JPH04327535A - Allergic inflammation-depressing composition - Google Patents

Abstract

PURPOSE:To provide an allergic inflammation-depressing composition comprising a liposoluble solvent extract obtained from a liquid phase separated from the culture solution of a specific Actinomyces bacterium. CONSTITUTION:An Actinomyces bacterium: Actinomyces.crysomallus (e.g. JCM 4355 stored in Institute of Physical and Chemical Research) or Actinomyces bacterium: Streptomyces.tumemacerans (e.g. JCM 5050 stored in Institute of Physical and Chemical Research) is cultured in a liquid medium containing nutrients, preferably a medium containing starch and ammonium compounds, at a pH of 5-9 at 20-40 deg.C for 3-10 days. The culture solution is subjected to a solid-liquid separation treatment, and the separated liquid phase is brought into contact with an organic solvent (preferably ethyl acetate or methanol) such as an ester, alcohol, ether or ketone to obtain an allergic inflammation- depressing ingredient.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to the actinomycete Streptomyces chrysomalus (Streptomyces Chrysomalus).
rysomallus) (hereinafter abbreviated as S. chrysomalus) or the actinomycete Streptomyces tumemacerans (Streptomyces tumemac).
The present invention relates to a composition for suppressing allergic inflammation comprising a fat-soluble solvent extract obtained from the separated liquid phase of a culture of S. erans (hereinafter abbreviated as S. tumemycerans).

0002

BACKGROUND OF THE INVENTION Allergy is a type of pathological symptom in which the immune response caused by second contact with a certain antigen is expressed in an excessive or inappropriate manner depending on the individual.
Type I, type II, type III due to differences in the properties of the antibodies involved
It has been classified into type and type IV reactions. Among these four types, allergic inflammatory reactions involved in type IV (delayed hypersensitivity reactions: DTH reactions) play an important role in the development and development of various inflammatory diseases such as rheumatoid arthritis, nephritis, and infectious diseases. It has become clear that he is playing the role of However, to date, no substance that suppresses allergic inflammation has been found from microorganisms.

By the way, various antibiotics have been found in actinomycete culture filtrates, and the culture filtrates are said to be a treasure trove of physiologically active substances.

[0004] However, no substance has yet been found that exhibits an allergic inflammation suppressing effect from actinomycete culture filtrate.

[0005]

[Problems to be Solved by the Invention] Conventional anti-inflammatory drugs such as aspirin and indomethacin have a problem in that their suppressive effects on allergic inflammation are extremely weak.

[0006] In view of the above-mentioned circumstances, the object of the present invention is to use actinomycetes S. Chrysomalus or Streptomyces S. An object of the present invention is to provide a composition for suppressing allergic inflammation obtained using Tumemacerans.

[0007]

[Means for Solving the Problems] The present inventors used the mouse delayed-type hypersensitivity (DTH) reaction, which is an experimental animal model of type IV allergy, in order to find an allergic inflammation suppressant for type IV allergy. As a result of screening, actinomycetes S. Solvent extract of culture filtrate of Chrysomalus and Actinomycetes S. The inventors have discovered the surprising fact that the solvent extract of the culture filtrate of P. trumena contains substances that exhibit allergic inflammation-suppressing activity, and have completed the present invention.

[0008] That is, the allergic inflammation suppressing composition according to the present invention is produced by the actinomycete S. Chrysomalus or Actinomycetes S
．． It consists of a fat-soluble solvent extract obtained from the separated liquid phase of the culture filtrate of P. tuberans.

[0009] Actinomycetes S., which is a raw material for the composition of the present invention. Chrysomalus and Actinomycetes S. All species of Streptomyces spp. are available from public archives, such as Actinomycetes S.
Chrysomalus is a preserved bacterium of RIKEN (JCM 4).
355), etc., are used, and actinomycetes S. Preserved bacteria from RIKEN (JCM 5050) and the like are used as maceration.

[0010] Actinomycetes S. Chrysomalus and Actinomycetes S.
Cultivation of Tumemerans is usually carried out by liquid culture using a culture solution containing appropriate nutrients, but solid culture is also applicable.

