JP6587057B2 - Anti-degranulation agent - Google Patents

Description

  The present invention relates to a degranulation inhibitor containing black vinegar as an active ingredient. The present invention also relates to an agent for preventing or improving allergic symptoms containing black vinegar as an active ingredient.

  Black vinegar has been handed down as a healthy food for about 200 years and is now widely used as a seasoning, as well as beverages, supplements and foods for specified health use. Brown rice vinegar and rice black vinegar are vinegars that are brewed by the stationary fermentation method, which is a traditional brewing method transmitted to Fukuyama Town, Kagoshima Prefecture. The raw materials are only steamed rice, rice bran, and groundwater, and it is produced using 6-9 times brown rice or rice as a raw material compared with general brewed vinegar and taking fermentation and aging period of more than one year. Therefore, the type and amount of amino acids contained are large.

Black vinegar has long been popular as a health food, and many functionalities have been reported, such as lipid metabolism improving action, hypertension inhibiting action, sugar metabolism improving action, liver function improving action, and antioxidant action. For example, Patent Document 1, drying of organic solvent extracts of black vinegar, cytosolic phospholipase A2 inhibitory activity and O ₂ ^- to have anti-inflammatory activity are disclosed by the production inhibitory activity. However, with regard to black vinegar, sufficient knowledge about allergic symptom relief and immunity promoting effects has not been obtained yet.
Although Patent Document 2 describes that black vinegar has an antiallergic action, it only confirms the effect of reducing cholesterol and neutral fat, and no specific examination has been made on the antiallergic action.

Japanese Patent No. 4344030 JP 2007-29020 A

  An object of the present invention is to confirm the antiallergic action of black vinegar and provide an agent for preventing or improving allergic symptoms comprising black vinegar as an active ingredient.

  The present inventor has intensively studied for the purpose of solving the above-mentioned problems, and found that black vinegar has an anti-allergic action of degranulation, and has completed the present invention. By utilizing this action of black vinegar, it becomes possible to provide a degranulation inhibitor containing black vinegar as an active ingredient and an agent for preventing or improving allergic symptoms.

That is, this invention relates to the degranulation inhibitor etc. which are shown by following (1) or (2).
(1) A degranulation inhibitor containing black vinegar as an active ingredient.
(2) An agent for preventing or improving allergic symptoms comprising black vinegar as an active ingredient.

  According to the present invention, a degranulation inhibitor containing black vinegar as an active ingredient can be provided as an antiallergic agent. By providing this agent, it is possible to suppress the release of histamine, and it is possible to prevent or improve symptoms of type I allergy such as hay fever associated with IgE.

It is the figure which showed the degranulation inhibitory effect by black vinegar (Example 2). It is the figure which showed the degranulation inhibitory effect in each fraction (Example 3). It is the figure which showed the evaluation result of thermal stability (Example 3). It is the figure which showed the evaluation result of protease resistance (Example 3). It is the figure which showed the influence of black vinegar with respect to intracellular calcium ion concentration (Example 4). It is the figure which showed the influence of black vinegar on the degranulation signal transduction pathway (Example 4). It is the figure which showed the effect of the oral administration of black vinegar to a passive skin anaphylaxis model mouse (Example 5).

The black vinegar used in the present invention is rice black vinegar defined by the JAS method, and any black vinegar as defined in the present invention can be used in the present invention. In particular, the black vinegar used in the present invention is a brown color obtained by further ripening steamed rice, rice bran, groundwater in a potter's pot and proceeding with saccharification, alcohol fermentation, and acetic acid fermentation in one pot. Or it is preferable that it is a vinegar which is colored black brown and has a unique deep taste.
As a black vinegar origin component of this invention, the component etc. which remove | excluded the acetic acid component from black vinegar other than black vinegar itself are mentioned.
In the examples described below, each test was performed on the component obtained by removing acetic acid from black vinegar, in order to clarify the difference from the action of the acetic acid component that is the main component of black vinegar, As a result, it has been found that the component obtained by removing acetic acid from black vinegar has a degranulation inhibitory effect, and therefore, it can be said that this effect is present in components other than acetic acid among components derived from black vinegar.

