JP2014009184A - Antiallergic substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antiallergic substance having a novel antiallergic action component as an active ingredient.SOLUTION: An antiallergic substance contains a compound represented by following chemical formula 1 as an active ingredient, where Ris CHor a sugar residue, and Ris H or a sugar residue of saccharide such as monosaccharide or disaccharide. It is preferable that the compound is derived from a leaf of Spinacia oleracea.

Description

  The present invention relates to an antiallergic substance containing an antiallergic component isolated and purified from a plant extract.

  In recent years, the number of allergic diseases has been increasing, and about 30% of people in Japan suffer from some allergic diseases. Among them, in particular, type I allergy represented by pollinosis causes allergens such as cedar pollen or ticks and is a symptom such as sneezing and stuffy nose, which has hindered daily life. For this reason, a fundamental response is required as a medical / health problem.

  The flow leading to the onset of allergic diseases starts when allergens invade and allergen uptake and antigen presentation by antigen-presenting cells (dendritic cells) in the body occur. The presented antigen induces type 2 helper T cells (Th2 cells) and increases production of IL-4 (interleukin 4). In addition, production of allergen-specific IgE antibodies by B cells occurs, and IgE antibodies bind to and sensitize FcεRI of mast cells and basophils. When the allergen re-enters here, the allergen is captured by the specific IgE antibody and cross-linked to activate mast cells and basophils. Activated mast cells and basophils release (degranulate) granules containing chemical mediators such as histamine and serotonin, causing energy inflammation such as eosinophil migration, vasodilation, or increased vascular permeability. Trigger. Among them, type I allergic reaction is due to the fact that allergic reaction occurs in mast cells or basophils sensitized with allergen-specific IgE antibody, where IgE antibody is cross-linked, degranulation and chemical mediator are released.

  As a means for preventing type I allergy, there are methods for suppressing the propagation of ticks by wearing, cleaning, or drugs that prevent allergens from entering the body. In addition, a method has been used in which an antihistamine or the like is used to inhibit a reaction by a chemical mediator such as sneezing or runny nose at the receptor level. However, it is necessary to wear the mask every time it develops, and the use of drugs also causes problems due to cost and side effects. Therefore, in these methods, troublesomeness, economic burden, and the like become problems.

  In contrast to these symptomatic treatments, radical treatment methods are also being studied. For example, there is an immunotherapy (desensitization therapy) that permanently attenuates or normalizes excessive cell signaling specific to a sensitizing allergen. By increasing the production of IgG antibodies against natural allergens over a long period of time, the amount of IgE antibodies is reduced and the symptoms are alleviated. However, desensitization therapy, which can be curatively treated, has its drawbacks, and there is a risk of inducing anaphylactic shock due to administration of pollen and mite crude extracts containing allergens. Furthermore, a time burden arises when the concentration is gradually increased from a low concentration allergen and it takes 3 years or more to reach the final dose. In order to avoid such drawbacks, there is a method of improving allergic disease symptoms using an antiallergic component in plants as a method attracting attention from the viewpoint of prevention or alleviation rather than treatment.

  For example, Patent Document 1 describes an in vivo antiallergic agent mainly composed of a flavonoid glycoside extracted, separated and purified from a plant of the genus Kawaratakemei. Patent Document 2 discloses a novel benzophenone-based substance having a therapeutic effect on allergic diseases contained in a dry powder of a leaf of the genus Vanilla genus plant (guava) or an extract extracted from the dry powder with a solvent, It describes sesquiterpene substances or flavonoid substances and antiallergic agents having them. Further, Patent Document 3 discloses a novel flavonoid glycoside obtained by performing extraction such as extraction, concentration, and purification using a asteraceae plant as a raw material, and an antibacterial comprising the novel flavonoid glycoside as an active ingredient. Allergic agents are described.

  On the other hand, the spinach plant components have been reported to have antioxidant activity and antitumor activity as physiological activities, but reports on antiallergic activity or degranulation inhibitory activity have been reported. Not done.

