JPH06305974A - Composition for inflammation-suppressing agent - Google Patents

Abstract

PURPOSE:To obtain a composition for suppressing agents effective also on every inflammation reactions of allergic and non-allergic using Streptomyces nobilis of Actinomyces as a raw material. CONSTITUTION:This composition for inflammation-suppressing agents consists of a fraction eluted between 85min and 95min after starting elution by subjecting a substance obtained by extracting cultured mixture of Streptomycesnobilis of Actinomyces or its dried material with an organic solvent to high- performance liquid chromatography using an ODS column for collection and changing the ratio of eluting solvents in the range of ratio of water: acetonitrile of (80:20) to (0:100) at a definite rate over 80min and then further continuing elution in a ratio of water to acetonitrile of 0:100.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to Streptomyces nobilis actinomycete (hereinafter abbreviated as "S. nobilis").
The substance extracted with an organic solvent from the culture broth or its dried product (hereinafter abbreviated as "crude extract") is subjected to high performance liquid chromatography (hereinafter abbreviated as "HPLC") using a preparative ODS column. ) And the elution solvent is water:
Acetonitrile = 80: 20 to 0: 100 was changed at a constant rate over 80 minutes, and then elution was continued with water: acetonitrile = 0: 100 to elute between 85 and 95 minutes after the start of elution. Consists of fractions,
The present invention relates to a composition for an allergic and non-allergic inflammation inhibitor (hereinafter abbreviated as "HPLC effective fraction").

[0002]

2. Description of the Related Art Inflammatory reactions are roughly classified into allergic inflammation and non-allergic inflammation. An allergy is a kind of pathological condition in which an immune reaction that occurs upon the second contact with an antigen is excessively or inappropriately expressed by an individual, and due to the difference in the nature of the antibody involved, type I, Type II, II
It is classified into type I and type IV reactions. Of these four types, allergic inflammatory reactions related to type III reaction (immune complex reaction: Arthus reaction) and type IV reaction (cellular immune reaction: delayed type hypersensitivity reaction) are self-induced diseases such as rheumatoid arthritis. It has become clear that it plays an important role in the development of immune diseases and various inflammatory diseases such as hepatitis, nephritis and infectious diseases.

[0003] Conventionally, for the treatment of inflammatory skin diseases, particularly for the treatment of intractable allergic skin diseases, an external preparation containing an adrenocortical hormone as an active ingredient has been widely used and its pharmacological effect is known to be high ( Monthly Pharmaceutical Affairs, Vol. 26, No. 8,
56, 1985).

However, since drugs containing adrenocortical hormones generally have significant side effects, there is a strong demand for alternatives to them.

By the way, conventionally, various antibiotics have been found in the actinomycete culture filtrate, and the culture filtrate is said to be a treasure trove of physiologically active substances. However, no substance that suppresses allergic inflammation has so far been found in the actinomycete culture filtrate.

Further, conventional anti-inflammatory agents such as aspirin and indomethacin have a problem that their inhibitory action against allergic inflammation is extremely weak.

Therefore, the inventors of the present invention used S. As a result of searching for substances having an allergic inflammation-suppressing action using Nobilis, it was obtained by subjecting a substance extracted from a culture solution or a dried solid product thereof with an organic solvent to ODS column chromatography and silica gel column chromatography. The effective fraction was confirmed to have a remarkable inhibitory effect and safety on allergic and non-allergic inflammatory reactions, and succeeded in finding a composition which is extremely useful as an anti-inflammatory agent and has no safety problems. .

[0008]

However, since the above composition is considered to contain various impurities that do not have an allergic and non-allergic inflammation inhibitory action, it is unavoidable to increase the dose. It was

In view of such circumstances, the object of the present invention is to purify the above-mentioned composition for allergic and non-allergic inflammation inhibitors to remove impurities, and to suppress allergic and non-allergic inflammation by administration in a small amount. Is to enable.