The “degranulation inhibitor” of the present invention is a histamine producing cell such as a basophil or a mast cell, and the granules stored in the cell are exposed to the outside by stimulation such as binding of an antigen to an antibody on the cell surface. An agent that suppresses release (degranulation).
In addition, the “preventive or ameliorating agent for allergic symptoms” of the present invention refers to an agent for preventing the occurrence of allergic symptoms associated with degranulation caused by IgE, or for alleviating and improving the symptoms. Say.

The “degranulation inhibitor” or “prevention or amelioration agent for allergic symptoms” of the present invention may be black vinegar itself or the remaining component itself obtained by removing the acetic acid component from black vinegar.
The remaining component obtained by removing the acetic acid component from black vinegar can be fractionated, and only the fraction having a high degranulation inhibitory effect can be used. In the case of such a fraction, it is preferable to use a fraction having a molecular weight of 500 or more, more preferably 3,500 or more.
Moreover, what mixed these with the other component which does not affect the effect | action of this invention may be used.

  The form of the “degranulation inhibitor” or “prevention or amelioration agent for allergic symptoms” of the present invention may be a liquid or a dried solid. The solid may be a solid or a powder. In the case of solids, it is dissolved or dispersed in a suitable liquid, or mixed with a suitable powder carrier or adsorbed thereto, and in some cases, further emulsifiers, dispersants, suspending agents, spreading Additives, penetrants, wetting agents, stabilizers and the like can be used as preparations such as emulsions, oils, wettable powders, powders, tablets, capsules, and liquids. When used as a preparation, the amount of the extract to be used varies depending on the form of the preparation, and there is no problem with safety, so there is no particular upper limit.

  Examples of foods and beverages include chewing gum, candy, tablet confectionery, gummy jelly, chocolate, biscuits or snacks, frozen confectionery such as ice cream, sherbet or ice confectionery, beverages, pudding, jam, dairy products, seasonings, etc. Daily intake of these foods and beverages to which the “degranulation inhibitor” or “allergy symptom prevention or amelioration agent” of the invention is added can provide a degranulation suppression effect and an allergy symptom prevention or improvement effect. The content of the agent of the present invention in food and drink may be contained within a range that does not impair the taste and flavor as a favorite product of the food and drink, and varies depending on the type and form of the food and drink. Moreover, it can administer by mixing with the feed normally given as a feed. In addition, as a result of investigating the drug interaction, the black vinegar of the present invention was found to have no effect on the expression of genes and proteins related to drug metabolism, so there are side effects even when other drugs are used in combination. And can be taken safely.

  The “degranulation inhibitor” or “allergic symptom prevention or amelioration agent” of the present invention is preferably intended for humans or animals other than humans, such as dogs, cats, rabbits, hamsters, guinea pigs, squirrels, etc. It is also possible to target animals such as pets (companion animals and companion animals), livestock such as cattle and pigs, and animals such as mice and rats.

The method of administering the “degranulation inhibitor” or “allergic symptom prevention or amelioration agent” of the present invention to humans or animals other than humans is desirable to be administered orally, and may be formulated, or may be used as food, drink or feed Ingestion is also possible.
The amount of ingestion in the case of oral administration is not particularly limited as long as the effect of suppressing degranulation and the effect of preventing or improving allergic symptoms. For example, as black vinegar extract (as black vinegar 10-fold concentrated solution) 6 to 200 mg / kg body weight / day is preferred.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

[Example 1]
Preparation of black vinegar 1000 ml of black vinegar (manufactured by Sakamoto Shuzo Co., Ltd., Sakamoto's Kurozu (product name)) was freeze-dried and powdered. Distilled water was added thereto and freeze-dried again. This operation was repeated four times to completely remove acetic acid in black vinegar. A solution obtained by dissolving the obtained powder in 100 ml of distilled water was used as a black vinegar 10-fold concentrated solution and used in tests in Examples described later. In these tests, the black vinegar 10-fold concentrated solution may be simply referred to as black vinegar.

