JPH09286733A - Composition for inflammation suppressing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a composition for an inflammation suppressing agent, effective for suppressing allergic inflammation and non-allergic inflammation and having high utility by using a precipitate obtained by adding an organic solvent to a cultured liquid of a specific actinomycete. SOLUTION: An organic solvent is added to a cultured liquid of Streptomyces kanamyceticus belonging to actinomycete and the obtained precipitate is used as an active component of the objective composition. Preferably, the precipitate is further subjected to gel-filtration column chromatography and the fraction having a molecular weight of 1,000,000-10,000,000 in terms of dextran is used as the active component. The actinomycete Streptomyces kanamyceticus is easily available e.g. as a strain (JCM4433) stocked by the Institute of Physical and Chemical Research. The administration dose of the inflammation suppressing agent is about 1ng/kg to 1g/kg in the case of a composition containing the precipitate as the active component and about 0.01ng/kg to 10mg/kg in the case of a composition containing the gel-filtrated fraction as the active component. The amount of the active component in the composition is about 0.1-50wt.%.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The invention according to claim 1 (hereinafter referred to as "the invention")
The present invention 1) is an allergenicity comprising a precipitate obtained by adding an organic solvent to a culture solution of Streptomyces kanamyceticus (hereinafter abbreviated as “S. kanamyceticus”) And a composition for an anti-inflammatory agent effective for non-allergic inflammation, the invention according to claim 2 (hereinafter referred to as the present invention 2) is obtained by subjecting the precipitate of the present invention 1 to gel filtration column chromatography. , A composition for an anti-inflammatory agent, which is effective for allergic and non-allergic inflammation, comprising a fraction having a dextran-equivalent molecular weight of 1,000,000 to 10,000,000.

[0002]

2. Description of the Related Art Inflammatory reactions are roughly classified into non-allergic inflammation and allergic inflammation. For non-allergic inflammation represented by carrageenin edema, non-steroids such as aspirin (Nikkei Science, 3, 70, 1991) and indomethacin (Ther. Res., 3, 1057, 1985) Effective anti-inflammatory agents and steroidal anti-inflammatory agents such as prednisolone are effective. On the other hand, allergic inflammation is roughly classified into types I to IV, and the type I reaction is involved in various allergic diseases other than atopic dermatitis, asthma, rhinitis, and this reaction is associated with adrenal glands such as prednisolone. Steroids and antiallergic agents such as sodium cromoglycate are effective. In addition, type III reaction (immune complex reaction: Arthus reaction) and type IV reaction (cellular immune reaction:
Delayed hypersensitivity reaction) plays an important role in the development of autoimmune diseases such as rheumatoid arthritis and various inflammatory reaction diseases such as hepatitis, nephritis, dermatitis, and infectious diseases. Has become. Steroidal anti-inflammatory agents such as prednisolone are effective for these allergic inflammations.

[0003]

[Problems to be Solved by the Invention] Steroidal anti-inflammatory agents effective for allergic inflammation have problems in terms of side effects such as reduced infection defense function, gastrointestinal tract disorder, inhibition of pituitary adrenal cortex function, and decreased bone formation. is there. In addition, the non-steroidal anti-inflammatory drug, which is considered to be safer than the steroid drug, has a problem that the inhibitory effect on allergic inflammation is extremely weak. Further, although an antiallergic agent has a preventive effect on type I allergic inflammation, it has a weak effect on seizures that have already occurred and is ineffective on other inflammatory reactions. 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 composition for an anti-inflammatory agent which is effective for allergic inflammation and non-allergic inflammation and is highly useful.

[0005]

Means for Solving the Problems The present inventors have found that actinomycete S. We have found the surprising fact that the precipitate obtained by adding an organic solvent to the culture fluid of Kanamyceticus contains a substance exhibiting an anti-inflammatory effect on allergic inflammation and non-allergic inflammation, and further said precipitate The fact that the substance having an inflammation suppressing action against allergic inflammation and non-allergic inflammation is contained in the fraction having a dextran-equivalent molecular weight of 1,000,000 to 10,000,000, which is obtained by subjecting the above to gel filtration column chromatography. Based on this finding, the present invention has been completed based on this finding.

That is, the composition for an anti-inflammatory agent according to the present invention 1 is composed of a precipitate obtained by adding an organic solvent to a culture solution of Streptomyces kanamyceticus. .

The composition for inflammation suppressant according to the present invention 2 comprises a fraction having a dextran-equivalent molecular weight of 1,000,000 to 10,000,000, which is obtained by subjecting the precipitate of the present invention 1 to gel filtration column chromatography. is there.

Actinomycetes S. cerevisiae used for producing a culture solution containing the composition for inflammation suppressant of the present invention 1 and 2. Kana Myceticus is available from public preservation agencies,
For example, a bacterium such as a preserved bacterium (JCM 4433) from RIKEN can be used.

