JPS62209023A - Antiallergic agent - Google Patents

Abstract

PURPOSE:To provide an antiallergic agent effective in directly suppressing allergic reaction, by culturing mycelia of Fomes japonicus in a liquid medium containing glucose and wheat embryo, extracting the culture product with chloroform and using a specific extracted fraction as an active component. CONSTITUTION:Mycelia of Fomes japonicus are cultured in a liquid medium containing 0.2-10w/v% glucose and 0.2-2w/v% wheat embryo as essential components. The obtained culture produce is extracted with chloroform, subjected to silica gel column chromatography and developed with benzene-ethyl acetate mixture (8:2) and a fraction having Rf value of 0.3-0.4 is collected. The purified fraction thus obtained is composed mainly of a mixture of oleic acid and one or more kinds of fatty acids selected from palmitic acid, stearic acid and linoleic acid and exhibits antiallergic activity. Especially, the agent remarkably suppresses histamine-liberation reaction from mast cell caused by antigen-antibody reaction.

Description

[Detailed description of the invention] field of invention The present invention relates to a novel antiallergic agent.

Various anti-allergic drugs have been developed for the treatment of allergic diseases, but as the mechanisms of allergic reactions that cause allergic diseases have been elucidated, direct drug therapy has become more important than symptomatic drugs. There is a growing desire for the development of anti-allergic agents that suppress allergic reactions.

While investigating the pharmacological effects of a liquid culture of the mycelium of C. chinensis, known as Ganoderma lucidum and prized as a herbal medicine since ancient times, the present inventors discovered that its extracted fraction, which mainly consists of certain fatty acids, It has been found that this compound exhibits an extremely excellent effect on suppressing allergic reactions, particularly suppressing histamine release reactions from mast cells due to antigen-antibody reactions, and is useful as an anti-allergic agent that directly suppresses allergic reactions.

1. The present invention is a culture obtained by culturing C. chinensis mycelium in a liquid medium containing 0.2 to low/v% glucose and 0.2 to 2 w/v% wheat germ as essential components. A purified fraction of the chloroform extract of (a) having an Rf value of 0.3 to 0.4 in thin layer chromatography on silica gel using benzene-acetic acid (8:2) as a developing solvent.
and (b) oleic acid and at least one selected from the group consisting of palmitic acid, stearic acid and linoleic acid.
The present invention provides an anti-allergic agent characterized in that the active ingredient is a fraction whose main component is a mixture of various fatty acids.

The liquid culture of Cinnamon mycelium for obtaining the fraction used as the active ingredient of the antiallergic agent of the present invention is already known from Japanese Patent Application Laid-Open No. 60-43318 by the present applicant.
In the present invention, the Mycelium mycelium is also cultured accordingly.

That is, the hyphae of C. monocytogenes were exposed to 0.2 to 1 liters of glucose.
ov/v%, preferably I-8w/v% and wheat germ 0.2-2w/v%, preferably 0.5-1. Ow
/v% and cultured under aerobic conditions.

It is preferable that the amounts of glucose and wheat germ in the liquid medium be in the above-mentioned ratio from the viewpoint of culturing efficiency and economical efficiency of the Cinderella mycelium, and in particular, the weight ratio of wheat germ nigercose should be 1:1 to 8. is preferred. If desired, the liquid medium may contain mineral components such as salts such as phosphorus, manganese, magnesium, calcium, and iron, as well as other grain germs, rice bran, corn staple liquor, vitamins, nucleic acids, starch, and amino acids. , yeast extract, peptone, and other nutritional ingredients may be added as appropriate.

The liquid medium can be prepared according to a conventional method. For example, each predetermined component is added to water, dispersed and dissolved, and then
It is sterilized at 130° C. for 15 to 30 minutes and then cultured.

The seed hyphae to be used may be any species as long as they belong to the family Aridaceae, which belongs to the Basidiomycetes order. Usually, the inoculum amount of seed hyphae is about 5-10x9/100x
(about 10 to 300 r, p, m.

Culture is carried out in the dark for 7 to 21 days at 25 to 35° C. with aeration at a total rate of 50 to 200 Q/min under stirring.