JP 2002-154970 A JP 2001-316398 A Japanese Patent Laid-Open No. 2001-233889

  Thus, at present, the use of antiallergic components in plants has been attracting attention, and various plants and food resources such as tea or vegetables are screened for those having antiallergic components using degranulation inhibitory activity as an index. Has been done.

  The present invention has been made in view of the above circumstances, and aims at screening a new plant having an antiallergic component and providing an antiallergic substance containing the novel antiallergic component as an active ingredient. To do.

  As a result of intensive studies by the present inventors, it was confirmed that spinach juice showed strong degranulation inhibitory activity. Furthermore, as a result of isolating and purifying the degranulation inhibitory active substance from the spinach hot water extract using the degranulation inhibitory activity as an index, among the active substances, the characteristic methylenedi of 6- and 7-positions of A ring A particularly strong antiallergic action was confirmed for flavonoid glucuronic acid glycosides having an oxy structure or aglycone thereof.

Therefore, the antiallergic substance according to an embodiment of the present invention is a compound represented by the following chemical formula 1 (wherein R ₁ is CH ₃ or a sugar residue, R ₂ is H or a sugar residue of a monosaccharide or a disaccharide). Is contained as an active ingredient.

Preferably, the compound is represented by Formula 2 below.

Preferably, the compound is represented by the following chemical formula 3.

  More preferably, the compound is a compound derived from spinach (Spinacia oleracea) leaves.

  ADVANTAGE OF THE INVENTION According to this invention, the antiallergic substance which uses a novel antiallergic action component as an active ingredient can be provided.

It is a figure which shows isolation | separation of the active ingredient from the spinach (Spinacia oleracea) leaf extract which concerns on a preparation example. It is a figure which shows the comparison of the degranulation inhibitory activity by the beta-hexosaminidase release | release from the RBL-2H3 cell by the antigen stimulation which concerns on a preparation example. It is a figure which shows the degranulation inhibitory activity by the beta-hexosaminidase release | release from the RBL-2H3 cell by the antigen stimulation which concerns on Example 1. FIG. It is a figure which shows the degranulation inhibitory activity by the beta-hexosaminidase release | release from the RBL-2H3 cell by the antigen stimulation which concerns on a comparative example. It is a figure which shows the degranulation inhibitory activity of SO-1 and SO-1 aglycone by the beta-hexosaminidase release | release from the RBL-2H3 cell by the antigen stimulation which concerns on Example 2. FIG. It is a figure which shows the cytotoxicity of SO-1 and SO-1 aglycone in the RBL-2H3 cell which concerns on Example 3. FIG. It is a figure which shows the antiallergic activity (Evans blue amount) of SO-1 aglycone in the mouse | mouth PCA reaction which concerns on Example 4. FIG.

  Hereinafter, embodiments of the present invention will be described in detail. In this specification, expressions such as “having”, “including”, or “containing” also mean “consisting of” or “consisting of”.

The antiallergic substance according to the embodiment of the present invention contains the compound represented by the above chemical formula 1 as an active ingredient. In the formula, R ₁ is CH ₃ or a sugar residue, and R ₂ is H or a sugar residue of a monosaccharide or a disaccharide. In the present invention, the “sugar residue” includes, for example, glucose, mannose, galactose, etc. for monosaccharides, and maltose, sucrose, lactose, etc. for disaccharides. Preferably, the compound is a flavonoid glucuronic acid glycoside represented by the above-described chemical formula 2 (also referred to as SO-1) or the above-described chemical formula 3 (also referred to as SO-1 aglycone) or an aglycone thereof.

  SO-1 and SO-1 aglycone are chemicals commonly used in the art, such as extraction, concentration, and purification using a solvent from leaves of spinacia oleracea (or other spinach plants) as a raw material. It can be easily obtained by performing separation and purification means (for example, various types of chromatography such as fractionation, column chromatography, or high performance liquid chromatography (HPLC)). A person skilled in the art will be able to prepare the compound by chemical synthesis using a commercially available flavonoid glycoside or an aglycon thereof with reference to the chemical formula in the present invention.

