JPS61227528A - Agent for controlling function of mastocyte - Google Patents

Abstract

PURPOSE:To provide the titled controlling agent containing a substance composed of the constituent component of eurocidin and exhibiting a specific absorption peak in ultraviolet absorption spectrum, as an active component. CONSTITUTION:An agent for controlling the function of mastocyte is obtained by using a substance composed of the constituent component of eurocidin and exhibiting an absorption peak (including shoulder) at about 300-350nm in ultraviolet absorption spectrum, as an active component. The above substance can be separated from eurocidin or a filtrate of a cultivation product of an eurocidin-producing microorganism using a high-performance liquid chromatography (HPLC). As an alternative method, the substance can be produced by culturing Streptoverticillium albireticuli in a conventional medium and separating from the culture medium by conventional method. The separation of the substance from the culture liquid can be carried out e.g. by ion-exchange resin process, or an adsorption and desorption using an adsorbent.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to an agent for regulating the function of isolated mast cells.

[Prior art]

Mast cells exist in the connective tissue of animals, and contain 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 serotonin. Mast cells have attracted attention as cells that have receptors for immunoglobulin E, and elucidating the stimulation transmission mechanism and degranulation reaction mechanism in mast cells will directly lead to understanding allergic reactions. It is desired to develop a substance that has functional regulating effects such as degranulation and degranulation inhibiting effects.

〔the purpose〕

Therefore, the present inventors extensively investigated whether a substance with such an effect could be produced, and found that it was a component of the known eurocidin (Journal of the Japanese Society of Agricultural Chemistry, Vol. 29, pp. 650-652, 1955). It has been recognized that the substance whose ultraviolet absorption spectrum shows an absorption maximum (including the shoulder) in the range of about 300 to 350 nm has a mast cell function regulating effect, and the commercially available Streptoverticillium eurocidicum (
Streptoverticilliua + euroc
idicum, IFO & 13491) strain, which produced a substance that has a strong mast cell function regulating effect, and as a result of isolating and purifying this substance, it was determined that it is a component of the same eurocidin as above. Admitted.

Therefore, an object of the present invention is to provide a novel mast cell function regulating agent.

〔composition〕

The present invention provides a mast cell function regulating agent containing as an active ingredient a substance that is a constituent of Eurocidin and exhibits an absorption maximum (including the shoulder) in the ultraviolet absorption spectrum at about 300 to 350. The present inventors are the first to discover that a component of Eurocidin has a functional regulatory effect on the function of mast cells. In the following, the mast cell function regulating substance contained in this Eurocidin component will be referred to as a single substance.

This substance can be isolated and obtained from eurocidin or eurocidin-producing microorganism culture filtrate using high-performance liquid chromatography (HPLC).
When HPLC separation is performed using a column (using Nucleosil 5C8 as packing material) with a length of 250 ++ us,
It can be separated as shown in Figure 1(b), approximately 300 to 350 nm.
Four types of substances exhibiting ultraviolet absorption spectrum absorption poles, namely:
Substances Nos. 1 to 4 corresponding to peaks No. 1 to No. 4 can be obtained. The ultraviolet absorption spectra of these substances No. 1 to No. 4 are shown in Figure 2 (
Shown in a) to (d). Each of these substances is about 30
2nm, about 316nm, about 332nm, and about 349nm
The absorption maximum (including the shoulder) is shown at m.

In addition, Figure 1 shows the results of IPLc separation of one substance,
FIG. 1(a) shows the results for this substance separated and obtained from the culture filtrate of eurocidin-producing microproducts, and FIG. 1(b) shows the results for eurocidin.

In addition, this substance can be used for Streptoverticillium eurocidicum (Streptoverticillium
eurocidicum.

IFONn13491) strain or Streptoverticillium albireticulli (Streptoverti
cillium albireticuli, IFOF
&112737) strain can be cultured in a general medium, produced in the medium, and collected by a conventional method. For example, glucose, sucrose as a carbon source.

Maltose, dextrin, starch, glycerin, etc., and peptone, meat extract, yeast extract as nitrogen sources.

Using malt extract etc., NaCQ is further added as an inorganic salt.

K2) Cultured in a neutral liquid medium containing IFO4, NazHPO4, MgSO4, etc. by aeration and stirring. The pl+ of the medium is 5-8. Preferably, it is cultured at around 7. The culture temperature is usually in the range of 20-35°C.

Preferably around 30°C 1 For example, glucose 1.5%, peptone 0.5%, meat extract 0.5%, yeast extract 0.5
%, NaCQ O, 30°C in a pH 7.0 liquid medium containing 5%.

This substance can be produced by culturing for 40 hours. Collection from the culture solution can be performed using an ion exchange resin method, an adsorption/desorption method using an adsorbent, a high performance liquid chromatography method, or the like.

