JPH0474336B2 - - Google Patents

Description

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

Background of the Invention Various anti-allergic agents have been developed for the treatment of allergic diseases, but as the mechanisms of allergic reactions that cause allergic diseases have been elucidated, it has become easier to treat allergic reactions directly, rather than symptomatically. There is a growing desire to develop 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.

DISCLOSURE OF THE INVENTION The present invention relates to a purified chloroform extract of a culture obtained by culturing Moscus mycelium in a liquid medium containing 0.2 to 10 w/v% of glucose and 0.2 to 2 w/v% of wheat germ as essential components. In minutes,
(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) oleic acid, palmitic acid, stearic acid and linoleic acid are used. The present invention provides an anti-allergic agent characterized in that the active ingredient is a fraction whose main component is a mixture with at least one fatty acid selected from the group consisting of:

The liquid culture of C. chinensis 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. 43318/1983 by the present applicant, and the present invention also uses the method of culturing the mycelium of C. monocytogenes in accordance with this method. Cultivate the mycelium.

That is, glucose
0.2 to 10 w/v%, preferably 1 to 8 w/v% and wheat germ 0.2 to 2 w/v%, preferably 0.5 to
It is inoculated into a liquid medium containing 1.0 w/v% and cultured under aerobic conditions.

The amounts of glucose and wheat germ in the liquid medium are preferably set to the above-mentioned ratios from the viewpoint of culturing efficiency and economic efficiency of the L. chinensis mycelium,
In particular, the weight ratio of wheat germ:glucose is 1:1 or more.
It is preferable to set it to 8. 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, by adding each predetermined component to water, dispersing it,
After dissolution, it is sterilized at 120-130°C for 15-30 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 amount of inoculation of seed hyphae is about 5~
About 10 mg/100 ml, stirring at 10 to 300 rpm, 25
Cultivation is carried out in the dark at ~35° C. for 7 to 21 days at an aeration rate of 50 to 200/min.

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 extract the C. chinensis mycelium culture obtained above with chloroform as it is, the mycelium is usually removed according to a conventional method, and the culture solution is concentrated under reduced pressure and dried. , is preferably subjected to chloroform 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, developed with benzene-ethyl acetate (8:2), and the Rf value was 0.3 to 0.4.
A desired purified fraction is obtained.

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 10% of oleic acid is present in the fatty acid mixture. It accounts for ~40%. This purified fraction generally exhibits a liquid to gel state, and is soluble in chloroform, ethyl ether, benzene, acetone, ethyl acetate, and ethanol, sparingly soluble in water, and insoluble in hexane.
Shows color reactions with iodine, 10% sulfuric acid, 1% vanillin sulfate, phosphomolybdic acid, dichlorophenol indophenol, and bromcresol green.

The purified fraction is mainly composed of fatty acids, which are known as food ingredients, and is derived from C. chinensis, which has been used as a herbal medicine since ancient times.
Its toxicity is very low and it can be suitably used as a medicine.

Thus, the antiallergic agent of the present invention can be prepared by adding an effective amount of the purified fraction to a pharmaceutically acceptable carrier or diluent, such as an excipient, a binder, a disintegrant, a lubricant, etc., according to conventional formulation techniques. , solvents, tonicity agents, emulsifiers, suspending agents, stabilizers to form dosage forms for oral or parenteral administration, such as tablets, powders, granules, capsules, aerosols, syrups, injections, and dots. nasal medicine,
It can be an ointment, cream, emulsion, lotion, etc. Preferably, it is in dosage unit form containing 1 to 80 mg of the purified fraction.

The antiallergic agent of the present invention can be used to treat allergic diseases such as bronchial asthma, allergic rhinitis, vasomotor rhinitis, acute rhinitis, combing, nasal discharge and cough associated with upper respiratory infections such as the common cold, hay fever, etc. Administered to mammals orally or parenterally. In the case of oral administration, the appropriate dose is about 10 to 80 mg of the purified fraction per day for an adult human.