In the case of liquid culture, the components of the medium are an aqueous solution containing one or more of sugars such as glucose, proteins such as peptone and malt extract, vitamins, nucleic acids, amino acids, and complex carbohydrates. is preferably used. Examples of particularly suitable media include starch/ammonium-based liquid media (soluble starch, K2 HPO4, NH4 C
(including l). The pH of the liquid medium is preferably in the range of 5 to 9, and the culture temperature is preferably 20 to 40°C. Further, the preferred culture time for liquid culture is 3 to 10 days. In the case of solid culture, grains such as rice grains and root vegetables such as potatoes are mainly used, but the culture conditions for solid culture are almost the same as those for liquid culture. As a culture method, a shaking culture method is applicable for liquid culture, and a static culture method is applicable for solid culture.

[0012] Thus, actinomycetes S. Chrysomalus or Streptomyces S. After culturing each of the cultivars, the culture solution is solid-liquid separated, the separated liquid phase is subjected to solvent extraction treatment, and the active ingredient of the allergic inflammation suppressant is obtained from the liquid phase. As the solid-liquid separation means, centrifugation, filtration, etc. are used as appropriate.

[0013] Solvent extraction is carried out by a method in which the liquid phase is directly brought into contact with a solvent, or by a method in which the liquid phase is evaporated to dryness and the dried product is contacted with a solvent.

[0014] Since the allergic inflammation suppressing component in the separated liquid phase is fat-soluble, an organic solvent is preferably used as the solvent for extraction. Typical examples of solvents include esters such as ethyl acetate; alcohols such as methanol, ethanol, and propanol; ethers such as ethyl ether and dioxane; and ketones such as acetone and methyl ethyl ketone. is not limited to these. Moreover, a mixture of the above-mentioned solvents can also be used. Particularly suitable solvents are ethyl acetate, methanol and the like.

The ratio of the separated liquid phase of the culture solution to the solvent is not particularly limited, but from the viewpoint of extraction efficiency and ease of operation, it is preferably in the range of 0.5 to 2 volumes of solvent per 1 volume of separated liquid phase. Solvent extraction may be carried out at room temperature or under heat, although the latter is more efficient. Heating is carried out at a suitable temperature below the boiling point of the solvent under normal pressure. The extraction time varies depending on the type of solvent, extraction temperature, etc., but is preferably in the range of 3 to 60 minutes. Also, during extraction, the liquid is allowed to stand or is left to stand while being stirred from time to time. Preferably, the extraction operation is repeated multiple times on the same separated liquid phase.

[0016] In order to formulate the composition of the present invention into an allergic inflammation suppressant, it is usually formulated into a pharmaceutical composition together with a pharmaceutical carrier. As carriers, diluents or excipients such as fillers, disintegrants, bulking agents, binders, wetting agents, surfactants, and lubricants that are commonly used to prepare drugs according to the dosage form can be used. Illustrated. Furthermore, by selecting an appropriate solvent, the obtained solvent extract or its concentrate can be used as it is as a liquid preparation for external use.

The dosage unit form of the allergic inflammation suppressant formulated using the composition of the present invention includes, in addition to the above-mentioned liquid preparations for external use, tablets, pills, drinking liquid preparations, powders, suspensions,
Examples include emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.), and ointments.

The amount of the composition of the present invention to be contained in the allergic inflammation suppressant is not particularly limited and can be appropriately selected within a wide range, but preferably 0.1% is contained in the allergic inflammation suppressant.
-50% by weight.

The allergic inflammation suppressant obtained from the composition of the present invention is administered in various ways depending on its use. For example, in the case of external liquid preparations as mentioned above, it is applied directly to the required area such as the skin or mucous membranes, and in the case of tablets, pills, drinking solutions, suspensions, emulsions, granules, and capsules, it is administered orally. Injections are administered intravenously, intramuscularly, intradermally, subcutaneously, or intraperitoneally; suppositories are administered rectally; and ointments are applied.

The dosage of the allergic inflammation suppressant obtained from the composition of the present invention is appropriately selected depending on the purpose of use, symptoms, etc., but is usually in the range of about 5 to 100 mg/kg per day. Furthermore, it is of course possible to administer the above pharmaceutical composition separately 3 to 4 times/day.

There is no problem with the safety of the allergic inflammation suppressant obtained from the composition of the present invention for humans and animals.

[0022]

Effects of the Invention According to the present invention, actinomycetes S. Chrysomalus or Streptomyces S. It is possible to provide a medicament comprising a fat-soluble solvent extract obtained from the separated liquid phase of a culture of P. tuberculosis and exhibiting remarkable allergic inflammation suppressing activity.