[Example 2]
Evaluation of degranulation inhibitory action In evaluating degranulation inhibitory action, degranulation activity of basophils was examined using as an index the release inhibitory activity of β-hexosaminidase present in granules together with histamine.
The black vinegar prepared in Example 1 was used as a sample, and 10 mM sodium phosphate buffer pH 7.4 (NaPB) was used as a control.

1. Culture of rat basophil cell line RBL-2H3 cell Rat basophil cell line RBL-2H3 cell pre-cultured in 10% fetal bovine serum (FBS) -DMEM medium (from JCRB Cell Bank, Incorporated Administrative Agency The number of cells in the distribution was adjusted to about 2.0 × 10 ⁵ cells / mL to prepare a cell suspension. 200 μL of this cell suspension was dispensed into each well of a 96-well plate and cultured overnight at 37 ° C., humidity 100%, 5% CO ₂ .

2. Degranulation induction 1) Anti-dinitroferol-IgE (manufactured by Sigma (hereinafter sometimes referred to simply as anti-DNP-IgE)) was diluted with 10% FBS-DMEM to 50 ng / mL and anti-DNP-IgE A solution was obtained.
Above 1. RBL-2H3 cells cultured overnight in PBS were washed with PBS, and 120 μL of anti-DNP-IgE solution was added to each well and cultured for 2 hours to sensitize the cells (final concentration of anti-DNP-IgE: 25 ng) / ML). Moreover, the medium which did not contain anti-DNP-IgE was put into the blank.
2) After the above 1), after washing the cells in each well, the cells were treated for 10 minutes with a Tyrode buffer to which serially diluted black vinegar was added, and then DNP-HSA (Sigma) as an antigen. And the mixture was incubated for 30 minutes. The amount of black vinegar added was shown using the protein concentration as an index (each protein concentration: 325 μg / mL, 650 μg / mL, 1300 μg / mL or 2600 μg / mL).
3) After the above 2), degranulation was evaluated using the release inhibitory activity of β-hexosaminidase existing inside and outside the cell as an index.

2) Results FIG. 1A shows the degranulation-inhibiting action by black vinegar using the release inhibitory activity of β-hexosaminidase as an index. As a result, it was shown that black vinegar suppresses degranulation of RBL-2H3 cells in a concentration-dependent manner.
Moreover, although the cell survival rate of RBL-2H3 cell was shown in FIG. 1, B, it became clear that the inhibitory action of the degranulation by black vinegar is not due to cytotoxicity.
Therefore, it was confirmed that black vinegar has an antiallergic effect by suppressing degranulation.

Example 3
Examination of degranulation inhibitor contained in black vinegar Fractionation of active ingredient and activity evaluation of each extracted fraction 1) Fractionation of active ingredient In order to infer what kind of substance is degranulation inhibitor contained in black vinegar, the molecular size of Attempted estimation.
In the following steps (1) to (3), black vinegar was dialyzed using a dialysis membrane having a molecular weight cut of 3,500. Thereafter, the release inhibitory activity of β-hexosaminidase was measured by the same method as in Example 2.
(1) A cellulose dialysis membrane (manufactured by Spectrum) having a molecular weight cut of 3,500 was stirred and washed in a beaker containing 10 mM sodium phosphate buffer (NaPB) for 30 minutes.
(2) The sample was injected into the dialysis membrane washed in (1) above, and dialyzed with 4 liters of 10 mM NaPB as an external solution while stirring at 4 ° C. for 12 hours or more. During the dialysis, the external solution was changed twice.
(3) After completion of the dialysis of (2) above, the dialysis membrane fluid was recovered.

