JPH08198764A - Allergic inflammation-inhibiting substance - Google Patents

Abstract

PURPOSE: To obtain an allergic inflammation-inhibiting substance comprising the extract of an actinomycete, Streptomyces variabilis, low in toxicity and high in usefulness. CONSTITUTION: The allergic inflammation-inhibiting substance comprises the extract of the culture solution of an actinomycete, Streptomyces variabilis, or the cells with an organic solvent. The extract is prepared into an allergic inflammation-inhibiting preparation containing the extract in an amount of 10<-8> to 50wt.%. The allergic inflammation-inhibiting preparation is administered at a does not of 1pg to 500mg/kg as the allergic inflammation-inhibiting substance once to four times a day. The culture is performed in a culture medium having nutritive ingredients (e.g. a liquid medium containing glucose, malt extract and yeast extract and having a pH of 2-9) at 14-42 deg.C for 1-14 days. The extraction is performed by leaving or stirring the mixture of the culture solution with an organic solvent (e.g. ethyl acetate) for 3-120min.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to suppression of allergic inflammation, which comprises a culture solution of Streptomyces variabilis actinomycete (hereinafter abbreviated as "S. variabilis") or a substance extracted from an bacterium thereof with an organic solvent. Regarding substances.

[0002]

2. Description of the Related Art Anti-inflammatory actions are roughly classified into non-allergic inflammation and allergic inflammation. Furthermore, allergic inflammation is roughly classified into types I to IV, and type I is involved in various atopic dermatitis, asthma, rhinitis and various other allergic diseases.
Type III reaction (immune complex reaction: Arthus reaction) and type IV reaction (cellular immune reaction: delayed hypersensitivity reaction) are autoimmune diseases such as chronic indirect rheumatism, as well as hepatitis, nephritis, dermatitis and infection. It has become clear that it plays an important role in the development of various inflammatory diseases such as illness.

Conventionally, as anti-inflammatory agents, steroidal anti-inflammatory agents such as hydrocortisone and prednisolone,
There are nonsteroidal anti-inflammatory drugs such as aspirin (Nikkei Science 3:70 (1991)) and indomethacin (Ther. Res. 3: 1057 (1985)).

[0004]

However, although steroidal anti-inflammatory agents are effective for allergic inflammation, they have a decreased infection defense function, gastrointestinal tract disorder, suppression of pituitary-adrenal cortex function, and decreased bone formation. There is a problem in that it causes side effects such as.
Further, the non-steroidal anti-inflammatory drug is considered to have higher safety than the steroid drug, but has a problem that the inhibitory effect on allergic inflammation is extremely weak. Therefore, more effective and less toxic anti-inflammatory agents are desired.

In view of the above points, an object of the present invention is to provide a novel anti-inflammatory substance effective for allergic inflammation and highly useful.

[0006]

In order to find out allergic inflammation-inhibiting substances for type III and type IV allergies, the present inventors have developed an animal model of type III allergic reaction in rat 4-hour heterologous passive skin anaphylaxis (4 As a result of screening using a rat tuberculin reaction, which is an animal model of type IV allergic reaction, as a result of the actinomycete S. The surprising phenomenon that a substance exhibiting an allergic inflammation-suppressing action is contained in a culture solution of Barrier-Bilis or a solvent extract of the bacterial cells has been found, and the present invention has been completed based on this finding.

That is, the allergic inflammation-inhibiting substance according to the present invention comprises a culture solution of Streptomyces variabilis or an extract obtained by organic solvent extraction from the cells.

The actinomycete S. Barrier bilis is available from public preservation institutions, for example, preservation bacteria of RIKEN (JCM 4422).
Bacteria such as ATCC 19815 in the US and CBS 568.68 in the Netherlands can be used.

Actinomyces S. Cultivation of Barrier bilis is performed using a medium containing appropriate nutrients. For liquid culture,
As a component of the medium, an aqueous solution containing one or several kinds of sugars such as glucose, proteins such as peptone and malt extract, vitamins, nucleic acids, amino acids, and complex carbohydrates is preferably used. As a typical medium example,
A YM liquid medium (including glucose, malt extract, yeast extract) can be mentioned. The pH of the liquid medium is preferably in the range of 2 to 9, and the culture temperature is preferably 15 to 42 ° C. The preferable culture time for liquid culture is 1 to 14 days. In the case of solid culture, the above-mentioned liquid culture medium containing agar is mainly used, but the culture conditions for solid culture are almost the same as those for liquid culture. Thus, S. After culturing the barrier bilis, solvent extraction treatment is performed. Solvent extraction is a method of directly contacting the culture solution with the solvent, a method of evaporating the culture solution to dryness and contacting the dried product with the solvent, a method of contacting the culture solution salting out product with the solvent, or contacting the cells with the solvent. Then, the active ingredient of the allergic inflammation-suppressing substance is obtained from the extraction phase.