  When spinach (Spinacia oleracea) leaves are used as a raw material, examples of the extraction method include extraction with water or an organic solvent usually at 3 to 100 ° C. Examples of organic solvents used for extraction include petroleum ether, hydrocarbons such as cyclohexane, toluene or benzene, halogenated hydrocarbons such as carbon tetrachloride, dichloromethane or chloroform, ethers, esters such as ethyl acetate, acetone, etc. Ketones, alcohols such as methanol, ethanol, propanol, butanol, polyethylene glycol, propylene glycol or butylene glycol, or pyridine. The extraction solvent may be used alone or in combination of two or more.

  Preferably, a water-containing alcohol such as water-containing ethanol or water-containing methanol is used and extracted with stirring at room temperature to 100 ° C. for 2 to 24 hours. Furthermore, the obtained spinach (Spinacia oleracea) leaf extract may be used as it is, but it may be used after concentration, filtration, purification, lyophilization or the like if necessary.

  As described above, the antiallergic substance according to the present embodiment contains a compound represented by Chemical Formula 1 having an antiallergic effect as an active ingredient. In the present invention, the “substance” refers to any substance that can contain the compound represented by the chemical formula 1 as an active ingredient for antiallergic action, such as pharmaceuticals, foods (health foods or beverages) or cosmetics. Show. The antiallergic substance according to the present embodiment can be used, for example, as a pharmaceutical product for the purpose of treating atopic dermatitis, contact dermatitis, hay fever, and the like. For example, as food, for the purpose of alleviating or preventing symptoms such as atopic dermatitis, contact dermatitis or hay fever, foods for specified health use, dietary supplements or health foods should be fully utilized as functions of food. For example, it can be used by blending as a food additive that is expected to have an antiallergic effect. Moreover, as cosmetics, it can utilize for skin care products, foundations, makeup products, etc. for the purpose of alleviating or preventing symptoms such as atopic dermatitis or contact dermatitis.

  More specifically, for example, in the case of a pharmaceutical, it is used in combination with not only the compound represented by the chemical formula 1 but also one or more other pharmaceutically acceptable compositions, molding agents or carriers. It may be an active ingredient or an antiallergic substance. The form of the substance depends on the type and extent of the disease in the case of pharmaceuticals, but for example, suitable for oral, parenteral (topical use) or intranasal administration, tablets, dragees, sublingual tablets, gelatin capsules , Lozenges, suppositories, creams, ointments, or gels for skin. The effective amount of the compound contained in the pharmaceutical is not particularly limited, and an effective amount may be appropriately selected according to the age and weight of the patient, the type and severity of the disease, and the route of administration. The same applies to food or cosmetics.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using a preparation example, an Example, and a comparative example, these do not limit this invention.

(Preparation example)
In this preparation example, a preparation example relating to isolation and purification of spinach (Spinacia oleracea) leaf-derived degranulation inhibitor will be described.

  First, raw spinach (Spinacia oleracea) leaves were made into a paste using a mixer, and twice the amount of pure water heated to 80 ° C. was added to the spinach paste and stirred for 3 hours, followed by gauze filtration. Next, fractionation was performed on the spinach extract.

  FIG. 1 is a diagram showing the separation of active ingredients from a spinach (Spinacia oleracea) leaf extract according to a preparation example. According to the chart shown in FIG. 1, the spinach extract was fractionated by column chromatography (Diaion HP20 and Sephadex LH-20) and thin layer chromatography. Finally, Fr. 4 (408 mg) was obtained and further fractionated therefrom, and the active fraction Fr. 4-2, Fr. 4-3 and Fr. 4-4 was obtained.

  Fr. 4-2, Fr. 4-3 and Fr. In order to confirm the activity of 4-4, degranulation inhibitory activity was evaluated using a rat basophilic leukemia cell line (RBL-2H3 cells). RBL-2H3 cells were purchased from Human Science Cell Bank (JCRB) and used. The degranulation inhibitory activity was evaluated by measuring the amount of β-hexosaminidase released from the cells by antigen-antibody stimulation in the cells.

