JP6332737B2 - Testing method for antioxidant activity - Google Patents

Description

  The present invention relates to a method for testing antioxidant activity.

  Active oxygen generated by metabolism, external stress, etc. has extremely high reactivity, reacts with proteins, lipids, DNA, etc. in the human body, promotes onset of lifestyle-related diseases and aging, ischemia reperfusion It is thought to bring about the expression of cancer cells due to subsequent tissue damage and DNA damage.

  In order to prevent these oxidative damages, various foods, cosmetics, pharmaceuticals and the like (hereinafter collectively referred to as antioxidant compositions) that eliminate active oxygen have been proposed.

  And, according to these antioxidant compositions, it is said that the antioxidant ability can be caused in the body, the skin surface, etc., and the environment of the body and the skin can be made healthy.

  By the way, it is desirable that the antioxidant ability of these antioxidant compositions can be specifically evaluated as numerical values and ratios.

  Thus, several methods for evaluating the antioxidant ability of the antioxidant composition have been proposed.

  For example, a method known as DPPH radical scavenging activity measurement is a method in which 1,1-diphenyl-2-picrylhydrazyl radical is reacted with an antioxidant for a certain period of time, then the absorbance at 520 nm is measured, and the decrease in the absorbance value is known. It is calculated as a relative value with respect to the absorbance of Trolox.

  In addition, a method known as the active oxygen absorption capacity (ORAC method) uses AAPH (2,2'-azo-bis (2-amidinopropane) dihydrochloride) to generate peroxy radicals (ROO), which are decomposed by these radicals. The fluorescence intensity of fluorescein is measured over time, and the area under the phenomenon curve (AUC) is obtained. Then, the difference (net AUC) obtained by subtracting the Blank AUC from the AUC measured in the presence of the antioxidant is calculated, and the relative value to the net AUC in Trolox with a known concentration is calculated and displayed in terms of Trolox equivalent. (For example, refer nonpatent literature 1.).

Tomoyuki OKI: ORAC Method, Food Functionality Evaluation Manual Collection II, p79-86 (2008)

  However, in the above-described DPPH radical scavenging activity measurement, there is a problem that when anthocyanins are contained, the absorbance cannot be measured and it is necessary to measure after separation by HPLC.

  In the ORAC method described above, there are hydrophilic H-ORAC and lipophilic L-ORAC, both of which require dissolution in an organic solvent, and there is a problem in that antioxidants such as carotenoids cannot be measured.

  A problem common to both methods is that, in order to measure the decomposition of radicals, the measurement must be performed at a pH of around 14.

  Currently, fluorescein is used as a standard probe in the ORAC method, but in this method, uric acid in plasma is measured as the main antioxidant. Problems have been pointed out, such as the ORAC level in the plasma of hyperureaemia patients being evaluated higher than that of healthy subjects (Reference 1: Akio Futaki "Topics on Antioxidant Activity and Efficacy: ORAC Database by USDA Vitamin 86 (9), pp. 519-520. Etsuo Niki (2012) Topics on capacity and benefitial effects of antioxidants: Removal of ORAC data base by USDA. VITAMINS 86 (9), 519-520.) .

  The USDA Nutrition Data Laboratory (NDL) announced on May 16, 2012 that the ORAC database will be withdrawn because the ORAC values of certain antioxidants are not directly related to human health as described above. (Reference 2: http://www.ars.usda.gov/Services/docs.htm?docid=15866).

  In addition, it has been pointed out that the ORAC method cannot distinguish the rate and amount (concentration) of radical capture by antioxidants (Reference 3: Niki E. (2010) Assessment of antioxidant capacity in vitro and in vivo. Free Radical Biology and Medicine 49 (4): 503-515.).

  Furthermore, foods and pharmaceuticals taken orally and pharmaceuticals administered into blood vessels cause antioxidant activity in the body, and the pH of the environment in which the antioxidant activity is exerted is in the neutral to acidic range. . Moreover, also about cosmetics used on the skin surface, the pH of the environment where the antioxidant activity is exhibited is in a neutral to weakly acidic region.

  In other words, all of the above-described methods for evaluating antioxidant activity measure the antioxidant activity in a strongly alkaline environment, and how much antioxidant activity these antioxidant compositions actually exhibit in the body and skin surface. As a method of evaluating whether or not.

  The present invention has been made in view of such circumstances, and the antioxidant activity of the antioxidant composition can be evaluated in a neutral region closer to the human body, and the reaction rate of the radical scavenging factor. And a method for testing antioxidant activity capable of detecting concentrations. The present invention also provides a medium on which data obtained by this test method is recorded, and a method for determining an index dose of an antioxidant active agent whose dosage is substantially unknown in vivo.

In order to solve the above-mentioned conventional problems, in the invention according to claim 1 of the present application, in the method for testing antioxidant activity, the active oxygen species is contained at a concentration of 1 μM or more in terms of at least potassium superoxide (KO ₂ ). Prepare a sample solution to be measured in the presence of a sample to be evaluated for antioxidant activity in a luminescent base solution in which active oxygen-containing water and luciferins that emit light in the presence of the active oxygen species are mixed. By measuring the intensity of chemiluminescence derived from the luciferins emitted from the sample liquid and the luminescent base liquid not containing the analyte, respectively, the intensity of chemiluminescence from the measurement sample liquid is determined from the luminescent base liquid. when the lower intensity of the chemiluminescence, wherein the analyte is a method of inspecting a determining antioxidant activity has an antioxidant activity, and oligo DNA and copper having a repeating sequence of cytosine and guanine It was decided, which comprises using a complex as an indication substance scavenger of superoxide anion radicals.

  Further, in the invention according to claim 2 of the present application, in the method for testing antioxidant activity according to claim 1, a plurality of measurement sample solutions having different analytes are prepared, and the chemiluminescence intensity from each measurement sample solution is compared. Thus, the strength of the antioxidant activity in the specimen added to the predetermined measurement sample liquid among the plurality of measurement sample liquids is determined with respect to the antioxidant activity in the specimen added to the other measurement sample liquid.

  In the invention according to claim 3 of the present application, in the method for testing antioxidant activity according to claim 1 or claim 2, the active oxygen-containing water is obtained by bringing water into contact with the excited photocatalyst. It was prepared by dispersing active oxygen species generated on the surface of the photocatalyst in water.

  Further, in the invention according to claim 4 of the present application, in the method for testing antioxidant activity according to claim 3, the water to be brought into contact with the photocatalyst is supplied through a supply water treatment unit for adding a predetermined substance to the water. The predetermined substance is at least one selected from oxygen gas, ozone gas, chlorine gas, nitrogen monoxide gas, ammonia gas, and metal salt.

In the invention according to claim 5 of the present application, in the method for testing antioxidant activity according to claim 4, the metal salt is at least one selected from NaCl, CaCl ₂ , KCl, MgCl ₂ , and CuSO _4. The concentration in the water is 0.05 mM to 50 mM for NaCl and CaCl ₂ , 0.05 mM to ₅ mM for KCl and MgCl ₂ , and 50 μM for CuSO _4. ˜200 μM.

  In the invention according to claim 6 of the present application, in the method for testing antioxidant activity according to any one of claims 1 to 5, the luciferin is a Cypridina Luciferin Analog. It was decided that.

Further, in the invention according to claim 7 of the present application, the second anti-oxidant active agent whose dose is substantially effective in vivo and the second anti-oxidant whose dose is substantially effective in vivo are known. A method for determining a dose that serves as an index for producing a medicinal effect equivalent to that of an oxidatively active agent, comprising active oxygen-containing water containing active oxygen species at a concentration of 1 μM or more in terms of at least potassium superoxide (KO ₂ ) In the luminescent base solution in which the first antioxidant active agent is present as a specimen in the luminescent base solution mixed with the luciferins that emit light in the presence of the active oxygen species, and in the luminescent base solution A second measurement sample solution containing the second anti-oxidant active agent as a sample, and the luciferin derived from each measurement sample solution and the luminescent base solution not containing the sample The intensity of chemiluminescence And is included within an error range of ± 25% of the ratio of the area under the time-dependent curve of the luminescence intensity of the second antioxidant active agent to the area under the time-dependent curve of the luminescence intensity of the luminescent base solution. The index dose determination method is characterized in that the concentration corresponding to the area under the time-dependent change curve of the luminescence intensity of the first antioxidant active drug is used as the index dose of the first antioxidant active drug.