Organic solvents are preferable as the solvent used for extracting the allergic inflammation-suppressing active ingredient, and typical examples thereof include esters such as ethyl acetate; alcohols such as methanol, ethanol and propanol; ethyl ether, dioxane and the like. Ethers; ketones such as acetone and methyl ethyl ketone; halogenated hydrocarbons such as dichloromethane and chloroform; but usable solvents are not limited to these. Also, a mixed solution of the above solvents can be used. Particularly suitable solvents are ethyl acetate, dichloromethane, acetone and the like.

The extraction time varies depending on the type of solvent and the extraction temperature, but is preferably in the range of 3 to 120 minutes. The liquid is allowed to stand during extraction or left with occasional stirring. Preferably, the extraction operation is repeated a plurality of times for the same sample. The extraction temperature is not particularly limited.

In formulating the allergic inflammation-suppressing substance according to the present invention, it is usually in the form of a pharmaceutical composition together with a pharmaceutical carrier. As a carrier, a filler, a disintegrating agent, a bulking agent, a binder, a coloring agent, a flavoring agent, a pH adjusting agent, a solubilizing agent, a suspending agent, which are usually used for preparing a drug depending on the dosage form, Buffer, stabilizer, preservative, moisturizer,
Examples thereof include surface active agents, lubricants and excipients. Further, by selecting an appropriate solvent, the obtained solvent extract or its concentrate can be used as it is as an external liquid preparation.

As the dosage unit form of the allergic inflammation suppressant obtained by using the substance of the present invention, in addition to the above external preparations, tablets, pills, drinking solutions, powders, suspensions, emulsions, granules , Extract, fine granule, syrup, dip, decoction, eye drop, troeach, supple, liniment,
Lotions, eye ointments, plasters, capsules, suppositories,
Examples include injections (solutions, suspensions, etc.), patches, ointments, inhalants, creams, sprays, nasal drops and the like.

The amount of the active substance of the present invention to be contained in the allergic inflammation inhibitor is not particularly limited and may be appropriately selected within a wide range, but preferably 10 in the allergic inflammation inhibitor.
^-8 to 50% by weight.

The allergic inflammation-suppressing agent obtained from the substance of the present invention is administered by a method suitable for various forms in its use. For example, in the case of the above external preparation, it is directly applied to a required site such as skin or mucous membrane, and orally in the case of tablets, pills, drinking solutions, suspensions, emulsions, granules and capsules. It is administered intravenously, intramuscularly, intracutaneously, subcutaneously or intraperitoneally in the case of injections, rectal in the case of suppositories, and applied in the case of patches, ointments and creams. , Inhalants, nasal drops are inhaled, and sprays are inhaled or applied.

The dose of the allergic inflammation suppressant obtained from the substance of the present invention is appropriately selected depending on the purpose of use, symptoms and the like, but usually 1 pg to 5 as the substance of the present invention per day.
The range is about 00 mg / kg. Further, it is of course possible to administer the above-mentioned pharmaceutical composition 1 to 4 times / day separately.

Regarding the safety of the allergic inflammation suppressant obtained from the substance of the present invention to humans and animals, there is no problem at all, as is clear from the results of toxicity test described later.

[0018]

EXAMPLES Next, examples of the present invention will be given to demonstrate the above effects.

Example (Preparation of Solvent Extract) Actinomyces S. cerevisiae purchased from RIKEN Barrier Billis (JC
M4422) in a 500 ml Erlenmeyer flask containing 100 ml of YM medium (containing 0.4% glucose, 1.0% malt extract and 0.4% yeast extract) at a temperature of 27.
Shaking culture (seed culture) was carried out at 4 ° C. for 4 days. Then, the above YM
A 50-liter jar containing 25 liters of medium was inoculated with 1.3 liters of the seed culture solution, and the aeration rate was 1 at 27 ° C.
The culture was carried out at 0 liter / min for 4 days at 200 rpm with shaking. The culture was centrifuged at 3,000 rpm for 20 minutes, 50 liters of the separated supernatant was adjusted to pH 2.0, and extracted twice with 50 liters of ethyl acetate. The cells obtained by centrifugation were extracted twice with 5 liters of acetone, the extract was concentrated, the pH of the concentrate was adjusted to 2.0, and 2 liters of ethyl acetate were used. Extraction was performed twice. The ethyl acetate extracts were combined and concentrated to dryness to obtain 5 g of extract.