Furthermore, this substance can also be obtained by extracting the bacterial cells using an organic solvent such as methanol, followed by ordinary purification operations such as concentration and crystallization. 6. Separating the substances contained in the culture solution. To recover, 1. For example, add 1/10 volume of Amberlite
After filtering Amberlite XAD-7 and washing it,
Elute with 80% methanol solution. After concentrating the eluate under reduced pressure, redissolving it in water and adjusting the pH to 4.0, CM-
When Toyopearl ion exchange chromatography is performed, 0
．． Elution begins at around pH 5.7 using a p)l gradient of 05M acetic acid-ammonium acetate. When this eluate is further adjusted to pH 8.5 and subjected to DEAR-Toyopearl ion exchange chromatography, elution begins at around pH 7.5. Furthermore, the active fraction was collected using MCI gel CHP-20.
Substance No. 3 of this substance can be obtained by adsorbing it on a P adsorption column, collecting a 40% acetonitrile elution fraction, and drying it under reduced pressure.

Next, the test method and results for the mast cell degranulation inhibitory activity of this substance are shown below.

Ascitic fluid was collected from the abdominal cavity of a rat and centrifuged for 10 minutes at 4° C. and 150×g, and the precipitated cells were separated by a method such as bovine serum albumin multilayer centrifugation to obtain a mast cell fraction. This was suspended in Tyrode's solution containing 0.2% bovine serum albumin at a density of 10' mast cells/IIIQ to obtain a mast cell suspension.

Next, 20 μμ of the mast cell suspension was added to 20 μΩ of physiological saline containing the test compound, mixed, and reacted at 37°C for 10 minutes. (
1μg/mQ) was added and reacted for 10 minutes.
The supernatant was collected by centrifugation using a water cooler. The histamine contained in the supernatant was analyzed by HPLC method (Onda et al.).
J, Med, Sci.

27, pp. 93-97, 1978).

In addition, as the degranulation inducing agent, compound 4g/
In addition to 80 (Sigma II), any commonly known drugs such as concanavalin A (manufactured by Wako Hyakuyaku Kogyo Co., Ltd.), ionophore A23187 (Calbiochem Behring Co., Ltd.), and antigen-antibody complexes are used. be able to. Next, measurements were carried out with various concentrations of the test compound, and a compound of 48/80 with lμgemΩ was measured using a single measurement system.
The concentration of the compound (ICsoI value) that inhibits 50% of mast cell degranulation induced by compound 48/80 was determined. The function regulation effect of the substance on mast cells was also measured, and the results showed that one substance had a strong mast cell function regulation effect as shown below.

(1) All of these substances exhibited mast cell degranulation effects at low concentrations, and their C5o values were as shown in Table 1 below.

(2) This substance independently exhibited a mast cell degranulation effect in a concentration range higher than that exhibiting the effect in (1) above, and this effect was dependent on the calcium ion concentration of the reaction solution.

(3) It was also possible to reduce the strength of the effects of (1) and (2) above by adding a substance having a sterol skeleton.

In addition, 1μg/2 servings of dung pound 4878 with 1 measurement system
The concentration of the compound that inhibits the mast cell degranulation effect induced by 0 by 50% is 1 unit/■Q [1 unit/so Ω]
And so.

Table-1 〔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.
100 mQ of a pH 7.0 liquid medium containing 5% yeast extract, 0.5% yeast extract, and 5% NaCQO is dispensed into a 500 van Sakalo flask and sterilized at 120°C for 20 minutes. One platinum loop of the actinomycete Streptoverticillium eurocidicum strain was inoculated onto this medium from a slanted agar medium, and 30
℃, and reciprocating incubation was performed for 3 days. A medium 20Q having the same composition as this culture solution was placed in a jar fermentor for 3 (N2), and 300Ω of a preculture solution of Streptoverticillium dilosidicum strain was transferred to this medium.
Culture was carried out for 40 hours at 0.00 rpm and 1 aeration volume per minute.

After the culture is completed, the bacterial cells and the culture solution are dispersed by continuous centrifugation at 7,000 rpm, and a dark brown culture solution of 18fi is obtained.
I got it. The mast cell degranulation inhibitory activity of this culture solution is 641
, 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 off, and the methanol extract was concentrated and dried. The obtained residue was redissolved using methanol,
Only the soluble fraction was collected.

After concentration again, the resulting crystalline substance Eurocidin was obtained. The activity of this substance against mast cells was approximately 113,000 units.

Example 3 Eurocidin was dissolved in methanol and separated by HPLC. The separation conditions in this case are as follows.

