JP2017057185A - Novel nadph oxidase activator and method for producing the same, and method for activating nadph oxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel NADPH oxidase activator and a method for producing the same, and a method for activating NADPH oxidase, by using PQQ (pyrroloquinoline quinone).SOLUTION: A NADPH oxidase activator comprises a compound represented by formula 1, 2, or 3 or salt thereof.SELECTED DRAWING: None

Description

  The present invention relates to a novel NADPH oxidase activator, a method for producing the same, and a method for activating NADPH oxidase.

  NADPH oxidase is an enzyme that oxidizes reduced nicotinamide adenine dinucleotide phosphate (NADPH) to generate superoxide and hydrogen peroxide as reactive oxygen species. In humans, Nox 1 to 5 and Duox 1 and 2 are known as NADPH oxidases and are called Nox families. Reactive oxygen species produced from these enzymes are used as signal transduction molecules and induce various biological responses including homeostasis. This enzyme is called the Nox family and Nox 1 to 5 and Duox 1 and 2 are known in humans. This enzyme generates superoxide and hydrogen peroxide, which are reactive oxygen species, and is thought to function to use them as cellular signals.

Reissue 2004/089412 JP-T-2006-520326 Special table 2010-521522 gazette JP 2008-231060 A

Oxidative Stress Medical Revision 2nd Edition p10-20, 2014, Diagnosis and Treatment Company Bioscience, Biotechnology and Biochemistry, 2015 DOI: 10.1080 / 091684511.2015.10627715 Zhou et al. Analyt. Biochem. (1997) 253, 162-168. Moribe et al. PLOS Genet. (2012) 8, e1002957

  Reactive oxygen species work in a variety of situations in the body, such as increased blood pressure, defense against infection, and thyroid hormone synthesis, and the decreased activity of this enzyme can cause abnormal blood pressure, opportunistic infection, and hormonal abnormalities. . Therefore, a substance that activates NADPH oxidase is required. In particular, hydrogen peroxide produced by Duox2 is essential for the synthesis of thyroid hormone, and when this becomes abnormal, thyroid function becomes abnormal (Non-patent Document 1). In addition, this enzyme is present in epithelial cells such as the respiratory system and digestive system, and plays a role in protecting each infection locally. As functions of the NADPH oxidase activator, immune enhancement, thyroid function normalization, and blood pressure normalization can be expected. So far, diphenylene iodonium (DPI) and many compounds are known as NADPH oxidase inhibitors (Patent Documents 1 to 4), but little research has been conducted on activators. Therefore, there is a demand for a material and method that can activate the function of NADPH oxidase and that is inexpensive and highly safe.

  Pyrroloquinoline quinone (hereinafter also referred to as “PQQ”) is a coenzyme molecule found in microorganisms, and various functions are known (Non-patent Document 2). However, it is not known to activate NADPH oxidase as various physiologically active actions of PQQ that have been clarified so far.

  The activity of NADPH oxidase can be quantified by the method described in the literature (Non-Patent Documents 3 and 4). This method makes it possible to search for an activator of NADPH oxidase.

  An object of the present invention is to provide a novel NADPH oxidase activator, a method for producing the same, and a method for activating NADPH oxidase using the PQQ.

  The present inventors diligently studied the influence of PQQ on a living body. As a result, as a result of studies by the present inventors using human cells, it was found that PQQ is involved in activation of NADPH oxidase. This invention is made | formed by the said knowledge.

  That is, the present invention is as follows.

  ADVANTAGE OF THE INVENTION According to this invention, the novel NADPH oxidase activator, its manufacturing method, and the NADPH oxidase activation method can be provided.

It is a figure which shows the measurement result of the hydrogen peroxide generation amount in Example A. It is a figure which shows the time change of the hydrogen peroxide generation amount in Example B. It is a figure which shows the measurement result of the hydrogen peroxide generation amount in the human Dual Oxidase expression cell in Example C. It is a figure which shows the time change of the hydrogen peroxide generation amount in Example D. It is a figure which shows the result analyzed by the Western blot in Example D.