Actinomyces S. The culture of Kanamyceticas is
It is performed using a medium containing appropriate nutrients. In the case of liquid culture, the components of the medium include sugars such as glucose, proteins such as peptone and malt extract, vitamins,
An aqueous solution containing one or more of nucleic acids, amino acids, and glycoconjugates is preferably used. A typical example of a culture medium is a starch / ammonium-based liquid medium (including soluble starch, dibasic potassium phosphate, and ammonium chloride). 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 preferred culture time of the liquid culture is 1 to 14 days. The culture solution thus obtained may be used as it is in order to obtain a composition for an inflammation suppressant, or may be subjected to S. cerevisiae by filtration or centrifugation. It may be used after removing the Kanamyceticus bacterial cells.

The organic solvent added to the culture solution to obtain the composition for inflammation suppressant is preferably an organic solvent miscible with water, and typical examples thereof include alcohols such as methanol, ethanol and propanol; acetone. Examples thereof include ketones, but usable organic solvents are not limited to these. Also, a mixed solution of the above solvents can be used. Particularly suitable solvents are acetone, methanol, ethanol and the like.

The amount of the organic solvent added to the culture medium to obtain the composition for inflammation suppressor is not particularly limited. After adding the organic solvent, let stand until a precipitate is sufficiently formed. The standing may be carried out at room temperature or under cooling, but cooling is more efficient. The time required to sufficiently form a precipitate depends on the type of organic solvent and the standing temperature. Further, it is preferable to wash the formed precipitate with an organic solvent or an organic solvent containing water a plurality of times.

[0012] As described above, the precipitate obtained by adding the organic solvent to the culture solution of "S. kanamyceticus" has an inflammation-inhibiting effect which is remarkable for allergic inflammation and non-allergic inflammation. It is a composition for inhibitors.

To obtain the composition for inflammation suppressant of the present invention 2, the precipitate obtained as described above is subjected to gel filtration column chromatography. The type of filler used for gel filtration is not particularly limited as long as it is generally used for aqueous gel filtration.

The gel filtration column chromatography operation will be described in detail below. First, fill the chromatography column with the packing material for gel filtration, and allow the solvent used for elution (such as water or an appropriate buffer solution) to flow sufficiently to completely equilibrate and use this column for the following chromatography. To do.

The sample obtained by dissolving the precipitate obtained as described above in a solvent used for elution (water or a suitable buffer solution) is charged into the above column. At this time, the concentration of the sample is not particularly limited, but is 1 to 1000 mg / m 2.
l is preferred. The amount of sample is not particularly limited,
It is preferably 0.01 to 10% of the column bed volume. Water or an appropriate buffer solution is used as the elution solvent, but one having an appropriate ionic strength is preferable in order to prevent the interaction between the sample and the packing material, for example, 0.01
.About.0.5 M potassium chloride aqueous solution, phosphate buffer, etc. may be mentioned. The flow rate at the time of elution is not particularly limited, but preferably 1 to ²⁰ ml / column bed area 1 cm ²
h. The elution temperature is not particularly limited, but is preferably a constant temperature of 20 to 80 ° C.

Before carrying out the chromatography of the precipitate, a standard dextran having a known molecular weight is used to examine the relationship between the dextran-equivalent molecular weight and the eluate volume in order to test the separation characteristics of the column. It is preferable to keep. Then, the precipitate is subjected to gel filtration column chromatography to collect eluates having a dextran-equivalent molecular weight in the range of 1 to 10,000,000 and evaporate to dryness using an evaporator.

As described above, the precipitate obtained by adding an organic solvent to the culture solution of "S. kanamyceticus" is subjected to gel filtration column chromatography to obtain a dextran-equivalent molecular weight of 1,000,000. The 10 to 10 million fraction is a composition for an anti-inflammatory agent, which has a remarkable anti-inflammatory effect on allergic inflammation and non-allergic inflammation.

The composition for an inflammation inhibitor of the present invention 1 and 2 can be used as an active ingredient of the inflammation inhibitor. Invention 1
In order to formulate an anti-inflammatory agent from the anti-inflammatory composition of (2) and (2), it is usually in the form of a pharmaceutical composition together with a pharmaceutical carrier. As the carrier, a filler, a disintegrating agent, a bulking agent, a binder, which is usually used for preparing a drug depending on the dosage form,
Coloring agents, flavoring agents, pH adjusting agents, solubilizing agents, suspending agents, buffering agents, stabilizing agents, preservatives, moisturizing agents, surface active agents,
Lubricants, excipients, etc. are exemplified. Further, by selecting an appropriate solvent, the obtained composition for inflammation inhibitor of the present invention 1 and 2 can be used as it is as a liquid preparation for external use.