[0010]

[Means for Solving the Problems] In order to find out allergic inflammation-inhibiting substances for type III allergy, the present inventors have developed an animal model of type III allergic reaction in rat 4 hours heterologous passive skin anaphylaxis (4 hours hete
As a result of screening using the rologous PCA) reaction, the actinomycete S. A crude extract of Nobilis culture or its dried solid was subjected to HPLC using a preparative ODS column, and the elution solvent was changed from water: acetonitrile = 80: 20 to 0: 100 at a constant rate over 80 minutes. After that, further elution was continued with water: acetonitrile = 0: 100 to confirm that the fraction eluted between 85 and 95 minutes after the start of elution contained allergic and non-allergic inflammation inhibitory substances. Based on this finding, the present invention has been completed based on this finding.

That is, the composition for an allergic and non-allergic inflammation suppressant according to the present invention is used for preparative extraction of a substance extracted from an actinomycete Streptomyces nobilis culture liquid or a dried solid thereof with an organic solvent. It was subjected to HPLC using an ODS column, the elution solvent was changed from water: acetonitrile = 80: 20 to 0: 100 over a period of 80 minutes at a constant ratio, and then water: acetonitrile =
By continuing the elution at 0: 100, 85
Min-95 min.

The actinomycete S. Nobilis is available from public preservation agencies, for example, RIKEN's Preservation Bacteria (JCM 4274) (this is the AT
Bacteria such as CC19252 and preserved as CBS 198.65 in the Netherlands) can be used.

In the following, Actinomyces S. Cultivation of Nobilis and solvent extraction from the culture solution are described.

Actinomycete S. Cultivation of Nobilis is performed using a medium containing appropriate nutrients. That is, in the case of liquid culture, as the component, 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 sugars is preferable. Used for. As a typical medium example,
A starch-ammonium-based liquid medium (including soluble starch, dipotassium hydrogen phosphate, and ammonium chloride) can be used. 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. The preferable culture time for liquid culture is 3 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 Nobilis, a solvent extraction treatment is performed. The solvent extraction is carried out by a method of bringing the culture solution into contact with the solvent as it is, or a method of evaporating the culture solution to dryness and bringing the dried product into contact with the solvent, and obtaining the active ingredient of the inflammation inhibitor from the extraction phase. In the solvent extraction treatment, S. Nobilis cells may or may not be present. When the extraction is carried out without the presence of bacterial cells, the culture solution is subjected to solid-liquid separation to remove the bacterial cells, and the separated liquid phase is used as it is or after being dried to solvent extraction treatment. Centrifugation, filtration, etc. are appropriately used as the solid-liquid separation means.

An organic solvent is used as an extraction solvent for the inflammation suppressing active ingredient. Typical examples thereof include esters such as ethyl acetate and ethyl propionate; alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol; ethers such as ethyl ether and dioxane; ketones such as acetone and methyl ethyl ketone. Examples of the solvent that can be used include, but are not limited to. Also, a mixed solution of the above solvents can be used. Particularly suitable solvents are ethyl acetate, methanol and the like.

In the case of extraction using the culture solution as it is without being dried, the ratio of the culture solution to the solvent is not particularly limited,
From the viewpoint of extraction efficiency and easiness of operation, the volume of the culture medium is preferably 0.5 to 2 volumes per volume. In the case of extraction using a dried product of the culture solution, the amount of solvent used is not particularly limited. The solvent extraction may be performed at room temperature or under heating, but the latter is more efficient. The heating is performed in the temperature range below the boiling point of the solvent under normal pressure. The extraction time varies depending on the type of solvent and the extraction temperature, but is preferably 3 to 12
It is in the range of 0 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 culture solution or a dried solid product thereof.

Next, the above S. HPLC for Nobilis broth solvent extract is described.

ODS as a column packing material for HPLC
(Octadecylsilanes such as octadecyldimethylchlorosilane) are used. The filling amount is not particularly limited, but preferably S. The amount is 100 to 5000 times the weight ratio to the Nobilis culture extract. S. When the column is charged with the ethyl acetate extract of Nobilis culture, that is, the crude extract, it is first suspended in a small amount of solvent and then solid-liquid separated. Centrifugation, filtration or the like is appropriately used as a means for solid-liquid separation, but this operation is preferably repeated several times. However, the crude extract may be directly subjected to HPLC without the above solvent suspension and solid-liquid separation.

