JPH021498A - Polyene substance and agent for controlling mast cell function - Google Patents

Abstract

NEW MATERIAL:The compound of formula (R<1> is H or alkyl; R<2> is H, OH, alkyl, alkoxy or ketone; R<3> is H, salt-forming cation or alkyl). USE:An agent for controlling mast cell function. PREPARATION:The objective substance can be produced by culturing Streptoverticillium eurocidicum IFO No.13491 strain or Streptoverticillium albireticuli IFO No.12737 strain preferably at about pH 7 and about 30 deg.C for, e.g., 40hr.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a polyene substance and a mast cell function regulator.

[Prior art]

Mast cells are present in the connective tissue and gastrointestinal mucosa of animals,
Inside the cell, there are many granules containing biologically active substances called chemical mediators.

Both inflammatory and allergic reactions begin when stimulated mast cells undergo a degranulation reaction and release chemical mediators such as histamine and sero-1-nin. Mast cells are attracting attention as cells that have receptors for immunoglobulin E, and elucidation of the stimulus transmission mechanism and degranulation reaction mechanism in mast cells will directly lead to understanding allergic reactions. There is a desire to develop substances that have functional regulating effects such as cell degranulation and degranulation inhibiting effects.

[Problem that the invention seeks to solve]

Therefore, the present inventors extensively investigated the development of a substance with such an effect, and found for the first time that a new polyene substance represented by the general formula below has a mast cell function regulating effect, and the present inventors Completed the invention.

Therefore, it is an object of the present invention to provide a novel polyene material and an obese cell function regulator.

[Means for solving problems]

According to the present invention, a polyene substance and a mast cell function regulator represented by the following general formula are provided.

In the above formula, R1 is a hydrogen atom or an alkyl group, and examples of the alkyl group include lower alkyl groups such as methyl, ethyl, and propyl. R2 is hydrogen, water lf!
J! , an alkyl group, an alkoxy group or a ketone J, (where the alkyl group is methyl, ethyl.

As lower alkyl groups such as propyl, alkoxy groups,
Examples include lower alkoxy groups such as methoxy, ethoxy, and propoxy. R2 is particularly preferably a hydrogen atom or a hydroxyl group. R3 is a hydrogen atom, a salt-forming cation or an alkyl group. Examples of salt-forming cations include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, as well as aluminum and ammonium.

Examples include organic ammonium. Examples of the alkyl group include lower alkyl groups such as methyl, ethyl, and propyl.

In the general formula (1), the polyene substance (hereinafter also referred to as the present substance) in which 1 (3) is a hydrogen atom is Streptovcillium eurocidicum (Streptovcillium eurocidicum).
rticillium eurocidicum, IF
ON (L13491) strain or Streptoverticillium albireticulli (Streptoverticillium
illjum albireticuli, IFON
(112737) strain can be cultured and produced in a general medium. In this case, the culture uses, for example, glucose, sucrose, maltose, dextrin, lIU powder, glycerin, etc. as a carbon source.

As the nitrogen source, pub 1-, meat extract, yeast extract, malt extract, etc. are used, and as an inorganic salt, NaCQ,
This can be carried out by aeration and stirring in a neutral liquid medium to which K2H1)04, Na211PO, MgSO4, etc. are added. Culture is carried out at a pH of 5 to 8, preferably around 7. The culture temperature is usually in the range of 20 to 35°C, preferably around 30°C. For example, PH7 containing 1.5% glucose, 0.5% peptone, 0.5% meat extract, 0.5% yeast extract, 5% NaCQO,
By culturing in a liquid medium of 0 at 28°C for 40 hours,
This substance can be produced.

The present substance can be collected from the culture filtrate using an organic solvent extraction method, an ion exchange resin method, an adsorption/desorption method using an adsorbent, a high performance liquid chromatography method, or the like.

For example, after adding 1/10 volume of Amberlite XAD-7 to the culture filtrate and performing a dressing treatment at room temperature for about 4 hours with occasional stirring, Amberlite XAD-7 is filtered and washed. Afterwards, elution treatment is performed with an 80% methanol solution. After concentrating the eluate under reduced pressure, it was redissolved in water, p 114
After adjusting the pH to 0, C-Toyopearl ion exchange chromatography is performed, and elution begins around pl+5.0 due to a pH gradient of 0.05M acetic acid-ammonium acetate.