Examples of the dosage form of the anti-inflammatory agent formulated using the composition for anti-inflammatory agent of the present invention 1 and 2 include tablets, pills, liquid solutions for drinking, powders, and suspensions in addition to the above-mentioned external preparations. Turbidity agents, emulsions, granules, extracts, fine granules, syrups,
Immersion, decoction, eye drop, troche, poultice, liniment, lotion, eye ointment, plaster, capsule,
Suppositories, injections (solutions, suspensions, etc.), patches, ointments,
Examples include inhalants, creams, sprays, nasal drops and the like.

The amount of the composition for inflammation inhibitor of the present invention 1 and 2 to be contained in the above inflammation inhibitor is not particularly limited and may be appropriately selected within a wide range, but preferably 0.1 in the inflammation inhibitor. Is in the range of 50% by weight.

The anti-inflammatory agents obtained from the compositions for anti-inflammatory agents of the present inventions 1 and 2 are administered at the time of their use according to various dosage forms. 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 anti-inflammatory agent obtained from the compositions for anti-inflammatory agent of the present inventions 1 and 2 is appropriately selected according to the purpose of use, symptoms, etc., but usually the anti-inflammatory agent of the present invention 1 per day The composition of the present invention has a range of about 1 ng / kg to 1 g / kg, and the composition of the present invention 2 has a range of about 0.01 ng / kg to 10 mg / kg. Further, it is of course possible to administer the above-mentioned inflammation inhibitor in 1 to 4 times per day.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION Next, examples of the present invention will be given to demonstrate the effects described above.

Example 1 (Preparation of Composition for Inflammatory Inhibitor) Actinomyces S. cerevisiae purchased from RIKEN One platinum loop of Kanamyceticus (JCM4433) was added to a starch / ammonium medium containing 0.2% by weight of yeast extract prepared in a 500 ml Sakaguchi flask (1 g of soluble starch and 100 ml of dipotassium hydrogen phosphate in 100 ml of distilled water). The mixture was inoculated into 125 ml of the mixture (containing 0.05 g of ammonium chloride and 0.05 g of ammonium chloride) and cultured with shaking (seed culture) at 30 ° C. for 68 hours.

Then, 6 liters of the starch / ammonium medium prepared in a 10 liter jar fermenter was inoculated with 40 ml of the above-mentioned seed culture bacterium solution, and aerated and stirred at a temperature of 30 ° C. for 4 days.

Next, the culture solution was suction-filtered, and the culture solution (about 5 liters) and S. Kanamycetica cells (about 10 g in wet weight) were separated.

To 100 ml of the culture solution obtained above,
An equal volume of acetone (final concentration of acetone 50% by volume) was added with slow stirring and left overnight at 20 ° C. After sufficient precipitation was generated, centrifugation was performed under conditions of 3000 rpm and 10 minutes to obtain a precipitate. 200 ml of the obtained precipitate
After washing three times with 50% by volume of acetone containing water, 50 ml of
Dissolved in water, concentrated to dryness using an evaporator,
350 mg of the composition of the invention 1 was obtained.

Test Example 1 (Action against Type III Allergic Inflammation) 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) Effect on rat PCA reaction for 4 hours (type III allergic skin reaction) The composition of the present invention 1 obtained in Example 1 was used at a final concentration of 1
A test solution was prepared by dissolving it in physiological saline so that the concentration was 0 mg / ml. As test animals, male Wistar rats weighing 120 to 200 g were used.

First, 0.05 ml of an injection solution prepared by diluting the above rabbit anti-ovalbumin serum four-fold with physiological saline.
Was intradermally injected into the back of the rat, and the rat was sensitized with the antiserum.

Then, 1 hour after the injection of the antiserum, the test solution was intravenously administered to the sensitized rats at 2 ml / kg rat (20 mg / kg rat as the composition of the present invention 1).

Then, 3 hours after administration of the test solution, 0.5 mg containing 2 mg / ml ovalbumin as a corresponding antigen.
A 2.5% / kg intravenous injection of wt% Evans blue saline was performed to induce a heterogeneous PCA reaction for 4 hours.

The leakage pigment at the site where the intradermal reaction was induced 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)
_{2 in} a mixed solution of 3 volume of Na ₂ SO ₄ aqueous solution and 7 volume of acetone.
It was left to soak for 4 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, a physiological saline solution containing no composition of the present invention 1 was used in place of the test solution described above, and the leaked pigment amount was determined in the same manner as in the above procedure.

As a control test, instead of the test solution of the present invention 1, 5% by weight of arabic gum aqueous solution was added with 5% by weight of dimethyl sulfoxide, and the final concentration of prednisolone was 5 mg / ml. 2 ml / kg rat (prednisolone) was used instead of using the suspension of the present invention 1 and intravenously administering the test solution of the present invention 1 at 2 ml / kg rat. (10 mg / kg rat) was intraperitoneally administered, and the amount of leaked pigment was determined in the same manner as in the case of using the composition of the present invention 1 as a test solution.