  DESCRIPTION OF EMBODIMENTS Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. However, the present invention is not limited to this, and various modifications can be made without departing from the gist thereof. Is possible.

[NADPH oxidase activator]
The NADPH oxidase activator of the present embodiment includes a compound represented by the following formula (1), (2), or (3) or a salt thereof.
(In the above formulas (1), (2), and (3), R independently represents a hydrogen atom, a substituent having 1 to 10 carbon atoms, or an alkali metal, and R ′ represents the number of carbon atoms. 1 to 10 substituents are shown.)

  As used herein, “activation” refers to upregulating a particular factor. “Upregulation” also refers to, for example, the expression or activity of one or more genes and the resulting protein (s) encoded by those genes in response to a signal or agent. Point to increase.

  Hereinafter, examples of the compound or salt thereof that can be used in the NADPH oxidase activator include a compound represented by the above formula (1), (2), or (3) or a salt thereof. Although it does not specifically limit as a salt, For example, an alkali metal salt is mentioned. Among these, pyrroloquinoline quinone sodium salts such as pyrroloquinoline quinone monosodium salt, pyrroloquinoline quinone disodium salt, and pyrroloquinoline quinone trisodium salt are preferable, and pyrroloquinoline quinone disodium is more preferable. By using such a salt, the NADPH oxidase activation effect tends to be further improved. Hereinafter, the compound represented by the following formula (1), (2), or (3) and a salt thereof will be described in more detail.

  Although it does not specifically limit as a C1-C10 substituent shown by R, For example, alkyl and an allyl are mentioned. Among these, alkyl is preferable from the viewpoint of synthesis. In addition, the C1-C10 substituent shown by R may contain an oxygen atom, a nitrogen atom, a hydrogen atom, a sulfur atom, and a phosphorus atom other than a carbon atom.

[Compound represented by formula (1) or a salt thereof]
In the above formula (1), a compound in which R is all hydrogen atoms is referred to as oxidized pyrroloquinoline quinone. The salt of the oxidized pyrroloquinoline quinone is not particularly limited, and examples thereof include tricarboxylic acid, tricarboxylic acid di-salt, tricarboxylic acid mono-salt, and tricarboxylic acid tri-salt. Examples of the salt include, but are not limited to, alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, strontium salt and barium salt; ammonium salt and alkylammonium salt And salts with such cationic compounds.

  In the above formula (1), one or more R are preferably hydrogen atoms. A compound in which one or more R is a hydrogen atom forms a salt in the solution and becomes ionic, so that the water solubility tends to be improved as compared with the tricarboxylic acid form.

  Although it does not specifically limit as a compound represented by the said Formula (1) or its salt, For example, oxidized pyrroloquinoline quinone; oxidized pyrroloquinoline quinone monosodium, oxidized pyrroloquinoline quinone disodium, oxidized pyrroloquinoline quinone Examples thereof include alkali metal salts of oxidized pyrroloquinoline quinone such as trisodium, oxidized pyrroloquinoline quinone dipotassium, oxidized pyrroloquinoline quinone tripotassium.

  Among these, from the viewpoint of easy availability, oxidized pyrroloquinoline quinone monosodium such as oxidized pyrroloquinoline quinone monosodium, oxidized pyrroloquinoline quinone disodium, oxidized pyrroloquinoline quinone trisodium is preferable.

[Compound represented by formula (2) or a salt thereof]
The compound represented by the above formula (2) or a salt thereof is a derivative of pyrroloquinoline quinone. Although it does not specifically limit as a C1-C10 substituent shown by R ', For example, an acetyl group, an ethoxy group, ketoalkyl, and hydroxyalkyl are mentioned. Among these, an acetyl group and a ketoalkyl group are preferable from the viewpoint of stability. In addition, the C1-C10 substituent shown by R 'may contain an oxygen atom, a nitrogen atom, a hydrogen atom, a sulfur atom, and a phosphorus atom other than a carbon atom.

