amount of flavin, an electron donor, a spin trap agent, a sample for evaluating superoxide elimination ability, and an aqueous solvent are injected into the evaluation solution storage unit 103 to prepare an evaluation solution.
  • the evaluation solution storage part 103 the structure similar to the solution storage part 23 for determination mentioned above can be mentioned, for example.
  • the evaluation solution preparation means 10 is configured to be controllable by a control signal output from the control signal output unit 62.
  • the evaluation spin adduct generation unit 11 only needs to have a configuration and a function capable of appropriately irradiating the evaluation solution with light.
  • a configuration similar to the above-described determination spin adduct / radical generation means 3 can be exemplified.
  • the spectrum acquisition means for evaluation 12 only needs to have a configuration and a function capable of acquiring a spectrum by detecting a spin adduct obtained by irradiating the evaluation solution with light by electron spin resonance.
  • the structure similar to the spectrum acquisition means 4 for determination mentioned above can be mentioned, for example.
  • the comparative evaluation means 13 only needs to have a configuration and a function capable of evaluating the superoxide elimination ability by comparing the superoxide standard spectrum and the evaluation spectrum.
  • the structure similar to the similarity determination means 5 mentioned above can be mentioned, for example.
  • the superoxide erasing ability can be evaluated by comparing the shape of the superoxide standard spectrum displayed on the display unit 52 with the shape of the evaluation spectrum.
  • the comparative evaluation means 13 can be configured by a personal computer or the like.
  • the storage means R separately stores a comparative evaluation program for executing a comparative evaluation process.
  • the arithmetic processing means C has an evaluation spectrum data acquisition unit 131 and a spectral comparison evaluation unit 132 in addition to the components of the arithmetic processing means C in the superoxide production apparatus 1 described above, and functions. It has become.
  • control signal output unit 62 when it is determined that the spectrum comparison determination unit 55 is similar, the control signal output unit 62 outputs a control signal indicating that the evaluation solution is prepared based on the flavin concentration acquired by the flavin concentration acquisition unit 61. The data is transmitted to the preparation means 10.
  • the evaluation spectrum data acquisition unit 131 acquires the evaluation spectrum acquired by the evaluation spectrum acquisition means 12 as data.
  • the spectrum comparison / evaluation unit 132 compares the evaluation spectrum with the standard spectrum to evaluate the superoxide erasing ability. Specifically, the spectrum comparison / evaluation unit 132 acquires the evaluation spectrum data acquired by the evaluation spectrum data acquisition unit 131 and the standard spectrum data acquired by the standard spectrum data acquisition unit 54, and compares them. Superoxide scavenging ability is evaluated according to a predetermined evaluation standard.
  • the determination solution preparation means 2 is also configured as the evaluation solution preparation means 10, and the determination spin adduct / radical generation means 3 is also configured as the evaluation spin adduct generation means 11.
  • the determination spectrum acquisition unit 4 may also serve as the evaluation spectrum acquisition unit 12, and the similarity determination unit 5 may also serve as the comparison evaluation unit 13 or may be a separate configuration.
  • the superoxide erasing ability evaluation apparatus 9 of the present embodiment may be configured to also serve as the superoxide production apparatus 1.
  • the superoxide in vivo production method according to the present invention is a method in which the superoxide production method according to the present invention is applied to a biological sample or a non-human animal body.
  • the amount of flavin (optimum amount) is obtained instead of obtaining the concentration of flavin, and the portion where the administered flavin and electron donor are present is irradiated with light to generate superoxide This is different from the superoxide production method according to the present invention.
  • a raw material administration step of administering an arbitrary amount of flavin, electron donor and spin trap agent into a biological sample or the body of a non-human animal (B) Superoxide spin adduct, electron donor radical spin adduct and / or electron donor by irradiating light in the biological sample or non-human animal body where the administered flavin and electron donor are present Spin adduct for determination / radical generation process for generating radicals, (C) A spectrum acquisition step for determination, in which the generated superoxide spin adduct, electron donor radical spin adduct and / or electron donor radical is detected by electron spin resonance to acquire a spectrum; (D) a similarity determination step of determining whether the standard spectrum of the superoxide spin adduct is similar to the determination spectrum; (E) Optimum flavin acquisition step of acquiring an optimal amount of flavins similar by the similarity determination, (F) and (G); (A) A raw material administration step of administering an arbitrary amount of flavin, electron donor and spin trap agent into a
  • a biological sample separated and collected from animals including humans, particularly mammals can be used.
  • biological samples separated and collected from affected mammals, old mammals, laboratory animals, etc. Can be used.
  • a solid sample such as a tissue section or a cell is desirable.