This eluate was further adjusted to pH 9.0 and DEAE-
When Toyopearl ion exchange chromatography is performed, the pH
Elution begins around 7.5. Furthermore, the active fraction was
This substance can be obtained by adsorbing it on an I-gel CHP-20P adsorption column, collecting a 40% acetonitrile elution fraction, and then using high performance liquid chromatography (IIPLC).

Furthermore, this substance can also be obtained by extracting the bacterial cells using an organic solvent such as methanol, then using adsorption chromatography using an ODS column, etc., and performing normal purification operations such as concentration and crystallization of the eluted fraction. be able to.

In the above general formula (1), 1<3 represents a salt-forming cation and an alkyl group, R
It can be obtained by reacting a substance in which 3 represents a hydrogen atom with a base containing a corresponding cation and an esterifying agent in a conventional manner.

Further, the present substance can also be obtained by a commonly used chemical synthesis method or a synthesis method using an e-cord. This substance can be synthesized by using a polyene substance or a similar substance and mycose or mycosamine.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Glucose 1.5%, peptone 0.5%, meat extract 0.
5%, t! /p mother extract 0.5%, NaCQ O, 5%
500 m of 100 m of liquid medium containing pH 7.0
Dispense into a Q Sakalo flask and sterilize at 120°C for 20 minutes. One platinum loop from a slanted agar medium of the actinomycete Streptoverticillium eurocidicum strain was inoculated into this medium, and cultured in a reciprocating manner at 28°C for 3 days. A medium 20Q with the same composition as this culture solution was placed in a jar fermenter for 30Q, and 300 mQ of a preculture solution of Streptoverticillium eurocidicum strain was transferred to this medium.
Culture was carried out for 40 hours at 200° C. and 1 air flow per minute. After the culture was completed, the bacterial cells and the culture filtrate were separated by continuous centrifugation at 7000 rpm to obtain 18 g of a dark brown culture filtrate. The mast cell degranulation inhibitory activity of this culture filtrate was 642,500 units.

Example 2 Five times the amount of methanol was added to the bacterial cells obtained in Example 1, stirred, and then left at room temperature. After storing for 24 hours with occasional stirring, the bacterial cells were filtered out, and the methanol extract was mixed with the same volume of water, and then added to an ODS adsorption column (manufactured by Nippon Millipore, column volume 778, methanol/water = 171). (equilibration). Using the eluate (methanol/water = 2/1), both fractions 1, 2 and lit were sequentially obtained, and by concentration and recrystallization operations, Nal of the present substance corresponding to each fraction: 5.0 mg,
Na2: 8.7 mH and Na3: loOmg were obtained.

Example 3 (1) In 5Q of the culture filtrate obtained in Example 1, adsorbent Amberlite XAD corresponding to L/10 volume was added
-7 was added, and adsorption treatment was carried out at room temperature for 4 hours with occasional shaking. The adsorbent was separated by suction filtration, washed with purified water, and then washed with a 20% aqueous methanol solution equivalent to twice the volume of the adsorbent. Thereafter, it was eluted with an 80% methanol-'-qEy' aqueous solution corresponding to 3 times the volume of the adsorbent, and the eluate was concentrated under reduced pressure.

Dissolved in purified water. Almost 100% activity was recovered.

(2) Next, pl+4.
A sample solution adjusted to 0 was adsorbed and eluted with 0.05M acetic acid-ammonium acetate (ρ116.0). The active fraction was eluted from the eluate at pH 5.0. The mast cell degranulation inhibitory activity of the active fraction was 25,250 units.

(3) Furthermore, the active fraction of (2) above is added to ρII! 1
, 0, and then adsorbed on a DEAE-Toyopearl 650M column [manufactured by Tosoh, column volume 179 mQ, equilibrated with 0.05M ammonium acetate-ammonia water to pl-19.0], and 0.05M ammonium acetate-ammonia Elution was performed with water (pH 7.5). The activity of this active fraction was 16,500 units.

(4) Furthermore, an adsorption column (column volume 40 mff) packed with MCI gel (CIIP-20P, Mitsubishi Kasei W)
) to adsorb the active fraction obtained in (3) above, and
After washing with a 40% acetonitrile aqueous solution, the mixture was eluted with a 40% acetonitrile aqueous solution and concentrated under reduced pressure to obtain a white substance.