  Although it does not specifically limit as a salt of the compound represented by Formula (2), For example, a tricarboxylic acid, a tricarboxylic acid di salt, a tricarboxylic acid mono salt, and a tricarboxylic acid tri salt are mentioned. Examples of the salt include, but are not limited to, alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, strontium salt and barium salt; ammonium salt and alkylammonium salt And salts with such cationic compounds.

  Although it does not specifically limit as a compound or its salt represented by the said Formula (2), For example, R 'is an acetyl group or an ethoxy group, Tricarboxylic acid, tricarboxylic acid disodium salt, tricarboxylic acid trisodium salt, tricarboxylic acid Acid dipotassium salt, tricarboxylic acid tripotassium salt.

[Compound represented by formula (3) or a salt thereof]
In the above formula (3), a compound in which R is all hydrogen atoms is referred to as a reduced pyrroloquinoline quinone formed by reducing an acid value type pyrroloquinoline quinone. The salt of reduced pyrroloquinoline quinone is not particularly limited, and examples thereof include tricarboxylic acid, tricarboxylic acid di-salt, tricarboxylic acid mono-salt, and tricarboxylic acid tri-salt. Examples of the salt include, but are not limited to, alkali metal salts such as lithium salt, sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, strontium salt and barium salt; ammonium salt and alkylammonium salt And salts with such cationic compounds.

  In the above formula (3), one or more R are preferably hydrogen atoms. In the formula (3), a compound in which one or more Rs are hydrogen atoms forms a salt in the solution and becomes ionic, so that the water solubility tends to be improved as compared with the tricarboxylic acid form.

  Although it does not specifically limit as a compound represented by the said Formula (3) or its salt, For example, reduced pyrroloquinoline quinone; reduced pyrroloquinoline quinone monosodium, reduced pyrroloquinoline quinone disodium, reduced pyrroloquinoline quinone Examples thereof include alkali metal salts of reduced pyrroloquinoline quinone such as trisodium, reduced pyrroloquinoline quinone dipotassium, and reduced pyrroloquinoline quinone tripotassium.

  Among these, from the viewpoint of ease of production, reduced pyrroloquinoline quinone disodium such as reduced pyrroloquinoline quinone monosodium, reduced pyrroloquinoline quinone disodium, and reduced pyrroloquinoline quinone trisodium is preferable.

  Among the compounds represented by formulas (1), (2), and (3), from the viewpoint of NADPH oxidase activation effect, oxidized pyrroloquinoline quinone disodium, reduced pyrroloquinoline quinone, pyrroloquinoline quinone monomethyl Ester and pyrroloquinoline quinone trimethyl ester are preferable, and pyrroloquinoline quinone monomethyl ester and pyrroloquinoline quinone trimethyl ester are more preferable.

[Method for producing the above compound or a salt thereof]
Although it does not specifically limit as a manufacturing method of pyrroloquinoline quinone, For example, the method of organic-chemical synthesis | combination, or a fermentation method is mentioned. Among them, the fermentation method is, for example, a method for producing PQQ by culturing a bacterium having an ability to assimilate methanol and capable of producing pyrroloquinoline quinone using methanol as a carbon source. .

  A derivative of pyrroloquinoline quinone, specifically, an ester of pyrroloquinoline quinone or a salt of pyrroloquinoline quinone (the above compound or a salt thereof) is obtained according to a conventional method using the pyrroloquinoline quinone thus obtained as a starting material. Can be synthesized. The pyrroloquinoline quinone and derivatives thereof can be separated and purified from the reaction solution by a usual method such as column chromatography, recrystallization method, or solvent extraction method. In addition, various means such as elemental analysis, NMR spectrum, IR spectrum, and mass spectrometry are used for identification.

[Usage: Use of PQQ]
This embodiment includes the use of the above compound or a salt thereof for the production of an NADPH oxidase activator.