Column: Nucleosil 5C8 packed, diameter 10mm,
Length 250mm Solvent used: Acetonitrile/ammonium acetate buffer (0.01M, pH 5.0) = 35/65 Flow rate:
4taQ/min Detection: Absorbance measurement at ultraviolet wavelength 350nffi Sample concentration: 0.2a+g/■Q Injection amount: 200μ When the constituent components of Eurocidin were separated under the above conditions, each component was found as shown in Figure 1 (b). The separation results are shown, and both substances and 1-NQ4 corresponding to the corresponding peaks were obtained. After concentrating both parts under reduced pressure, freeze-drying was repeated and the substance &1
~Na4 was obtained. The ultraviolet absorption spectra of this substance Nal~&4 are shown in Figure 2 (a) to (d), and each substance has wavelengths of approximately 302 nm, approximately 316 nm+m, and approximately 332 nm, respectively.
and showed an absorption maximum (including a shoulder) at about 349 nm.

Example 4 (1) Example! In 512 of the culture solution obtained, the adsorbent Amberlite
-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% aqueous methanol solution equivalent to three times the volume of the adsorbent, and the eluate was concentrated under reduced pressure and then dissolved in purified water. Habo 100%
Similar activity was recovered.

(2) Next, CM-Toyopearl 650M column [Toyo Soda Kogyo layer, column volume 533jl, 0.05M acetic acid]
Equilibrate with ammonium acetate (p) 14.0) to pH 4.
A sample solution adjusted to pH 6.0 was adsorbed and eluted with 0.05M acetic acid-ammonium acetate (pH 6.0). The most strongly active fraction was eluted from the eluate at pH 5.7.

The mast cell degranulation inhibitory activity of the active fraction was 25,250 units.

(3) Further, the active fraction from (2) above was adjusted to pH 8.5, and then loaded onto a DEAE-Toyopearl 650M column [
Toyo Soda industrial layer, column volume 179+s Q, 0.05
Equilibrated to pH 8.5 with M ammonium acetate-aqueous ammonia] and eluted with 0.05M ammonium acetate-aqueous ammonia (pH 7.5). The activity of this active fraction was 16,500 units.

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

This substance was dissolved in methanol, an ultraviolet absorption spectrum was measured, and HPLC separation was performed under the separation conditions shown in Example 3 above. The results are shown in FIG. 1(a) and FIG. 3, respectively. The obtained substance was the same as substance &3 among the constituent components of Eurocidin. The activity amount is 12,
It was 500 units.

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. The cells were suspended in Tyrode's solution 2IIIQ, layered over 4 tn (l) of physiological saline containing bovine serum albumin prepared to a specific gravity of 1.068, and centrifuged for 12 minutes at 4°C and 1100X. After washing twice with Tyrode's solution,
Mast cells were suspended in Tyrode's solution containing 0.2% bovine serum albumin at approximately 10 6 cells/IIQ to obtain a mast cell suspension.

(2) 20 μQ of phosphate buffer containing the test compound and 20 μQ of mast cell suspension were reacted for 10 minutes at 37°C, and compound 48/8 was added to the final concentration of 1 μg/raQ.
0 μa of solution was added and the reaction was continued for an additional 10 minutes. After the reaction, the mixture was cooled with water for 1 minute, and then centrifuged at 3000 x g for 4 minutes to separate the supernatant and cells. The supernatant is the 0nd
Fluorescence quantification of histamine was performed according to the HPLC method of A et al., and the value was expressed as a relative value, with the amount of histamine taken as 100 when the cells were completely disrupted with 0.5% Triton X-100.

(3) The effects of substances Nα1 to Nα4 on the mast cell degranulation effect induced by compound 4g/80 at 1 μg/mQ were as follows.

(3) -(i) Substances Nα1 to Nα4 all exhibit an inhibitory effect on mast cell degranulation at low concentrations, and the IC6 of substances Nα1 and Nα3 is
The o values were both 4 μg/llQ. Substance Nα2 and Blood 4 were trace components and difficult to measure quantitatively, but they had similar activities to Substance N (11 and Nci3).

(3) - (ii) Substances &1 to h4 exhibit mast cell degranulation effects when used alone in the high concentration range, and this effect occurs when the calcium ion concentration of the reaction solution is approximately 1 mM or higher, and It showed calcium ion dependence, which was not expressed when the calcium ion concentration was close to O.

(3) -(iii) After the reaction, add cholesterol to a constant concentration (approximately 2μg10+Q
) By adding the above, the above (3) - (i) and (
The effects of 3)-(ii) decreased in a concentration-dependent manner on cholesterol.