The invention according to claim 8 of the present application is a complex of copper and oligo DNA having cytosine and guanine repetitive sequences, and is superposed by the antioxidant activity testing method according to any one of claims 1 to 6. The antioxidant was evaluated as an oxide anion radical scavenger.

According to the invention of claim 1 of the present application, active oxygen-containing water containing active oxygen species at a concentration of 1 μM or more in terms of at least potassium superoxide (KO2) and luciferin that emits light in the presence of the active oxygen species. A sample solution to be evaluated is prepared in the presence of a sample to be evaluated for antioxidative activity in a luminescent base solution mixed with a liquid, and is emitted from the same sample solution and the luminescent base solution not containing the sample. By measuring the intensity of chemiluminescence derived from the luciferins, when the intensity of chemiluminescence from the measurement sample solution is lower than the intensity of chemiluminescence from the luminescent base solution, the sample has antioxidant activity. Then, since it decided to determine, the antioxidant activity test | inspection method which can be evaluated in the neutral range nearer to the human body about the antioxidant activity of an antioxidant composition can be provided. In addition, since a complex of oligo DNA having a repetitive sequence of cytosine and guanine and copper is used as an indicator substance for the superoxide anion radical scavenging substance, a solid test can be performed.

  According to the invention of claim 2 of the present application, a plurality of measurement sample liquids having different specimens are prepared, and the intensity of chemiluminescence from each measurement sample liquid is compared. Since it was decided to determine the strength of the antioxidant activity of the sample added to the measurement sample solution against the antioxidant activity of the sample added to the other measurement sample solution, among the plurality of measurement sample solutions, This sample can be compared with the antioxidant activity in other measurement sample liquids.

  According to the invention of claim 3 of the present application, the active oxygen-containing water disperses the active oxygen species generated on the surface of the photocatalyst by bringing the water into contact with the excited photocatalyst. Therefore, the inspection can be performed by containing a high concentration of active oxygen species.

  According to the invention according to claim 4 of the present application, the water to be brought into contact with the photocatalyst is supplied via a supply water treatment unit that adds a predetermined substance to the water, and the predetermined substance is Since it is at least one selected from oxygen gas, ozone gas, chlorine gas, nitric oxide gas, ammonia gas, and metal salt, it can be inspected by containing a higher concentration of active oxygen species. .

According to the invention of claim 5, the metal salt is at least one selected from NaCl, CaCl ₂ , KCl, MgCl ₂ and CuSO ₄ , and the concentration in the water is NaCl and CaCl. in the ₂ is the concentration of 0.05MM～50mM, in the KCl and MgCl ₂ at a concentration of 0.05MM～5mM, since it was decided in the CuSO ₄ is 50Myuemu～200myuM, efficient high concentration The active oxygen species can be included in the inspection.

  Further, according to the invention of claim 6, since the luciferin is a Cypridina Luciferin Analog, the presence / absence of the specimen in the luminescent base solution, or between a plurality of specimens It is possible to detect with high sensitivity the strength of antioxidant activity.

In addition, according to the invention of claim 7 of the present application, the second anti-oxidant active agent whose dose that is substantially effective in vivo is known and the dose that is substantially effective in vivo is known. Is a method for determining a dose which is an index for producing an effect equivalent to that of an antioxidant active agent, and contains active oxygen species at a concentration of 1 μM or more converted to at least potassium superoxide (KO ₂ ) A first measurement sample solution in which the first antioxidant active agent is present as a specimen in a luminescent base solution in which water and luciferins that emit light in the presence of the active oxygen species are mixed, and the luminescent group The second measurement sample liquid in which the second antioxidant active agent is present as a specimen in the liquid is prepared, and the luciferins emitted from each measurement sample liquid and the luminescent base solution not containing the specimen The intensity of chemiluminescence derived from each Measured over time and included within an error range of ± 25% of the ratio of the area under the time-dependent change curve of the luminescence intensity of the second antioxidant active agent to the area under the time-change curve of the light-emitting intensity of the luminescent base solution. Since the concentration corresponding to the area under the time-dependent change curve of the luminescence intensity of the first antioxidant active drug is used as the index dose of the first antioxidant active drug, there is a dose that is substantially effective in vivo. The index dose of the unknown first antioxidant active agent can be easily determined.

Moreover, according to the invention which concerns on this-application Claim 8 , it is a composite_body | complex of oligo DNA which has a repeating sequence of cytosine and guanine, and copper, The test method of antioxidant activity of any one of Claims 1-6 Thus, since the antioxidant can be evaluated as a superoxide anion radical scavenger, an accurate test result can be obtained in the method for testing antioxidant activity.

It is explanatory drawing which showed the active oxygen species density | concentration in the active oxygen containing water at the time of adding a metal salt. It is explanatory drawing which showed the verification result of the effectiveness in antioxidant activity evaluation of active oxygen containing water. It is explanatory drawing which showed the evaluation result of the antioxidant activity of glutathione. It is explanatory drawing which showed the evaluation result of the antioxidant activity of ascorbic acid. It is explanatory drawing which showed the evaluation result of the density ^| concentration dependence antioxidant activity by Z-type oligo DNA * Cu2 ⁺ complex. By Z-type oligo DNA and Cu ² is an explanatory view showing evaluation results of antioxidant activity exerted by complex formation. It is explanatory drawing which showed the evaluation result of the antioxidant activity of an edaravone formulation. It is explanatory drawing which showed the evaluation result of antioxidant activity, such as lemon. It is explanatory drawing which showed the evaluation result of antioxidant activity, such as tomato. It is explanatory drawing which showed the evaluation result of the antioxidant activity of soft drink.

  The present invention relates to an evaluation object of antioxidant activity in a luminescent base solution in which active oxygen-containing water containing active oxygen species at a concentration of at least 1 μM and luciferin that emits light in the presence of the active oxygen species are mixed. By preparing a measurement sample solution in the presence of the analyte, and measuring the intensity of chemiluminescence derived from the luciferins emitted from the measurement sample solution and the luminescent base solution not containing the sample, When the intensity of chemiluminescence from the measurement sample solution is lower than the intensity of chemiluminescence from the luminescent base solution, the method provides a test method for antioxidant activity that determines that the specimen has antioxidant activity.

  As described above, conventionally, the antioxidant ability of medicines, cosmetics, foods and beverages has been evaluated using the ability to eliminate radical species generated in the reaction process of organic compounds. A typical method for evaluating antioxidant capacity is to measure water-soluble formazan WST-1 produced in the reduction reaction of superoxide anion radical and tetrazolium salt WST-1 generated when xanthine oxidase is reacted with pipexanthine by absorbance. Of radical scavenging antioxidants using chemical reactions such as the method of evaluating enzyme activity (superoxide dismutase activity) using superoxide anion radical as substrate and ORAC method, DPPH radical scavenging activity (SET) method, etc. There are evaluation methods.

  However, in the DPPH radical scavenging activity (SET) method, by using organic radicals, the entire sample cannot be evaluated in a water-containing state, and evaluation is performed under conditions such as lyophilization. Therefore, polyphenol activity, anthocyanin-containing samples and the like must be separately measured for their analytical amounts, and cannot be evaluated in a state inoculated and administered. (Evaluation by itself is not possible)

In the ORAC method, there is a problem that activities such as high-concentration uric acid that should not be contained in the living body or food of a healthy person are highly evaluated, and superoxide dismutase (SOD) using xanthine oxidase and formazan formation reaction. In the evaluation method of activity and SOD-like activity, it is not possible to distinguish the effect of an inhibitory substance on the superoxide anion radical (O ₂ ⁻ ) and an inhibitory substance on the xanthine oxidase which is an O ₂ ⁻ producing enzyme.