As a control in the above control test, the above-mentioned dimethyl sulfoxide-containing arabic gum aqueous solution containing no prednisolone was used in place of the above suspension.
Otherwise, the amount of leaked dye was determined by operating in the same manner as when the above suspension was used as the test solution.

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 results of this test are shown in FIG.

As is clear from this figure, the above-mentioned present invention 1
In the group administered with the test solution containing the composition of (20 mg / kg rat as the composition of the present invention 1), the amount of pigment leaked to the skin of the heterogeneous PCA reaction site in the rat for 4 hours was significantly reduced as compared with the control group. The effect was equal to or higher than that of prednisolone (administered to 10 mg / kg rat). That is, the composition of the present invention 1 was found to have a remarkable allergic inflammation suppressing activity.

Test Example 2 (Action against type IV allergic inflammation) Next, the action of the composition of the present invention 1 on rat type IV allergic inflammation (tuberculin reaction) was examined.

A final concentration of the composition of the present invention 1 is 25 mg.
A solution dissolved in physiological saline to give a test solution was prepared. As test animals, male Wistar rats weighing 170 to 250 g were used.

The specific test procedure was as follows.
The tuberculin reaction is based on the method of Kuriyama et al.
Vol., 113-118, 1989).
The test solution containing the composition of the present invention was administered 2 times at a total of 2 times 1 hour before and 6 hours after the induction, each of which was 2 ml / kg rat (50 mg / k per time as the composition of the present invention 1).
(g rat).

24 hours after the induction, the erythema intensity at the reaction induction site was measured according to the Draize criterion, and the diameter was 1.
The reaction site skin was punched out with an 8 cm punch and the skin weight was measured.

As a control for this test, physiological saline containing no composition of the present invention 1 was used in place of the test solution described above, and otherwise the same as in the above-mentioned procedure to determine the erythema strength and skin weight at the reaction site. It was measured.

As a control test, instead of the test solution of the present invention 1, 5% by weight of arabic gum aqueous solution was added with 5% by weight of dimethyl sulfoxide, and a final concentration of prednisolone was 5 mg / ml. Was used as a test solution, and the test solution of the present invention 1 was used for a total of 2 hours 1 hour before and 6 hours after induction.
Instead of being administered intravenously once in 2 ml / kg rat, a suspension containing 2 ml / kg rat (prednisolone 10 mg) was prepared 6 hours after induction.
1 / kg rat), the present invention 1 except that it was intraperitoneally administered (unlike the case of the test solution of the present invention 1, the administration was performed only once).
The erythema strength and the skin weight of the reaction site were measured by operating in the same manner as when the composition of 1 was used as a test solution.

As a control of the above control test, the above dimethyl sulfoxide-containing arabic gum aqueous solution containing no prednisolone was used in place of the above suspension.
In other respects, the erythema strength and skin weight at the reaction site were measured by operating in the same manner as when the suspension was used as the test solution.

Each of these tests was carried out using 5 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 apparent from this figure, the above-mentioned present invention 1
In the group to which the test solution containing the composition was administered (50 mg / kg rat × 2 times as the composition of the present invention 1), the erythema strength and skin weight at the rat skin tuberculin reaction site were significantly reduced as compared with the control group. The effect was equal to or higher than that of prednisolone (administered to 10 mg / kg rat). That is, the substance of the present invention 1 was confirmed to have a remarkable allergic inflammation inhibitory activity.

Test Example 3 Action on type I allergic inflammation (1) Rat anti-2,4-dinitrobenzenesulfonic acid-
Preparation of Ascaris suum Extract (DNP-As) Serum Method of Tada and Okumura (Journal of
Immunology; 106, 1002, 1971), rat anti-DNP-As serum was prepared. Ascaris suu
m) of the extract from Strejan and Campbe
11 (Journal of Immunology; 98, 893, 19
67) and prepared according to the method of Eisen et al. (Journal
of American Chemical Society; Vol. 75, p. 4583, 1953.
Year), 2,4-dinitrobenzene sulfonic acid (DN
P) and DNP-bound Ascaris suum
(DNP-As) was obtained.

1 mg of the above DNP-As was dissolved in 1 ml of physiological saline in which 1 × 10 ¹⁰ pertussis killed bacteria were suspended, and the solution was subcutaneously injected into the footpad of a female rat weighing about 200 g. Five days later, 0.5 mg of DNP-As was dissolved in 0.5 ml of physiological saline and injected into the right and left dorsal muscles.
Eight days after the first injection, blood was collected from the abdominal aorta and the serum was separated to obtain rat anti-DNP-As serum. The titer of this antiserum in the rat homologous PCA reaction for 48 hours was 1: 512.

