JPH09110857A - New isoflavon derivative and histidine decarboxylase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound useful as a medicine, a functional food, etc., having inhibitory activity against histidine decarboxylase. SOLUTION: This isoflavon derivative is shown by the formula (R is H or OH). The compound, for example, is obtained by culturing a fungus such as a strain belonging to the genus Aspergillus (e.g. Aspergillus oryzae IFO4206) in a medium and collecting the compound from the culture product. The objective histidine decarboxylase inhibitor participating in biosynthesis of histamine to cause inflammatory diseases such as tumor and allergic diseases comprises the compound of the formula as an active ingredient. A dose of the compound of the formula is 0.01-20g as an active ingredient per adult daily and can be properly mixed with another medicine, etc., and used. When the compound of the formula is used as a functional food, the compound of the formula added is >=0.01g, preferably 0.5-10g per kg of a food and is applicable to seasonings, marine product foods, processed foods of animal meat, liquors, beverages, cakes, cans, foods of daily dishes, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel isoflavone derivative having histidine decarboxylase inhibitory activity and a histidine decarboxylase inhibitor containing the isoflavone derivative as an active ingredient.

[0002]

2. Description of the Related Art Allergic diseases such as gastric ulcer, duodenal ulcer and the like, asthmatic bronchitis, atopic dermatitis and the like are inflammatory diseases caused by excessive production of histamine in a living body. Histamine is produced by the decarboxylation of histidine. The enzyme that catalyzes this reaction is histidine decarboxylase (hereinafter referred to as “HDC”).

[0003] As therapeutic agents for the above-mentioned inflammatory diseases, those that inhibit histamine biosynthesis, that is, HDC inhibitors are known. Examples of such a drug include (+)-catechin, a histidine derivative, cyanidanol, and tritoqualine.

[0004]

DISCLOSURE OF THE INVENTION The present invention is to provide an excellent HDC inhibitor containing a novel substance as an active ingredient.

[0005]

As a result of intensive studies to solve the above problems, the present inventors have found that the novel isoflavone derivative represented by the following formula has excellent HDC inhibitory activity, The present invention has been completed. That is, the present invention provides the following general formula:

[0006]

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(Wherein R is H or OH) is a novel isoflavone derivative. The present invention is also a histidine decarboxylase inhibitor containing the above isoflavone derivative as an active ingredient. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The starting material for obtaining the isoflavone derivative of the present invention (hereinafter referred to as "the substance of the present invention") may be any as long as it contains the substance of the present invention or a precursor thereof. The substance of the present invention can be obtained, for example, by culturing a microorganism capable of producing the substance of the present invention in a medium and collecting the substance of the present invention from the culture.

The microorganism used in the present invention may be any strain as long as it has the ability to produce the substance of the present invention. For example, a microorganism belonging to the genus Aspergillus, specifically Aspergillus oryzae.
(IFO 4206) and the like. Further, the microorganism used in the present invention may be not only the above-mentioned strain but also a mutant strain obtained artificially or naturally. As a method for artificially creating a mutant strain of the above strain, for example,
A method of mutating the original strain using a radioisotope, ultraviolet rays, nitrosoguanidine, etc. can be used.

The medium used in the present invention is an ordinary medium for culturing microorganisms, that is, a substance containing a carbon source, a nitrogen source, an inorganic substance and other nutrients in an appropriate ratio and capable of serving as a precursor of the substance of the present invention. Either a synthetic medium or a natural medium can be used as long as it contains it. Examples of such a medium include a mixture of steamed soybeans and wheat crushed after roasting.

The culture of the microorganism is carried out at 25 to 35 ° C., preferably around 30 ° C. for 30 to 80 hours, preferably 40 to 45.
Just go on time. By the above operation, the substance of the present invention is produced and accumulated in the culture. After the completion of the culture, in order to purify the substance of the present invention from the culture, usual methods including solvent extraction and distillation can be used. The substance of the present invention is, for example,
It can be purified using the following method.

First, after adding distilled water to the culture, 35-35
The substance of the present invention is extracted from the culture by standing at 45 ° C. overnight. Then, the culture is subjected to gauze filtration and centrifugation,
The centrifugation supernatant obtained is filtered. An organic solvent such as ethyl acetate, 1-propanol, etc. is added to the above-mentioned centrifugal supernatant, and the mixture is shaken and then separated into an upper layer and a lower layer. For this upper layer, the same organic solvent treatment as above is performed twice,
Get the lower layer. Combine all the obtained lower layers, and add pH with hydrochloric acid.
After adjusting to about 2.0, add an organic solvent and shake,
Divide into upper and lower layers. The upper layer is concentrated using a rotary evaporator (manufactured by Tokyo Rika Co., Ltd.) to obtain a concentrated solution.