First, RBL-2H3 cells were seeded on a 96-well culture plate (NUNC) at 5 × 10 ⁴ cells / 90 μl / well. Subsequently, 10 μl of an anti-DNP-IgE antibody solution was added, followed by culturing at 37 ° C. for 24 hours to adhere the cells. The supernatant medium was removed by aspiration, and the wells were washed twice with Tyrode-HEPES buffer. Thereafter, Fr. 4-2, Fr. 4-3 and Fr. 80 μl of each sample solution of 4-4 (sample concentrations 1 μg / ml and 10 μg / ml) was added and incubated at 37 ° C. for 30 minutes. Thereafter, 20 μl of DNP-HSA was added and incubated at 37 ° C. for 1 hour. Finally, 20 μl of cell supernatant was collected in a 96-well assay plate (IWAKI). 100 μl of Lysis buffer was added to blank (naturally-released) cells, and after confirming cell lysis, 20 μl of the cell solution was recovered in a 96-well assay plate. 50 μl of the substrate solution was added to the cell supernatant and cell lysate, and incubated at 37 ° C. for 90 minutes. Brote buffer (0.2 M, pH 9.8) 100 μl was added, and after mixing, the absorbance at 415 nm was measured using a microplate reader.

From the measured absorbance, the inhibition rate against β-hexosaminidase release by sample addition, which is an evaluation of degranulation inhibitory activity, was calculated based on the following formula. S is the absorbance in the presence of the sample solution at the time of DNA-HSA stimulation, B is the absorbance at the blank (natural release), and C is the absorbance at the time of DNA-HSA stimulation.
Inhibition rate (%) = [1- (SB) / (CB)] × 100

  FIG. 2 is a graph showing a comparison of degranulation inhibitory activity due to β-hexosaminidase release from RBL-2H3 cells by antigen stimulation according to Preparation Example. As data, in each test of degranulation inhibitory activity, 3 wells were used for each sample solution and sample concentration, and the average value was measured three times, and the average ± standard error (SE) was calculated. Asked. As shown in FIG. 2, the Fr. 4-2, Fr. 4-3 and Fr. 4-4, Fr. A particularly strong degranulation inhibitory activity was confirmed in 4-4.

Next, Fr. 4-2, Fr. 4-3 and Fr. Each of 4-4 was purified using high performance liquid chromatography (HPLC). The purified compounds were designated as SO-1 (Fr. 4-4), SO-2 (Fr. 4-2), and SO-3 (Fr. 4-1), respectively. MS, NMR ( ¹ H-NMR (700 MHz, C ₅ D ₅ N), ¹³ C-NMR (175 MHz, C ₅ D ₅ N)), HMBC for purified SO-1, SO-2 and SO-3 And structural analysis by ROESY.

The structural analysis of SO-1 was as follows.

The structural analysis of SO-2 was as follows.

The structural analysis of SO-3 was as follows.

Further, the purified SO-1 was hydrolyzed with snail-derived β-glucuronidase to purify the SO-1 aglycone (see the following chemical formula 7). In addition, confirmation of the purification of SO-1 aglycone was performed by comparing the SO-1 before decomposition with the UV spectrum, as predicted from the retention time in high performance liquid chromatography (HPLC).

Example 1
This Example 1 demonstrates the Example which concerns on the degranulation inhibitory effect by the beta-hexosaminidase release | release from RBL-2H3 cell by antigen stimulation about SO-1, SO-2, and SO-3.

  Regarding SO-1, SO-2, and SO-3 prepared by the above-described method, the degranulation inhibitory activity due to the release of β-hexosaminidase from RBL-2H3 cells using DNA-HSA stimulation was evaluated. For the evaluation method (absorbance measurement and calculation procedure), see Fr. 4-2, Fr. 4-3 and Fr. This is the same as the evaluation method described above for each of 4-4.

FIG. 3 is a graph showing the degranulation inhibitory activity of β-hexosaminidase release from RBL-2H3 cells by antigen stimulation according to Example 1. As shown in FIG. 3, SO-1 showed a concentration-dependent strong degranulation inhibitory activity, and IC ₅₀ = 1.76 μM. SO-2 and SO-3 showed weak degranulation inhibitory activity inferior to SO-1.