(2) Effect on rat homologous PCA reaction (type I allergic skin reaction) for 48 hours. The composition of the present invention 1 was dissolved in physiological saline to a final concentration of 25 mg / ml, and 5% by weight was added. Prednisolone was suspended in a solution prepared by adding 5% by weight of dimethyl sulfoxide to an aqueous solution of gum arabic so that the final concentration was 5 mg / ml, and the obtained solutions were used as test solutions. As test animals, male Wistar rats weighing 120 to 200 g were used.

First, 0.05 ml of an injection solution prepared by diluting the above anti-DNP-As serum with physiological saline 20 times was injected into the dorsal skin of the rat to sensitize the rat with the above antiserum.

Then, 48 hours after the administration of the antiserum, the OD of 2 mg / ml DNP-As containing 2 mg / ml as the corresponding antigen was obtained.
A PCA reaction was induced by intravenously injecting 5 wt% Evans' blue physiological saline at a rate of 2.5 ml / kg.

In the rats administered with the composition of the present invention 1 described above, 2 ml of the test solution containing the composition of the present invention 1
kg (50 mg / kg as the composition of the present invention 1) was intravenously administered 3 hours before the induction, and in the rats of the prednisolone administration group, 2 ml / kg (10 mg / k as prednisolone) of the test solution containing prednisolone was administered.
g) was intraperitoneally administered 3 hours before the induction.

The leakage pigment at the site where the intradermal reaction was induced in this manner was determined by the method of Harada et al. (J. Pharm. Pharmacol. 23, 2).
Page 18, 1971). That is, the animal was sacrificed 1 hour after the antigen injection, and the skin of the allogeneic PCA reaction part was cut into small pieces for 48 hours, and this was cut at 0.3% (w /
v) Dipped in the mixed solution of 3 volume of Na ₂ SO ₄ aqueous solution and 7 volume of acetone for 24 hours to extract the leaked dye. The thus-extracted dye was colorimetrically determined at 620 nm to determine the amount of leaked dye, which was measured at the site where the rat anti-DNP-As serum was injected.
It was expressed as the amount of leaked dye (μg) per (site).

As a control for this test, a physiological saline solution containing no composition of the present invention 1 or the above-mentioned aqueous solution of gum arabic gum containing dimethyl sulfoxide was used in place of the above-mentioned test solution. The amount of leaked pigment was determined in the same manner as in (Saline solution was intravenously administered, and dimethylsulfoxide-containing aqueous gum arabic solution was intraperitoneally administered).

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

The results of this test are shown in FIG.

As is clear from this figure, the above-mentioned present invention 1
In the group administered with the test solution containing the composition of the present invention (50 mg / kg rat as the composition of the present invention 1), the amount of pigment leaked to the skin of the allogeneic PCA reaction site in the rat for 48 hours was significantly reduced as compared with the control group. The effect was equal to or higher than that of prednisolone (administered to 10 mg / kg rat). That is, the composition of the present invention 1 had a remarkable allergic inflammation suppressing activity.

Test Example 4 Action on non-allergic inflammation The action of the substance of the present invention 1 on rat carrageenin footpad edema reaction was examined by the following method.

First, the composition of the present invention 1 was added to a final concentration of 10 m.
A test solution was prepared by dissolving the solution in physiological saline so as to be g / ml. As a control test, a suspension of aspirin in a solution of 5% by weight of arabic gum aqueous solution and 5% by weight of dimethyl sulfoxide was added so that the final concentration was 50 mg / ml. And Wistar male rats weighing 120 to 200 g were used as test animals.

The volume of the right hind limb of the rat was measured using a Plethysmometer, and then 0.1 ml of a 1% (w / v) carrageenan physiological saline solution was subcutaneously injected into the right hind paw of the rat to give a carrageenin footpad edema reaction. Was triggered. To examine the degree of swelling of the right hind footpad of the rat, the volume of the right hind paw of the rat was measured by Plet every 1 hour up to 5 hours after the induction of the footpad edema reaction.
It was measured using a hysmometer and the difference from the volume before the induction of the reaction was determined (this difference is shown as the right hind limb edema volume (ml) in FIG. 4 described later).

In the rats of the present invention 1 composition-administered group, 1 ml before the induction, a test solution containing the above composition was added to 2 ml / kg rat (20 mg / k as the above composition).
(g rat). In the rats in the aspirin administration group, the test solution containing aspirin was intraperitoneally administered at 2 ml / kg rat (100 mg / kg rat as aspirin) 1 hour before induction.

The present invention 1 was used as a control for this test.
A physiological saline containing no composition and an aqueous solution of dimethyl sulfoxide-containing arabic gum containing no aspirin were used, and the other points were the same as the above-mentioned operations (saline was intravenously administered, dimethyl sulfoxide-containing arabic gum was used). The aqueous solution was administered intraperitoneally) to examine the degree of swelling of the right hind footpad of the rat.