  The NADPH oxidase activator can be used as a food, functional food, pharmaceutical or quasi drug for humans or animals. The functional food here means foods taken for the purpose of maintaining health or supplementing nutrition instead of dietary foods such as health foods, nutritional supplements, functional nutritional foods, and nutrition insurance foods. Specific forms include, but are not limited to, capsules, tablets, chewable tablets, drinks and the like.

  For pharmaceutical use, NADPH oxidase activator can be used by methods such as oral administration, injection, and skin absorption. In the case of oral administration, the NADPH oxidase activator can be used by mixing with other substances in the form of hard capsules, soft capsules, and tablets. NADPH oxidase activator can also be used as a beverage, drip solution, or injection solution by utilizing its high water solubility. Furthermore, the NADPH oxidase activator can be mixed with an emulsion and blended into a cosmetic cream or cake, or can be easily mixed with rice or wheat flour and used in foods using the same.

  When the NADPH oxidase activator is used against nematodes which are model organisms for research, a method of mixing in a medium can be mentioned. Specifically, a NADPH oxidase activator is dissolved in a medium containing a large amount of amino acids, such as peptone, cholesterol, and inorganic ions. Can be absorbed orally or percutaneously absorbed from the medium. The amount to be mixed in the medium is preferably 1 to 100 mM, more preferably 1 to 15 mM.

  When the NADPH oxidase activator is used for an animal, the dosage varies depending on the subject animal. When the target animal is a human, the dosage varies depending on various factors such as the disease applied, the age, sex or weight of the patient, the severity of symptoms, and the route of administration. In the case where the subject animal is a human, typically, an ADAPH oxidase activator of 0.25 to 1000 mg, preferably 1 to 250 mg, more preferably 10 to 100 mg per day for an adult weighing about 60 kg. Is administered. The NADPH oxidase activator can be administered once or multiple times per day. The NADPH oxidase activating effect is effective for eukaryotes, preferably animals.

  According to the NADPH oxidase activation method of this embodiment, symptoms and diseases related to NADPH oxidase, such as involvement in blood pressure increase, infection protection, and thyroid hormone synthesis, can be improved.

  Although it does not specifically limit as a specific method, For example, the method of administering the said NADPH oxidase activator for the purpose of a treatment or a treatment by the method as described in the said [use] to an animal or a patient. Can be mentioned. That is, a method of activating NADPH oxidase in a subject or animal by administering an NADPH oxidase activator to the subject or animal.

  In this specification, “animal” or “patient” can be used for vertebrates such as birds, fish, and mammals, and invertebrates such as insects and nematodes. In addition, when the NADPH oxidase activation method of the present embodiment is a therapeutic method, humans are excluded from target animals.

In the present specification, the “treatment” or “treatment” includes the following, but is not particularly limited.
(A) preventing the occurrence of a disease state in a mammal, specifically if such a mammal is susceptible to the disease state but has not yet been diagnosed as affected;
(B) suppressing the disease state, eg, stopping the progression thereof; and / or (c) reducing the disease state, eg, causing the regression of the disease state to a desired end point. Includes treatment. Treatment also includes recovery of disease symptoms (eg, relief of pain or discomfort), and such recovery may or may not have a direct effect on the disease (eg, cause, transmission, manifestation, etc.).

[Activation method of NADPH oxidase]
The NADPH oxidase activation method of the present embodiment uses a compound represented by the above formulas (1), (2), and (3) or a salt thereof.

  The specific activation method is not particularly limited, and examples thereof include a method of using (administering) the NADPH oxidase activator to animals by the method described in the above [Use].

  The NADPH oxidase activation method is effective for symptoms and diseases related to NADPH oxidase. More specifically, it can contribute to blood pressure elevation, infection protection, thyroid hormone synthesis, and the like. The NADPH oxidase activation method is used to prevent or treat a disease or disorder associated with NADPH oxidase expression and / or function. Here, the disease or disorder includes those associated with abnormal expression and / or function of NADPH oxidase mutants or normal NADPH oxidase.

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples. The present invention is not limited in any way by the following examples.

