JP2007070251A - Compound exhibiting antioxidant activity and antiallergic activity, and manufacturing method of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To search an antioxidant substance and an antiallergic substance contained in Thelephora vialis, to determine the chemical structure thereof and to develop a novel application of the compound. <P>SOLUTION: A dibenzofuran compound represented by formula (1) (wherein R<SB>1</SB>and R<SB>2</SB>are simultaneously or independently a 2-10C acyl group or a hydrogen atom) is provided. The dibenzofuran compound represented by formula (1) exhibits antioxidant activities and antiallergic activities. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a compound having antioxidant activity and antiallergic activity, and a method for producing the compound, and in particular, a compound having antioxidant activity and antiallergic activity derived from Thelephora vialis belonging to the family Basidiomycetes The present invention relates to a method for producing the compound.

  Celefora Vaialis (Chinese name “gan-ba-jun”, Japanese name “Tsuboibotake”) belonging to the family Basidiomycetes is one of the most popular edible mushrooms since ancient times. One. Since the fragrance enhances appetite, research on the volatile components of the celephora moth has been vigorously conducted in relation to the fragrance.

On the other hand, research on non-volatile components has not progressed as much as volatile components. However, in recent years, attention has been focused on the relationship with bioregulatory functions. Examples of non-volatile components of the genus Cerephora that have been reported so far include, for example, phellanic acid (ferroellaleucus) as an antibiotic and 5-O-methyl-xylerythrin, peniophorin as a pigment component. peniophorin and peniosanguin (Peniophora sanguinea). Further, as antiallergic substances, 16 or more kinds of amino acids, D-glucose and D-mannose extracted from the mycelium or fruiting body of edible mushrooms belonging to the genus Capriformae, D-glucose and D-mannose are antiallergic with very few side effects. It has been reported that it has an action (Patent Document 1).
JP-A-6-183992

  In recent years, rapid oxidation by free radicals and active oxygen produced in an environment surrounding oxygen has been a problem because it adversely affects foods and living bodies. Therefore, compounds having antioxidative activity such as radical scavenging action are expected to be applied to prevention of food degradation, prevention and treatment of oxidative damage to living bodies. Therefore, it can be said that characteristic antioxidant substances having various chemical structures are widely demanded as bioregulatory functional substances.

  Further, in the prior art, it was known that mushrooms of the genus Cerephora belong to an antiallergic substance, but the main component compound and its structure were not specified.

  Therefore, the present invention aims to search for antioxidants and antiallergic substances contained in Celeforia variaris and determine their chemical structures, and to develop new uses of the compounds and methods for producing the compounds. is there.

  As a result of intensive studies by the present inventors, the present inventors have found the presence of a plurality of antioxidants such as a green substance exhibiting a strong antioxidant activity in the dried fruiting bodies of Celeforia vialis. This invention is made | formed based on this knowledge, and provides the dibenzofuran compound shown by following formula (1). The dibenzofuran compound represented by the following formula (1) has an antioxidant action and an antiallergic action.

（Ｒ₁及びＲ₂は同時に又は各々独立して炭素数２〜１０のアシル基または水素原子を示す。）

(R ₁ and R ₂ each simultaneously or independently represents an acyl group having 2 to 10 carbon atoms or a hydrogen atom.)

  The present invention also includes a step of extracting Celephora vials with acetone to obtain an acetone extract, a step of concentrating the acetone extract to obtain a concentrate of the acetone extract, and a step of adding the concentrate to pH 2. Adjusting to 0-4.0, performing solvent extraction, separating the aqueous layer and the organic layer, concentrating the organic layer to obtain a middle / acidic fraction, and separating the middle / acidic fraction And a step of separating and purifying the obtained fraction, and a method for producing a dibenzofuran compound.

  The dibenzofuran compound represented by the above formula (1) has a radical scavenging action, β-hexosaminidase release inhibition, and TNF-α production inhibition action. Therefore, it can be used as a radical scavenger, a β-hexosaminidase release inhibitor, and a TNF-α production inhibitor.

  In order to use a dibenzofuran compound and a pharmacologically acceptable salt thereof for therapeutic purposes, the compound and its non-toxic salt are used as active ingredients and are administered orally or parenterally. The dose varies depending on symptoms, age, sex, body weight, dosage form, etc., but for example, when administered orally to an adult, the daily dose is usually 0.1-1000 mg.