The separated liquid-phase aggregated liquid is dried to obtain
Add a small amount of solvent again to dissolve, and charge the column. The solvent used at this time is not particularly limited, but is preferably methanol, acetonitrile or the like.
The elution solvent is not particularly limited, but a solvent having a polarity of water: acetonitrile = 80: 20 to 0: 100 is preferably used.

To formulate the composition of the present invention into an anti-inflammatory agent, it is usually in the form of a pharmaceutical composition together with a pharmaceutical carrier. Examples of the carrier include fillers, disintegrators, extenders, binders, moisturizers, surfactants, diluents and diluents commonly used for preparing a drug according to the dosage form. It is illustrated. Further, by selecting an appropriate solvent, the obtained solvent extract or its concentrate can be used as it is as an external liquid preparation.

The dosage unit form of the anti-inflammatory agent formulated using the composition of the present invention is, in addition to the above-mentioned external liquid preparation, tablets, pills, drinkable liquids, powders, suspensions, emulsions, granules. , Capsules, suppositories, injections (solutions, suspensions, etc.),
Ointments and the like are exemplified.

The amount of the composition of the present invention to be contained in the anti-inflammatory agent is not particularly limited and may be appropriately selected from a wide range, but is preferably 0.1 to 50% by weight in the anti-inflammatory agent.

The inflammation inhibitor obtained from the composition of the present invention is
When it is used, it is administered by a method depending on various 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 injection, intravenous, intramuscular,
It is administered intradermally, subcutaneously or intraperitoneally, rectally in the case of suppositories, and applied in the case of ointments.

The dose of the anti-inflammatory agent obtained from the composition of the present invention is appropriately selected according to the purpose of use, symptoms, etc.
Usually, 0.2 to 50 mg / day as the composition of the present invention
It is in the range of about kg. Further, it is of course possible to administer the above-mentioned pharmaceutical composition 3 to 4 times / day separately.

[0026]

EXAMPLES Next, the present invention will be concretely demonstrated by giving examples of the present invention.

Example 1 (Preparation of crude extract and elution by ODS column chromatography, silica gel column chromatography, HPLC) Actinomyces S. cerevisiae purchased from RIKEN. Nobilis (JCM427
4) is cultivated in 100 ml of starch / ammonium medium containing 0.2% yeast extract for 40 to 45 hours with shaking (pre-preculture)
Then, 3 liters of the same medium was inoculated with 60 ml of the pre-preculture liquid, and shake culture (seed culture) was performed for 25 to 30 hours. Furthermore, 285 liters of starch-ammonium medium (containing 1 g of soluble starch, 0.05 g of dipotassium hydrogen phosphate and 0.05 g of ammonium chloride in 100 ml of distilled water) were inoculated with the whole seed culture, and inoculated at 3 ° C at 3 ° C. Culture was performed with shaking for 10 days.

The culture was centrifuged to obtain 250 liters of the separated supernatant, which was concentrated under reduced pressure to 28 liters. 3.4 L of this culture concentrate was placed in a separating funnel, an equal amount of ethyl acetate was added thereto, and the whole liquid was shaken for 10 minutes. After standing still for 5 minutes, the aqueous phase and the ethyl acetate phase were separated. An equal amount of ethyl acetate was added to the separated aqueous phase, the above operation was repeated, and the aqueous phase and the ethyl acetate phase were separated again.
An equal amount of ethyl acetate was added to the separated aqueous phase, the above operation was repeated once more, and the third extraction was performed. Thus 3
The ethyl acetate phases obtained by the operation once were collected, and the collected liquid was concentrated to dryness with an evaporator to obtain 1 g of a crude extract.

Next, 1 g of the crude extract obtained above
To ODS (octadecyldimethylchlorosilane) 1.
After being adsorbed on 5 g and suspended in a small amount of solvent (pure water), an ODS column (150 g of ODS was suspended in methanol and charged in a column having an inner diameter of 32 mm and a length of 305 mm and then water: methanol = 1: 9 was added. The solvent was gradually changed to 9: 1 and the solvent in the column was charged with water: methanol = 9: 1).