2) Results FIG. 2 shows the degranulation inhibitory action in each fraction using the β-hexosaminidase release inhibitory activity as an index. As a result, it was confirmed that the dialysis fraction composed of a substance having a molecular weight of 3,500 or more has an increased release inhibitory activity of β-hexosaminidase compared with non-dialyzed black vinegar.
Therefore, from this result, it was suggested that the active substance contained in black vinegar is not a low molecular substance such as a free amino acid but a substance having a molecular weight larger than 3,500.
Moreover, since the release inhibitory activity of β-hexosaminidase was similarly increased in a fraction consisting of a substance having a molecular weight of 500 or more, which was dialyzed using a dialysis membrane having a molecular weight cut of 500 (manufactured by Spectrum), It was also suggested that the active substance contained in black vinegar may be a substance having a molecular weight greater than 500.

2. Evaluation of Thermal Stability and Protease Resistance 1) Evaluation of Thermal Stability As an evaluation of thermal stability, black vinegar prepared in Example 1 was heated at 100 ° C. for 30 minutes, and this was used as a sample in Example 2. In the same manner as described above, the release inhibitory activity of β-hexosaminidase was evaluated.
As a result, as shown in FIG. 3, it was confirmed that even when black vinegar was heat-treated, no change was observed in the β-hexosaminidase release inhibitory activity.

2) Evaluation of protease resistance As an evaluation of resistance to proteolytic enzymes, trypsin (sum of various concentrations (0 μg / mL, 10 μg / mL, 100 μg / mL or 500 μg / mL) of black vinegar prepared in Example 1 was used. And processed with Proteinase K (manufactured by Wako Pure Chemical Industries, Ltd.) by the following method.

Trypsinization (1) Trypsin was prepared with 10 mM NaPB at 500 μg / mL and 50 μg / mL.
(2) An equal amount of the trypsin prepared in (1) above was added to each concentration of black vinegar and allowed to react at 37 ° C. for 15 minutes.
(3) After the reaction for 15 minutes in (2) above, heating was performed at 100 ° C. for 10 minutes to inactivate trypsin.
(4) After heating for 10 minutes in the above (3), it was placed on ice to obtain a trypsin-treated sample.

Proteinase K treatment (1) Proteinase K was adjusted to 500 μg / mL and 50 μg / mL with 10 mM NaPB.
(2) An equal amount of Proteinase K prepared in (1) above was added to each concentration of black vinegar and allowed to react at 37 ° C. for 12 hours.
(3) After the reaction for 12 hours of (2) above, Proteinase K was deactivated by heating at 100 ° C. for 10 minutes to obtain a Proteinase K-treated sample.

Thereafter, using this as a sample, the release inhibition activity of β-hexosaminidase was evaluated by the same method as in Example 2.
As a result, as shown in FIGS. 4, A and 4, B, it was confirmed that even when black vinegar was treated with a proteolytic enzyme, no change was observed in the release inhibitory activity of β-hexosaminidase. .

  From the results of 1 and 2 above, it is suggested that the active substance in black vinegar has a molecular weight of about 500 or more, more preferably 3,500 or more, and its activity is not changed by heat treatment and proteolytic enzyme treatment. It was.

Example 4
Elucidation of the mechanism of action of black vinegar Effect of black vinegar on intracellular calcium ion concentration It is known that degranulation by basophils is induced by an increase in intracellular calcium ion concentration when stimulation by allergen is transmitted into the cell. Therefore, in order to elucidate how the increase in intracellular calcium ion is changed by the action of black vinegar, Fluo3 (produced by Dojindo Laboratories), which is a calcium ion fluorescent probe, is used to It examined by the process of 1)-(10).