(Comparative example)
In this comparative example, ketotifen fumarate, which is a commercially available antiallergic agent, was evaluated for the degranulation inhibitory activity of β-hexosaminidase release from RBL-2H3 cells by antigen stimulation using the same evaluation method. .

  FIG. 4 is a diagram showing degranulation inhibitory activity due to β-hexosaminidase release from RBL-2H3 cells by antigen stimulation according to a comparative example. When FIG. 3 and FIG. 4 were compared, it was confirmed that the degranulation inhibitory activity of SO-1 was significantly stronger than the degranulation inhibitory activity of ketotifen fumarate (the inhibition rate was high even at low concentrations).

(Example 2)
In this Example 2, an example relating to the degranulation inhibitory activity of β-hexosaminidase release from RBL-2H3 cells by antigen stimulation of the prepared SO-1 aglycone will be described. About the evaluation method of the degranulation inhibitory effect, it is the same as that of the above-mentioned method performed with respect to each of SO-1, SO-2, and SO-3.

FIG. 5 is a diagram showing the degranulation inhibitory activity of SO-1 and SO-1 aglycone in the release of β-hexosaminidase from RBL-2H3 cells by antigen stimulation according to Example 2. As shown in FIG. 5, SO-1 aglycone also showed strong degranulation inhibitory activity. As described above, SO-1 had an IC ₅₀ = 1.76 μM, and SO-1 aglycone had an IC ₅₀ = 1.47 μM. It was.

As shown in the results of Example 1 and Example 2 described above, SO-1 and SO-1 aglycone exhibit particularly strong degranulation inhibitory activity. The 7-position methylenedioxy structure is thought to be strongly related to the activity. Therefore, not only the compound having the structure of SO-1 and SO-1 aglycone itself, but, for example, when CH _{3 of} C ring is a sugar residue as shown in Chemical Formula 1 above, H or glucuronic acid of B ring is It is suggested that even if it is a sugar residue of other monosaccharide or disaccharide, strong inhibitory activity is exhibited.

(Example 3)
In Example 3, an example relating to confirmation of cytotoxicity for SO-1 and SO-1 aglycone will be described.

  Specifically, for SO-1 and SO-1 aglycone, cytotoxicity (cell viability compared to control) in RBL-2H3 cells in a concentration range showing degranulation inhibitory activity was confirmed by the WST-1 method. did. FIG. 6 is a graph showing the cytotoxicity of SO-1 and SO-1 aglycone in RBL-2H3 cells according to Example 3. As shown in FIG. 6, SO-1 and SO-1 aglycone showed almost no cytotoxicity in the concentration range showing degranulation inhibitory activity. That is, when SO-1 and SO-1 aglycone are used as active ingredients of antiallergic substances, it is suggested that there is little possibility of giving side effects to other cells.

Example 4
In Example 4, an example relating to measurement of the antiallergic activity of SO-1 aglycone in passive skin anaphylactic reaction (PCA reaction) of mice will be described.

  Samples used for measurement of antiallergic activity were prepared SO-1 aglycone and a commercially available antiallergic drug oxatomide (Wako Pure Chemical Industries, Ltd.). The oxatomide used was suspended in 5% gum arabic so that the oral dose was 100 μmol / kg and homogenized using a mixer so as to be uniform. As for SO-1 aglycone, a suspension obtained by suspending in 5% gum arabic so as to be 100 μmol / kg and making it uniform was further diluted with 5% gum arabic to 25 μmol / kg.

  As the mice for effect evaluation, 11-week-old Jcl: ICR male mice (CLEA Japan) were used for each group (control group, SO-1 aglycone group and oxatomide group). In a breeding room with constant temperature and humidity, a solid feed (CE-2, CLEA Japan) was given at 5 g / mouse / day, and tap water was freely taken for breeding. After about 1 week of preliminary breeding, it was subjected to an experiment. The handling of experimental animals was in accordance with the ethics regulations for research on humans and animals at the prefectural Hiroshima University (enforced July 8, 2005).