The elution solvent was water: methanol = 9: 1 (5).
00 ml, 400 ml of water: methanol = 6: 4, water:
400 ml of methanol = 3: 7, 400 ml of methanol
ml, ethyl acetate 400 ml, flow rate 3 ml / in this order
Flow in minutes and the liquid eluted with these elution solvents is
When dried using an evaporator, respectively, water: methanol = 9: 1 elution fraction was 465 mg, water: methanol = 6: 4 elution fraction was 85 mg, and water: methanol = 3: 7.
Elution fraction 102 mg, methanol elution fraction 131 m
and 117 mg of ethyl acetate-eluted fractions were obtained.

Next, 131 mg of the ODS column methanol elution fraction obtained above was adsorbed on 250 mg of silica gel (manufactured by Merck) and a small amount of solvent (hexane) was added.
Suspended in silica gel column (silica gel 1.5
g, suspended in ethyl acetate and packed, 10 mm inner diameter, 4 length
0 mm column). As an elution solvent, ethyl acetate, ethyl acetate: methanol = 95: 5, ethyl acetate: methanol = 90: 10, ethyl acetate: methanol = 80: 20, ethyl acetate: methanol = 60: 4.
0, ethyl acetate: methanol = 40: 60, methanol were sequentially flowed in this order at a flow rate of 1 ml / min, 6 ml each, and the liquids eluted with these elution solvents were dried using an evaporator. Elution fraction is 47 mg, ethyl acetate: methanol = 95: 5 Elution fraction is 8.6 mg, ethyl acetate: methanol = 90:
10 elution fractions 13 mg, ethyl acetate: methanol = 8
16 mg of the fraction eluted with 0:20, 21 mg of the fraction eluted with ethyl acetate: methanol = 60: 40, 11 mg of the fraction eluted with ethyl acetate: methanol = 40: 60, and 5.8 mg of the fraction eluted with methanol were obtained.

Next, the ethyl acetate obtained above:
21 mg of the elution fraction of methanol = 60: 40 was suspended in a small amount of methanol, centrifuged at 3000 rpm for 10 minutes, and the obtained supernatant was dried and dissolved in a small amount of methanol again to collect ODS for fractionation. (Octadecyldimethylchlorosilane) column (manufactured by Tosoh Corporation, ODS-80TM,
The inner diameter was 21.5 mm and the length was 300 mm).
Then, the solvent flow rate was set to 5 ml / min, and the elution solvent was changed from water: acetonitrile = 80: 20 to 0: 100 at a constant ratio over 80 minutes, and then elution was continued with water: acetonitrile = 0: 100. Thus, a fraction eluted between 85 minutes and 95 minutes after the start of elution was obtained. The collected liquid of this fraction was dried to dryness using an evaporator, and HPL
C effective fraction of 6.6 mg was obtained. In addition, 14 mg of a dry solid was obtained in the same manner from the collected liquid obtained by elution at other times.

Example 2 (Elution of crude extract by HPLC) 1 g of the crude extract obtained in the same manner as in Example 1 was directly used for fractionation without solvent suspension and solid-liquid separation in Example 1. By operating in the same manner as in Example 1 except that it was subjected to HPLC using an ODS column, 85
The HPLC effective fraction of 20 mg was obtained from the fraction eluted between minutes and 95 minutes. In addition, from the collected liquid of the liquids obtained by eluting at other times, the other eluted fractions 98
0 mg was obtained.

Test Example 1 (Action against 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).

Each 0.5 ml of this antigen solution weighs about 3 k
g of New Zealand White male rabbit was injected into the left and right gluteus muscularis 4 times per 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 hours heterologous passive skin anaphylaxis (4 hours heterologous PC
A) The titer of the reaction was 1:32.

Ii) Rat 4-hour heterogeneous PCA reaction (III
Type of allergic skin reaction) The dry solidified HPLC fractions obtained in Example 2 correspond to 25 mg / ml as the crude extract amount, that is, the HPLC effective fraction is 0.5 mg / ml and the other eluted fractions are 2.45 mg
The amount of dimethyl sulfoxide was 5% by weight to a 5% by weight aqueous solution of gum arabic and the suspension was suspended in a solution of 5% by weight. As a control experiment, a crude extract 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 as to have a final concentration of 25 mg / ml. did.

The suspension thus obtained was used as a test solution.
As test animals, male Wistar rats weighing 120 to 200 g were used.