The fraction used as the active ingredient of the antiallergic agent of the present invention is obtained by extracting the obtained culture with chloroform and purifying it by chromatography.

That is, although it is possible to directly extract the C. chinensis mycelium culture obtained above with chloroform, the mycelium is usually removed according to a conventional method, the culture solution is concentrated under reduced pressure, dried, and extracted with chloroform. Preferably, it is subjected to extraction.

Chloroform extraction can be carried out according to a conventional method, but it is preferable to repeat the extraction several times under heat for about 1 to 24 hours, and then remove the chloroform under reduced pressure to obtain a chloroform extract.

This chloroform extract is subjected to silica gel column chromatography and eluted with benzene, chloroform and chloroform-methanol. The fractions eluting last with chloroform and the fractions eluting first with chloroform-methanol are combined and chromatographed again with chloroform on a silica gel column.

The last eluted fraction is again subjected to silica gel column chromatography and eluted with benzene and benzene-ethyl acetate. The eluted fraction was subjected to preparative silica gel thin layer chromatography, and benzene-ethyl acetate (8:
2), and [(io, 3-0°4 fraction was collected,
Obtain the desired purified fraction.

The obtained purified fraction mainly contains a mixture of oleic acid and at least one fatty acid selected from the group consisting of palmitic acid, stearic acid, and linoleic acid, and usually
In the fatty acid mixture, oleic acid accounts for 10-40%. This purified fraction is generally liquid to gel-like, soluble in chloroform, ethyl ether, benzene, acetone, ethyl acetate, ethanol, sparingly soluble in water, insoluble in hexane, iodine, 10% sulfuric acid, 1% Shows color reaction with vanillin sulfate, phosphomolybdic acid, dichlorophenolindophenol and bromcresol green.

The purified fraction is mainly composed of fatty acids known as food ingredients, and is also derived from Cinnamon mushroom, which has been used as a herbal medicine since ancient times, and its toxicity is very low, so it can be suitably used as a medicine.

Thus, the antiallergic agent of the present invention can be prepared by adding the purified fraction of active ingredient 1 to a pharmaceutically acceptable carrier or diluent, such as an excipient, a binder, a disintegrant, a lubricant, or a solvent. , tonicity agents, emulsifiers, fP, E agents, and stabilizers for oral or parenteral administration, such as tablets, powders, granules, capsules, aerosols, syrups, injections, and dots. It can be used as a nasal spray, ointment, cream, emulsion, lotion, etc. Preferably, 1 to 80xg of the purified fraction
in dosage unit form containing.

The antiallergic agent of the present invention can be used to treat allergic diseases, such as bronchial asthma, allergic rhinitis, vasomotor rhinitis, acute rhinitis, and sneezing, nasal discharge, and cough rash associated with upper respiratory infections such as the common cold.
It is administered orally or parenterally to humans or mammals for the treatment of hay fever, etc. For oral administration, the dosage is
Approximately 10 to 80 to 9 of the purified fraction is suitable for each adult human.

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

Example 1 Preparation of Bamboo shoots mycelium culture 0.75 g of wheat germ, 3 g of glucose and 150 d of water
A liquid medium consisting of the following was inoculated with Moscus mycelium and cultured with shaking at room temperature for 4 days. The obtained culture was further inoculated into the same medium and cultured with shaking at room temperature for 4 days to obtain seed culture 2.412.

The obtained seed culture 2.2Q was added to a liquid medium consisting of wheat germ 7509, glucose 30009 and water 15012, and stirred at 64-95 r, p, m, 8.
14 at 30-32°C at an air flow rate of 0-16012/min
The cells were cultured in the dark for 1 day.

After culturing, 50Q of water was added to the culture, stirred, and then the mycelium was removed using a continuous centrifugal filter to reduce the culture solution to about 13ml.
Obtained 0Q. The mycelium was washed with water 60 (2), and the culture solution and washing solution were combined and concentrated to dryness under reduced pressure to obtain the desired culture 5009 in the form of a tan powder.