Each test was carried out using 5 rats each, and the degree of swelling of the foot pads was the average of the values obtained for these rats.

The results of this test are shown in FIG.

As is clear from this figure, the above-mentioned present invention 1
In the group to which the test solution containing the composition was administered (20 mg / kg rat as the composition of the present invention 1), the swelling degree of the right hind footpad was obviously suppressed as compared with the control group, and the effect was administration of aspirin ( 100 mg as aspirin
/ Kg rats). That is, the composition of the present invention 1 had a remarkable non-allergic inflammation inhibitory activity.

Test Example 5 Effect on Adjuvant Arthritis The composition of the present invention 1 was dissolved in physiological saline to a final concentration of 25 mg / ml, and 5% by weight of dimethyl sulfoxide was added to a 5% by weight aqueous solution of gum arabic. Prednisolone was suspended in the added solution so that the final concentration was 5 mg / ml, and the resulting solutions were used as test solutions. Wistar Lewis male rats weighing 120-200 g were used as test animals.

First, Mycobact suspended in liquid paraffin
erium tuberculosis H37RA (6 mg / ml) 0.1
ml was injected subcutaneously in the right hind limb of rats to induce arthritis. To determine the degree of swelling of the left and right hind limbs of rats, the volume of the left and right hind limbs of rats was
The difference with the volume before the induction of arthritis was determined (this difference is shown as the edema volume (ml) in FIG. 5 described later).

In the rats administered with the composition of the present invention 1 described above, 2 ml of the test solution containing the composition of the present invention 1
kg / day (50 mg / kg / day as the above composition),
It was administered intravenously once a day for 20 days after Mycobacterium tuberculosis injection, and in rats of the prednisolone administration group, 2 ml / k of the above test solution containing prednisolone was administered.
g / day (10 mg / kg / day as prednisolone)
And 20 days after Mycobacterium tuberculosis injection
It was administered once intraperitoneally.

As a control for this test, a physiological saline solution containing no composition of the present invention 1 or the above aqueous solution of arabic gum containing dimethyl sulfoxide was used in place of the above-mentioned test solution, and other points were the same as the above-mentioned procedure. The same procedure as described above (intravenous administration of physiological saline, intraperitoneal administration of dimethyl sulfoxide-containing aqueous gum arabic solution) was carried out to examine the degree of swelling of the left and right hind footpads of rats.

This test was carried out using 5 rats each, and the degree of swelling was the average of the values obtained for these rats.

The test results are shown in FIG.

As is apparent from this figure, the above-mentioned present invention 1
In the group to which the test solution containing the composition was administered (50 mg / kg / day as the composition of the present invention 1), the swelling degree of the left and right hind footpads was obviously suppressed as compared with the control group, and the effect was prednisolone (10 mg / Kg / day) or more. That is, the composition of the present invention 1 had remarkable allergic inflammation and non-allergic inflammation suppressing activity.

Example 2 (Preparation of Inflammatory Inhibitor Composition) Toyopearl HW-65F (manufactured by Toso Co., Ltd.), a gel filtration filler, was packed in a chromatography column to give an inner diameter of 2.
A gel filtration column having a length of 6 cm and a length of 30 cm was prepared.
In addition, a peristaltic pump was installed on the inflow side of the column, water was sufficiently flowed through the column to completely equilibrate the column, and the columnar pump was used for the following chromatography. First, a standard dextran (Pharmacia) was used to test the separation characteristics of this column. As a result, a substance having a dextran-equivalent molecular weight of 1 to 10 million was found to have an elution volume of 50 to 100.
It was confirmed to be eluted in ml.

Then, the composition of the present invention 1 obtained in Example 1 was dissolved in water to a concentration of 30 mg / ml, and 1 ml of the solution was charged into the column. Water was used as an elution solvent, and the elution was performed at 25 ° C. at a flow rate of 1 ml / min. At this time, the eluate eluted in an elution volume of 50 to 100 ml was collected and dried under reduced pressure using an evaporator to obtain 1 mg of the composition of the present invention 2.

Next, the action of the composition of the present invention 2 on type III allergic inflammation was examined. Test Example 1 (Action on Type III Allergic Inflammation) In Test Example 1 of Example 1, "the composition of the present invention 1 obtained in Example 1 was adjusted to a final concentration of 10 mg / ml with physiological saline. “Dissolved in water to give a test solution”, and “1 hour after the injection of antiserum, the sensitized rat was treated with 2 ml / kg of the test solution (20 m as a composition of the present invention 1
g / kg rat). ”
"The composition of the present invention 2 obtained in Example 2 was dissolved in physiological saline to obtain a test solution so that the final concentration was 0.1 mg / ml," and "of antiserum injection" 1
After a lapse of time, 2 ml / kg rat (0.2 mg / kg rat as the composition of the present invention 2) was applied to the sensitized rat with the above test solution.
It was administered intravenously in the same manner as in Example 1, except that the amount of leaking dye was determined in the same manner as in Test Example 1 of Example 1, and the amount of leaking dye was determined per site injected with rabbit anti-ovalbumin serum. Expressed as (μg).