  As the PQQ disodium salt used in this example, BioPQQ manufactured by Mitsubishi Gas Chemical was used. For other compounds, Wako Pure Chemical reagents (special grade) were used unless otherwise specified. Moreover, the PQQ free body (the thing which has not formed the salt) was obtained by adding the concentrated hydrochloric acid to the pyrroloquinoline quinone disodium aqueous solution, and drying the solid which precipitated when pH was set to 1.

[Production Example 1: PQQTME (PQQ trimethyl ester)]
32 g of PQQ free body was dissolved in 300 g of N, N-dimethylformamide heated to about 30 ° C. When 30 g of potassium carbonate was added to this solution and 350 g of dimethyl sulfate was mixed, the solution temperature rose to 50 ° C. after 30 minutes. When the solution temperature dropped to room temperature, 30 g of potassium carbonate was further mixed. The solution was stirred at room temperature for 3 days, the resulting solution in 1 L of water was added and 30 g of 2N HCl was mixed. The solution was filtered and the filtrate (PQQTME) was washed with isopropanol. The result of NMR is shown below.
1H-NMR (CDCl ₃ ): 3.98 ppm, 4.01 ppm, 4.18 ppm, 7.49 ppm, 8.90 ppm

[Production Example 2: Monomethyl PQQ]
Monomethyl PQQ was obtained by improving the method described in JP-A-5-70458. Specifically, 4 g of PQQTME was mixed with 400 g of acetonitrile, and a 400 g water solution containing 5.5 g of K ₂ CO ₃ was added thereto. Thereafter, the mixture was mixed at room temperature for 2 days, 12.5 g of concentrated hydrochloric acid was added to the resulting solution, and then acetonitrile was removed with an evaporator. The solid precipitated in the solution was filtered and dried under reduced pressure to obtain 4.07 g of solid. The result of NMR is shown below. The NMR of the obtained solid was consistent with the literature (Japanese Patent Laid-Open No. 5-70458).
1H-NMR (dmso-d ₆ ): 3.87 (Me) pmm, 7.27 pmm, 8.61 pmm

[Production Example 3: Reduced PQQ]
An aqueous solution was prepared by mixing 3.0 g of PQQ disodium salt (Mitsubishi Gas Chemical BioPQQ) and 1.2 L of water. On the other hand, 30 g of ascorbic acid, 120 g of water, and 2.5 g of 2N hydrochloric acid were mixed, and the temperature was adjusted to 12 ° C. The aqueous solution was added with stirring over 2 hours. After mixing, the mixture was stirred at 20 ° C. for 18 hours. To the obtained solution, 2.5 g of 2N hydrochloric acid was mixed and stirred for 1 hour. Thereafter, the solid precipitated in the aqueous solution was filtered, and washed with 2 mL of 2N hydrochloric acid, 50% ethanol, and 8 mL of water. Drying under reduced pressure was performed at room temperature for 20 hours to obtain 3.35 g of a hydrous crystal of reduced pyrroloquinoline quinone represented by the following formula (6).

[Method for measuring NADPH oxidase activity by cells]
As a cell, human fibrosarcoma HT1080 was used. For measurement of hydrogen peroxide, Amplex red reagent (Life Technologies) that reacts with hydrogen peroxide in the presence of peroxidase to form fluorescent Resorbin was used. Experiments were carried out based on Non-Patent Documents 3 and 4 regarding a cell line that stably expresses each gene and a hydrogen peroxide measurement method using Amplex red. The reagent medium used was Nacalai Tesque special grade.

  First, according to Non-Patent Document 3, a cell line that stably expresses BLI-3, which is a nematode Dual Oxidase, and TSP-15 and DOXA-1 necessary for its activation, was prepared (cell line A). Subsequently, a cell line stably expressing human dual oxidase 1 and dual oxidase activator 1 required for its activation, or dual oxidase activator 2 and dual oxidase activator 2 required for its activation was prepared (cell line B). .