  • non-human animals can include animals other than humans, such as cattle, monkeys (monkeys minus humans), pigs, goats, dogs, cats, guinea pigs, rabbits, hamsters. , Mammals such as rats and mice, poultry such as chickens and turkeys, reptiles, amphibians and fish.
  • a method for administering a substance such as flavin and an electron donor into a biological sample or a non-human animal can be performed according to a conventional method.
  • a method for administering to a biological sample flavin and electron donor
  • methods for administering a membrane-encapsulated liposome or microinjection method to a non-human animal include oral administration methods and subcutaneous injection methods.
  • the body fluid includes not only extracellular fluid but also intracellular fluid.
  • tissue fluid such as interstitial fluid, intercellular fluid, interstitial fluid, serous cavity fluid, brain
  • spinal fluid joint fluid
  • body cavity fluid such as aqueous humor, digestive fluid, urine, semen, vaginal fluid, amniotic fluid, and milk.
  • the superoxide in vivo scavenging ability evaluation method according to the present invention is a method to which the superoxide scavenging ability evaluation method according to the present invention is applied in the body of a biological sample or non-human animal.
  • Use of body fluids as an aqueous solvent, acquisition of flavin amount (optimum amount) instead of obtaining flavin concentration, and administered flavin and electron donor, or administered flavin, electron donor, spin The method differs from the superoxide production method according to the present invention in that a superoxide is generated or a spin adduct is generated by irradiating an evaluation specimen including a trap agent and a sample for evaluating superoxide elimination ability with light.
  • a raw material administration step of administering an arbitrary amount of flavin, electron donor and spin trap agent into a biological sample or the body of a non-human animal (B) Superoxide spin adduct, electron donor radical spin adduct and / or electron donor by irradiating light in the biological sample or non-human animal body where the administered flavin and electron donor are present Spin adduct for determination / radical generation process for generating radicals, (C) A spectrum acquisition step for determination, in which the generated superoxide spin adduct, electron donor radical spin adduct and / or electron donor radical is detected by electron spin resonance to acquire a spectrum; (D) a similarity determination step of determining whether the standard spectrum of the superoxide spin adduct is similar to the determination spectrum; (E) Optimum flavin acquisition step of acquiring an optimal amount of flavins similar by the similarity determination, (H
  • a sample for evaluating flavin, electron donor, spin trap agent and superoxide scavenging ability is directly administered to a biological sample or a non-human animal body, and the specimen to be evaluated is placed in the biological sample or non-human animal body. It may be prepared.
  • Example 1 Measurement by electron spin resonance of radicals generated by irradiating an aqueous solution containing riboflavin, EDTA, and CYPMPO by means of electron spin resonance Riboflavin as a redox reaction catalyst, EDTA as an electron donor, and CYPMPO as a spin trap agent An aqueous solution was prepared, and this was irradiated with light to generate radicals, and the generated radicals were identified and quantified by electron spin resonance (ESR).
  • ESR electron spin resonance
  • aqueous solution Riboflavin, EDTA, and CYPMPO (Radical Research Co., Ltd.) were added to a phosphate buffer solution of 50 mmol / L and pH 7.4 to give 1 ⁇ mol / L, 5 mmol / L, and 10 mmol / L, respectively.
  • a solution (a SOD-free aqueous solution) was prepared.
  • SOD superoxide dismutase
  • an enzyme that specifically eliminates superoxide was added at 10 U / mL, and an SOD-added aqueous solution was separately prepared.
  • Example (2) Measurement by ESR
  • the aqueous solutions (the SOD-free aqueous solution and the SOD-added aqueous solution) prepared in Example (1) were respectively introduced into the sample tubes of an electron spin resonance apparatus JES-RE1X (JEOL Ltd.).
  • the sample tube was irradiated with 1500 lux of visible light using a xenon lamp for 30 seconds, and then measured by ESR under the following measurement conditions to obtain a spectrum.
  • the spectrum of the SOD-free aqueous solution was designated as spectrum A
  • the spectrum of the SOD-added aqueous solution was designated as spectrum B.
  • the result is shown in FIG.
  • the shape of the spectrum A in FIG. 10 and the shape of the superoxide standard spectrum in the upper diagram of FIG. It was confirmed that the signal intensity of the standard spectrum hardly changed. Moreover, it was confirmed that the shape of the spectrum B of FIG. 10 and the shape of the EDTA radical standard spectrum of the lower figure of FIG. 11 are the same. Further, as shown in FIG. 10, it was confirmed that the signal intensity of the spectrum B was smaller than the signal intensity of the spectrum A, and the peak observed in the spectrum A was not observed in the spectrum B.