  There are no restrictions on the dosage form for formulating dibenzofuran compounds and their pharmacologically acceptable salts, and solids such as tablets, pills, capsules, powders, granules, solutions, suspensions, emulsions, etc. These liquid preparations can be used orally, and intravenous, intramuscular and subcutaneous injections, suppositories, patches and the like can be used parenterally.

  When used as a solid agent, excipients such as starch, lactose, glucose, calcium phosphate, magnesium stearate, carboxymethyl cellulose can be used, and if necessary, lubricants, disintegrants, coating agents, coloring agents, etc. Can be used. In the case of injections and liquid preparations, stabilizers, solution aids, suspending agents, emulsifiers, buffers, preservatives and the like can be contained.

[Production example]
Next, production examples of the dibenzofuran compound represented by the general formula (1) will be described. 420.0 g of dried fruit bodies of Thelephora vialis were extracted with 8.0 L of 80% acetone to obtain an acetone extract of Celefora vials. It was concentrated under vacuum conditions to obtain 1.6 L of an acetone extract concentrate. The concentrated solution was adjusted to pH 3.0 with 1N hydrochloric acid, extracted with an equal amount of ethyl acetate, and separated into an aqueous layer and an organic layer.

Among these, the organic layer was concentrated under vacuum conditions to obtain 36.5 g of a middle / acidic fraction. Then, 10.0 g of the medium and acidic fractions were separated using Sephadex LH-20 column chromatography (CHCl ₃ / CH ₃ OH = 6: 4) to obtain two fractions A and B.

The A fraction is purified by silica gel chromatography (CHCl ₃ / CH ₃ OH = 49: 1), followed by medium pressure liquid chromatography (CH ₃ CN: 0.15% KH ₂ PO ₄ (pH 3.5) = 6: 4). Then, four substances of TVEA-a (160.3 mg), TVEA-b (79.8 mg), TVEA-c (87.9 mg) and TVEA-d (5.8 mg) were isolated.

  Of these, the compound TVEA-d (5.8 mg) is the compound of the present invention. The structural formula (2) of TVEA-d is shown below.

The molecular formula of TVEA-d is given as C ₃₄ H ₂₄ O _9, and 20 H and 34 min from ¹ H-NMR (600 MHz, CD ₃ OD) and ¹³ C-NMR spectrum (150 MHz, CD ₃ OD). The C signal was given. Table 1 shows the physicochemical data, Table 2 shows the ¹ H-NMR data, and Table 3 shows the ¹³ C-NMR spectrum.

Further, ¹ H- ¹ H COZY, HMQC, from the analysis of two-dimensional NMR spectrum, such as HMBC spectrum, the chemical structure of TVEA-d is 3- (4-hydroxyphenyl) dibenzofuran -1,2,4,7,8- It was determined as pentanol 1,2-O-diphenyl acetate.

[Comparative manufacturing example]
Of the two fractions A and B obtained by separating 10.0 g of the medium / acidic fraction using Sephadex LH-20 column chromatography (CHCl ₃ / CH ₃ OH = 6: 4), The structural formula of TVEA-c of the A fraction is shown in the following formula (3). The chemical structure of TVEA-c is, ¹ H- ¹ H COZY, HMQC, from the analysis of two-dimensional NMR spectrum, such as HMBC spectrum, was identified as Ganbajunin B (ganbajunin B).

On the other hand, 10.0 g of the medium and acidic fractions were separated using Sephadex LH-20 column chromatography (CHCl ₃ / CH ₃ OH = 6: 4), and two fractions A and B were obtained. Among these, the B fraction was purified by preparative high performance liquid chromatography (CH ₃ CN: 0.15% KH ₂ PO ₄ (pH 3.5) = 8: 2), and TVEB-b (1.0 mg), TVEB- Four active substances were isolated: c (3.2 mg), TVEB-d (8.2 mg) and TVEB-e (8.9 mg).

Among these, the structural formula of TVEB-e is shown in the following formula (4). The chemical structure of TVEB-e is, ¹ H- ¹ H COZY, HMQC, from the analysis of two-dimensional NMR spectrum, such as HMBC spectrum, was identified as cyclo leuco melon (cycloleucomelone).

[Test Example 1] Measurement of Antioxidant Activity Using TVEA-d (Example 1) as a sample, the antioxidant activity was measured as follows. Mixing 0.5 mL of 0.5 mM 1,1-diphenyl-2-picrylhydrazyl (1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution and 1.0 mL of 0.1 M sodium acetate buffer (pH 5.5) To the solution, 2.0 mL of a sample ethanol solution whose concentration was adjusted stepwise was added, stirred, and allowed to stand for 30 minutes under light shielding.The absorbance at 517 nm of the obtained reaction solution was measured with a spectrophotometer. The DPPH radical scavenging activity value of each sample was obtained by the following formula: 2.0 mL of ethanol was used as a control, and the sample concentration showing 50% absorbance of the control was 50% capture concentration (EC ₅₀ value). The results are shown in Table 4.