In addition, with regard to the scavenging ability of superoxide anion radicals (O ₂ ⁻ ), any method is a low molecular weight antioxidant that reacts non-selectively directly with active oxygen such as O ₂ ⁻ as in ascorbic acid. it is difficult to identify the decomposition reaction polymer antioxidant group activity that catalyzes the ^- selective O ₂ as the group and superoxide dismutase (SOD).

  Further, the pH at the time of evaluation is evaluated with strong alkalinity, and there is a possibility that the sample is denatured. In addition, the use of organic radicals may cause direct reaction with the sample, which may prevent accurate evaluation.

  Moreover, although it is sold as a kit, a problem may occur in stability reproducibility.

  Furthermore, since it is not a comprehensive evaluation, the antioxidant activity of food is expressed by the total amount of substances that are said to have antioxidant ability, and the strength of the antioxidant activity as a comprehensive evaluation of the target sample is expressed. I can't show it. (For example, vitamin C 1000 mg, citric acid 3000 mg, polyphenol 10 mg, etc.)

  Therefore, the present inventors have conducted intensive research and have detected long-time active oxygen that has been able to be generated constantly with a photocatalytic water generator and a water generator having redox activity. The idea was to evaluate the antioxidant capacity of the samples using water that can be used.

The evaluation method is a chemiluminescence method using Cypridina luciferin analog (CLA). After adding 5 μL of the sample to 5 μL of the saturated CLA solution, 490 μL of active oxygen-containing water that has reached a steady state is added, and fluorescence emission is measured for 5 minutes. It is defined that the steady state is reached when the emission intensity with a spike intensity of 1 μmol / L (KO ₂ concentration) or more can be detected continuously for 5 minutes or more.

  The luminescence intensity of the sample to which the sample is not added is compared with that of the sample, and the antioxidant ability of the target sample is evaluated.

  The reactive oxygen species that can be primarily evaluated by this method are singlet oxygen or superoxide anion radicals. Since evaluation can be performed using two types of reactive oxygen species, it is possible to evaluate both cationic antioxidants and anionic antioxidants.

Antioxidants that remove reactive oxygen species include known superoxide anion radical (O ₂ ⁻ ) scavenging activity, hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (HO ⁻ ), singlet It can be broadly classified into those known to act only on other active oxygen species such as term oxygen ( ¹ O ⁻ ). As antioxidants known to have an ability to eliminate O ₂ ⁻ , superoxide dismutase (SOD), ascorbic acid, bilirubin and the like are known.

On the other hand, uric acid, which is evaluated as a substance having high antioxidant activity by the ORAC method, has no superoxide anion radical (O ₂ ⁻ ) scavenging ability. Therefore, the problem that is pointed out as a problem of the ORAC method, such as the ORAC value in the plasma of hyperureaemia patients being evaluated higher than that of healthy subjects (the above-mentioned reference 1), is the superoxide anion radical (O ₂ ^-) can be avoided by carrying out the evaluation reflecting higher scavenging ability against.

  As an evaluation index, it can be quantified by the area ratio of the area under the decreasing curve due to the difference between the target control curve (area under curve) and the curve exhibited by the sample addition group. Evaluation is the most persuasive.

  Reactive oxygen species originally played an important role in maintaining health, such as being involved in biological defense including the pathogen elimination mechanism of macrophages during infection with viruses and bacteria. However, because it is extremely reactive, once it is excessive, it reacts with proteins, lipids, or DNA macromolecules in the body, causing protein modification, lipid peroxide generation, genetic disorders, etc. It is said to cause onset and aging, and carcinogenic effects. In addition, at the time of ischemia / reperfusion injury of organs such as cerebral infarction and myocardial infarction, tissue damage due to reactive oxygen species is enhanced, resulting in tissue edema and irreversible degeneration.

  For this reason, it is necessary to appropriately evaluate the antioxidant capacity and simply evaluate the concentration effect including the prediction of the efficacy and dynamics of the sample. In this method, a hydrophilic substance can be used for comparative evaluation of a large number of samples without requiring pretreatment of the target sample. For example, the antioxidant activity comparison for each variety and the antioxidant activity of drinking water can be easily reproducible.

  Advances in the development of beverages, drugs, and foods that have been appropriately evaluated through the use of this law will contribute to the promotion of health in a super-aging society.

Here, as the reactive oxygen species in the present specification, for example, superoxide anion radical (O ₂ ⁻ ), hydroxy radical (· OH), lipid peroxide (LOOH, LOO ·), halogenated oxygen (ClO ⁻ ), Cyclic peroxides and hydroperoxides generated from singlet oxygen ( ¹ O ₂ ) and singlet oxygen include what are called free radicals such as nitrogen monoxide radical (NO ·) and oxygen-containing organic radical species (eg phenoxy radical). And hydrogen peroxide is not included.

Further, the active oxygen-containing water used in the inspection method according to the present embodiment contains such active oxygen species at a concentration of 1 μM or more in terms of at least potassium superoxide (KO ₂ ). A concentration of less than 1 μM in terms of potassium superoxide (KO ₂ ) is not preferable because it is similar to luminescence expressed by a small amount of ozone metabolite contained in tap water, for example. Containing reactive oxygen species at a concentration of 1 μM or more in terms of potassium superoxide (KO ₂ ) has been confirmed to be a reactive oxygen species mainly composed of superoxide anion radicals by adding Tiron, and by adding DABCO. It can be confirmed that the active oxygen species contains singlet oxygen.

  The active oxygen-containing water is not particularly limited, but is prepared by bringing water into contact with the excited photocatalyst to disperse the active oxygen species generated on the surface of the photocatalyst in this water. It is desirable to be a thing.

When such a method is used for preparing active oxygen-containing water, it is possible to make active oxygen species of 1 μM or more in water relatively easily converted to potassium superoxide (KO ₂ ). Inspection can be performed efficiently. Moreover, it can be set as the active oxygen containing water which can be detected continuously for a long time.

In addition, the luciferins used in the inspection method according to the present embodiment emit light in the presence of active oxygen species. For example, the superoxide anion radical (O ₂ ⁻ ), the hydroxy radical (.OH ), Lipid peroxides (LOOH, LOO ·), halogenated oxygen (ClO ⁻ ), nitric oxide radicals (NO ·), oxygen-containing organic radical species (eg phenoxy radicals), so-called free radicals, single Any material may be used as long as it emits light by at least one of cyclic peroxide and hydroperoxide generated from singlet oxygen ( ¹ O ₂ ) and singlet oxygen.

As a specific example of such luciferins, for example, Cypridina Luciferin Analog can be used. By using luciferin as a Cypridina luciferin analog, it is possible to detect with high sensitivity the presence / absence of the analyte in the luminescent base solution and the strength of the antioxidant activity between a plurality of analytes. Incidentally, Cypridina luciferin analogs, O ₂ ^- and a chemiluminescent probe to react to the light emitting specifically to ¹ O _2, O ₂ ^- is and ¹ O ₂ effective to evaluate the specialized scavenging activity in .

  And in the test | inspection method which concerns on this embodiment, the liquid with which this luciferin and the above-mentioned active oxygen containing water were mixed is used as a luminescent base solution. The luminescent base solution is a solution that serves as a base of a measurement sample solution for measuring the antioxidant activity of a specimen, and also serves as a control solution for comparison with the measurement sample solution.

  That is, a measurement sample solution is prepared by allowing a sample to be measured for antioxidant activity to be present in the luminescent base solution. In addition, the mixing order of the active oxygen-containing water, luciferins, and the specimen in the preparation of the measurement sample solution is not particularly limited, and can be appropriately determined according to the properties of the specimen. For example, if the specimen is liquid, first prepare the measurement sample solution by dispensing the specimen into a luminescence measurement cuvette, etc., then adding luciferins, and finally adding water containing active oxygen. Also good. According to such a method, the start of luminescence by luciferins can be suppressed until just before the measurement, and the stirring of the specimen in the measurement sample solution can be omitted in some cases. Such a procedure is particularly effective when the amount of the specimen or the total amount of the measurement sample solution is small.

  The measurement sample solution thus prepared is immediately subjected to chemiluminescence measurement. The instrument used for the measurement of chemiluminescence is not particularly limited as long as it is an apparatus that can detect the intensity of the wavelength of light emitted from luciferins by reactive oxygen species. For example, a luminometer or luminometer manufactured by Ato Corporation. A net sensor or the like can be used.