Cell lines A and B of 5 × ¹⁰ 4 / in 100μL concentration seeded in 96 well black plates were incubated overnight at Dulbecco's modified Eagle medium (DMEM) medium containing 10% bovine serum. As a negative control, HT1080 cells into which no gene was introduced were used.

As buffers, Dulbecco's composition buffered saline (D-PBS) containing 0.9 mM CaCl ₂ , 0.49 mM MgCl ₂ , 1 g / L glucose, or Hanks balanced salt solution (HBSS) containing 10 mM HEPES pH 7.4 did.

  After the culture, the medium was removed and the cells were washed several times with the above buffer solution. Then, the sample was added at each measurement concentration to a buffer solution containing a final concentration of 50 μM Amplex red, 0.1 U / mL horseradish peroxidase (HRP), and cultured at 37 ° C. for 1 hour. Since human Dual Oxidase requires calcium stimulation, ionomycin was added to a final concentration of 1 μM.

  Excitation light with a wavelength of 544 nm was irradiated, and fluorescence with a wavelength of 590 nm was measured. A standard calibration curve was prepared using hydrogen peroxide solution whose concentration was known in advance in each experiment. Then, the concentration of hydrogen peroxide present in the culture supernatant was converted from the fluorescence value of each well using a calibration curve.

[Example A]
[Example A1: NADPH oxidase expressing cells in 10 μM pyrroloquinoline quinone disodium]
Nematode NADPH oxidase, Dual Oxidase gene BLI-3, tsp-15 gene that binds to BLI-3 and encodes tetraspanin, and doxa-encodes Dual Oxidase activating protein 10 μM pyrroloquinoline quinone disodium was added to HT1080 cells (TSP / DOX / BLI) co-expressed with one gene, and the amount of hydrogen peroxide generated was measured. As a result, 11.8 μM of hydrogen peroxide was generated, while 2.2 μM of hydrogen peroxide was generated in HT1080 cells (non-trf) that did not express these gene groups.

[Comparative Example A1: NADPH oxidase-expressing cells without addition of pyrroloquinoline quinone disodium]
No additive was added to the HT1080 cells (TSP / DOX / BLI) used in Example A1, and the amount of hydrogen peroxide generated was measured. As a result, 1.9 μM hydrogen peroxide was generated.

[Comparative Example A2: NADPH oxidase expressing cells added with imidazoloquinone]
To the HT1080 cells (TSP / DOX / BLI) used in Example 1, 10 μM imidazoloquinone was added, and the amount of hydrogen peroxide generated was measured. As a result, 2.5 μM hydrogen peroxide was generated.

  From the results of Example A1 and Comparative Example A1 above, when pyrroloquinoline quinone disodium is added to cells, hydrogen peroxide more than 5 times the amount of hydrogen peroxide normally generated in cells may be generated. I understood. Further, from the results of Example A1 and Comparative Example A2, it was also found that imidazoloquinone has almost no effect of promoting hydrogen peroxide generation, despite being an analog of pyrroloquinoline quinone.

[Example A2: NADPH oxidase expressing cells in 50 μM pyrroloquinoline quinone disodium]
50 μM pyrroloquinoline quinone disodium was added to the HT1080 cells (TSP / DOX / BLI) used in Example A1, and the amount of hydrogen peroxide generated was measured. As a result, 12.4 μM hydrogen peroxide was generated. On the other hand, in HT1080 cells (non-trf) not expressing these gene groups, 3.4 μM of hydrogen peroxide was generated.

[Example A3: NADPH oxidase inhibitor DPI added]
To HT1080 cells (TSP / DOX / BLI) used in Example A1, 50 μM pyrroloquinoline quinone disodium and NADPH oxidase inhibitor (DPI) were added, and the amount of hydrogen peroxide generated was measured. As a result, 0.9 μM of hydrogen peroxide was generated.

  Comparison of Examples A2 and A3 indicated that the generation of hydrogen peroxide was greatly inhibited by the NADPH oxidase inhibitor. This result shows that the NAPH oxidase activator of this embodiment activates NADPH oxidase.

