JPH064669B2 - Polyene substance and obesity function regulator - Google Patents

Description

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 tissues of animals and in the gastrointestinal mucosa,
It has many granules containing biologically active substances called chemical mediators in its cells. Both an inflammatory reaction and an allergic reaction start when stimulated mast cells undergo a degranulation reaction and release chemical mediators such as histamine and serotonin. Mast cells are attracting attention as cells that have receptors for immunoglobulin E,
Since elucidation of the stimulus transmission function and the mechanism of action of degranulation reaction in mast cells directly leads to understanding of allergic reaction, it has function-regulating actions such as degranulation action and degranulation inhibition action of mast cells. Development of substances is desired.

[Problems to be solved by the invention]

Therefore, the present inventors have extensively studied the development of a substance having such an action, and have revealed for the first time that a novel polyene substance represented by the following general formula has a mast cell function-regulating action, Completed the invention.

Therefore, an object of the present invention is to provide a novel polyene substance and a mast 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, R ¹ is a hydrogen atom or an alkyl group, and examples of the alkyl group include lower alkyl groups such as methyl, ethyl and propyl. R ² is a hydrogen atom or a hydroxyl group.
R ³ is a hydrogen atom, a salt-forming cation or an alkyl group. Examples of the salt-forming cation include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, as well as aluminum, ammonium and organic ammonium. Examples of the alkyl group include lower alkyl groups such as methyl, ethyl and propyl.

In the general formula (I), a polyene substance in which R ³ is a hydrogen atom (hereinafter, also referred to as this substance) is a Streptoverticillium eurocidicum (IFO No. 13491) strain or Streptoverticillium arubi Reticuli
The (Streptoverticillium albireticuli, IFO No. 12737) strain can be cultured in a general medium and produced in the medium. The culture in this case is performed by, for example, glucose, sucrose, maltose, dextrin, starch as a carbon source,
Glycerin, etc. as the nitrogen source, peptone, meat extract,
It can be carried out by using yeast extract, malt extract and the like, and aerating and stirring in a neutral liquid medium to which NaCl, K ₂ HPO ₄ Na ₂ HPO ₄ , MgSO _{4 and the} like have been added as inorganic salts. The medium is cultured 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, glycol 1.5%, peptone 0.5%,
PH 7.0 containing 0.5% meat extract, 0.5% yeast extract, 0.5% NaCl
This substance can be produced by culturing at 28 ° C. for 40 hours in the above liquid medium.

Collection of this substance from the culture filtrate can be carried out by an organic solvent extraction method, an ion exchange resin method, an adsorption / desorption method using an adsorbent, a high performance liquid urography method, or the like.
For example, 1/10 volume of Amberlite XAD- in the culture filtrate.
After adding 7 and adsorbing at room temperature for about 4 hours with occasional stirring, Amberlite XAD-7 is filtered off, washed, and then eluted with 80% methanol solution. The eluate was concentrated under reduced pressure, redissolved in water, adjusted to pH 4.0, and subjected to CM-Toyopearl ion exchange chromatography to give 0.05M.
PH with an acetic acid-ammonium acetate pH gradient
Elution starts from around 5.0. When this eluate is further adjusted to pH 9.0 and subjected to DEAE-Toyopearl ion exchange chromatography, elution begins around pH 7.5. Further, the active fraction is adsorbed on an MCI gel CHP-20P adsorption column, a 40% acetonitrile-eluted fraction is collected, and then this product can be obtained by using high performance liquid chromatography (HPLC).

Furthermore, this substance can also be obtained by subjecting the bacterial cells to an extraction treatment using an organic solvent such as methanol, and then using adsorption chromatography using an ODS column or the like to concentrate the elution fraction and carry out ordinary purification operations such as crystallization. be able to.

In the above general formula (I), R ³ represents a salt-forming cation and an alkyl group, a substance having a cation corresponding to R ³ in the general formula (I) is a base and esterification containing a corresponding cation. It can be obtained by reacting the agent by a conventional method.

The substance can also be obtained by a commonly used chemical synthesis method or a synthetic method using an enzyme. This substance can be synthesized by using a polyene substance or the like and mycose or mycosamine.