  The light emission intensity may be measured by measuring the intensity at a predetermined timing after the start of light emission, or measuring the change in intensity over time until the predetermined time after the start of light emission.

  Then, the luminescent base solution not containing the sample is used as a control (positive control), compared with the luminescence intensity of the measurement sample solution, and the sample is resistant to the behavior of the luminescence intensity of the measurement sample solution compared to the control. It can be determined whether or not it has oxidation activity.

  Specifically, if the emission intensity of the measurement sample solution is lower than that of the control, the action of the active oxygen species derived from the active oxygen water is suppressed by the specimen, and the specimen has antioxidant activity. Can be determined. Of course, a negative control may be provided as necessary. The negative control may be, for example, tap water, phosphate buffer solution, distilled water, or active oxygen-containing water only.

  By the way, in the above-described inspection method, the comparison between the control and the measurement sample solution is mentioned. However, the measurement sample solution can be compared with other measurement sample solutions.

  That is, by preparing a plurality of measurement sample liquids with different specimens, comparing the intensity of chemiluminescence from each measurement sample liquid, the antioxidant activity in the specimen added to the predetermined measurement sample liquid among the plurality of measurement sample liquids The strength of the antioxidant activity in the specimen added to another measurement sample solution may be determined.

  By setting it as such a method, it becomes possible to compare and examine which health food exhibits the higher antioxidant activity about the commercially available health food X and the health food Y, for example. For example, when trying to provide vegetable juice with extremely high antioxidant activity, it has the best taste, high food nutrition, and also exhibits antioxidant activity due to changes in the composition ratio and differences in composition varieties. It becomes possible to mix and adjust vegetable juice.

  In addition, the data obtained by the above-mentioned luminescence measurement is recorded on a predetermined medium, so that data for determining the presence or absence of the antioxidant activity in the specimen or the antioxidant activity in the specimen in the predetermined sample solution It can also be used as data for determining the strength of a sample in another sample solution, and information can be provided as an application.

  That is, it becomes an evaluation target of antioxidant activity in a luminescent base solution in which active oxygen-containing water containing active oxygen species at a concentration of at least 1 μM and luciferins that emit light in the presence of the active oxygen species are mixed. Data for determining the presence or absence of antioxidant activity in the specimen, in which the ratio of the chemiluminescence intensity derived from luciferins to the luminescent base solution not containing the specimen of the measurement sample liquid prepared in the presence of the specimen is known. It may be a recorded medium.

  In addition, it becomes an evaluation target of antioxidant activity in a luminescent base solution in which active oxygen-containing water containing active oxygen species at a concentration of at least 1 μM and luciferins that emit light in the presence of the active oxygen species are mixed. The ratio of the intensity of chemiluminescence derived from luciferins to other sample liquids of a predetermined sample liquid among a plurality of measurement sample liquids prepared in the presence of the specimen is different. A medium on which data for determining the strength of the antioxidant activity in the sample with respect to the sample in the other sample solution may be recorded.

  Here, the medium may be, for example, a paper medium or a medium that is electrically, magnetically, or optically recorded. According to the medium on which such data is recorded, it is possible to easily check how much antioxidant activity the specimen has.

  In addition, the present embodiment also provides a method for determining an index dose of an antioxidant active drug whose dose is substantially in vivo (hereinafter also referred to as a first antioxidant active drug).

  For example, there is an antioxidant active drug (hereinafter, also referred to as a second antioxidant active drug) whose dosage is substantially effective in vivo, and the first antioxidant is used as a generic drug having the same or different active ingredient. When conducting an administration test of an oxidatively active agent, a number of different concentrations of the first antioxidative agent are used to determine the dose required to produce an effect substantially equivalent to that of the second antioxidatively active agent. In some cases, preparations are prepared, and each of them is administered to observe the efficacy and to estimate an appropriate dose.

  However, this operation requires many subjects and labor, and is extremely complicated.

Therefore, in the present embodiment, the first antioxidant active agent whose dosage that is substantially effective in vivo is unknown, and the second antioxidant active agent whose dosage that is substantially effective in vivo are known A method for determining a dose that serves as an index for causing an equivalent medicinal effect, comprising active oxygen-containing water containing active oxygen species at a concentration of 1 μM or more in terms of at least potassium superoxide (KO ₂ ) and the active oxygen species The first measurement sample solution in which the first antioxidant active agent is present as a sample in the luminescent base solution mixed with the luciferin that emits luminescence in the presence of the second luminescent base solution in the second luminescent base solution A second measurement sample solution containing an antioxidant active agent as a specimen is prepared, and the intensity of chemiluminescence derived from the luciferins emitted from each measurement sample liquid and the luminescent base solution not containing the specimen is prepared. Each measured over time The first antioxidant within an error range of ± 25% of the ratio of the area under the time-dependent curve of the luminescence intensity of the second antioxidant active agent to the area under the time-dependent curve of the luminescence intensity of the luminescent base solution. The concentration corresponding to the area under the time-dependent change curve of the luminescence intensity of the active agent is used as the index dose of the first antioxidant active agent.

  Then, according to the index dose determining method according to the present embodiment, the dose of the first antioxidant drug, which is a guideline necessary for producing an effect substantially equivalent to that of the second antioxidant active drug, is obtained. The administration test can be made extremely efficient, and the above-described work can be saved.

  Hereinafter, the test method and medium for antioxidant activity according to the present embodiment will be described more specifically with reference to the drawings.

[1. Preparation of water containing active oxygen)
(1-1. About apparatus for generating active oxygen-containing water)
The active oxygen-containing water used in the method for testing antioxidant activity according to this embodiment is obtained by bringing water into contact with an excited photocatalyst and dispersing the active oxygen species generated on the surface of the photocatalyst into the water. Can be prepared.

  The simplest method for producing such active oxygen-containing water, that is, the simplest active oxygen-containing water generating device, is necessary to excite the photocatalyst by immersing the photocatalyst in water stored in a predetermined container. And those having a configuration for irradiating the photocatalyst with electromagnetic waves such as ultraviolet rays and microwaves. According to such an active oxygen-containing water generating apparatus, active oxygen-containing water can be prepared by dispersing active oxygen species from the surface of the photocatalyst into water.

  In addition, an ultrasonic source that can irradiate the photocatalyst with ultrasonic waves of 100 kHz to 500 kHz, or 500 kHz or more is installed in the container to improve the dispersion efficiency of active oxygen species from the photocatalyst surface to water. You may make it let it. In particular, when the photocatalyst is a fibrous photocatalyst described in (1-2. Configuration of catalyst) described later, active oxygen species can be more efficiently contained in water at a high concentration.

  Moreover, the water brought into contact with the photocatalyst body is not limited to the stored water, and may be running water. In the case of running water, a one-pass system configured so that the water supplied through the container containing the photocatalyst body passes only once, or a circulation system configured to pass through the container multiple times by forming a circulation path. It can also be.

  Specifically, a water supply port for supplying water to the container containing the photocatalyst body and a water discharge port for discharging water in the container are formed. In the case of the one-pass method, running water is supplied from the water supply port and from the water discharge port. The discharged water is obtained as active oxygen-containing water.

  In the case of the circulation system, a connection flow path that connects the water supply port and the water outlet is formed, and water discharged from the water outlet is again supplied from the water supply port into the container through a pump or the like. A flowing water branching mechanism such as a three-way valve is provided in the middle of the connection channel, and a part of the circulating water can be discharged from the flowing water branching mechanism to obtain active oxygen-containing water.

Such an active oxygen-containing water generating device is described in detail in the international publication WO2010 / 032765 previously filed by the present inventor and used in the method for testing antioxidant activity according to the present embodiment. It can be suitably used as a production apparatus for active oxygen-containing water.

  If it adds, the active oxygen containing water production | generation apparatus A described in FIG.1 and FIG.2 of international publication WO2010 / 032765 and the active oxygen containing water production | generation apparatus B described in FIG. 3 can also be used suitably.