Examples 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 g of water
ml of liquid medium was inoculated with Moscus mycelia 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 2.4.

The obtained seed culture 2.2 was mixed with 750 g of wheat germ,
Add to a liquid medium consisting of 3000 g of glucose and 150 g of water, stir at 64-95 rpm, and stir at 80-160 rpm.
The cells were cultured in the dark for 14 days at 30-32°C with an aeration volume of 10 min.

After culturing, add 50% water to the culture and stir.
Next, the mycelium was removed using a continuous centrifugal filter,
Approximately 130 g of culture liquid was obtained. The mycelium was washed with 60 g of water, and the culture solution and washing solution were combined and concentrated to dryness under reduced pressure to obtain 500 g of the desired culture in the form of a yellowish brown powder.

Chloroform extraction and preparation of purified fractions 200 g of the obtained powdered culture was extracted with chloroform.
The extract was extracted twice with 1.5 liters of water for 1 hour each while hot, the extracts were combined, and the chloroform was distilled off under reduced pressure to obtain 4 g of a chloroform extract as a dark brown oil.

This chloroform extract was loaded onto a 500 g column of silica gel, and then benzene 2 (fraction 1), chloroform 6 (fraction 2, 3, 4 and 5), and then
Elution was carried out sequentially with chloroform-methanol (95:5) 6 (fractions 6 and 7), and the solvent of each fraction was distilled off to obtain brown oily substances (fraction 1: 54 mg,
Fraction 2: 437 mg, Fraction 3: 27 mg, Fraction 4: 8 mg,
Fraction 5: 315mg, Fraction 6: 594mg and Fraction 7:
508mg).

Fractions 5 and 6 were combined, dissolved in a small amount of chloroform, placed on a 100 g column of silica gel, and chloroform 13 (fractions 5-1, 5-2 and 5-
3), and the solvent of each fraction was distilled off to obtain oily substances (Fraction 5-1: 124 mg, Fraction 5-1: 124 mg, Fraction 5-1: 124 mg,
2: 205 mg and fraction 5-3: 235 mg).

Fraction 5-3 was dissolved in a small amount of benzene, placed on a column containing 20 g of silica gel, and 880 ml of benzene (fractions 5-3-1, 5-3-2 and 5-3-3) and benzene-ethyl acetate (95 :5) 1070ml (fractions 5-3-4, 5-3-5 and 5-3-6) were sequentially eluted and the solvent of each fraction was distilled off to obtain an oily substance (fraction 5-3-1: 7 mg, fraction 5-3-
2: 6 mg, fraction 5-3-3: 38 mg, fraction 5-3-
4: 35 mg, fraction 5-3-5: 17 mg and fraction 5-
3-6: 10 mg).

Fractions 5-3-2 to 5-3-4 were combined, subjected to preparative silica gel thin layer chromatography, and developed with benzene-ethyl acetate (8:2) to give an Rf value of 0.3 to 0.4.
69 mg of the desired purified fraction in the form of a white translucent gel was obtained.

This purified fraction is soluble in chloroform, ethyl ether, benzene, acetone, ethyl acetate, ethanol, slightly soluble in water, insoluble in hexane, and contains iodine, 10% sulfuric acid, 1% vanillin sulfuric acid, phosphomolybdic acid, and dichlorophenol. Shows color reaction with indophenol and bromucquesol green. In addition, it does not have an absorption band in the ultraviolet region and shows a nuclear magnetic resonance spectrum (CDCl ₃ ) as shown in the attached Figure 1.
5% Unisol 400 (60-80 mesh Chromosorb W, 2m x 3mm) column, column temperature 240℃,
FID detection) shows a chromatogram as shown in the attached Figure 2.

Tablet for oral administration Using the obtained purified fraction, according to the following recipe,
Antiallergic tablets for oral administration were obtained by direct compression.

Ingredients Purified fraction by weight 30 Calcium phosphate 490 Crystalline cellulose 350 Carboxymethylcellulose 120 Magnesium stearate 10 Example 2 Using the purified fraction obtained in Example 1, according to the following recipe, anti-allergic product was produced by wet granulation using a conventional method. Granules for oral administration were obtained.