DPPH radical scavenging activity (%) = [(BA) / B] x 100
(A: sample absorbance, B: control absorbance)

  In addition, as comparative examples, TVEA-c (Comparative Example 1) and TVEB-e (Comparative Example 2) separated from the dried fruiting bodies of Celefora Vialis, and Bayrinin A (Comparative Example 3), which is the main product of this mushroom, Antioxidant activity was measured in the same manner as described above using BHT (butylhydroxytoluene) (Comparative Example 4) used as a synthetic antioxidant.

[Test Example 2] Measurement of anti-allergic activity Anti-allergic activity was measured in the following manner using TVEA-d (Example 1) as a sample. That is, a β-hexosaminidase release inhibition test using RBL-2H3 cells and a TNF-α production inhibition test, which is an inflammatory cytokine, were performed as anti-allergy test models.

  In addition, as comparative examples, TVEA-c (Comparative Example 1) and TVEB-e (Comparative Example 2) isolated from the dried fruiting bodies of Cerefora Vialis and FK-506 (Comparative Example 5) used as an immunosuppressant And anti-allergic activity was measured in the following manner using staurosporine (Comparative Example 6).

(1) β-hexosaminidase release inhibition test
RBL-2H3 cells were sensitized with anti-DNP-IgE antibody, seeded in 24-well plates at 2.0 × 10 ⁵ cells / mL, and cultured at 37 ° C. for 24 hours. After washing each well, the sample was added and kept at 37 ° C. for 15 minutes. Thereafter, DNP-BSA was added and reacted at 37 ° C. for 3 hours. The culture supernatant of each well was collected, and the inhibition rate of degranulation was determined from the released amount of β-hexosaminidase released from the cells during degranulation. The results are shown in Table 5.

(2) TNF-α production inhibition test Using the culture supernatant obtained in (1), TNF-α released in the culture supernatant was subjected to ELISA using Rat TNF-α Immunoassay kit (Biosource). It was measured by (enzyme immunoassay). The results are shown in Table 5.

(3) Anti-allergic activity suppression pathway identification test Using TVEA-d (Example 1) as a sample, anti-allergic activity suppression pathway was identified as follows.

RBL-2H3 cells adjusted to 2.0 × 10 ⁵ cells / mL were seeded in 24-well plates and cultured at 37 ° C. for 24 hours. After washing each well, the sample was added and kept at 37 ° C. for 15 minutes. After that, TPA (12-o-tetradecanoylphorbol-13-acetate phorbol ester), an activator of protein kinase C, and DTBHQ (2,5-di-tert-butyl-1,4-hydroquinone), a kind of calcium ionophore ) Was added and reacted at 37 ° C. for 3 hours. The culture supernatant of each well was collected, and the inhibition rate of degranulation was determined from the released amount of β-hexosaminidase released from the cell during degranulation, and compared with the result of (1). The results are shown in Table 6.

  In addition, as a comparative example, TVEA-c (Comparative Example 1) and TVEB-e (Comparative Example 2) separated from the dried fruiting bodies of Cerefora Vialis were used, and the same measurement as described above was performed.

Claims (5)

The dibenzofuran compound shown by following formula (1).

(R ₁ and R ₂ each simultaneously or independently represents an acyl group having 2 to 10 carbon atoms or a hydrogen atom.)   A radical scavenger comprising the dibenzofuran compound according to claim 1 or a pharmacologically acceptable salt as an active ingredient.   The beta-hexosaminidase release inhibitor which uses the dibenzofuran compound or pharmacologically acceptable salt of Claim 1 as an active ingredient.   A TNF-α production inhibitor comprising the dibenzofuran compound according to claim 1 or a pharmacologically acceptable salt as an active ingredient. The process of extracting Celefora Vialis Thelephora vialis with acetone to obtain an acetone extract,
Concentrating the acetone extract to obtain a concentrate of the acetone extract;
Adjusting the concentrate to pH 2.0 to 4.0, performing solvent extraction, and separating the aqueous layer and the organic layer;
Concentrating the organic layer to obtain a medium / acidic fraction;
Separating the intermediate / acidic fraction and separating and purifying the obtained fraction;
A process for producing a dibenzofuran compound.