(1-2. Configuration of catalyst body)
The catalyst body used for producing the active oxygen-containing water is not particularly limited as long as the active oxygen species can be contained in the water contacted with the catalyst body. However, more preferably, a catalyst body formed in a fiber aggregate shape can also be used.

  For example, it is an aggregate of metal fiber bodies having a diameter of about 50 to 200 μm (in this embodiment, 100 μm), and the fiber bodies are made of titanium oxide on alumina fibers formed by sintering an alumina coating on the surface of aluminum fibers. Can be used.

Specifically, a plurality of aluminum fibers formed of aluminum having a fiber length of 5 mm to 20 cm are entangled and stored in a predetermined mold, and the aluminum fibers are 0.5 g / cm per unit volume. Forming an aluminum fiber aggregate arranged in a desired shape that aggregates at a density of ^{3 to 3} g / cm ³ , and the aluminum fiber aggregate is reduced to about a half of its melting point temperature per minute. After heating while maintaining a temperature gradient of 5 ° C. or less, the film thickness of the oxide film is increased to 5 nm or more by maintaining the temperature of about one half of the melting point temperature for 30 minutes to 3 hours, and then the aluminum fiber aggregate After heating to a temperature close to the melting point temperature, maintaining the temperature in the vicinity of 30 minutes to 12 hours to produce an alumina fiber aggregate with an oxide film thickness of 50 nm or more, the alumina solution is contained in a sol solution containing a titania compound. Fiber collection The titania fiber aggregate is manufactured by forming a titania thin film on the surface of the alumina fiber by immersing the body and pulling it up, drying the sol solution adhering to the surface of the alumina fiber constituting the alumina fiber aggregate and then firing it. can do.

  In addition, before drying, a gas may be blown to the alumina fiber aggregate pulled up from the sol solution so that the gas is vented to the contact between the alumina fibers constituting the alumina fiber aggregate. Thereby, it is possible to prevent the dried sol solution from being crystallized (dama) on the fiber surface, and it is possible to obtain a uniform coating solution even in the vicinity of the contact. The sol solution also contains 15 to 25 wt% titanium diisopropoxybisacetylacetonate, 5 to 10 wt% isopropanol, 55 to 75 wt% ethanol, and 5 to 10 wt% water. You may make it do. By carrying out titania coating with the sol liquid having such a composition, it is possible to perform titania coating that is uniform and difficult to peel off. In the sol solution, the mixing ratio of titanium diisopropoxybisacetylacetonate, ethanol, and water may be 3.5: 9: 1. Thereby, a stronger titania thin film can be formed. In addition, the immersion of the alumina fiber aggregate in the sol liquid is performed using a sol liquid at 35 ° C. to 60 ° C. in a sealed container having a volume larger than the volume of the sol liquid to be accommodated. It is good also as immersing, raising the pressure of the gaseous phase in an airtight container, volatilizing the contained alcohol. By carrying out in this way, it is possible to cause fine precipitation on the fiber surface while preventing precipitation of titania in the sol solution, and to form a uniform titania thin film.

(1-3. About water supplied to the active oxygen-containing water generator)
The water to be contacted with the catalyst body may be pure water, tap water, well water, ozone water, etc., but by adjusting the additive in water, dissolved oxygen concentration, dissolved ozone concentration, temperature, pH, viscosity, etc. It is possible to control the activity of the reactive oxygen species.

  Specifically, a supply water treatment unit for adding a predetermined substance or the like to the water may be provided upstream of the water supply port so that various components are added to the water.

Examples of the components to be added include oxygen gas, ozone gas, chlorine gas, nitric oxide gas, ammonia gas, metal salts (NaCl, CaCl ₂ , KCl, MgCl _2, CuSO ₄ and other compounds, iron and its compounds, etc. And at least one selected from zinc and its compounds, aluminum and its compounds, and the like.

  In FIG. 1, the example of the result of having examined about content of the active oxygen species at the time of adding metal salts to water is shown. The vertical axis represents the difference in luminescence intensity derived from luciferins before and after the addition of metal salts, and the horizontal axis represents each metal salt. In addition, the density | concentration of each metal salt has shown the final concentration in the water supplied to an active oxygen containing water production | generation apparatus.

  As can be seen from FIG. 1, when NaCl is used as the metal salt, the emission intensity is enhanced by about 4000 when 0.05 mM is added, and about 9000 when 0.5 mM is added. There was a slight increase in emission intensity. In addition, when 5 mM was added, the degree of enhancement of the luminescence intensity started to decrease, and was about 2000, and when 50 mM was added, it was about 800. From these results, it was shown that when NaCl is used as the metal salt, it is better to add it at a concentration of 0.05 mM to 50 mM, more desirably 0.05 mM to 5 mM.

In addition, when CaCl ₂ is used as the metal salt, the emission intensity is enhanced by about 5000 when 0.05 mM is added, and the degree of enhancement of the emission intensity when 0.5 mM is added, compared to the case where CaCl ₂ is not added. Once it turned down, the emission intensity increased by about 3000. In addition, when 5 mM was added, an increasing trend was observed again, reaching about 6000, and when adding 50 mM, it was about 10000. From these results, it was shown that when CaCl ₂ is used as the metal salt, it should be added at a concentration of 0.05 mM to 50 mM, more preferably 5 mM to 50 mM.

  In addition, when KCl is used as the metal salt, the emission intensity is increased by about 4000 when 0.05 mM is added, and the emission intensity is increased by about 7000 when 0.5 mM is added. Was seen. In addition, when 5 mM was added, the degree of enhancement of the luminescence intensity decreased to about 3000, and when 50 mM was added, luminescence was suppressed as compared to the case where KCl was not added, and was about -3000. From these results, it was shown that when KCl is used as the metal salt, it should be added at a concentration of 0.05 mM to 5 mM.

In addition, when MgCl ₂ is used as the metal salt, the emission intensity is enhanced by about 100 with the addition of 0.05 mM, and the emission intensity of about 300 with the addition of 0.5 mM, compared with the case where MgCl ₂ is not added. The increase was seen. When 5 mM was added, it was about 1500, and when 50 mM was added, light emission was suppressed as compared to the case where MgCl ₂ was not added, and about -2000. From these results, it was shown that when KCl is used as the metal salt, it should be added at a concentration of 0.05 mM to 5 mM.

(1-4. Preparation of active oxygen-containing water)
Active oxygen-containing water was prepared by supplying water to the active oxygen-containing water generator and bringing water into contact with the excited photocatalyst to disperse active oxygen species generated on the surface of the photocatalyst in this water. The concentration of active oxygen species of the active oxygen-containing water prepared in this way was 1 to 5 μM.

(1-5. Effectiveness in evaluating antioxidant activity of water containing active oxygen)
Whether the active oxygen-containing water prepared in (1-4. Preparation of Active Oxygen-Containing Water) can evaluate the antioxidant activity is determined by using bovine erythrocyte-derived antioxidant superoxide dismutase (Bovin RBC SOD). It was examined using.

  Specifically, 5 μl of 25 mM potassium phosphate buffer (pH 7.0) saturated with Cypridina Luciferin Analog was dispensed into a 1.5 ml centrifuge tube, and then bovine erythrocytes as a sample solution After dispensing 5 μl of the aqueous antioxidant substance solution, 490 μl of active oxygen-containing water was added and mixed to prepare a measurement sample solution. In the following description of the present specification, a method of preparing a measurement sample solution by the same method regardless of the specimen is simply referred to as “measurement sample preparation method”. In this test, the superoxide dismutase aqueous solution derived from bovine erythrocytes is a total of six measurement sample solutions with different specimens so that the final concentrations are 0.045U, 0.45U, 4.5U, 45U, 450U, and 830U. Prepared.

  The prepared measurement sample solution was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferins and changes with time were measured. The results are shown in FIG.

  In FIG. 2, the vertical axis represents the intensity of luminescence derived from luciferins, and the horizontal axis represents time (seconds). As can be seen from FIG. 2, the luminescence intensity changes depending on the concentration of the antioxidant superoxide dismutase derived from bovine erythrocytes having antioxidant activity. The higher the concentration and the higher the antioxidant activity, the lower the emission intensity.