The obtained material was dissolved in methanol and separated by IIPLC. The separation conditions in this case are as follows.

Column: filled with Nucleosil 5C8, diameter 10mm,
Length 250mm Solvent used: Niacetonitrile/ammonium acetate buffer (
0.01M, , 115.0) = 35/65 flow rate:
4mQ/min Detection: Ultraviolet wavelength: Absorbance d at 350 nm
1q constant sample concentration = 0.2 mg/d Injection amount: 200 μQ/time Under the above conditions, fractionate both substances corresponding to Nnl, Nα2 and Nα3, and concentrate under reduced pressure to obtain Substance Nnl: 1.1 mg,
Nα2: 1.5 mg and Nn3: 10 mg were obtained.

Example 4 (1) About 1 each of the substances Ncil, Nn2 and Na3
．． ．． 5B is positive -1\'= 1 -r61'%, N=1.76%, substance Nn2:C
=57.70%, 1I=7.57%1, +3. N = 1.65%, substance Na3: C = 58T8-h
'A, l-1 = 8.10%, N = 1.86%, respectively, C15llsiOxtN (theoretical value C
=57.27%, H=7, 76%, N=1.71
%) C4゜11G50□iN (Theoretical value: C=5-7-i70%, I+=7.87%, N=1.6
8%), C,, HGsOl, N (C=58°88,
It was revealed that the composition was 8.03% (H = 8.03%, N = 1.72%).

(2) Substance Nα1. Mass spectrometry was performed on Nα2 and Nα3. The measurement results by FAB-MS method revealed that substance Nα1 was (Mill)”=782.36, and C3g+(,,0,SN (M=781°39). Substance &2 was (Mill)”= 796.47
So, C4゜)I,,01. N (M=795.40)
It turned out to be. The substance Nα3 is (Mill)”=780
．． 45, C4,)1. ．． 01. N (M=779.
41). The elemental analysis values are for dihydrate (
211□0).

(3) Substance Nα1. Nα2 and Nα3 were dissolved in methanol and subjected to ultraviolet absorption spectroscopy. The result is that the substance Nal, &2 and waste 3 are both 302 nm, 3
16nm.

It was revealed that the pentaene structure has maximum wavelengths at 331 nm and 349 nm, and the absorption strength is E (1%
.

4 (316r+n+), 1375 (331 nm) and 1
388 (349 nm), and in the material Nn2, it is 34
4 (302nm), 700 (316nm), 112
2 (33Lnm) and 1144 (349nm), and for the substance Na3: 162 (302nm), 76
0 (316 nm), 1210 (331 nm) and 12
32 (349 nm).

(4) About 10 each of the substances Nnl, Nα2 and Nα3
Measure out μg and mix with potassium bromide to make tablets.
Infrared absorption spectrum analysis using the tablet method was conducted on B. As a result, each substance had a
1700cm-", 1560am-', 1390cm
-1,1070 cm-' and 10010 O5'', indicating the characteristics of a polyene material.

(5) Substances Nα1, Nα2, and N(13) were dissolved in deuterium-substituted dimethylformamide (oMr-d) or deuterium-substituted dimethyl sulfoxide (DMSO-d), respectively, and nuclear magnetic resonance spectroscopy was performed. 1. Nuclear Magnetic Resonance Spectrum 13 c M Magnetic Resonance Spectrum, 1
3C-DEPT measurement and Jl-J (shift correlation two-dimensional nuclear magnetic resonance spectrum analysis were performed, and the structure of each substance was determined by the general formula %Nα3, R'=C113, P=11,)+3=l
It was determined that it indicates +.

Example 5 (1) Wistar female rats (body weight 150-250
g) The animal was bled to death, and 20 m of Tyrode's solution was administered intraperitoneally.
Q was injected, and the abdomen was lightly massaged for about 2 minutes. Ascites fluid was collected after laparotomy and centrifuged at 150×g for 10 minutes at 4° C. to collect precipitated cells. These cells were suspended in 2 mQ of Tyrode's solution, overlaid on 4 mQ of physiological saline containing bovine serum albumin adjusted to a specific gravity of 1.068, and placed at 4°C.
After centrifugation for 12 minutes at 1100X, the precipitated cells were collected. After washing twice with Tyrode's solution, 0.2%
There are approximately 1 mast cell in Tyroth's solution containing bovine serum albumin.
The cells were suspended at a concentration of 0.06 cells/mQ to obtain a mast cell suspension.