  • the peak seen in the spectrum A is derived from the spin adduct of superoxide, and that a large amount of superoxide was generated in the SOD-free aqueous solution.
  • the peak seen in the spectrum B is derived from the EDTA radical, and since the peak seen in the spectrum A is not seen in the spectrum B, a small amount of EDTA radical is generated in the SOD-added aqueous solution, It was shown that no superoxide was generated.
  • the aqueous solution according to this example generates two types of radicals, superoxide and electron donor radicals (interfering radicals, TH radicals), and selectively generates superoxide. It has been clarified that the generation of superoxide can be suppressed by adding a substance having superoxide elimination ability to this aqueous solution.
  • oxidation-reduction reaction catalyst and electron donor By changing the oxidation-reduction reaction catalyst or electron donor, radicals are generated by light irradiation, and radicals generated by ESR are identified and quantified to generate superoxide. Suitable redox reaction catalysts and electron donors were investigated.
  • Riboflavin, TMD, and CYPMPO were added to a phosphate buffer solution of 50 mmol / L and pH 7.4 so as to be 10 ⁇ mol / L, 10 mmol / L, and 10 mmol / L, respectively, to prepare an aqueous solution.
  • TMD was replaced with methionine
  • an aqueous solution was prepared in the same manner as in Example (1), and measurement by ESR was performed to obtain a spectrum.
  • the obtained spectrum is shown in FIG.
  • FMN, EDTA, and CYPMPO were added to a phosphate buffer solution of 50 mmol / L and pH 7.4 to give 5 ⁇ mol / L, 5 mmol / L, and 10 mmol / L, respectively, thereby preparing an aqueous solution.
  • An aqueous solution was prepared in the same manner as in Example (1) by replacing riboflavin with fluorescein and TMD with methionine, and a spectrum was obtained by measurement by ESR. The obtained spectrum is shown in FIG.
  • riboflavin is used as the redox reaction catalyst and TMD is used as the electron donor
  • riboflavin is used as the redox reaction catalyst and as the electron donor.
  • methionine is used, FMN is used as the oxidation-reduction reaction catalyst, TMD is used as the electron donor, fluorescein is used as the oxidation-reduction reaction catalyst, and methionine is used as the electron donor.
  • Example 3 Examination of relationship between riboflavin concentration and amount of superoxide generated and purity of superoxide Generation of superoxide and electron donor radicals (interfering radicals, TH radicals) by changing the concentration of riboflavin in the aqueous solution The amount change was confirmed.
  • Example 1 (1) In the aqueous solutions prepared in Example 1 (1) (SOD-added aqueous solution and SOD-free aqueous solution), the riboflavin concentrations were 0.1 ⁇ mol / L, 0.5 ⁇ mol / L, 1 ⁇ mol / L, and 2.5 ⁇ mol / respectively. Aqueous solutions were prepared so as to be L, 5 ⁇ mol / L, 10 ⁇ mol / L, 15 ⁇ mol / L, 25 ⁇ mol / L, and 50 ⁇ mol / L.
  • radicals generated in each aqueous solution were identified or the amount of generated radicals was quantified. That is, the generated EDTA radical (interfering radical) amount was quantified based on the spectrum of the SOD-added aqueous solution, and the generated superoxide amount was quantified based on the spectrum of the SOD-free added aqueous solution. The amount of superoxide was quantified when it was determined that it had a spectrum shape unique to superoxide based on the shape of the superoxide standard spectrum (upper figure in FIG. 11). Further, based on the numerical results, the purity of superoxide in the radicals generated in the aqueous solution of each riboflavin concentration was calculated by the following equation 4. The result is shown in FIG.
  • EDTA radicals interfering radicals
  • the purity of superoxide Were determined to be 87.5%, about 83.3%, and about 76.5%, respectively.
  • the signal derived from the EDTA radical (interfering radical) was strong, so the amount of generated superoxide was not quantified and the purity of superoxide was not calculated. .
  • Example 4 Confirmation of relationship between light irradiation time and superoxide generation amount Time from light irradiation start to superoxide generation, time from light irradiation stop to superoxide generation stop, light irradiation time and superoxide generation amount Confirmed the relationship.
  • aqueous solution Riboflavin, EDTA, and CYPMPO (Radical Research Co., Ltd.) were added to a phosphate buffer solution of 50 mmol / L and pH 7.4 so as to be 1 ⁇ mol / L, 3 mmol / L, and 10 mmol / L, respectively.
  • a solution was prepared.
  • the prepared aqueous solution was divided into 16 samples, which were designated as Sample 1 to Sample 16, respectively.