[Example A4-7: Experiment with different concentrations]
To the HT1080 cells (TSP / DOX / BLI) used in Example A1, pyrroloquinoline quinone 0.1 to 2.5 μM was added, and the amount of hydrogen peroxide generated in each cell was measured.

  In addition, as a result of adding pyrroloquinoline quinone 0-50 μM or imidazoloquinone 0-50 μM to HT1080 cells (TSP / DOX / BLI) and measuring the amount of hydrogen peroxide generated in each cell, HT1080 cells (TSP / DOX / BLI) were measured. As a result of adding pyrroloquinoline quinone 50 μM or imidazoloquinone 50 μM and NADPH oxidase inhibitor (DPI) to BLI), and adding only NADPH oxidase inhibitor (DPI) to HT1080 cells (TSP / DOX / BLI) The results are summarized in FIG.

[Example A8-15, Comparative Example A3, 4]
HT1080 cells (DOX / BLI) were prepared which expressed the dual Oxidase gene BLI-3, which is an NADPH oxidase, and the doxa-1 gene that is necessary for the activation of BLI-3. Pyrroloquinoline quinone disodium 0-50 μM was added to HT1080 cells (DOX / BLI), and the amount of hydrogen peroxide generated in each cell was measured. Further, as Comparative Example A3, the amount of hydrogen peroxide generated in a cell to which pyrroloquinoline quinone disodium was not added was measured. The amount of hydrogen peroxide generated was measured. The result is shown in FIG. It was also found that pyrroloquinoline quinone disodium promotes the generation of hydrogen peroxide in these HT1080 cells (DOX / BLI).

  In addition, pyrroloquinoline quinone 0-50 μM or imidazoloquinone 0-50 μM was added to HT1080 cells (DOX / BLI), and the amount of hydrogen peroxide generated in each cell was measured, and HT1080 cells (DOX / BLI) The results of adding pyrroloquinoline quinone 50 μM or imidazoloquinone 50 μM and NADPH oxidase inhibitor (DPI) are collectively shown in FIG.

  Further, pyrroloquinoline quinone 0-50 μM or imidazoloquinone 0-50 μM was added to HT1080 cells (non-trf), and the amount of hydrogen peroxide generated in each cell was measured. HT1080 cells (DOX / BLI) The results of adding pyrroloquinoline quinone 50 μM or imidazoloquinone 50 μM and NADPH oxidase inhibitor (DPI) are collectively shown in FIG.

[Example B]
[Examples B1 to 4, Comparative Example B1: Influence of time change]
HT1080 cells (TSP / DOX / BLI) used in Example 1 were used to change the amount of hydrogen peroxide generated over time according to the concentration of pyrroloquinoline quinone disodium, and pyrroloquinoline quinone disodium and NADPH oxidase. The change over time in the amount of hydrogen peroxide generated when an inhibitor (DPI) was added was measured. The result is shown in FIG. As shown in FIG. 2, it was found that hydrogen peroxide was generated in a very short time. This indicates that there is a high possibility that pyrroloquinoline quinone directly increases the activity of NADPH oxidase. As an enzyme activation method, there is a method of increasing the expression level of the enzyme, but in this embodiment, it can be seen that PQQ directly increases the enzyme function.

[Example C]
HT1080 cells (D1A1) expressing a human-derived Dual Oxidase 1 and a Dual Oxidase Activator 1 gene necessary for its activation were prepared. At the same time, HT1080 cells (D2A2) expressing Dual Oxidase 2 and Dual Oxidase Activator 2 gene necessary for its activation were also prepared. To each of HT1080 cells (D1A1) and HT1080 cells (D2A2), pyrroloquinoline quinone 0 to 50 μM was added, and the amount of hydrogen peroxide generated in each cell was measured. Further, the amount of hydrogen peroxide generated in cells to which pyrroloquinoline quinone or imidazoloquinone and NADPH oxidase inhibitor (DPI) were added, and the peroxidation of cells to which 0 to 50 μM of imidazoloquinone was added instead of pyrroloquinoline quinone. The amount of hydrogen generation was measured. The result is shown in FIG. As can be seen from this result, pyrroloquinoline quinone generates hydrogen peroxide in a concentration-dependent manner, and it is understood that NADPH oxidase is also activated in human genes.