  From this result, it was shown that the antioxidant activity of the antioxidant composition can be evaluated according to the method for testing antioxidant activity according to the present embodiment.

[2. (Evaluation of antioxidant activity)
(2-1. Evaluation of drugs-glutathione)
[1. Using the active oxygen-containing water prepared in [Preparation of Active Oxygen-Containing Water], the emission intensity when the concentration of glutathione in the system of the method for testing antioxidant activity according to this embodiment was changed was examined. Glutathione is an amino acid that is usually present in cells as reduced glutathione and is converted to oxidized glutathione by acting as an electron donor during oxidative stress, thereby reducing cell damage.

  First, a glutathione aqueous solution was used as a sample liquid, and a measurement sample liquid was prepared according to the above-described measurement sample preparation method. In this test, the glutathione aqueous solution was added so as to have final concentrations of 1 mM, 100 μM, 10 μM, 1 μM, and 100 nM, and a total of five types of measurement sample solutions with different specimens were prepared.

  The prepared measurement sample solution was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferins and changes with time were measured. The result is shown in FIG.

  As can be seen from FIG. 3, the luminescence intensity varies depending on the concentration of glutathione having antioxidant activity. Specifically, the higher the glutathione concentration and the higher the antioxidant activity, the lower the luminescence intensity. It was observed.

  From this result, it was shown that the antioxidant activity of the antioxidant composition can be evaluated according to the method for testing antioxidant activity according to the present embodiment. Moreover, all the measurement sample liquids were neutral, and it was shown that the antioxidant activity measurement in a neutral region closer to the human body is possible.

(2-2. Evaluation of drugs-ascorbic acid)
Next, the emission intensity when the concentration of ascorbic acid was changed was examined. Ascorbic acid is widely known as an antioxidant, vitamin N, and is also used as an antioxidant in food additives. However, its active time is short.

  First, an ascorbic acid aqueous solution was used as a sample solution, and a measurement sample solution was prepared according to the above-described measurement sample preparation method. In this test, the ascorbic acid aqueous solution was added so that the final concentrations were 1 mM, 100 μM, 10 μM, 3 μM, and 1 μM, and a total of five types of measurement sample solutions with different specimens were prepared.

  The prepared measurement sample solution was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferins and changes with time were measured. FIG. 4 shows the result.

  As can be seen from FIG. 4, the luminescence intensity varies depending on the concentration of ascorbic acid having antioxidant activity. Specifically, the higher the concentration of ascorbic acid and the higher the antioxidant activity, the lower the luminescence intensity. The tendency to become was seen. In particular, as with glutathione, ascorbic acid at a high concentration of 10 μM or more tends to have a low emission intensity depending on the concentration, and exhibits high antioxidant activity.

  It should also be noted that when comparing 1 μM and 3 μM, the emission intensity was almost the same up to about 50 seconds, but 1 μM suddenly lost its antioxidant activity by 100 seconds, and the control was lost. While high luminescence intensity that approximates the curve is shown, 3 μM shows the behavior that the antioxidant activity is lost in around 100 seconds while maintaining the antioxidant activity. Thus, according to the method for testing antioxidant activity according to the present embodiment, it is possible to know how the antioxidant activity changes over time by observing the luminescence intensity over time.

  In addition, this result indicates that the antioxidant activity of the antioxidant composition can be evaluated according to the method for testing antioxidant activity according to the present embodiment. It was also shown that concentration effects can be easily predicted, including sample efficacy prediction and kinetic prediction.

Furthermore, ascorbic acid can be judged to be a low molecular weight antioxidant that directly reacts with active oxygen such as O ₂ ⁻ in terms of scavenging ability of superoxide anion radical (O ₂ ⁻ ). On the other hand, glutathione and superoxide dismutase (SOD) can be identified as polymer-type antioxidants that catalyze a selective O ₂ ⁻ decomposition reaction.

(2-3. Evaluation of chemicals-Evaluation of antioxidant activity of Z-type DNA / Cu2 ⁺ complex)
As a novel polymer-type biocatalyst, 5′-CGCGCG-3 ′, a 6-base pair double-stranded oligo DNA that forms a double strand and takes a Z-type structure, was prepared. As controls, 5′-ATATAT-3 ′ forming a non-Z-type double-stranded DNA structure and 5′-CCCCCC-3 ′ and 5′-GGGGGG-3 ′, which are single-stranded oligo DNAs, were also prepared.

First, using a Z-type DNA / Cu ²⁺ complex solution as a sample solution, a measurement sample solution was prepared according to the above-described measurement sample preparation method. The Z-type DNA / Cu ²⁺ complex solution is a 1: 1 concentration of 6 base pair double-stranded DNA having the base sequence shown in FIG. 5 (a) in an aqueous copper sulfate (CuSO ₄ ) solution having a predetermined final concentration. It was prepared by adding. In this test, the Z-type DNA / Cu ²⁺ complex solution was added to a final concentration of 30 μM, 10 μM, 3 μM, 1 μM, and 0.3 μM, and a total of 5 types of measurement sample solutions with different specimens were prepared. went.

  The prepared measurement sample solution was set in a measuring apparatus (Lumin sensor manufactured by ATTO) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferin and changes with time were measured. The result is shown in FIG.

  As can be seen from FIG. 5B, the luminescence intensity derived from luciferin was significantly lower than that of the positive control in any of the prepared measurement sample solutions, indicating that it has antioxidant activity.

In addition, the luminescence intensity changes according to the concentration of the Z-type DNA / Cu ²⁺ complex having antioxidant activity. Specifically, the concentration of the Z-type DNA / Cu ²⁺ complex is high and the antioxidant activity is high. The higher the value, the lower the emission intensity.

Further, using a Z-type DNA and a copper sulfate (CuSO ₄ ) solution as a specimen solution, a measurement sample solution was prepared according to the above-described measurement sample preparation method. Each solution was added so as to have a final concentration of 100 μM, 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, and 0.1 μM, and a total of seven types of measurement sample solutions with different specimens were prepared.

  Immediately after addition of active oxygen-containing water, the prepared measurement sample solution is set in a measuring device (ATTO's luminescence sensor) and measured for the intensity of luminescence from luciferin and changes over time. The ratio between the indicated emission intensity value and the maximum value of the emission intensity of active oxygen-containing water as a control was calculated. FIG. 6A shows the result.

  From these results, it was shown that the aqueous solution of copper sulfate and the Z-type DNA alone did not have strong antioxidant activity, and both of them exhibited strong antioxidant activity when bound in an enzyme-coenzyme manner. On the other hand, although not shown, no activity was observed with non-Z-type oligo DNA or single-stranded oligo DNA.

  Furthermore, for the purpose of evaluating whether this test method can obtain the same results as other antioxidant activity methods, the reaction with WST-1 formazan reagent was conducted in a 100 μM concentration solution that showed the strongest antioxidant activity. The sample was tested for antioxidant activity according to this embodiment using absorbance measurement. FIG. 6B shows the result (difference spectrum compared with the target section).

As a result, as described above, the copper sulfate aqueous solution and the Z-type DNA alone did not exhibit the antioxidant activity, and both of them bound as an enzyme + coenzyme, thereby forming a Z-type DNA / Cu ²⁺ complex. It was revealed that it exhibits antioxidant ability. Thus, according to this method, it was shown that the antioxidant activity of the antioxidant composition can be evaluated for time and concentration.

(2-4. Evaluation of drugs-Evaluation of antioxidant activity of Edaravone preparation)
Next, the antioxidant activity of the Edaravone preparation was evaluated. Edaravone (edaravone) is an active oxygen species (often expressed as free radicals) that occurs during ischemia-reperfusion injury caused by cerebral infarction. It is used to reduce organizational problems. However, this agent has a strong removal action against anionic reactive oxygen species, and it may be impossible to detect even when the spin trap method is used in the method based on the detection of antioxidant activity by cationic active oxygen species typified by hydroxy radicals.

  When Edaravone is administered to the human body, it is intravenously injected into the blood vessel as a 0.3 mg / ml solution over 30 minutes and slowly administered 30 mg systemically. Furthermore, this drug often contains L-cysteine hydrochloride hydrate having the same antioxidant action at a concentration of 0.1 mg / ml.