[Brief explanation of the drawing]

Figure 1 is a graph showing the results of separation by high performance liquid chromatography; Figure 1 (a) is a graph showing the results for mast cell function regulators purified from culture filtrate;
) shows the results for eurocidin. In FIG. 1, the vertical axis shows the intensity of ultraviolet absorption at 350 nm, the horizontal axis shows the elution time (minutes), and Nα1 to Nα4 in FIG. 1(b) indicate the constituent components of eurocidin corresponding to each peak. Second
The figure shows the ultraviolet absorption spectra of substances Nα1 to Nα4 in the constituent components of Eurocidin, and the vertical axis is the intensity of ultraviolet absorption,
The horizontal axis indicates wavelength (nm). Diagrams (a) to (d) in FIG. 2 show ultraviolet absorption spectra of substances Nα1 to Nα4 corresponding to the respective peaks in FIG. 1(b), respectively. Figure 3 shows an ultraviolet absorption spectrum diagram, where curve 1 represents the ultraviolet absorption of a substance in the constituent components of eurocidin (substance equivalent to Nα3), and curve a represents the ultraviolet absorption of a mast cell function regulating substance (substance equivalent to Nα3) purified from the culture filtrate. The spectrum is shown. The vertical axis shows the intensity of ultraviolet absorption, and the horizontal axis shows the wavelength (nm). Designated agent: Agency of Industrial Science and Technology, Institute of Microbial Technology, Chodai
Toshiaki Ikeura, Sub-Attorney, Patent Attorney, Yamatsugu Department Figure 1 (a) (b) Elution time Figure 2 (c) (d) Procedures Amendment Book July 2, 1986 Director-General of the Patent Office U Tono Ka Michibe 1゜Indication of the case Patent Application No. 69559 of 19852, Name of the invention Mast cell function regulator 3, Relationship to the amended person case Patent applicant address 1-3 Kasumigaseki, Chiyoda-ku, Tokyo Number 1 name
(114) Kozo Iizuka, Director of the Agency of Industrial Science and Technology (and 1 other person) 4. Successor Rito 151 Address 1-58-10 Yoyogi, Shibuya-ku, Tokyo -
Nishiwaki Building 113 Name (7450) Patent Attorney Toshiaki Ikeura Telephone (370) 2533 5 Date of amendment order Proprietor 6 Number of inventions increased by amendment 07 Column for detailed explanation of the invention in the specification to be amended 8. Contents of amendment The following amendments will be made to the specification of this application. (1) "Culture filtrate" on page 2, line 15 and page 3, line 14. Corrected to "culture solution". (2) “About 300-350” on page 3, lines 17 to 18
"Exhibits an ultraviolet absorption spectrum absorption pole at about 30 nm."
The ultraviolet absorption spectrum shows an absorption maximum in the range 0 to 350 nm.'' (3) "Microproducts" in line 6 of page 4 will be corrected to "microorganisms." (4) Page 5, lines 6 to 11 [This substance can be produced. , , , , (omitted) , , , , In addition, this substance has been amended to read as follows: ``This substance does not contain bacterial cells in methanol, etc.'' [This substance can be produced.] The six substances can be collected from the culture filtrate using organic solvent extraction method, ion exchange resin method, adsorption/desorption method using adsorbent, high performance liquid chromatography method, etc. etc.” (5) Page 5, line 14 and 1
Correct "in culture medium" in line 5 to "in culture filtrate". (6) On page 7, line 7, change “measured by” to “
The degranulation state was measured by this. ” will be corrected. (7) "Antigen-antibody complexes, etc." on page 7, lines 11 and 12 will be corrected to "products that utilize antigen-antibody reactions." (8) On page 7, line 12, "any drug" should be corrected to "any drug or method." (9) On page 10, line 4, "disperse the bacterial cells and culture solution" is corrected to "separate the bacterial cells and culture filtrate." (10) On page 10, line 5, "Culture solution 18I2 was obtained. Of this culture solution" should be corrected to "Culture filtrate 18Q was obtained. of this culture filtrate." (11) “Substance Eurocidin” on page 10, line 13,
Corrected to "Eurocidin-containing substance." (12) Correct "each fraction" in line 10 of page 11 to "each fraction". (13) Correct "culture solution" in line 16 of page 11 to "culture-filtrate." (14) “Prepared” on page 13, lines 16 to 17
is corrected to "adjusted." (15) Correct “phosphate buffer” in line 3 of page 14 to “physiological saline”. (16) Page 14, line 7, “After water cooling, 3000
Correct gJ to "1500Xg after cooling on ice." (17) Page 14, line 10, ro, s%”, ro, 0
Corrected to 5%. (18) “After reaction” on page 15, line 11 is replaced with “reaction liquid”
I will correct it. (19) On page 16, line 15, "indicates the messenger." is corrected to "indicates."

Claims (1)

[Claims] (1) A mast cell function regulating agent containing as an active ingredient a substance that is a constituent of Eurocidin and exhibits an absorption maximum at a wavelength of about 300 to 350 nm in the ultraviolet absorption spectrum.