Chloroform extraction and preparation of purified fractions The obtained powdered culture 2009 was extracted with chloroform 1. The extracts were extracted twice for 1 hour each time with 100 ml each, the extracts were combined, and the chloroform was distilled off under reduced pressure to obtain a dark brown oily chloroform extract.
I got it.

This chloroform extract was placed on a 500 g column of silica gel, and benzene 2Q (fraction 1), chloroform 6f2
(Fractions 2.3.4 and 5), then chloroform-
Elution was carried out sequentially with methanol (95:5) 6 (2 (fractions 6 and 7), and the solvent of each fraction was distilled off to give brown oily substances (fraction 1: 54R9, fraction 2: 437 scraps 2, fraction 3: 27Jl1g, fraction 4: 8B, fraction 5: 3153!
9, fraction 6:594 and fraction 7:50819).

Fractions 5 and 6 were combined and dissolved in a small amount of chloroform;
Place it on a column of 100y of silica gel and chloroform 13
f2 (fraction 5-1.5-2 and 5-3), and the solvent of each fraction was distilled off to obtain an oily substance (fraction 5-1.5-2 and 5-3).
1:124xy, fraction 5-2:205g and fraction 5
-3:235 decay).

Fraction 5-3 was dissolved in a small amount of benzene and silica gel 20
9 column, benzene 880m(!(Fraction 5-3
-1,5-3-2 and 5-3-3) and benzene-
Ethyl acetate (95:5) 1070 shoulder Q (fraction 5-3-4
．． 5-3-5 and 5-3-6), and the solvent of each fraction was distilled off to obtain an oily substance (fraction 5-3-6).
1 near m9, fraction 5-3 = 2:6 m9, fraction 5-3-3
:381119, Fraction 5-3-4: 351g, Fraction 5-
3-5:17 ziF and fraction 5-3-6:10ziF).

Fractions 5-3-2 to 5-3-4 were combined, subjected to preparative silica gel thin layer chromatography, and benzene-ethyl acetate (
8:2) and fractions with an Rf value of 0.3 to 0°4 were collected to obtain the desired purified fraction 69R9 in the form of a white translucent gel.

This purified fraction contains chloroporum, ethyl ether, benzene, acetone, ethyl acetate, soluble in ethanol, slightly soluble in water, insoluble in hexadiene, iodine, 10% sulfuric acid, 1% vanillin sulfuric acid, phosphomolybdic acid, dichlorophenol. Shows color reaction with indophenol and bromcresol green. In addition, it does not have an absorption band in the ultraviolet region, and shows a nuclear magnetic resonance spectrum (CDC (!3) as shown in the attached Figure 1.
5% Ninisol 400 (60-80 mesh Chromosorb W, 2mX3mm) column, column temperature 240℃, F
ID detection) shows a romatogram as shown in the attached Figure 2.

Tablets for Oral Administration Using the obtained purified fraction, anti-allergic tablets for oral administration were obtained by direct keystroke according to the recipe of GG.

Component Weight part Purified fraction
30 calcium phosphate
490 crystalline cellulose
350 carboxymethyl cellulose
120 Magnesium Stearate 1
Example 2 Using the purified fraction obtained in Example 1, anti-allergic granules for oral administration were obtained by wet granulation in a conventional manner according to the recipe of GG.

Component Weight part Purified fraction (
Example 1) 380 lactose
480 polyvinylpyrrolidone
45 Hydroxypropyl Cellulose 95 Example 3 Using the purified fraction obtained in Example I, an anti-allergy ointment was obtained in a conventional manner according to the recipe of GG.

Component Weight part Purified fraction (
Example 1) 20 beeswax
100 paraffin wax
60 lanolin
30 isopropyl myristate
60 Squalane 80 Liquid paraffin 250 Polyoxyethylene sorbitan monostearate 18 Propylene glycol 50 Borax
7 water
325 Example 4 Example! Using the purified fraction obtained in step 1, an anti-allergic emulsion was obtained in a conventional manner according to the following recipe.

Component Weight part Purified fraction (
Example 1) 50 stearic acid
20 cetanol
5 lanolin
20 isopropyl myristate
20 Squalane 30 Liquid paraffin 80 Polyoxyethylene cetyl ether 17 Triethanolamine IO glycerin
40 Fragrances and Preservatives
Adjust the appropriate amount of water to 1000 parts Example 5 Using the purified fraction obtained in Example 1, according to the following recipe,
An antiallergic injection solution was obtained according to a conventional method.