  First, 5 μl of 25 mM potassium phosphate buffer (pH 7.0) saturated with Cypridina Luciferin Analog was dispensed into a 1.5 ml centrifuge tube, and then the sample solution as the sample solution was dispensed from the container. After dispensing 5 to 100 μl of Edaravone drip solution opened and taken out, the measurement sample solution was prepared by measuring up to 500 μl while adding and mixing active oxygen-containing water. In this study, Edaravone preparations were added so that the final concentrations were 60 μg / ml, 30 μg / ml, 9 μg / ml, 6 μg / ml, 3 μg / ml, 1.8 μg / ml, and 0.6 μg / ml. A total of seven different measurement sample solutions were prepared.

  The prepared measurement sample solution was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferins and changes with time were measured. FIG. 7 shows the result.

  As can be seen from FIG. 7, it can be seen that the Edaravone final concentration of 3 μg / ml corresponds to the one whose luminescence intensity decreases according to the concentration of Edaravone and the area under curve of the control curve shows 50% attenuation. Specifically, Edaravone with a high concentration of 3 μg / ml or more in the final concentration has a low emission intensity depending on the concentration and exhibits a high antioxidant activity. However, Edaravone with an Edaravone final concentration of 3 μg / ml or less has its antioxidant activity abruptly reduced after 100 seconds and approximates a control curve.

  In the future, it is reasonable to evaluate the administration effect based on the evaluation of this method based on the 50% attenuation of the Area under curve in the development of drugs aimed at antioxidant activity in the future. It was shown that there is. Of course, this standard is a standard for systemic administration of drugs, and when treating acute myocardial infarction, when administered locally through a catheter, it can be administered at a lower concentration, resulting in renal excretion. It is possible to reduce the occurrence of side effects during systemic administration due to being a drug.

  In addition, the Edaravone formulation used in this study is 5 μl of potassium phosphate buffer (pH 7.0), 5 μl of Edaravone drip solution, and mixed with active oxygen-containing water. In FIG. 7, the concentration is set to draw a curve shown as 3 μg / ml, that is, 0.3 mg / ml.

  When this Edaravone preparation is regarded as the second antioxidant drug, the first anti-dose whose dosage is unknown in the living body (substantially the same effect as the second antioxidant drug) is unknown. To determine the index dose of the oxidatively active drug, the concentration that produces an area under the curve of approximately 50%, which is the ratio of the 3 μg / ml area under the curve of Edaravone to the area under the curve of the positive control. What should I do?

  That is, the concentration in the measurement sample of the first anti-oxidant drug that draws an area under the curve showing the ratio of Edaravone's 3 μg / ml area under the curve to the area under the curve of the positive control is the same. When the experimental condition was Xμg / ml, the concentration of the first anti-oxidant drug was 0.1 x Xmg / ml to bring about the same effect as the edaravone concentration of 0.3mg / ml in the edaravone preparation. Some, index dose, index formulation concentration, index dose concentration, etc. can be easily determined in an in vitro system.

  In determining the index dose of the first anti-oxidant drug that produces the same effect as the second anti-oxidant drug, the area of the first anti-oxidant drug against the positive control area under the curve The ratio of the under-the curve (hereinafter also referred to as the first ratio) is not necessarily the ratio of the area under the curve of the second antioxidant active agent to the area under the curve of the positive control (hereinafter also referred to as the second ratio). Need not be exactly the same as.). According to the experience of the present inventor, it is known that if the first ratio is included in the range of ± 25% of the second ratio, the antioxidant activity and the drug effect are almost the same. I think it works well.

  Moreover, it was shown from this test result that the antioxidant activity of the antioxidant composition can be evaluated according to the method for testing antioxidant activity according to this embodiment. Moreover, all the measurement sample liquids were neutral, and it was shown that the antioxidant activity measurement in a neutral region closer to the human body is possible.

Comparison of the results of glutathione, ascorbic acid and edaravone with the results of superoxide dismutase and DNA / Cu ²⁺ complex (curve pattern), and the mechanism of reactive oxygen scavenging by adjusting the dilution system of the sample appropriately but if a low molecular type antioxidant group that reacts with active oxygen-selective, selective O ₂ ^- or a polymer type antioxidant group that catalyze the decomposition reaction of readily identify It is possible.

(2-5. Evaluation of food-Evaluation of antioxidant activity such as lemon)
Next, the antioxidant activity in a commercially available lemon and a commercially available lemon drink was evaluated.

  First, lemon juice or lemon drink was used as a sample liquid, and a measurement sample liquid was prepared according to the above-described measurement sample preparation method. In this test, lemon drinks are used in two types, one containing vitamin C at a concentration of 2 mg / ml and one containing 7.14 mg / ml. Prepared. Since 3 μM ascorbic acid is roughly equivalent to 0.5 mg / ml vitamin C, this sample can be expected to exhibit high antioxidant activity without being attenuated.

  The prepared measurement sample solution was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferins and changes with time were measured. FIG. 8 shows the result.

  As can be seen from FIG. 8, the luminescence intensity derived from luciferins was significantly lower than that of the positive control for both lemon juice and the two lemon beverages, indicating that it has antioxidant activity.

  Moreover, when each measurement sample liquid was compared, it was suggested that lemon juice containing vitamin C at a concentration of 7.14 mg / ml has almost the same antioxidant activity as lemon juice.

  In addition, lemon juice containing vitamin C at a concentration of 2 mg / ml was shown to have lower antioxidant activity than lemon juice and lemon juice containing vitamin C at a concentration of 7.14 mg / ml. . Although not shown, soft drinks in which vitamin C 2 mg / ml, citric acid 2 mg / ml, and polyphenol 20 μg / ml are dissolved have a high antioxidant activity equivalent to lemon juice due to the additive effect of antioxidant active ingredients. Indicated.

Similarly to this method, as an evaluation of the antioxidant activity in vegetables, for example, leafy vegetables such as kaiware radish, broccoli sprout, lettuce and herbs can be directly evaluated for antioxidant capacity using vegetable juice. In addition, in a food group having a low water content such as root vegetables, it is possible to make a comparative evaluation using a solution that defines a sample weight and is compressed by a stomacher with respect to a phosphate buffer solution. Furthermore, according to this method, it is possible to easily evaluate differences in cooking methods, for example, differences in antioxidant activity due to heating in a hot water bath and a microwave oven.

In addition, the decrease in antioxidant activity by storage methods such as freeze-freeze method and freeze-dry method is not antioxidant activity by vitamins and polyphenols, but is inactivated by enzyme freezing damage. The sample can be easily detected by adding a freezing operation immediately before the measurement.

(2-6. Evaluation of food-Evaluation of antioxidant activity such as tomato)
Next, the antioxidant activity in commercially available cherry tomatoes and commercially available tomato beverages was evaluated.

  First, the measurement sample liquid was prepared according to the above-mentioned measurement sample preparation method, using a small tomato juice or a tomato drink as a sample liquid. In this test, two types of tomato beverages were used, and a total of three types of measurement sample solutions with different specimens were prepared.

  The prepared measurement sample solution was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the luminescence intensity derived from luciferins and changes with time were measured. FIG. 9 shows the result.

  In FIG. 9, the vertical axis represents the intensity of luminescence derived from luciferins, and the horizontal axis represents time (seconds). As can be seen from FIG. 9, the luminescence intensity derived from luciferins was significantly lower than that of the positive control for both the tomato juice and the two tomato beverages, indicating that it has antioxidant activity.

  Interestingly, when the measured sample solutions are compared, the antioxidant activity of the raw cherry tomatoes is the highest, and in the comparison of the two tomato beverages, the straight tomato beverage is more than the concentrated reduced tomato beverage. It was suggested that the antioxidant activity is high. In the presented sample, the result was reminiscent of the antioxidant activity by the concentration reduction method, but in other samples not presented, there are also samples that exhibit excellent antioxidant activity in the concentration reduction method, It was suggested that the antioxidant activity can be kept high by the production method and materials.