Test Example 1 (Effect on Type III Allergic Reaction) i) Preparation of Rabbit Anti-Ovalbumin Serum Eda et al.'S method (Nippon Jpn Jpn., Vol. 66, p. 237, 1970)
Yearly), rabbit anti-ovalbumin serum was prepared by the following method. That is, an antigen solution was prepared, which consisted of an emulsified solution of a 2 mg / ml solution of ovalbumin (Sigma) dissolved in physiological saline and complete Freund's adjuvant (Difco). 0.5 ml of this antigen solution
Each of them was injected into the left and right gluteus muscularis of a New Zealand white male rabbit weighing about 3 kg four times a week. Seven days after the final injection, blood was collected from the carotid artery, and only serum was separately obtained and used as rabbit anti-ovalbumin serum. This antiserum rat 4 hour xenopathic skin anaphylaxis (heterolo
The titer of the gous PCA) reaction was 1:32.

Ii) Rat 4-hour heterogeneous PCA reaction (III
Type allergic skin reaction) to a final concentration of 25 m
The suspension was suspended in a solution prepared by adding 5% by weight of dimethyl sulfoxide to a 5% by weight aqueous solution of gum arabic so that the concentration would be g / ml. The suspension thus obtained was used as a test solution. As test animals, male Wistar rats weighing 120 to 200 g were used.

First, the above test solution was applied to rats at 2 ml / kg.
(S. 50 as a biliary broth ethyl acetate extract
(mg / kg rat) was intraperitoneally administered.

Then, 20 hours after the administration of the test solution, 0.05 ml of an injectable solution prepared by diluting the rabbit anti-ovalbumin serum with physiological saline four times was injected into the dorsal skin of the rat to inject the rat. It was sensitized with the above antiserum.

Further, 3 hours after the injection of the antiserum, the test solution was intraperitoneally administered again to the sensitized rat at 2 ml / kg (50 mg / kg as the extract amount rat).

Then, 1 hour after the second administration of the test solution, 0.5% by weight of Evans blue physiological saline containing 2 mg / ml of ovalbumin as a corresponding antigen was added to 2.
An intravenous injection of 5 ml / kg was performed to induce a heterogeneous PCA reaction for 4 hours.

The leaking pigment at the site where the intradermal reaction was elicited in this manner was determined by the method of Harada et al. (J. Pharm. Pharmacol. 23, 2).
Page 18, 1971). That is, the animals were sacrificed 1 hour after the antigen injection and the heterologous P
Cut the skin of the CA reaction area into small pieces, 0.3% (w / v)
_{4 in} a mixture of 3 volume of Na ₂ SO ₄ aqueous solution and 7 volume of acetone.
It was left to soak for 8 hours and the leaked dye was extracted. The thus-extracted dye was colorimetrically determined at 620 nm to obtain the amount of leaked dye, which was measured at the site injected with rabbit anti-ovalbumin serum.
It was expressed as the amount of leaked dye (μg) per (site).

As a control for this test, the aqueous solution of dimethyl sulfoxide-containing gum arabic gum containing no solvent extract was used in place of the test solution, and the other points were the same as in the above-mentioned procedure to determine the amount of leaked dye. I asked.

As a control test, instead of the above test solution, prednisolone was suspended at 25 mg / ml in a solution prepared by adding 5% by weight of dimethyl sulfoxide to 5% by weight aqueous solution of gum arabic, The resulting suspension was intraperitoneally administered at 2 ml / kg 3 hours before the induction, and the amount of leaked pigment was determined by performing the other operations in the same manner as above.

Each of these tests was carried out using 5 rats, and the amount of leaked pigment (μg / site) was the average of the values obtained for these rats.

The test results are shown in FIG.

As is apparent from this figure, in the group administered with the test solution containing the solvent extract of the Example, compared with the control group using the solution not containing the extract, the 4-hour heterogeneous PCA reaction part in the rat was used. The amount of pigment that leaks into the skin of the animal is significantly reduced, and remarkable type III allergic inflammation suppressive activity is observed.