The concentrate is subjected to high performance liquid chromatography using a reversed phase column (hereinafter referred to as “reverse phase HPLC”). Wakosil-II5 was used as a reversed phase column.
C18HG (manufactured by Wako Pure Chemical Industries), TSKgel ODS-
120T (manufactured by Tosoh Corporation) and the like. The adsorption fraction is
It can be eluted with a gradient of acetonitrile containing trifluoroacetic acid. After fractionating the eluate, the HDC inhibitory activity of each fraction is measured according to the measurement method described below.
The fraction having HDC inhibitory activity is concentrated by a rotary evaporator to obtain a concentrate. After the concentrated solution is dissolved in distilled water, ethanol, etc., gel filtration chromatography is performed to collect fractions having an HDC inhibitory activity. Examples of the gel filtration support include Biogel P-2 resin (manufactured by Bio-Rad), Toyopearl CW-35 (manufactured by Tosoh), and the like.

By the above operation, the highly purified substance of the present invention can be obtained. The substance of the present invention is excellent in HDC.
It shows inhibitory activity and high safety, and is useful as a drug or functional food. When the substance of the present invention is used as a therapeutic agent for inflammatory diseases, it can be administered alone, but usually,
Tablets, powders, wettable powders, etc. using excipients, lubricants, diluents, pH adjusters, preservatives, sweeteners, aromatics, emulsified powders, etc.
It can be formulated into emulsions, capsules, injections, drops, suppositories, etc., and administered orally or parenterally. Examples of the excipient include water, gelatin, starch, crystalline cellulose, magnesium stearate, lactose, acacia, vegetable oil and the like.

The dose of the substance of the present invention varies depending on the method of administration and the degree of symptoms, the age and weight of the patient, etc., but is usually 0.01 to 20 g, preferably 0.5 to 10 g as an active ingredient per day for an adult. It is selected in the range of. Further, the substance of the present invention can be used by appropriately mixing with other pharmaceuticals and the like. When the substance of the present invention is used as a functional food, it can be used as it is or added to other foods. When the substance of the present invention is added to other foods, examples thereof include a method of mixing into the food and a method of applying it to the surface of the food.
In that case, 0.01 g or more, preferably about 0.5 to 10 g, may be added per 1 kg of food.

Examples of applicable foods include seasonings such as soy sauce, miso, and soup, fish paste products such as kamaboko and chikuwa, processed meat products such as ham and sausage, alcoholic beverages such as sake, western sake, fruit wine, soft drinks, and fruits. Examples include drinks such as drinks, and confectionery such as cakes, puddings, and buns. Furthermore, the substance of the present invention can be used by adding it to fruits, vegetables, canned foods, prepared foods and the like.

The substance of the present invention can be used by appropriately mixing it with other food additives and the like. HDC of the substance of the present invention
The method for measuring the inhibitory activity is shown below. (1) Reagent Enzyme solution HDC was applied to mouse mast tumor cell P-815 (Bioch
im. Biophys. Acta. , 1133, 172
-178 (1992)), the method of Ohmori et al. (J. Biochem., 107, 834-839).
(1990)).

Substrate solution Phosphate buffer 100 mM Histidine 5 μg / ml Pyridoxal phosphate 5 μg / ml Enzyme solution 20 μl / ml Specimen solution Fractions obtained in each purification step of the substance of the present invention

(2) Reaction conditions 10 μl of a sample solution is added to 0.1 ml of the substrate solution.
Incubate at 7 ° C for 12 hours.