  • Example 2 Measurement by ESR
  • the aqueous solution prepared in Example (1) was sequentially introduced into a sample tube of an electron spin resonance apparatus JES-RE1X (JEOL Ltd.) and irradiated with visible light after changing the irradiation time.
  • a spectrum was obtained by measuring by ESR.
  • the irradiation time of visible light in each sample is shown below.
  • the ESR observation magnetic field was fixed at 335.7 mT (the same magnetic field as the magnetic field in which the peak derived from superoxide was confirmed in Example 1), and the measurement by ESR was performed.
  • Other light irradiation conditions and measurement conditions by ESR were the same as in Example 1 (2).
  • Visible light irradiation time sample 1 30 seconds before the start of irradiation (-30)
  • Sample 2 15 seconds before the start of irradiation (-15)
  • Sample 3 0 seconds before the start of irradiation (0)
  • Sample 4 Irradiation time 5 seconds (5)
  • Sample 5 irradiation time 10 seconds (10)
  • Sample 6 irradiation time 15 seconds (15)
  • Sample 7 irradiation time 20 seconds (20)
  • Sample 8 irradiation time 30 seconds (30)
  • Sample 9 irradiation time 45 seconds (45)
  • Sample 10 irradiation time 60 seconds (60)
  • Sample 11 irradiated for 60 seconds, 5 seconds after irradiation stopped (+5)
  • Sample 12 irradiated for 60 seconds, 10 seconds after irradiation stopped (+10)
  • Sample 13 irradiated for 60 seconds, 20 seconds after irradiation stopped (+20)
  • Sample 14 60 seconds irradiation, 30 seconds after irradiation stop
  • the signal intensity of the obtained spectrum was plotted on the vertical axis, and the visible light irradiation time was plotted on the horizontal axis. The result is shown in FIG.
  • Example 1 (1) Determination of radical generation amount by light irradiation of riboflavin SOD addition so that SOD concentrations of the SOD-added aqueous solution in Example 1 (1) are 0.5 U / mL, 1 U / mL, and 1.5 U / mL, respectively.
  • An aqueous solution and an SOD-free aqueous solution were prepared.
  • Each prepared aqueous solution was measured by ESR in the same manner as in Example 1 (2) to obtain a spectrum. Based on the obtained spectrum, the amount of radicals generated was quantified.
  • Example 2 After adding xanthine oxidase to the prepared SOD-free aqueous solution and SOD-added aqueous solution to 0.2 U / mL, respectively, measurement by ESR was performed in the same manner as in Example 1 (2), and the spectrum was obtained. Obtained. Subsequently, the amount of radicals generated was quantified based on the obtained spectrum. The measurement by ESR was performed 60 seconds after adding xanthine oxidase.
  • the radical generation inhibition rate by SOD represents the purity of superoxide. That is, in the generated radical, the higher the superoxide purity, the higher the radical generation inhibition rate by SOD.

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Abstract

La présente invention concerne un procédé destiné à produire facilement et de manière stable un superoxyde en générant sélectivement le superoxyde ou un radical contenant le superoxyde avec une pureté élevée ; un procédé pour évaluer facilement la capacité de piégeage du superoxyde sur un échantillon témoin ; un dispositif destiné à produire facilement et de manière stable un superoxyde en générant sélectivement le superoxyde ou un radical contenant le superoxyde avec une pureté élevée ; et un dispositif pour évaluer facilement la capacité de piégeage du superoxyde sur un échantillon témoin. Le procédé de production d'un superoxyde mentionné précédemment comprend : une étape (a) de préparation d'une solution destinée à la détermination ; une étape (b) de formation d'un adduit/radical destiné à la détermination ; une étape (c) d'acquisition d'un spectre destiné à la détermination ; une étape (d) de détermination de la similarité ; une étape (e) d'acquisition de la concentration en flavine ; une étape (f) de préparation d'une solution de produit de départ ; et une étape (g) de génération.
PCT/JP2010/072050 2009-12-09 2010-12-08 Procédé de production d'un superoxyde, procédé d'évaluation de la capacité de piégeage du superoxyde, dispositif de production d'un superoxyde, et dispositif d'évaluation de la capacité de piégeage du superoxyde WO2011071088A1 (fr)

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US13/514,876 US20120255853A1 (en) 2009-12-09 2010-12-08 Method for Producing Superoxide, Method for Evaluating Superoxide Scavenging Ability, Device for Producing Superoxide, and Device for Evaluating Superoxide Scavenging Ability
JP2011545231A JP5723294B2 (ja) 2009-12-09 2010-12-08 スーパーオキシド製造方法、スーパーオキシド消去能評価方法、スーパーオキシド製造装置およびスーパーオキシド消去能評価装置

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