JPH0415770B2 - - Google Patents

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 have many granules containing biologically active substances called chemical mediators inside the cells. 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. [Purpose] Therefore, the present inventors extensively investigated whether a substance with such an effect could be produced, and found that the known eurocidin produced by Streptoverteicillium (Japanese Society of Agricultural Chemistry Journal)
Vol. 29, pp. 650-652, 1955), it was recognized that substances whose ultraviolet absorption spectra exhibit maximum absorption in the wavelength range of about 300 to 350 nm (including the shoulder) have a mast cell function regulating effect, and commercially available Streptoverticillium eurocidicum (IFO No.
13491) was found to produce a substance that has a strong mast cell function regulating effect in the culture solution, and as a result of isolation and purification of this substance, it was confirmed that it is a component of the same eurocidin as above. . Therefore, an object of the present invention is to provide a novel mast cell function regulator. [Configuration] The present invention relates to eurocidin, which is the main component of the product of Streptoverteicillium bacteria, and whose ultraviolet absorption spectrum is approximately 302 nm, approximately 316 nm, and approximately 302 nm, approximately 316 nm, and approximately
The present invention provides a mast cell function regulator comprising at least one compound selected from a group of compounds exhibiting maximum absorption at 332 nm and about 349 nm. The present inventors are the first to discover that eurocidin has a functional regulatory effect on mast cell function. The eurocidin referred to herein is a compound produced by Streptoverteicillium bacteria, and is a general term for compounds having a basic skeleton structure represented by the following formula. (In the formula, R ¹ to ^{R 5} are hydrogen atoms or substituents.) The specific eurocidin compound according to the present invention is hereinafter referred to as the present substance. This substance can be isolated and obtained from commercially available eurocidin or a culture solution of eurocidin-producing microorganisms using high performance liquid chromatography (HPLC). For example, when HPLC separation is performed using a column with a diameter of 10 mm and a length of 250 nm (using Nucleosil 5C8 as a packing material), the separation shown in Figure 1b is obtained, and the ultraviolet absorption spectrum exhibits an absorption maximum at approximately 300 to 350 nm. Substances consisting of four types of single compounds, that is, substances No. 1 to No. 4 corresponding to each of peaks No. 1 to No. 4 can be obtained. The ultraviolet absorption spectra of these substances No. 1 to No. 4 were
Shown in Figures a-d. Each of these substances is approximately
It exhibits absorption maxima (including a shoulder) at 302 nm, about 316 nm, about 332 nm, and about 349 nm. Furthermore, Figure 1 shows the HPLC separation results of this substance, Figure 1a shows the results for this substance separated and obtained from the culture filtrate of a eurocidin-producing microorganism, and Figure 1b shows the results for commercially available eurocidin. . In addition, this substance is Streptoverticillium eurocidicum (Streptoverticillium
eurocidicum, IFO No. 13491) strain or Streptoverticillium albireticuli, IFO No.
12737) strain can be cultured in a general medium, produced in the medium, and collected by a conventional method. For example, glucose, sucrose,
Maltose, dextrin, starch, glycerin, etc. are used as nitrogen sources, peptone, meat extract, yeast extract, malt extract, etc. are used as nitrogen sources, and inorganic salts such as NaCl, K ₂ HPO ₄ , Na ₂ HPO ₄ , MgSO ₄ are added. The cells are cultured in a neutral liquid medium with aeration and agitation. The culture medium is cultured at a pH of 5 to 8, preferably around 7. Culture temperature is usually 20-35℃
The temperature is preferably around 30°C. For example, glucose 1.5%, peptone 0.5%, meat extract 0.5%,
This substance can be produced by culturing at 30°C for 40 hours in a pH 7.0 liquid medium containing 0.5% yeast extract and 0.5% NaCl. Collection from culture filtrate can be performed using organic solvent extraction method, ion exchange resin method, adsorption/desorption method using adsorbent,
This can be carried out using high performance liquid chromatography or the like. This substance can also be obtained by extracting bacterial cells using an organic solvent such as methanol, followed by conventional purification operations such as concentration and crystallization. To separate and recover the substances contained in the culture filtrate, for example, add 1/10 volume of Amberlite XAD-7 to the culture filtrate and perform adsorption treatment at room temperature for about 4 hours with occasional stirring. After filtering Amberlite XAD-7 and washing it, 80%
Elute with 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, elution begins at around pH 5.7 using a pH gradient of 0.05M acetic acid-ammonium acetate. This eluate was further adjusted to pH 8.5 and subjected to DEAE-Toyopearl ion exchange chromatography.
Elution begins around 7.5. Furthermore, the active fraction
Adsorbed on MCI gel CHP-20P adsorption column, 40%
Substance No. 3 of this substance can be obtained by separating the 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 rat's peritoneal cavity, 4℃, 150×
After centrifuging for 10 minutes under the conditions of g, the precipitated cells were
Bovine serum albumin was separated by a method such as multilayer centrifugation to obtain a mast cell fraction. ¹⁰⁶ mast cells were added to Tyrode's solution containing 0.2% bovine serum albumin.
A mast cell suspension was obtained by suspending the mast cells at cells/ml. Next, the test compound was mixed with 20 μl of the mast cell suspension in 20 μl of physiological saline, and the mixture was allowed to react at 37°C for 10 minutes. at final concentration
1 μg/ml) was added and reacted for 10 minutes, cooled on ice, centrifuged, and the supernatant was collected. The histamine contained in the supernatant
HPLC method (Onda et al., Hiroshima, J.Med.Sci.,
27, pp. 93-97, 1978). In addition, as the degranulation inducing agent, in addition to Compound 48/80 (manufactured by Sigma), Concanavalin A (manufactured by Wako Pure Chemical Industries, Ltd.), Ionophore A23187
(manufactured by Calbiochem Behring) and any commonly known drugs or methods such as those utilizing antigen-antibody reactions can be used.
Next, measurements were performed with various concentrations of the test compound, and the concentration [IC ₅₀ ] value of the compound that inhibited 50% of mast cell degranulation induced by 1 μg/ml Compound 48/80 using this measurement system was determined. Ta. In addition, we also measured the functional regulatory effect of this substance on mast cells under various conditions, including in the absence of Compound 48/80 and in the presence of cholesterol.
As shown in the results below, this substance was found to have a strong mast cell function regulating effect. (1) All of these substances exhibit an inhibitory effect on mast cell decondylation at low concentrations, and their IC ₅₀ values are shown in the table below.
It was as shown in 1. (2) This substance independently exhibited a mast cell degranulation effect in a concentration range higher than the concentration exhibiting the effect described 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 (1) and (2) above by adding a substance having a sterol skeleton. In addition, in this measurement system, compound 48/8 with a concentration of 1μg/ml
The concentration of the compound that inhibits 50% of the mast cell degranulation effect induced by 1 unit/ml [1 unit/ml]
ml].