[0023]

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

Example 1 (Preparation of S. chrysomalus solvent extract) Actinomycetes S. chrysomalus purchased from RIKEN. Chrysomalus (
JCM 4355) 1 loopful of starch/ammonium medium (1 g of soluble starch in 100 ml of medium)
, 0.05 g of K2 HPO4, and 0.05 g of NH4 Cl.
05g) and cultured with shaking at 30°C for 7 days. This culture solution was centrifuged at 3,000 rpm for 20 minutes to obtain 95 ml of separated supernatant. Pour this supernatant liquid into a separating funnel, add an equal amount of ethyl acetate to it, and add 10% of the entire liquid.
Shake for a minute. After standing still for 5 minutes, the aqueous phase and ethyl acetate phase were separated. An equal amount of ethyl acetate was added to the separated aqueous phase, and the above operation was repeated to separate the aqueous phase and ethyl acetate phase again. An equal amount of ethyl acetate was added to the separated aqueous phase, and the above operation was repeated once again to perform a third extraction. The ethyl acetate phase obtained in this three-time operation was collected, and the collected liquid was concentrated to dryness using an evaporator. 20 mg of Chrysomalus extract was obtained.

Example 2 (Preparation of S. tumemacerans solvent extract) As actinomycetes, S. tumemacerans purchased from RIKEN was used. S.C. was prepared by using Twome Macerance (JCM 5050) and performing other operations in the same manner as in Example 1. An extract of Tume macerans was obtained.

Efficacy test (effect on type IV allergy model) The effect of the solvent extracts of Examples 1 and 2 on the mouse DTH reaction was investigated in the following manner.

The aggregate of the ethyl acetate extract obtained in Example 1 was concentrated to dryness using an evaporator, and the obtained extract was
It was dissolved in a solution prepared by adding 5% by weight of dimethyl sulfoxide to a 5% by weight aqueous gum arabic solution so that the final concentration was 10 mg/ml. The solution thus obtained was used as a test solution. The test animal is an IC weighing 40-50 g.
R male mice were used.

First, 0.1 ml of the above test solution was intraperitoneally administered to a test animal mouse, and sheep red blood cells were added to the left leg pad of the mouse in physiological saline to a final concentration of 4 x 109 cells/ml. Inject 0.05 ml of diluted solution as follows,
Mice were sensitized with red blood cells.

Then, 4 days after sensitization, 0.0% of the above test solution was applied.
1 ml was administered intraperitoneally to sensitized mice.

Immediately after administering this test solution, 4 sheep red blood cells were added again.
A DTH response was induced by intracutaneously injecting 0.05 ml of ×109 cells/ml into the right foot pad of a sensitized mouse.

[0031]Furthermore, 6 hours after the induction of the DTH reaction, 0.1 ml of the above test solution was again intraperitoneally administered to the same mouse.

Finally, 18 hours after the second administration of the test solution, the thickness of the right foot pad of the mouse was measured and the degree of swelling thereof was examined.

As a control for this test, the above-mentioned dimethyl sulfoxide-containing gum arabic aqueous solution containing no solvent extract was used instead of the above-mentioned extract-containing solution, and the procedure was otherwise the same as above, and the right footpad was The degree of swelling was investigated.

The results of this test are shown in FIG.

The effect of the solvent extract obtained in Example 2 on the mouse DTH reaction was also investigated in the same manner as above. The test results are shown in FIG. 2.

As is clear from these figures, Example 1
The test solution containing the solvent extract of 2 and 2 showed a significant reduction in the swelling of the right footpad compared to the control using a solution not containing the same extract, and a remarkable allergic inflammation suppressing effect was observed.

[Brief explanation of drawings]

FIG. 1 shows S. It is a graph showing the swelling of the footpad of a chrysomalus-containing solution and its control.

FIG. 2 shows S. It is a graph showing the swelling of the footpad of a liquid containing two-memacerance and its control.

Claims (1)

[Claims] [Claim 1] Streptomyces chrysomalus
lus ) or the actinobacterium Streptomyces tumemac
A composition for suppressing allergic inflammation comprising a fat-soluble solvent extract obtained from the separated liquid phase of a culture solution of P. erans.