  Under anesthesia, 20 μl of 0.86% NaCl solution (left ear) or anti-DNP-IgE antibody (right ear) was subcutaneously administered to the auricle of each group of mice. 24 hours later, under anesthesia, the control group was orally administered with 0.86% NaCl at 0.1 ml / 10 g, and the SO-1 aglycone group and oxatomide group at 25 μmol / kg and 100 μmol / kg, respectively. It was. Two hours later, DNP-HSA solution was administered by 250 μl tail vein. After 30 minutes, the animals were sacrificed by cervical dislocation.

Next, the auricles after slaughter were cut and dissolved overnight using 500 μl of 1N KOH aqueous solution. Thereafter, 3.25 ml of an acetone-0.6N H ₃ PO ₄ mixed solution was added, stirred and then centrifuged (700 × g, 20 min), and the absorbance of the supernatant was measured at 620 nm.

  In order to determine the Evans blue amount (related to degranulation inhibitory activity) from the measured absorbance, a calibration curve was prepared in advance. As mice for the calibration curve, four 12-week-old Jcl: ICR male mice (CLEA Japan) were used. The calibration curve was subcutaneously administered to the auricles of the four ICR male mice at 10, 20, 40, or 60 μg / site, immediately sacrificed by cervical dislocation, and the above-mentioned group of mice for sample evaluation (sample administration) The absorbance at 620 nm was measured in the same procedure as in the mouse group, and a calibration curve was prepared.

  Based on the calibration curve thus prepared, the Evans blue amount in each mouse group for evaluating the action was determined from the measured absorbance. FIG. 7 is a graph showing the antiallergic activity (Evans blue amount) of SO-1 aglycone in the mouse PCA reaction according to Example 4. As shown in FIG. 7, as a result of the mouse PCA reaction, the amount of Evans blue in the SO-1 aglycone group was a blank value, and a strong inhibitory action was confirmed. Furthermore, compared with the oxatomide group, the dose of the oxatomide group is 100 μmol / kg, but the dose of the SO-1 aglycone group is 25 μmol / kg, which is four times different. It was confirmed that the action is much stronger than that of the allergic agent oxatomide.

Further, when the PCA reaction inhibition rate of the mouse group for each action evaluation was calculated from the obtained Evans blue amount using the following formula, SO-1 aglycone was 100% and oxatomide was 64.3%. It was.
Inhibition rate (%) = (pigment release amount by stimulation (without sample) −pigment release amount by stimulation (with sample)) / pigment release amount by stimulation (without sample) × 100

  In addition, the result by the statistical process in the present Example 4 was shown by the average value +/- standard deviation (SD). Student t-test was used for the significant difference test. A P value of less than 0.05% was determined to be significant.

  The present invention is not limited to the description of the embodiments, preparation examples, comparative examples and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

  The contents of published patent gazettes and the like specified in this specification are incorporated by reference in their entirety.

  As a result of intensive studies by the present inventors, it was confirmed that spinach juice showed strong degranulation inhibitory activity. Furthermore, as a result of isolating and purifying the degranulation inhibitory active substance from the spinach hot water extract using the degranulation inhibitory activity as an index, among the active substances, the characteristic methylenedi of 6- and 7-positions of A ring A particularly strong antiallergic action was confirmed for flavonoid glucuronic acid glycosides having an oxy structure or aglycone thereof. Therefore, according to the present invention, there is provided an antiallergic substance containing a flavonoid compound having a characteristic methylenedioxy structure at positions 6 and 7 of the A ring, which is a novel antiallergic agent, as an active ingredient. Can do.

Claims (4)

An antiallergic agent comprising a compound represented by the following chemical formula 1 (wherein R ₁ is CH ₃ or a sugar residue, R ₂ is H or a sugar residue of a monosaccharide or a disaccharide) as an active ingredient: Sex substances.
The antiallergic substance according to claim 1, wherein the compound is represented by the following chemical formula 2.
The antiallergic substance according to claim 1, wherein the compound is represented by the following chemical formula 3.
  4. The antiallergic substance according to claim 2, wherein the compound is a compound derived from spinach (Spinacia oleracea) leaves.