〔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
%, yeast extract 0.5%, and PH7.0 liquid medium containing 0.5% NaCl was dispensed into a 500 ml Sakaguchi flask.
Sterilize at 120℃ for 20 minutes. One platinum loop of the actinomycete Streptoverteicillium eurocidicum strain was inoculated onto this medium from a slanted agar medium, and cultured with reciprocating shakes at 30°C for 3 days. A medium 20 with the same composition as this culture solution was placed in a jar fermenter for 30, and 300 ml of the preculture solution of Streptoverteicillium eurocidicum strain was transferred to this medium, and the mixture was heated at 30°C.
Culture was carried out for 40 hours at 200 rotations and an air flow rate of 10 per minute.
After the culture was completed, the bacterial cells and the culture filtrate were separated by continuous centrifugation at 7000 rpm to obtain a black culture filtrate 18. 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 leaving 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, and only the soluble fraction was collected. After re-concentration, the resulting crystalline eurocidin-containing material was obtained. The activity of this substance against mast cells was approximately 113,000 units. Example 3 Commercially available Eurocidin (manufactured by Takeda Pharmaceutical Co., Ltd.) was dissolved in methanol and separated by HPLC. The separation conditions in this case are as follows. Column: Nucleosil 5C8 packed, diameter 10mn,
Length 250mm Solvent used: Acetonitrile/ammonium acetate buffer (0.01M, PH5.0) = 35/65 Flow rate: 4ml/min Detection: Absorbance measurement at ultraviolet wavelength 350nm Sample concentration: 0.2mg/ml Injection volume: 200μl When the constituent components of commercially available Eurocidin were separated under the above conditions, the separation results of each component were shown in Figure 1b, and the substance No. corresponding to the corresponding peak was shown.
Fractions No. 1 to No. 4 were obtained. After concentrating each fraction under reduced pressure, freeze-drying was repeated to obtain substances No. 1 to No. 4. The ultraviolet absorption spectra of these substances No. 1 to No. 4 are shown in Figure 2 a to d, each substance has a wavelength of about 302 nm,
It showed absorption maxima (including a shoulder) at about 316 nm, about 332 nm, and about 349 nm. Example 4 (1) Add the adsorbent Amberlite XAD-7 equivalent to 1/10 volume to 5 of the culture filtrate obtained in Example 1, and adsorb at room temperature for 4 hours with occasional shaking. Processed. 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. almost
Nearly 100% activity was recovered. (2) Next, CM-Toyopearl 650M column [manufactured by Toyo Soda Kogyo Co., Ltd., column volume 533ml, 0.05M acetic acid]
Equilibrate with ammonium acetate (PH4.0)] to PH4.0
Adsorb the sample solution adjusted to 0.05M acetic acid.
Elution was performed with ammonium acetate (PH6.0). The most strongly active fraction was eluted from the eluate at pH 5.7. The mast cell degranulation inhibitory activity of the active fraction is
It was 25,250 units. (3) Furthermore, after adjusting the active fraction from (2) above to pH 8.5, DEAE-Toyopearl 650M column [manufactured by Toyo Soda Kogyo Co., Ltd., column volume 179 ml, equilibrated to pH 8.5 with 0.05 M ammonium acetate-ammonia water. ]
It was adsorbed on the solution and eluted with 0.05M ammonium acetate-ammonia water (PH7.5). The activity of this active fraction was 16,500 units. (4) Furthermore, an adsorption column (column volume 40ml) packed with MCI gel (CHP-20P, manufactured by Mitsubishi Kasei)
The active fraction obtained in (3) above was adsorbed onto the
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. Dissolve this substance in methanol,
Ultraviolet absorption spectra were measured, and HPLC separation was performed under the separation conditions shown in Example 3 above. The results are shown in FIG. 1a and FIG. 3, respectively. The obtained substance was the same as substance No. 3 among the constituent components of commercially available Eurocidin. The amount of activity was 12,500 units. Example 5 (1) Female Wistar rat (weight 150-250g)
Bleed to death and inject 20 ml of Tyrode's solution intraperitoneally.
The abdomen was lightly massaged for about 2 minutes.
Ascites fluid was collected after laparotomy and centrifuged at 150 xg for 10 minutes at 4°C to collect precipitated cells. The cells were suspended in 2 ml of Tyrode's solution,
The cells were layered on 4 ml of physiological saline containing bovine serum albumin adjusted to a specific gravity of 1.068, and centrifuged at 4° C. and 100×g for 12 minutes, and the precipitated cells were collected.
After washing twice with Tyrode's solution, the mast cells were placed in Tyrode's solution containing 0.2% bovine serum albumin.
The mast cells were suspended at a concentration of 10 ⁶ cells/ml to obtain a mast cell suspension. (2) 20 μl of physiological saline containing the test compound and 20 μl of the mast cell suspension were reacted for 10 minutes at 37°C, then 10 μl of compound 48/80 solution was added to give a final concentration of 1 μg/ml, and the mixture was allowed to react for an additional 10 minutes. Ta. After the reaction, the mixture was cooled on ice for 1 minute and then centrifuged at 1500 xg for 4 minutes to separate the supernatant and cells. The supernatant was subjected to fluorescence quantification of histamine according to the HPLC method of Onda et al., and the value was determined using 0.05% Triton
The amount of histamine when cells are completely destroyed at 100
Expressed as a relative value set to 100. (3) The effects of substances No. 1 to No. 4 on the mast cell degranulation effect induced by Compound 48/80 at 1 μg/ml were as follows. (3) (i) Substances No. 1 to No. 4 all exhibit an inhibitory effect on mast cell degranulation at low concentrations;
Both IC ₅₀ values were 4 μg/ml. Substance No. 2 and
Although No. 4 was a trace component and difficult to quantitatively measure, it had the same activity as Substances No. 1 and No. 3. (3) (ii) Substances No. 1 to No. 4 independently exhibit a mast cell degranulation effect in the high concentration range, and this effect is expressed when the calcium ion concentration of the reaction solution is increased to approximately 1 mM or higher.
It showed calcium ion dependence, which was not expressed when the calcium ion concentration of the reaction solution was close to 0. (3) (iii) Add cholesterol to the reaction solution at a constant concentration (approximately 2 μg/
ml) or more, the above (3)-(i) and (3)-
The effect of (ii) on cholesterol decreased in a concentration-dependent manner.