The results of this test are shown in FIG.

As is apparent from this figure, the above-mentioned present invention 2
In the group administered with the test solution containing the composition (0.2 mg / kg rat as the composition of the present invention 2), the amount of pigment leaked to the skin of the heterogeneous PCA reaction site for 4 hours in the rat was significantly larger than that in the control group. The effect was equal to or higher than that of prednisolone (administered to 10 mg / kg rat). That is, the composition of the present invention 2 was confirmed to have a remarkable allergic inflammation suppressing activity.

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

In Test Example 2 of Example 1, "Invention 1
A solution prepared by dissolving the composition in physiological saline to a final concentration of 25 mg / ml was used as a test solution ", and" a test solution containing the composition of the present invention 1 was prepared 1 hour before and 6 hours after induction. 2 times in total in each 2 ml / kg rat (50 mg / kg rat per one time as the above-mentioned composition of the present invention 1) ”. A solution dissolved in physiological saline so as to have a concentration of 25 mg / ml was used as a test solution ", and" a test solution containing the composition of the present invention 2 was added twice, 1 hour before and 6 hours after the induction. 2 ml / each
erythema at the reaction site was performed in the same manner as in Test Example 2 of Example 1, except that the administration was performed intravenously in kg rats (0.5 mg / kg rat per dose as the composition of the present invention 2 above). The strength and skin weight were measured.

The results of this test are shown in FIG.

As is clear from this figure, the above-mentioned present invention 2
In the group to which the test solution containing the composition was administered (0.5 mg / kg rat as the composition of the present invention x 2 times), the erythema strength and skin weight of the rat skin tuberculin reaction site were significantly reduced as compared with the control group. However, the effect was equal to or higher than that of prednisolone (administered to 10 mg / kg rat). That is, the substance of the present invention 2 was confirmed to have a remarkable allergic inflammation suppressing activity.

Test Example 3 Action on Type I Allergic Inflammation Next, the action of the composition of the present invention 2 on rat type I allergic inflammation (tuberculin reaction) was examined. Example 1
In Test Example 3 of "the composition of the present invention 1 has a final concentration of 2
A solution dissolved in physiological saline so as to have a concentration of 5 mg / ml was used as a test solution ", and" the test solution containing the composition of the present invention 1 was used at 2 ml / kg rat (the above-mentioned present invention 1 Instead of being administered intravenously as a composition (50 mg / kg rat) ”, a solution prepared by dissolving the composition of the present invention 2 in physiological saline to a final concentration of 0.25 mg / ml was used as a test solution. 2 ml 3 hours before the induction, the test solution containing the composition of the present invention 2 was added.
/ Kg rat (0.5 mg / in the above-mentioned composition of the present invention 2)
(kg rat) was administered intravenously ”.
The amount of leaking dye was determined in the same manner as in Test Example 3 of 1. above, and this was expressed as the amount of leaking dye (μg) per site where the rat anti-DNP-As serum was injected.

The test results are shown in FIG.

As is clear from this figure, the above-mentioned present invention 2
In the group administered with the test solution containing the composition of the present invention (0.5 mg / kg rat as the composition of the present invention 2), the amount of dye leaked to the skin of the rat PCA reaction site for 48 hours was significantly larger than that in the control group. The effect was equal to or higher than that of prednisolone (administered to 10 mg / kg rat).
That is, the composition of the present invention 2 had a remarkable allergic inflammation suppressing activity.

Test Example 4 Action on non-allergic inflammation The action of the substance of the present invention 2 on rat carrageenin footpad edema reaction was examined. In Test Example 4 of Example 1, the final concentration of the composition of the present invention 1 obtained in Example 1 was 10 mg /
The test solution was prepared by dissolving the test solution in physiological saline so that the amount of the solution became 1 ml, and "1 hour before the induction, the test solution containing the composition was 2 ml / kg rat (20 mg / kg as the composition. Rat), the composition of the present invention 2 obtained in Example 2 was dissolved in physiological saline to a final concentration of 0.1 mg / ml. "Being a test solution" and "1 of induction"
2 hours / hour before the test solution containing the above composition is added.
Rats (0.2 mg / kg rat as the above composition) were intravenously administered ”, and the degree of swelling of the right hind footpad of the rat was examined in the same manner as in Test Example 4 of Example 1. .