Ingredients Part by weight Purified fraction (Example 1) 1 Polyoxyethylene sorbitan monooleate (20EO) 1 Distilled water for injection Adjusted to 100 parts Effect of the invention Antiallergic activity of the purified fraction obtained in Example 1 To investigate, a test for inhibiting histamine release from mast cells was conducted as follows.

Male Wistar rats weighing 2509 were bled to death and then intraperitoneally injected with physiological buffer solution (N
aC12154mM5KC122,7mM, CaCLo
.

911M% Glucose 5.6mM, HEPES 5m
M.

pH 7.4, hereinafter referred to as PS, was injected with about 9
After gentle abdominal surgery for 0 seconds, the abdomen was opened and the peritoneal cell fluid was collected. The cell suspension was centrifuged using TOOX9 at 4°C for 5 minutes, and the resulting cell pellet was washed twice with PS. After washing, it was resuspended in fresh r'55Rc and purified by Percoll density gradient centrifugation.
Mast cells were purified to a purity of 5% or higher. The purified mast cells were divided into 4 x 10' cells/x(l) concentration teps
l=@iEl, and put 5 in the test tube containing PSl and 6jIQ.
It was dispensed in 0μσ portions.

A 1 mM suspension of the purified fraction was prepared with a 0.25% sodium carboxymethylcellulose (hereinafter referred to as CMC-Na) aqueous solution, and after ultrasonication under water cooling for 10 minutes, 0.25% CMC was added. The purified fraction was diluted with a -Na aqueous solution to a predetermined concentration to prepare a sample suspension.

Add 0.2xQ of the sample suspension to the test tube containing the mast cell suspension and hold at 37°C for 15 minutes. Solution 0, 2xc (manufactured by Wellcome Inc.) (final concentration 0.35μg/x
σ) was added and further incubated at 37°C for 10 minutes. Then, the reaction was stopped by water cooling, and the reaction was heated at 200x.
One species was centrifuged at 4°C for 10 minutes to separate the supernatant and cell pellet.

0.8N perchloric acid 1xf2 was added to the supernatant 1tRQ to prepare a sample for quantifying the amount of free histamine. Meanwhile, 4tIQ of 0.4N perchloric acid was added to the cell pellets and
The cells were heated for 1 minute to completely release histamine remaining in the cells, centrifuged at 200 x g for 10 minutes, and 2 inches of the supernatant was used as a sample for quantifying the amount of remaining histamine. The amount of histamine in each sample was measured by a fluorescence method, and the histamine release rate was calculated from the following formula.

Similarly, the release rate of histamine was calculated using only the purified fraction without adding Compound 48/80. FIG. 3 shows histamine release rates with and without Compound 48/80m at various purified fraction concentrations.

Figure 3 shows the histamine release rate (%) on the vertical axis, and the purified fraction concentration (μ9/R (D) on the horizontal axis, where ○ indicates the case where compound 48/80 was added, and . show.

As is clear from Figure 3, the purified fraction is compound 4.
Histamine release by addition of 8/80 is well suppressed.

[Brief explanation of drawings]

Figure 1 is a nuclear magnetic resonance spectrum of the purified fraction obtained in Example 1, Figure 2 is a chromatogram of its gas chromatography, and Figure 3 is a graph showing the histamine release inhibiting effect of the purified fraction. .

Claims (1)

[Claims] (1) Purified fraction of the chloroform extract of the culture obtained by culturing C. chinensis mycelium in a liquid medium containing 0.2 to 10 w/v% glucose and 0.2 to 2 w/v% wheat germ as essential components. (a) The Rf value of thin layer chromatography on silica gel using benzene-ethyl acetate (8:2) as a developing solvent is 0.3 to 0.4, and (
(b) An anti-allergic agent characterized in that the active ingredient is a fraction containing a mixture of oleic acid and at least one fatty acid selected from the group consisting of palmitic acid, stearic acid and linoleic acid.