〔Example〕

 Next, the present invention will be described in more detail with reference to examples.

Example 1 500 ml of 100 ml of pH 7.5% liquid medium containing glucose 1.5%, peptone 0.5%, meat extract 0.5%, enzyme extract 0.5%, and NaCl 0.5%
Dispense into a ml Sakaguchi flask and sterilize at 120 ° C for 20 minutes.
This medium was inoculated with 1 platinum loop amount from a slope agar medium of Streptoverticillium eurosidicum strain of Streptomyces sp.
Reciprocating agitation culture was performed at 3 ° C for 3 days. A medium 20 having the same composition as this culture solution was charged into a jar fermenter for 30 and 300 ml of a preculture solution of the Streptoverticillium eurosidicum strain was transferred to this medium, and at 28 ° C., 200 rpm, and an aeration rate of 10 minutes, 40 hours. Culture was performed. 7,000 after culture
The cells were separated from the culture filtrate by continuous rotation centrifugation to obtain a black-brown culture filtrate 18. The mast cell degranulation inhibitory activity of this culture filtrate was 642,500 units.

Example 2 To the cells obtained in Example 1 was added 5 times the amount of methanol, the mixture was stirred, and then allowed to stand at room temperature. After leaving for 24 hours with occasional stirring, the bacterial cells were filtered off, and the methanol extract was mixed with an equal volume of water to obtain an ODS adsorption column (Nihon Millipore, column volume 7
78 ml, equilibrated with methanol / water = 1/1). Fractions I, II and III were sequentially obtained using the eluate (methanol / water = 2/1), and concentrated and recrystallized to give the corresponding substance NO.1: 5.0 mg, NO. 2: 8.7 mg and N
O. 3: 100 mg was obtained.

Example 3 (1) 1 out of 5 of the culture filtrates obtained in Example 1
/ 10 volume of adsorbent Amberlite XAD-7 is added,
Adsorption treatment was performed at room temperature for 4 hours with occasional shaking. In suction filtration, the adsorbent was filtered off, washed with purified water, and then washed with a 20% aqueous methanol solution corresponding to twice the adsorbent volume. Then, elute with 80% aqueous methanol solution corresponding to 3 times the adsorbent volume, and concentrate the eluate under reduced pressure.
It was dissolved in purified water. Almost 100% of the activity could be recovered.

(2) Next, a CM-Toyopearl 650M column (manufactured by Tosoh, column volume 533 ml, equilibrated with 0.05 M acetic acid-ammonium acetate (pH 4.0)) was adsorbed with a sample solution adjusted to pH 4.0, and 0.05 M was added.
It was eluted with acetic acid-ammonium acetate (pH 6.0). The active fraction began to elute at pH 5.0 after elution. The mast cell degranulation inhibitory activity of the active fraction was 25,250 units.

(3) After further adjusting the active fraction of (2) above to pH 9.0,
EAE-Toyopearl 650M column [Tosoh, column volume 179
ml, equilibrated to pH 9.0 with 0.05 M ammonium acetate-ammonia water], and eluted with 0.05 M ammonium acetate-ammonia water (pH 7.5). The activity of this active fraction is 1
It was 6,500 units.

(4) Furthermore, the active fraction obtained in (3) above was adsorbed to an adsorption column (column volume 40 ml) packed with MCI gel (CHP-20P, manufactured by Mitsubishi Kasei), and 20% aceto-tolyl aqueous solution was used. After washing, it was eluted with 40% aqueous acetonitrile solution and concentrated under reduced pressure to obtain a white substance.

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

Column: Nucleosyl 5C8 packed, diameter 10 mm, length 250 mm Solvent used: acetonitrile / ammonium acetate buffer (0.01 M, pH 5.0) = 35/65 Flow rate: 4 ml / min Detection: Absorbance measurement at UV wavelength 350 nm Sample concentration: 0.2 mg / ml Injection volume: 200 μl / times Under the above conditions, the fractions corresponding to substance NO.1, NO.2 and NO.3 were collected and concentrated under reduced pressure to obtain substance NO.1: 1.1 mg, NO .2: 1.5 mg
And No. 3: 10 mg were obtained.