(3) Quantification of histamine as a reaction product The histamine produced is quantified using a HPLC as a dimethylaminoazobenzene isothiocyanate derivative. HPLC conditions are as follows. Column: Cosmosil 5SL (manufactured by Nacalai Tesque, Inc.) Eluent: chloroform / dimethylformamide / water / acetic acid = 210/80/4/1 (vol./vol.) Detection wavelength: 435 nm

(4) Calculation of HDC inhibitory activity The method of calculating the HDC activity inhibitory rate is as follows. Inhibition rate (%) = (1-A / B) × 100 A: Histamine production amount when the sample solution is added to the substrate solution and reacted B: 10 μl of methanol is added instead of the sample solution, and the same Amount of histamine produced when reacted under the conditions

[0022]

EXAMPLES The present invention will be described more specifically by showing examples and test examples below. [Example] Acquisition of substance of the present invention (1) Cultivation of inoculum Aspergillus oryzae (IFO420
6) was subjected to slope culture using an MA medium (2.0% malt extract, 2.0% glucose, 0.1% peptone, 2.0% agar). One loopful of the culture was inoculated into 5 g of bran having a water content of 80% w / v, and cultured at 30 ° C. for 4 days, and the resulting culture was used as a seed.

(2) Main Culture The medium used for the main culture was prepared by the following method. That is, 300 g of whole soybeans were immersed in water for 12 hours and then steamed for 4 hours. To the obtained steamed soybeans, 335 g of wheat that had been roasted and ground was added. 2 g of a seed bacterium was added to the above medium, mixed, and cultured at 30 ° C. for one week. After 800 ml of distilled water was added to the obtained culture, the mixture was allowed to stand at 42 ° C. overnight, and then the culture was subjected to gauze filtration and centrifugation (7000).
rpm). The obtained centrifugal supernatant was filtered through a 0.8 μm filter (manufactured by Millipore), and then filtered at 0.45 μm.
With a bottle top filter (Falcon).

(3) Purification of the substance of the present invention Ethyl acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 500 ml of the above filtrate.
After adding 250 ml and shaking for 3 minutes, the mixture was separated into an upper layer and a lower layer. This upper layer was subjected to the same ethyl acetate treatment twice as described above to obtain a lower layer. The obtained lower layers were combined, adjusted to pH 2 with hydrochloric acid, added with 250 ml of ethyl acetate, shaken for 3 minutes, and separated into upper and lower layers. 250 ml of ethyl acetate (manufactured by Wako Pure Chemical Industries) was added to the obtained upper layer,
Shake for minutes and separate into upper and lower layers. The obtained upper layer is concentrated by a rotary evaporator (manufactured by Tokyo Rika Co., Ltd.),
10 mL of a concentrated solution was obtained.

The concentrated solution was added to a precolumn (column: Wak).
reverse-phase HPLC (column: Wakos) equipped with osil-II5C18HG 10 × 50 mm manufactured by Wako Pure Chemical Industries, Ltd.
il-II5C18HG 10 × 300 mm manufactured by Wako Pure Chemical Industries, Ltd.). The adsorbed fraction was eluted with a concentration gradient of acetonitrile containing 0.05% trifluoroacetic acid (FIG. 1). The eluate was fractionated by 15 mL, and fractions having an HDC inhibitory action were collected. Two fractions having a high HDC activity inhibitory effect, peak A and peak B, were further purified by the following method. That is, the peak A fraction was concentrated by a rotary evaporator to obtain a concentrated solution of 3 m.
1 was obtained. The concentrated solution was applied to a column (20 × 380 mm) packed with a biogel P-2 resin (manufactured by Bio-Rad) equilibrated with 1 M acetic acid, and subjected to gel filtration. The eluate was fractionated by 15 mL, and the sections having an HDC inhibitory action were collected. After concentrating this with a rotary evaporator, about 17 mg of a purified preparation of peak A was obtained. The same operation is performed for the peak B, and about 10 parts of the purified preparation of the peak B is prepared.
mg was obtained.

(4) Structural analysis The physical properties of peaks A and B are shown below. Peak A Properties; White powder Melting point (° C); 212-218 ° C (decomposition) Molecular weight and molecular formula; 386, C ₁₉ H ₁₄ O ₉ mass spectrum; m / z 387 (M + H) [FABmass, KRATOS CONCEPT-IIH (Shimadzu Kraitos IR spectrum; KBr tablet method, JASCO FT / IR-7300
(Manufactured by JASCO Corporation) (cm ^-1 ): 3420, 1630, 1540, 1520, 1450, 1250, 120
0, 1110, 970, 890, 840, 780