[Brief explanation of drawings]

Figure 1 is a graph showing the separation results by high-performance liquid chromatography; Figure 1a is the result for a mast cell function regulator purified from the culture filtrate, and Figure 1b is the result for commercially available Eurocidin. shows. In Figure 1, the vertical axis shows the intensity of ultraviolet absorption at 350 nm, and the horizontal axis shows the elution time (minutes).
No. 1 to No. 4 in FIG. 1b indicate the constituent components of commercially available Eurocidin corresponding to each peak. Figure 2 shows substances No. 1 to No. 4 in the constituent components of commercially available Eurocidin.
The figure shows the ultraviolet absorption spectrum of , where the vertical axis shows the intensity of ultraviolet absorption and the horizontal axis shows the wavelength (nm). Figures a to d in Figure 2 show the ultraviolet absorption spectra of substances No. 1 to No. 4 corresponding to the respective peaks in Figure 1 b, respectively. Figure 3 shows an ultraviolet absorption spectrum diagram, and curve b shows a substance in the constituent components of commercially available Eurocidin (No.
Curve a shows the ultraviolet absorption spectrum of the mast cell function regulating substance (substance equivalent to No. 3) purified from the culture filtrate. The vertical axis shows the intensity of ultraviolet absorption, and the horizontal axis shows the wavelength (nm).

Claims (1)

[Claims] 1. Eurocidin, which is the main component of Streptoverteicillium products, has an ultraviolet absorption spectrum of about 302 nm, about 316 nm, about 332 nm, and about
A mast cell function regulator comprising at least one compound selected from a group of compounds that exhibit maximum absorption at 349 nm.