Example 4 (1) About 1.5 mg of each of the substances NO.1, NO.2 and NO.3 was accurately measured and trace element analysis was performed. The contents of carbon, hydrogen and nitrogen are as follows: NO.1: C = 57.59%, H = 7.61%, N = 1.
76% and substance NO.2: C = 57.70%, H = 7.57%, N = 1.65%,
Material NO.3: C = 58.86%, H8.10%, N = 1.86%,
C ₃₉ H ₆₃ O ₁₇ N (theoretical value C = 57.27%, H = 7.76%,
N = 1.71%) C ₄₀ H ₆₅ O ₁₇ N (Theoretical value: C = 57.75%, H = 7.87
%, N = 1.68%), C ₄₀ H ₆₅ O ₁₆ N (C = 58.88%, H = 8.03%, N
= 1.72%).

(2) Mass spectrometry was performed on the substances NO.1, NO.2 and NO.3. FAB-MS measurement results show that substance No. 1 is (M +
H) ⁺ = 782.36, which proved to be C ₃₉ H ₅₉ O ₁₅ N (M = 781.39). Material NO.2 becomes (M + H) ⁺ = 796.47, and C ₄₀ H ₆₁ O ₁₅ N (M
= 795.40). The substance NO.3 became (M + H) ⁺ = 780.45, which was found to be C ₄₀ H ₆₁ O ₁₄ N (M = 799.41). The elemental analysis values were estimated to be dihydrate (2H ₂ O), respectively.

(3) The substances NO.1, NO.2 and NO.3 were dissolved in methanol and subjected to ultraviolet absorption spectrum analysis. The result is substance NO.
1, NO.2 and NO.3 are all 302 nm, 316 nm, 331 nm and 34
It was clarified that it has a pentaene structure with a maximum wavelength at 9 nm, and the absorption intensity is E (1%, MeOH).
In O.1, 398 (302 nm), 844 (316 nm), 1375 (331 nm) and 13
88 (349 nm), and for substance No. 2, 344 (302 nm), 700 (316 nm)
nm), 1122 (331 nm) and 1144 (349 nm) .For substance NO.3, 362 (302 nm), 760 (316 nm), 1210 (331 nm) and 1232 (34 nm).
9 nm).

(4) About 10 μg of each of the substances NO.1, NO.2 and NO.3 was weighed and mixed with potassium bromide to prepare tablets, and infrared absorption spectrum analysis by the KBr tablet method was performed. As a result, for each substance, 3380 cm ^-1 , 1700 cm ^-1 , 1560 cm ^-1 ,
1390 cm ^-1, an absorption in 1070 cm ^-1 and 1005cm ^-1, showed the characteristics of a polyene material.

(5) Substances NO.1, NO.2 and NO.3 were dissolved in deuterium-substituted dimethylformamide (DMF-d) or deuterium-substituted dimethylsulfoxide (DMSO-d), respectively, and nuclear magnetic resonance spectra were performed. . ¹ H nuclear magnetic resonance spectrum, ¹³ C nuclear magnetic resonance spectrum, ¹³ C-DEPT measurement and ¹ H- ¹ H shift correlation two-dimensional nuclear magnetic resonance spectrum analysis, the structure of each substance in the general formula (I), In the case of substance NO.1, R ¹ = H, R ² = OH, R ³
= H, substance NO.2, R ¹ = CH ₃ , R ² = OH, R ³ = H, substance N
In the case of O.3, it was determined that R ¹ = CH ₃ , R ² = H, and R ³ = H.

Example 5 (1) Female Wistar rats (body weight 150 to 250 g) were killed by exsanguination, 20 ml of Tyrode's solution was intraperitoneally injected, and the abdomen was gently massaged for about 2 minutes. After laparotomy, cover water and collect at 4 ℃
Were centrifuged at 150 xg for 10 minutes to collect the precipitated cells. Suspend these cells in 2 ml of Tyrode's solution,
Saline containing bovine serum albumin adjusted to a specific gravity of 1.068 4
The cells were layered on top of each other, and centrifuged at 4 ° C. and 100 × g for 12 minutes, and then the precipitated cells were collected. After washing twice with Tyrode's solution, the solution was suspended in Tyrode's solution containing 0.2% bovine serum albumin so that mast cells were about 10 ⁶ cells / ml to obtain a mast cell suspension.