Example D
Using HT1080 cells (TSP / DOX / BLI) used in Example 1, time-dependent change in the amount of hydrogen peroxide generated according to the concentration of pyrroloquinoline quinone disodium, and inhibition of pyrroloquinoline quinone disodium and protein synthesis Changes in the amount of hydrogen peroxide generated over time when an agent (cycloheximide, CHX) 10 μg / ml was added were measured. The result is shown in FIG. As shown in FIG. 4, the promotion of hydrogen peroxide production is not inhibited by the protein synthesis inhibitor, suggesting that pyrroloquinoline disodium does not promote the synthesis of new proteins.

  HT 1080 cells (TSP / DOX / BLI) used in Example A and comparative examples HT 1080 cells (non-trf, nTrf), HT 1080 cells (D1A1) and HT 1080 cells (D2A2) used in Example C were treated with pyrroloquinoline. Western blotting of BLI-3, TSP-15, DOXA-1, human DUOX1, human DUOX2, human DUOXA1, and human DUOXA2 expression levels on the cell membrane from the cell extract (lysate) after addition of 10 μM sodium and incubation for 1 hour The results analyzed in Fig. 5 are shown in Figs. Moreover, the cell surface molecule | numerator is labeled with the biotin which does not permeate cell membrane, The precipitate (StAv) pulled down with the streptavidin carrier was blotted with each antibody, and the result analyzed as a cell surface expression level is also shown collectively. GAPDH is shown as a cell endogenous control molecule, and NaKATPase is shown as a cell surface control molecule. Since there is no difference between pyrroloquinoline disodium added (-) and added sample (+), pyrroloquinoline disodium promotes protein synthesis of these hydrogen peroxide synthases and their function-related proteins. It is suggested that it is not.

Example E
[Example E1-3, Comparative Example E1-2: Activation of NADPH Oxidase by PQQ Derivative]
Table 1 shows the results of measuring the amount of hydrogen peroxide generated by adding 10 μM PQQ derivative or 10 μM ascorbic acid or imidazoloquinone (IPQ) to the BLI-3 cells (TSP / DOX / BLI) used in Example 1. Show. As shown in Table 1, NADPH oxidase activation was also observed in the PQQ derivatives.

  Reduced PQQ, monomethyl PQQ, and PQQTME have the ability to generate hydrogen peroxide. These substances are expected to return to pyrroloquinoline quinone in vivo, and the hydrogen peroxide generation capacity varies depending on the difference in absorbability to cells and the initial structure. In contrast, it did not occur with ascorbic acid, which is said to generate hydrogen peroxide. In addition, it was found that the IPQ in which the quinone is broken is very small and has no enzyme activity function.

  The NADPH oxidase activator and the method for activating NADPH oxidase of the present invention have industrial applicability in applications such as foods and pharmaceuticals.

Claims (4)

A NADPH oxidase activator comprising a compound represented by the following formula (1), (2), or (3) or a salt thereof.
(In the above formulas (1), (2), and (3), R independently represents a hydrogen atom, a substituent having 1 to 10 carbon atoms, or an alkali metal, and R ′ represents the number of carbon atoms. 1 to 10 substituents are shown.)   The NADPH oxidase activator according to claim 1, wherein the compound represented by the formula 1 is pyrroloquinoline quinone disodium. Use of a compound represented by the following formula (1), (2), or (3) or a salt thereof for the production of an NADPH oxidase activator.
(In the above formulas (1), (2), and (3), R independently represents a hydrogen atom, a substituent having 1 to 10 carbon atoms, or an alkali metal, and R ′ represents the number of carbon atoms. 1 to 10 substituents are shown.)   A method for activating NADPH oxidase using the NADPH oxidase activator according to claim 1.