The group to which the test solution containing prednisolone was administered also showed a similar effect on rat 4 compared to the control group.
The amount of pigment leaked to the skin at the time-different PCA reaction site was significantly reduced, and it exhibited type III allergic inflammation suppressive activity.

Test Example 2 (Action on Type IV Allergic Reaction) Next, the action of the substance of the present invention on rat type IV allergic inflammation (tuberculin reaction) was examined.

The dried extract obtained in the example was suspended in a solution prepared by adding 5% by weight of dimethyl sulfoxide to a 5% by weight aqueous solution of gum arabic so that the final concentration was 25 mg / ml. It became cloudy. The suspension thus obtained was used as a test solution. As test animals, male Wistar rats weighing 170 to 250 g were used.

The tuberculin reaction was induced according to the method of Kuriyama et al. (Journal of the Japanese Pharmacological Society, 94, 113-118, 1989). However, 2 ml / kg of the above test solution (50 mg / k as an ethyl acetate extract of S. barrierbilis culture solution)
g) was intraperitoneally administered 1 hour before and 6 hours after induction.
24 hours after the induction, the erythema intensity was measured according to the Draize criterion, and the reaction site was punched out with a punch having a diameter of 1.8 cm to measure the skin weight.

As a control for this test, the dimethyl sulfoxide-containing arabic gum aqueous solution containing no solvent extract was used in place of the test solution, and the other points were the same as in the above-mentioned procedure to determine the amount of leaked dye. I asked.

Further, as a control test, instead of the above-mentioned test solution, prednisolone was suspended in a solution prepared by adding 5% by weight of dimethyl sulfoxide to an aqueous solution of 5% by weight arabic gum to obtain a suspension ( (Final concentration 25mg / ml)
1 ml before 2 ml / kg (prednisolone 50 mg /
kg) was intraperitoneally administered, and otherwise the amount of leaked pigment was determined in the same manner as the above operation.

Each of these tests was carried out using 4 rats, and the erythema intensity and the skin weight were averaged from the values obtained for these rats.

The test results are shown in FIG.

As is clear from this figure, in the group to which the test solution containing the solvent extract of the example was administered, the erythema strength and the skin weight were significantly suppressed as compared with the control group.
It showed a strong inhibitory activity against type IV allergic inflammation (tuberculin reaction).

The group to which the test solution containing plendnisolone was administered also showed type IV allergic inflammation (tuberculin reaction) suppressing activity as compared with the control group.

Test Example 3 (Toxicity test) The toxicity of the above ethyl acetate extract was examined by the following method.

First, the ethyl acetate extract obtained in the Example was mixed with 5% so that the final concentration was 1000 mg / 5 ml.
It was suspended in a solution prepared by adding 5% by weight of dimethyl sulfoxide to an aqueous solution of arabic gum. The suspension thus obtained was used as a test solution. 25 ~ as a test animal
30 g of ICR male mouse was used.

The above test solution was intraperitoneally administered to a mouse at 5 ml / kg (corresponding to 10 times the effective amount of Test Examples 1 and 2). As a result, no toxic symptoms were observed, and the mortality rate 2 weeks after the administration of the test solution was 0%. No abnormality was observed in the autopsy of the surviving test animals. As is clear from this result, the ethyl acetate extract obtained in the example did not show toxicity at 10 times the effective amount. This test was carried out using 5 mice.

[0045]

EFFECTS OF THE INVENTION According to the present invention, a substance containing an active ingredient exhibiting a remarkable allergic inflammation-suppressing action is provided by extracting a culture solution of Streptomyces variabilis or its cells with an organic solvent. be able to.

[Brief description of drawings]

1 shows the results of Test Example 1, S. Ethyl acetate extract-containing liquid of Barrier-Bilis culture (100 mg / k
Fig. 6 is a graph showing the amount of leaked pigment in g) and its control (twice administered 24 hours before and 1 hour before induction).

2 shows the results of Test Example 2, S. Ethyl acetate extract-containing liquid of Barrier-Bilis culture (100 mg / k
3 is a graph showing the amount of leaked pigment in g) and its control (two administrations 1 hour before and 6 hours after induction).

Claims (1)

[Claims] 1. An allergic inflammation-inhibiting substance, which comprises a culture solution of Streptomyces variabilis or an extract obtained by organic solvent extraction from the fungus body of Streptomyces variabilis.