(1) A white 96-well plate was seeded with 4.0 × 10 ⁴ cells / well and incubated for 12 hours.
(2) After the incubation in (1) above, the culture supernatant was removed, and the cells were washed with PBS (−).
(3) An anti-DNP-IgE solution (1,000-fold dilution) was added to the washed plate and incubated at 37 ° C. for 2 hours.
(4) During the incubation of (3) above, Loading Buffer and Recording Medium were prepared with the compositions described in Tables 1 and 2.
(5) After the incubation in (3) above, the supernatant was removed and washed twice with PBS (−) heated at 37 ° C.
(6) 100 μL / well of Loading Buffer prepared in (4) was added to the washed plate at a temperature of 37 ° C. for 1 hour.
(7) After the incubation in (6) above, the supernatant was removed and washed twice with PBS (−) heated at 37 ° C.
(8) The Recording Medium prepared in (4) was added to the washed plate at 100 μL / well, and incubated at 37 ° C. for 10 minutes.
(9) After the incubation of (8) above, the fluorescence intensity (λ _ex = 480 nm, λ _em = 530 nm) was measured with a fluorescence plate reader, and the baseline was examined.
(10) After measuring the baseline in (9) above, 10 μL / well of DNP-HSA solution was added as an antigen substance, and the fluorescence intensity (λ _ex = 480 nm, λ _em = 530 nm) was measured with a fluorescence plate reader.

  As a result, as shown in FIG. 5, it was confirmed that the increase in intracellular calcium ion concentration induced by antigen stimulation was suppressed by the action of black vinegar. From these results, it was suggested that, in antigen-induced degranulation, intracellular signaling was inhibited by black vinegar, thereby suppressing degranulation by suppressing an increase in intracellular calcium ion concentration.

2. Effect of black vinegar on degranulation signaling pathway From the above results, the influence of black vinegar on the degranulation signaling pathway induced by antigen stimulation was evaluated by the following steps 1) to 3).
The primary antibodies used in this evaluation are as follows.
Anti-Lyn Antibody (manufactured by Cell Signaling Technology)
Anti-Phospho-Lyn (Tyr507) Antibody (manufactured by Cell Signaling Technology)
Anti-Syk Antibody (manufactured by Cell Signaling Technology)
Anti-Phospho-Syk (Tyr525 / 526) Antibody (manufactured by Cell Signaling Technology)
Anti-PLCγ1 (D9H10) Antibody (manufactured by Cell Signaling Technology)
Anti-Phospho-PLCγ1 (Tyr783) Antibody (manufactured by Cell Signaling Technology)
Anti-PLCγ2 Antibody (manufactured by Cell Signaling Technology)
Anti-Phospho-PLCγ2 (Tyr759) Antibody (manufactured by Cell Signaling Technology)
Anti-PI3 Kinase p85 (19H8) Antibody (manufactured by Cell Signaling Technology)
Anti-Phospho-PI3 Kinase p85 (Tyr458) / p55 (Tyr199) Antibody (manufactured by Cell Signaling Technology)
Anti-Actin (I-19) Antibody (manufactured by Santa Cruz Biotechnology)

1) Cell disruption and protein extraction RBL-2H3 cells were cultured under culture conditions with black vinegar added, and degranulation was induced by antigen stimulation. For comparison, RBL-2H3 cells (negative control) cultured under a culture condition in which NaPB was added instead of black vinegar, and RBL-2H3 cells (control) that had been cultured under this culture condition and subjected to antigen stimulation were collected. .
Each of these collected cells was washed twice with PBS (−) and then cell lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na ₄ P ₂ O ₇ , 1% v / v Triton-X). ) And an inhibitor (5 mg / mL Pefabloc SC AEBSF, 2 μg / mL Aprotinin, Complete; EDTA free, Phosphatase) were added, and the mixture was pipetted to prevent foaming and allowed to stand on ice for 30 minutes. Thereafter, the mixture was centrifuged at 12,000 × g for 15 minutes, the supernatant was collected, and the protein concentration between each cell lysate (sample) was made uniform.

