JPH08245407A - Allergic inflammation suppressor - Google Patents

Abstract

PURPOSE: To obtain the subject substance capable of exhibiting remarkable allergic inflammation suppressing action from an extract obtained by extraction of culture solution, etc., of specific Actinomyces with an organic solvent. CONSTITUTION: This substance comprises an extract obtained by culturing Streptomyces Karnatakensis (JCM-4485, ATCC-25463) which is an actinomyces, e.g. at pH 2-9 and 15-42 deg.C culture temperature for 1-14 days culturing time in a liquid medium and extracting the resultant cultured solution or its bacterial cell with an organic solvent such as ethyl acetate. Furthermore, the substance is preferably contained in an amount of 10<-8> to 50wt.% based on an allergic infilammation suppressor.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to suppression of allergic inflammation, which comprises a medium extracted from an actinomycete Streptomyces carnatakensis (hereinafter abbreviated as "S. carnatakensis") or an organic solvent thereof. 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 type I to type IV, of which type III reaction (immune complex reaction: Arthus reaction) and type IV reaction (cellular immune reaction: delayed hypersensitivity reaction) are similar to those of chronic indirect rheumatism. It has become clear that it plays an important role in the development of autoimmune diseases and various inflammatory diseases such as hepatitis, nephritis, dermatitis and infectious diseases.

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. We have found a surprising phenomenon that a substance exhibiting an allergic inflammation-suppressing action is contained in a culture solution of Carnatakensis or a solvent extract of the bacterial cells, and based on this finding, the present invention has been completed.

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

The actinomycete S. Karnatakensis is available from public preservation institutions, for example, preservation bacteria (JCM 4) of RIKEN.
485) (which is also preserved in ATCC 25463 in the US and CBS 696.69 in the Netherlands).

Actinomyces S. Cultivation of carnatakensis is performed using a medium containing appropriate nutrients. In the case of liquid culture, 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 preferable as a component of the liquid culture. Used. PH of liquid medium is 2-9
Is preferable, 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 carnatus kensis, 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, troche, suppository, liniment, lotion, eye ointment, plaster, capsule, suppository, injection (solution, suspension) Etc.), patches, ointments, inhalants,
Examples include 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 in the case of tablets, pills, drinking solutions, suspensions, emulsions, granules and capsules, it is orally administered. In the case of injections, it is administered intravenously, intramuscularly, intracutaneously, subcutaneously or intraperitoneally, in the case of suppositories, it is administered rectally, and in the case of patches, ointments and creams, patches or It is applied and inhaled in the case of inhalants and nasal drops, and inhaled or applied in the case of sprays.

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, etc., but usually it is 1 pg / k as the substance of the present invention per day.
The range is about g to 1000 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 Karnatake Kensis
(JCM4485) as seed medium [glucose 1.0%, soluble starch 2.0%, yeast extract 0.5%, NZ amine A
(Sheffield Products) 0.5% and CaC
50 ml of pH 7.2 containing 0.1% of O ₃ was added 2
Shaking culture (pre-preculture) was performed in a 50 ml Erlenmeyer flask at a temperature of 30 ° C. for 3 days.

Then, 1 ml of Erlenmeyer flask containing 400 ml of the same medium was inoculated with 50 ml of the above pre-precultured culture broth and shake-cultured (seed culture) at a temperature of 30 ° C. for 48 hours.

Then, the production medium [SAG 471 (Union
Carbide) 0.02%, polypropylene glycol (molecular weight 2,000) 0.01%, glucose 1.0%, black strap molasses 2.0
%, Bacto-peptone (manufactured by Difco) 0.5%, and CaCO ₃ 0.2%, pH 6.8] in a 14-liter jar fermenter containing 10 liters. 2 liters were inoculated, and the aeration rate and the rotation speed of the stirring blade were adjusted at a temperature of 30 ° C. so that the dissolved oxygen content was 50 to 80% of saturation, and the cells were cultured for 66 hours.

Next, the culture solution was filtered to obtain a filtrate 1
0 liter was extracted twice with 10 liter ethyl acetate.
The cells separated by filtration were extracted twice with 1 liter of acetone, the extract was concentrated with an evaporator, the pH of the concentrate was adjusted to 2.0, and the mixture was extracted twice with 200 ml of ethyl acetate. Extraction was performed. The ethyl acetate extracts were combined and concentrated to dryness to obtain 15 g of extract.

Test Example 1 (Effect on Type III Allergic Reaction) i) Preparation of Rabbit Anti-Ovalbumin (ovalbumin) Serum The method of Eda et al. (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 100% of the dried extract obtained in the example.
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 mg / 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. carnatakensyl culture solution 200 mg / kg rat as ethyl acetate extract) had been 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-fold 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 (200 mg / kg rat as the extract amount).

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 evoked 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 above-mentioned test solution was replaced by the above aqueous solution of dimethyl sulfoxide-containing acacia gum containing no solvent extract, 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-mentioned test solution, prednisolone was added to a 5% by weight aqueous solution of gum arabic in an amount of 5% by weight of dimethyl sulfoxide to give a final concentration of 25 mg / ml. The resulting suspension was intraperitoneally administered at 2 ml / kg 3 hours before the induction, and the amount of leaked pigment was determined in the same manner as in the above procedure except for the above points.

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 clear from this figure, in the group to which the test solution containing the solvent extract of the example was administered, as compared with the control group using the solution containing no 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.

Similarly, the group to which the test solution containing prednisolone was administered also showed rat 4 as compared with 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 100 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, Vol. 94, 113-118, 1989). However, the above test solution 2 ml / kg (200 mg as S. carnatakensis culture solution ethyl acetate extract)
/ Kg) 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 above test solution was replaced by the above aqueous solution of dimethyl sulfoxide-containing gum arabic gum which did not contain the above solvent extract. I asked.

As a control test, instead of the above test solution, prednisolone was suspended in a solution prepared by adding 5% by weight of dimethyl sulfoxide to a 5% by weight aqueous solution of gum arabic 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 results of this test are shown in FIG.

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

Test Example 3 (Toxicity Test) The toxicity of the 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 4000 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.

[0048]

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

[Brief description of drawings]

1 shows the results of Test Example 1, S. A solution containing an ethyl acetate extract of the carnatakensis culture solution (400 mg /
(kg) and its control showing the amount of leaked pigment (twice administered 24 hours before and 1 hour before induction).

2 shows the results of Test Example 2, S. A solution containing an ethyl acetate extract of the carnatakensis culture solution (400 mg /
(kg) and a control graph showing the amount of leaked pigment (twice administered 1 hour before and 6 hours after induction).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 465)

Claims (1)

[Claims] 1. An allergic inflammation-inhibiting substance consisting of a culture solution of Streptomyces Karnatakensis or an extract obtained by organic solvent extraction from the cells thereof.