(2-7. Evaluation of food-Evaluation of antioxidant activity of soft drinks)
Some soft drinks such as juices also have an antioxidant activity due to polyphenols and anthocyanins. However, in soft drinks, it is said that a Meillard-like reaction is induced by the reaction of reducing sugars and amino acids, and there is a possibility that a reaction of generating reactive oxygen species occurs.

  First, a measurement sample solution was prepared according to the above-described measurement sample preparation method using a soft drink opened and taken out from a container as a sample solution. In addition, the soft drink used in this test is said to contain 210 mg of polyphenol and 60 mg of anthocyanin using two kinds of fruits.

  Similarly, the measurement sample was set in a measuring device (ATTO Co., Ltd. luminescence sensor) immediately after the addition of active oxygen-containing water, and the intensity of luminescence derived from luciferins and changes with time were measured. FIG. 10 shows the result.

  As can be seen from FIG. 10, the luminescence intensity derived from the luciferins immediately after the start of the measurement shows a significantly higher value than that of the positive control, and exhibits strong luminescence as if producing a high concentration of active oxygen. Thereafter, the emission intensity decreases due to the antioxidant activity of polyphenols and anthocyanins contained therein.

  This indicates that some of the fruits that have special antioxidant activity may have a Maillard-like reaction due to the reaction of fructose and amino acids in the fruits depending on the storage state during transport. .

[3. (Create recording medium)
The results obtained by carrying out the above-mentioned method for testing antioxidant activity are written on the paper in letters and numbers, and data that shows the ratio between the luminescence intensity of the positive control and the luminescence intensity of each measurement sample solution is recorded. Paper on which data for determining the presence or absence of antioxidant activity in a given sample or for determining the strength of the antioxidant activity of a sample in a predetermined sample solution relative to the sample in the other sample solution is recorded Created media.

  Similarly, a data file is created by inputting the above data on a computer, and the data file is recorded on a CD-R, so that the emission intensity of the positive control, the emission intensity of each measurement sample solution, and In order to determine the presence or absence of antioxidant activity in a sample in which data with a known ratio is recorded, or to determine the strength of antioxidant activity in a sample in a predetermined sample solution relative to the sample in the other sample solution CD-R was prepared.

  Then, according to these paper media and CD-R, it is easy to determine whether or not a specimen has antioxidant activity and the strength of the antioxidant activity of a specimen in a predetermined sample liquid with respect to the specimen in another sample liquid. I can know.

Further, the medium in which these data are recorded contains the method for testing antioxidant activity according to the present embodiment, that is, the active oxygen species is converted to at least potassium superoxide (KO ₂ ) at a concentration of 1 μM or more. Prepare a sample solution to be measured in the presence of a sample to be evaluated for antioxidant activity in a luminescent base solution in which active oxygen-containing water and luciferins that emit light in the presence of the active oxygen species are mixed. By measuring the intensity of chemiluminescence derived from the luciferins emitted from the sample liquid and the luminescent base liquid not containing the analyte, respectively, the intensity of chemiluminescence from the measurement sample liquid is determined from the luminescent base liquid. When the intensity of chemiluminescence is lower, the test method display section is displayed on the paper to indicate that the specimen is the result of evaluation by the test method of antioxidant activity that is determined to have antioxidant activity. Or it may be provided as digital data in a predetermined visible state or the like via the software if the digital medium. By providing such an inspection method display unit, the inspection method can be clearly recognized. Note that the content described in the test method display section is not necessarily the above wording as long as it is understood that the test is performed by the method for testing antioxidant activity according to the present embodiment, and a simple display such as a common name is used. It may be.

As described above, according to the method for testing antioxidant activity according to the present embodiment, active oxygen-containing water containing active oxygen species at a concentration of 1 μM or more in terms of at least potassium superoxide (KO ₂ ) and the above-mentioned A measurement sample solution is prepared in the presence of a sample to be evaluated for antioxidant activity in a luminescent base solution mixed with luciferins that emit luminescence in the presence of reactive oxygen species. By measuring the intensity of chemiluminescence derived from the luciferin that is emitted from the luminescent base solution containing no luminescence, the intensity of chemiluminescence from the measurement sample solution is less than the intensity of chemiluminescence from the luminescent base solution. In this case, since the specimen is determined to have antioxidant activity, the antioxidant activity of the antioxidant composition can be evaluated in a neutral range closer to the human body. The law can be provided.

  Finally, the description of each embodiment described above is an example of the present invention, and the present invention is not limited to the above-described embodiment. For this reason, it is a matter of course that various modifications can be made in accordance with the design and the like as long as they do not depart from the technical idea according to the present invention other than the embodiments described above.

Claims (8)

In a luminescent base solution in which active oxygen-containing water containing active oxygen species at a concentration of at least 1 μM in terms of potassium superoxide (KO ₂ ) and luciferins that emit light in the presence of the active oxygen species are mixed A sample liquid to be evaluated is prepared in the presence of a sample to be evaluated for antioxidant activity, and the intensity of chemiluminescence derived from the luciferins emitted from the sample liquid and the luminescent base solution not containing the sample is measured. By each measuring, when the intensity of chemiluminescence from the measurement sample solution is lower than the intensity of chemiluminescence from the luminescent base solution , the test method for determining the antioxidant activity that determines that the specimen has antioxidant activity There,
A method for testing antioxidant activity, wherein a complex of oligo DNA having a repetitive sequence of cytosine and guanine and copper is used as an indicator substance for a superoxide anion radical scavenging substance .   A plurality of measurement sample liquids having different specimens are prepared, the chemiluminescence intensity from each measurement sample liquid is compared, and the antioxidant activity of the specimen added to a predetermined measurement sample liquid among the plurality of measurement sample liquids is measured. The method for testing antioxidant activity according to claim 1, wherein the strength of the antioxidant activity in a specimen added to another measurement sample solution is determined.   The active oxygen-containing water is prepared by bringing water into contact with an excited photocatalyst to disperse the active oxygen species generated on the surface of the photocatalyst in the water. The method for testing antioxidant activity according to claim 1 or 2. The water to be brought into contact with the photocatalyst body is supplied through a supply water treatment unit that adds a predetermined substance to the water,
4. The antioxidant activity test according to claim 3, wherein the predetermined substance is at least one selected from oxygen gas, ozone gas, chlorine gas, nitric oxide gas, ammonia gas, and metal salt. Method. The metal salt is at least one selected from NaCl, CaCl ₂ , KCl, MgCl ₂ , CuSO ₄ ,
The concentration in the water is 0.05 mM to 50 mM for NaCl and CaCl ₂ ,
For KCl and MgCl2, the concentration is 0.05 mM to ₅ mM.
5. The method for testing antioxidant activity according to claim 4, wherein CuSO ₄ is 50 μM to 200 μM.   The method for testing antioxidant activity according to any one of claims 1 to 5, wherein the luciferin is Cypridina Luciferin Analog. An index for producing a drug effect equivalent to that of a second antioxidant active agent whose dosage is substantially effective in vivo, of the first antioxidant active drug whose unknown effective dose in vivo is unknown A method for determining a dose of
In a luminescent base solution in which active oxygen-containing water containing active oxygen species at a concentration of at least 1 μM in terms of potassium superoxide (KO ₂ ) and luciferins that emit light in the presence of the active oxygen species are mixed A first measurement sample solution in which the first antioxidant active agent is present as a specimen, and a second measurement sample solution in which the second antioxidant active agent is present as a specimen in the luminescent base solution; The intensity of chemiluminescence derived from the luciferins emitted from each measurement sample solution and the luminescent base solution not containing the specimen is measured over time, and the luminescence intensity of the luminescent base solution changes over time. Under the time-dependent change curve of the luminescence intensity of the first antioxidant active agent included in the error range of ± 25% of the ratio of the area under the curve with time of the luminescence intensity of the second antioxidant active agent to the area under the curve The concentration to be the area Indicators dose determination method, characterized by an indicator dosage of 1 antioxidant active agent.   An anti-oxidant that can be evaluated as a superoxide anion radical scavenger by a method for examining antioxidant activity according to any one of claims 1 to 6, which is a complex of oligo DNA having a repetitive sequence of cytosine and guanine and copper. Oxidized material.