Ingredients Part by weight Purified fraction (Example 1) 380 Lactose 480 Polyvinylpyrrolidone 45 Hydroxypropylcellulose 95 Example 3 Using the purified fraction obtained in Example 1, an anti-allergic ointment was prepared in a conventional manner according to the following recipe. Obtained.

Ingredients Purified fraction by weight (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 Using the purified fraction obtained in Example 1, an antiallergic emulsion was obtained in a conventional manner according to the following recipe.

Ingredients Purified fraction by weight (Example 1) 50 Stearic acid 20 Cethanol 5 Lanolin 20 Isopropyl myristate 20 Squalane 30 Liquid paraffin 80 Polyoxyethylene cetyl ether 17 Triethanolamine 10 Glycerin 40 Fragrance and preservative Appropriate amount of water Adjust to 1000 parts Example 5 Using the purified fraction obtained in Example 1, an antiallergic injection solution was obtained in accordance with the following recipe and 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 Examining the antiallergic activity of the purified fraction obtained in Example 1 Therefore, a test for suppressing histamine release from mast cells was conducted as follows.

Male Wistar rats weighing approximately 250 g were bled to death and then intraperitoneally injected with a physiological buffer solution (NaCl 154mM, KCl 2.7mM, _CaCl2 0.9mM, glucose 5.6mM, HEPES 5mM, PH7.4;
After injecting 10 ml of PS (referred to as PS), the abdomen was gently massaged for about 90 seconds, the abdomen was opened, and the intraperitoneal cell fluid was collected. The cell suspension was centrifuged at 100 xg for 5 minutes at 4°C, and the resulting cell pellet was washed twice with PS. After washing, resuspend in 5 ml of fresh PS.
By Percoll density gradient centrifugation,
Mast cells were purified with a purity of over 95%. The purified mast cells were suspended in PS at a concentration of 4×10 ⁵ cells/ml, and 50 μl of the suspension was dispensed into test tubes containing 1.6 ml of PS.

Purified fractions were added to a 0.25% sodium carboxymethyl cellulose (hereinafter referred to as CMC-Na) aqueous solution.
A 1mM suspension was prepared, subjected to ultrasonication for 10 minutes under ice cooling, and then diluted with a 0.25% CMC-Na aqueous solution to a predetermined purified fraction concentration to prepare a specimen suspension.

Add 0.2 ml of the sample suspension to the test tube containing the above mast cell suspension and hold at 37°C for 15 minutes.
(Manufactured by Wellcome) 0.2ml of solution (final concentration 0.35μ)
g/ml) and further incubated at 37°C for 10 minutes. The reaction was then stopped by cooling on ice, and centrifuged at 200 xg for 10 minutes at 4°C to separate the supernatant and cell pellet. 0.8N per 1ml of supernatant
1 ml of perchloric acid was added to prepare a sample for quantifying the amount of free histamine. Meanwhile, 4ml of 0.4N perchloric acid was added to the cell pellets and heated in a boiling water bath for 10 minutes to completely release the histamine remaining in the cells.
The mixture was centrifuged at 200×g for 10 minutes, and 2 ml of the supernatant was used as a sample for quantifying the amount of residual 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.

Histamine release rate (%) = free histamine amount / free histamine amount + residual histamine amount In the same way, without adding compound 48/80,
The release rate of histamine was calculated using only the purified fraction. Compounds at various purified fraction concentrations
Figure 3 shows the histamine release rates with and without the addition of 48/80.

Figure 3 is a graph with the histamine release rate (%) on the vertical axis and the purified fraction concentration (μg/ml) on the horizontal axis, where ○ indicates the case where Compound 48/80 was added and ● indicates the case without addition. .

As is clear from FIG. 3, the purified fraction well inhibits histamine release due to the addition of compound 48/80.

[Brief explanation of the drawing]

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

Claims (1)

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