The test results are shown in FIG.

As is apparent from this figure, the present invention 2
In the group administered with the test solution containing the composition (0.2 mg / kg rat as the composition of the present invention 2), the swelling degree of the right hind footpad was obviously suppressed as compared with the control group, and the effect was aspirin-induced. Administration (100m as aspirin
(g / kg rat) was much stronger than the group. That is, the composition of the present invention 2 had a remarkable non-allergic inflammation inhibitory activity.

Test Example 5 Action on Adjuvant Arthritis Next, the action of the composition of the present invention 2 on adjuvant arthritis was examined. In Test Example 5 of Example 1, “Invention 1
A solution prepared by dissolving the composition in physiological saline so that the final concentration was 25 mg / ml was used as a test solution ", and" 2 ml / kg / day of the test solution containing the composition of the present invention 1 (the above-mentioned composition Instead of being administered intravenously at 50 mg / kg / day) as a product, a solution prepared by dissolving the composition of the present invention 2 in physiological saline to a final concentration of 0.25 mg / ml was used as a test solution. Done, and "the present invention 2"
The test solution containing the composition was intravenously administered at 2 ml / kg / day (0.5 mg / kg / day as the composition) ”except that the test solution of Example 1 was used. Then, the degree of swelling of the left and right hind foot pads of the rat was examined.

The test results are shown in FIG.

As is apparent from this figure, the present invention 2
In the group to which the test solution containing the composition was administered (0.5 mg / kg / day as the composition of the present invention 2), the swelling degree of the left and right hindlimb footpads was obviously suppressed as compared with the control group,
The effect was more than prednisolone (10 mg / kg / day). That is, the composition of the present invention 2 had remarkable allergic inflammation and non-allergic inflammation suppressing activity.

[0094]

The constitution of the present invention 1 is as described above. According to the present invention 1, a precipitate obtained by adding an organic solvent to a culture solution of Streptomyces kanamyceticus is used. As a result, it is possible to provide a composition containing an active ingredient exhibiting a remarkable allergic inflammation and non-allergic inflammation suppressing action.

The constitution of the present invention 2 is as described above. According to the present invention 2, the precipitate obtained by adding the organic solvent to the culture solution of Streptomyces kanamyceticus is subjected to gel filtration. To provide a composition containing an active ingredient exhibiting remarkable allergic inflammation and non-allergic inflammation suppressing action by using a fraction having a dextran-equivalent molecular weight of 1,000,000 to 10,000,000 obtained by subjecting to column chromatography. You can

[Brief description of drawings]

FIG. 1 is a graph showing the results of Test Example 1, and is a graph showing the amounts of leaked pigments of a liquid containing the composition of the present invention 1, prednisolone, and each control.

FIG. 2 is a graph showing the results of Test Example 2 and showing the erythema strength and skin weight of a liquid containing the composition of the present invention 1 and prednisolone, respectively.

FIG. 3 is a graph showing the results of Test Example 3, and is a graph showing the amounts of leaked pigments of the composition containing the composition of the present invention 1 and prednisolone, and the respective controls.

FIG. 4 shows the results of Test Example 4, and is a graph showing the volume of the right hind footpad edema in rat carrageenin edema of the composition containing the composition of the present invention 1 and aspirin.

FIG. 5 shows the results of Test Example 5, and is a graph showing the edema volume of the left and right hind limbs of the control solution-containing composition of the present invention 1 and prednisolone.

FIG. 6 is a graph showing the results of Test Example 1 and a graph showing the amounts of leaked pigments of a liquid containing the composition of the present invention 2 and prednisolone, and each control.

FIG. 7 shows the results of Test Example 2, and is a graph showing the erythema strength and skin weight of a liquid containing the composition of the present invention 2 and prednisolone, respectively.

FIG. 8 is a graph showing the results of Test Example 3 and a graph showing the amounts of leaked pigments of a liquid containing the composition of the present invention 2 and prednisolone, and each control.

FIG. 9 shows the results of Test Example 4, and is a graph showing the volume of the right hind footpad edema in rat carrageenin edema of the control solution containing the composition of the present invention 2 and aspirin.

FIG. 10 shows the results of Test Example 5, and is a graph showing the edema volume of the left and right hind limbs of the control solution-containing composition of the present invention 2 and prednisolone.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Sumi Kuriyama 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka 2-1 Sekisui Chemical Co., Ltd.

Claims (2)

[Claims] 1. A composition for an inflammation inhibitor, which comprises a precipitate obtained by adding an organic solvent to a culture solution of Streptomyces kanamyceticus. 2. A composition for an inflammation inhibitor, which is obtained by subjecting the precipitate according to claim 1 to gel filtration column chromatography and comprises a fraction having a dextran-converted molecular weight of 1,000,000 to 10,000,000.