2) Electrophoresis and blotting After each cell lysate with the same protein concentration as in 1) above was subjected to electrophoresis by SDS-PAGE, the protein was transferred to a PVDF membrane (manufactured by GE Healthcare). For the transfer from the polyacrylamide gel to the PVDF membrane, a blotting apparatus manufactured by Bio-Rad was used, and a constant current of 0.8 mA / cm ² was used for 1 hour.

3) Reaction with antibody and detection of band The PVDF membrane to which the protein was transferred in 2) above was blocked with 5% skim milk-Tris Buffered Saline with Tween 20 (TBS-t) for 1 hour at room temperature. .
After blocking, the plate was washed 3 times with TBS-t for 5 minutes. The primary antibody was diluted with 5% BSA-TBS-t and reacted with the membrane at 4 ° C. for 12 hours. The membrane after completion of the primary antibody reaction was washed 3 times with TBS-t for 5 minutes, and then, as a secondary antibody, goat anti-rabbit Immunoglobulin-HRP (1,000-fold dilution, manufactured by Cell Signaling Technology), or donkey anti -Goat IgG-HRP (4,000-fold dilution, manufactured by Santa Cruz Biotechnology) was diluted with 5% skim milk-TBS-t and allowed to react with the membrane at room temperature for 1 hour. After the reaction of the secondary antibody, it was washed 3 times with TBS-t for 5 minutes. Thereafter, a color development reaction was performed with a color developing solution in which 1 mL of Reagents 1 and 2 of Immunostar LD (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed at 1: 1, and the band was detected with an image analyzer (manufactured by Bio-Rad).

  As a result, as shown in FIG. 6, among RBL-2H3 cells cultured under the culture conditions to which black vinegar was added and degranulation was induced by antigen stimulation, among degranulation signal factors, Syk, PI3K, PLCγ1, AKT It was confirmed that the activation level was suppressed. Syk is a tyrosine kinase that exists directly under the high affinity IgE receptor (FcεRI) expressed on the surface of basophil cells and is located upstream of signal transduction, and is activated by phosphorylation of PI3K and PLCγ1. Is involved. Therefore, from this result, the active substance in black vinegar suppresses activation of PI3K, PLCγ1, and AKT downstream by suppressing the activation by phosphorylation of Syk, and suppresses the inflow of calcium ions, It was assumed that degranulation was suppressed.

Example 5
Effect of Oral Administration of Black Vinegar to Passive Skin Anaphylaxis Model Mice In order to clarify the antiallergic effect of black vinegar in vivo, the effect of oral administration to passive skin anaphylaxis model mice was examined.
That is, anti-DNP-IgE was administered subcutaneously to the auricles of mice (7 weeks old, female, BALB / c mice, n = 5), and after 23 hours, a low dose (6 mg protein / kg body weight) and a high dose ( 60 mg protein / kg body weight) black vinegar was orally administered. One hour later, DNP containing 0.5% Evans blue was administered from the tail vein, and 30 minutes later, Evans blue infiltration in the auricle was evaluated.

  As a result, as shown in FIG. 7, it was confirmed that pigment infiltration was significantly suppressed by oral administration of black vinegar. Therefore, it was shown that the allergic response in the ear pinna can be suppressed by oral administration of black vinegar, and the allergic response in the body can be suppressed by oral administration.

  According to the present invention, a degranulation inhibitor containing black vinegar as an active ingredient can be provided as an antiallergic agent. By providing this agent, it is possible to suppress the release of histamine, and it is possible to prevent or improve symptoms of type I allergy such as hay fever associated with IgE.

Claims (2)

A degranulation inhibitor comprising black vinegar from which the acetic acid component has been removed as an active ingredient. An agent for preventing or improving allergic symptoms associated with degranulation using black vinegar from which acetic acid components have been removed as an active ingredient.