(2) Physiological saline zOμQ containing the test compound and 20μQ mast cell suspension were reacted at 37°C for 10 minutes, and after dissolving compound 48/80 to a final concentration of Iμg/mA, cooling with water for 1 minute, 1. The supernatant and cells were separated by centrifugation at 500×g for 4 minutes. Histamine contained in the supernatant was analyzed using the IIPLC method of Onda et al.
ma J, Mad.

Sci, Vol. 27, pp. 93-07, 1978)
Degranulation was measured by quantification. In this case, the mast cell degranulation inhibitory activity was calculated by the following formula 6
A; Amount of histamine released when cells were reacted only with compound 48/80 B; Compound 4 was released after reacting the test compound with cells.
Amount of histamine released when cells were further reacted with 8/80 C; Amount of histamine released by Ti when cells were reacted only with buffer solution (or physiological saline) As a degranulation inducing agent, Compound 48/ 80 (Sigma Co., Ltd.), concanavalin A, ionophore A23187, and any other commonly known drugs can be used.

...1...1. m-“Measurements were made with various concentrations of the test compound.
The concentration (IC, ) of the compound that inhibits 50% of the mast cell degranulation induced by lμg/- of Compound 4g/80 was determined in a constant system.
We also measured the functional regulatory effect of this substance on mast cells under various conditions such as the absence of 80, the addition of cholesterol, and the addition of cholesterol.The results are as follows. As shown,
This substance was found to have a strong mast cell function regulating effect.

In addition, book 211! The mast cell degranulation effect induced by compound 48/80 at 1 μg/mA was
% inhibiting compound concentration by 1 unit/- [1 unit l-/
m1ll).

(3) Substances Nα1 and Nα2 against the mast cell degranulation effect induced by Compound 48/80 of 10112 on lμ
The effects of Na3 and Na3 were as follows.

(3) -(i) Substance Nα1. Na2 and Nα3 exhibit an inhibitory effect on mast cell degranulation at low concentrations, and their IC values are similar to that of substance-2.
'9'l: 3.6 μg/mfA, substance) lIa2: 1.
The content was 8 μg/min, and the substance Nα3 was 1.0 μg/−.

(3) -(ii) Substance Nα1. Nα2 and Nα3 independently exert a mast cell degranulation effect in the high concentration range, and the single effect is expressed when the calcium ion concentration of the reaction solution is lowered to about 1mM or more.
When the calcium ion concentration of the reaction solution was close to O, calcium ion dependence was not expressed.

(3) -(iii) Add cholesterol to the reaction solution at a constant concentration (approximately 2 μg/mQ)
By adding the above, the above (3) - (i) and (
3) The effect of -(ii) decreased in a concentration-dependent manner on cholesterol.

Example 6 Take 100 mg of the substance Nal, dissolve it in 2 mQ of dimethyl sulfoxide, and then add 0.2 d of anhydrous methanol.
was added and diluted. 1 mQ of tetrahydrofuran containing 0.3 molar concentration of diazomethane was added thereto, and the mixture was reacted at 4° C. for about 2 minutes to perform esterification. Nal-free ethyl ether 20+++9 was added to the reaction solution, and the precipitated material was collected by centrifugation, washed with a small amount of anhydrous ethyl ether, and then recrystallized with the same solvent. In formula (1), R1=C11J, R”
=11.1ce'=CII:I), 72 mg was obtained.

The methyl ester was identified by mass spectrometry and nuclear magnetic resonance spectrum analysis.

〔Effect of the invention〕

As described above, the present invention has revealed that a specific polyene substance has an effect of regulating obese X1 cell function.

Patent Applicant: Yukio Iizuka, Director, Agency of Industrial Science and Technology Designated Agent: Tomoo Suzuki, Director, Institute of Microbial Technology, Agency of Industrial Science and Technology

Claims (1)

[Claims] (1) A polyene substance represented by the following general formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 is a hydrogen atom or an alkyl group, R^2 is a hydrogen atom, hydroxyl group, alkyl group, alkoxy group, or ketone group, R^3 is a hydrogen atom or a salt (2) A mast cell function regulator comprising the polyene substance according to claim 1.