¹ H-NMR spectrum; 200 MH in DMSO-d ₆
z, JOEL JNM-FX200 (made by JEOL Ltd.) (ppm): 4.48 (1H, d), 5.13 (1H, d), 6.8 (2H, d), 7.09
(1H, dd), 7.13 (1H, dd), 7.39 (2H, d), 8.02 (1H, d), 8.
35 (1H, s), 9.53 (1H, br) ¹³ C-NMR spectrum; DMSO-d ₆ , 50MHz, JOEL JNM
-FX200 (made by JEOL Ltd.) δ (ppm): 71.2, 81.0, 102.2, 115.0, 115.3, 118.0,
122.4, 123.7, 127.0, 130.0, 153.1, 157.1, 162.4, 1
69.0,171.9,174.8 UV spectrum; absorption in aqueous solution at 248 nm and 302 nm

Peak B Properties; pale yellow powder Melting point (° C); 139-144 ° C (decomposition) Molecular weight and molecular formula; 402, C ₁₉ H ₁₄ O ₁₀ mass spectrum; m / z 403 (M + H) [FABmass, KRATOS CONCEPT- IIH (Shimadzu Kratos)] IR spectrum; KBr tablet method, JASCO FT / IR-7300
(Manufactured by JASCO Corporation) (cm ^-1 ): 3450, 1650, 1620, 1580, 1520, 1310, 125
0, 1180, 1160, 1070, 840

¹ H-NMR spectrum; 200 MH in DMSO-d ₆
z, JOEL JNM-FX200 (manufactured by JEOL Ltd.) δ (ppm): 4.51 (1H, d), 5.15 (1H, d), 6.37 (1H, d), 6.62
(1H, d), 6.82 (2H, d), 7.39 (2H, d), 8.38 (1H, s), 9.52 (1
H, br), 12.90 (1H, s) ¹³ C-NMR spectrum; DMSO-d ₆ in 50MHz, JOEL JNM
-FX200 (made by JEOL Ltd.) δ (ppm): 70.9, 79.3, 93.4, 98.8, 105.7, 115.0, 12
0.9, 122.5, 130.0, 154.3, 157.2, 157.4, 161.6, 16
3.5, 168.4, 171.5, 180.3 UV spectrum; absorption in water at 260 nm From the results of the above instrumental analysis, peak A is a compound represented by the following general formula (where R = H), and peak B is It was concluded that the compound was a compound represented by the general formula (where R = OH).

[0030]

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Test Example 1 HDC Inhibitory Activity Test The purified preparations of peak A and peak B were examined for HDC inhibitory activity by the method described above. Table 1 shows the results. Incidentally, (+)-catechin [Lorenz, W., Kusche, J., Barth, H., and Mathias, CH (19), which is a known HDC inhibitor as a control, is used.
73) in Histamine (Maslinski, CZed.) Pp.265-269, Dow
dew, Hutchinson and Ross, Stroudsburg, Pennsylvania)
Was used. The “50% inhibition value” in the table means the amount of the substance of the present invention (μg / m 2) required to bring the activity of HDC to 50%.
l).

[0032]

[Table 1]

From the results in Table 1, it is clear that the compounds of peaks A and B both have HDC inhibitory activity, and in particular, the compound of peak B exhibits HDC inhibitory activity comparable to that of (+)-catechin. .

[Test Example 2] Acute toxicity test “Revised Guidelines for Drug Toxicity Test Method GLP Standards” Purified preparations of peak A and peak B described in the examples were dissolved in distilled water for injection at a dose of 50 mg per 5 ml, and 5 animals were dissolved. C
rj: CD-1 (ICR) mice (5 weeks old, male) were orally administered at a rate of 1 ml per 25 g body weight (dose: 2.0 g / kg) and observed for 14 days.

Peak A administration group and peak B after the test
In the administration group, no abnormality was observed in autopsy and histopathological examination of each organ of the whole body. From the results of the test, the LD ₅₀ values of peak A and peak B were 2.0 g / kg (oral) or more, and the toxicity of both was judged to be extremely low.

[0036]

Industrial Applicability The novel isoflavone derivative of the present invention exhibits an excellent activity inhibiting effect on histidine decarboxylase, which is involved in histamine biosynthesis, which causes inflammatory diseases such as ulcers and allergic diseases, and has no side effects. This substance is very useful as medicines, functional foods, etc.

[Brief description of the drawings]

FIG. 1 shows an elution chart of the substance of the present invention by reverse phase HPLC.

Claims (2)

[Claims] 1. The following general formula: (In the formula, R is H or OH) A novel isoflavone derivative. 2. A histidine decarboxylase inhibitor containing the isoflavone derivative according to claim 1 as an active ingredient.