(2) 20 μl of physiological saline containing the test compound and 20 μl of mast cell suspension were reacted at 37 ° C. for 10 minutes to give a final concentration of 1 μg /
Add 10 μl of compound 48/80 solution to make up to ml,
The reaction was continued for another 10 minutes. After the reaction was cooled in ice for 1 minute, 1,
The supernatant was separated from the cells by centrifugation at 500 xg for 4 minutes. Histamine contained in the supernatant was analyzed by the Onda et al. HPLC method (Hiro
Chima J. Med. Sci, 27, 93-97, 1987) was used to measure degranulation. in this case,
The mast cell degranulation inhibitory activity was calculated by the following formula.

A: Amount of histamine released when cells were reacted only with compound 48/80 B; Compound 48 was reacted with the test compound and then reacted with compound 48/80
/ 80 added histamine amount when further reacted C; Histamine amount released when cells were reacted only with buffer (or physiological saline) Compound 48/80 as a degranulation inducer In addition to (manufactured by Sigma), concanavalin A, ionophore A23187 and any other generally known drug can be used.

The concentration of the test compound was changed variously, and the concentration [IC ₅₀ ] value of the compound that inhibits 50% of mast cell degranulation induced by 1 μg / ml of compound 48/80 in this measurement system was determined. Also, various conditions such as the absence of compound 48/80, the condition of adding cholesterol,
Measurement of the function-regulating action of this substance on mast cells under various conditions such as the addition of cholesterol was also performed, and the results show that this substance has a strong mast-cell function regulating action. Was recognized.

The concentration of the compound that suppresses 50% of the mast cell degranulation action induced by 1 μg / ml compound 48/80 in this assay system was 1 unit / ml [1 unit / ml].

(3) The effects of substances NO. 1, NO. 2 and NO. 3 on the mast cell degranulation effect induced by 1 mg / ml of compound 48/80 were as follows.

(3)-(i) Substances NO.1, NO.2 and NO.3 show mast cell degranulation inhibitory action in the low concentration region, and its IC ₅₀ value is substance NO.1: 3.6 μg
/ Ml, substance NO.2: 1.8 μg / ml, substance NO.3: 1.0 μg / ml
Met.

(3)-(ii) Substances NO.1, NO.2 and NO.3 showed mast cell degranulation action alone in the high concentration region, and this action increased the calcium ion concentration of the reaction solution to about 1 mM or more. There was a calcium ion dependency that it was sometimes expressed and was not expressed when the calcium ion concentration of the reaction solution was close to zero.

(3)-(iii) The action of (3)-(i) and (3)-(ii) above depends on the concentration of cholesterol by adding cholesterol to the reaction solution at a certain concentration (about 2 μg / ml) or more. Decreased.

Example 6 100 mg of the substance NO.1 was weighed and dissolved in 2 ml of dimethyl sulfoxide, and then 0.2 ml of anhydrous methanol was added to dilute it.
Tetrahydrofuran containing 0.3 molar diazomethane 1
Esterification was performed by adding ml to this and reacting at 4 ° C. for about 2 minutes. 20 ml of anhydrous ethyl ether was added to the reaction mixture, and the precipitated substance was collected by centrifugation, washed with a small amount of anhydrous ethyl ether, and recrystallized in the same solvent to give substance N.
The methyl ester of O.1 (in the general formula (I), R ¹ ═C
H ₃ , R ² = H, R ³ = CH ₃ ), 72 mg were obtained.

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

〔The invention's effect〕

As described above, according to the present invention, it was revealed that a specific polyene substance has a mast cell function regulating action.

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Claims (2)

[Claims] 1. A polyene substance represented by the following general formula. (In the formula, R ¹ represents a hydrogen atom or an alkyl group, R ² represents a hydrogen atom or a hydroxyl group, R ³ represents a hydrogen atom, a salt-forming cation or an alkyl group) 2. A mast cell function regulator comprising the polyene substance according to claim 1.