First, 2 ml / kg of the above test solution was applied to rats.
(HPLC effective fraction amount 1.0 mg / kg, other elution fraction amount 49 mg / kg, crude extract amount 5
(0 mg / kg) was intraperitoneally administered.

20 hours after the administration of the test solution, 0.05 ml of an injection solution prepared by diluting the rabbit anti-ovalbumin serum 4 times with physiological saline was injected into the dorsal skin of the rat. It was sensitized with the above antiserum.

Then, 4 hours after the administration of the antiserum, 0.
2.5 ml / k of 5 wt% Evans Blue physiological saline
g intravenous injection to induce a heterogeneous PCA reaction for 4 hours.

The leakage dye 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)
The leaked dye was extracted by immersing it in a mixed solution of 3 volumes of an aqueous solution of sodium sulfate and 7 volumes of acetone for 24 hours. 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 HP
The amount of leaked dye was determined by using the above dimethyl sulfoxide-containing arabic gum aqueous solution containing no LC fraction, and otherwise performing the same procedure as above.

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, in the group administered with the test solution containing the other eluted fractions of Example 2, compared to the control group, the amount of pigment leaked to the skin at the different PCA reaction site in the rat for 4 hours was higher than that in the control group. It has almost no effect. However, in the group administered with the test solution containing the HPLC effective fraction, along with the group administered with the test solution containing the crude extract,
Compared with the control group, the amount of pigment leaked to the skin at the different PCA reaction site for 4 hours in the rat was significantly reduced, and a remarkable allergic inflammation suppressing activity was observed. That is, it can be seen that the crude extract has an allergic inflammation-suppressing activity, and that activity is derived from the substances contained in the HPLC effective fraction of Example 2.

In the group to which the test solution containing the HPLC effective fraction of Example 2 was administered, the dose was 1/50 of that of the test solution containing the crude extract, compared to the control group for 4 hours in the rat. The amount of pigment leaked to the skin at the PCA reaction site was greatly reduced. That is, the specific activity of the HPLC effective fraction is about 50 times that of the ODS column effective fraction.

Test Example 2 (Action against type IV allergic reaction) i) Action against mouse footpad delayed edema reaction (type IV allergic footpad edema reaction) The above-mentioned HPLC effective fraction in type IV allergic reaction by the following method. Was investigated. First, a test solution containing an HPLC effective fraction having a concentration of 0.2 mg / ml was prepared in the same manner as in Test Example 1. As a control experiment, a crude extract was prepared by suspending the crude extract in the above aqueous solution of gum arabic containing dimethyl sulfoxide so that the final concentration was 10 mg / ml. In addition, as an animal to be tested, ICR weighing 40 to 50 g
Male mice were used.

Sheep red blood cells in physiological saline to a final concentration of 4 ×
Dilute to 10 ⁹ cells / ml, and dilute 0.0
5 ml was injected into the left footpad of the mouse, and the mouse was sensitized with sheep red blood cells.

Then, 4 days after the sensitization, the test solution of 0.
2 ml (0.8-1.0 mg as HPLC effective fraction amount)
/ Kg, and a crude extract amount of 40 to 50 mg / kg) were intraperitoneally administered to the sensitized mice.

Immediately after administration of this test solution, sheep red blood cells 4 × 1
0.05 ml of ⁰⁹ cells / ml was reinjected into the right footpad of sensitized mice to induce type IV aralgic reaction.

Furthermore, 6 of the above-mentioned type IV aralgic reaction induction
After a lapse of time, 0.2 ml of the above test solution was intraperitoneally administered to the same mouse again.

Finally, 18 hours after the second administration of the test solution, the degree of swelling of the right footpad of the mouse was examined by a macroscopic score.

As a control for this test, the HP
The swelling degree of the right footpad of the mouse was examined by using the above dimethyl sulfoxide-containing arabic gum aqueous solution containing no LC effective fraction and performing the other points in the same manner as the above operation.

This test was carried out using 7 mice each, and the degree of swelling was averaged from the values obtained for these mice.

The results of this test are shown in FIG.

As is clear from this figure, H of the second embodiment
In the group to which the test solution containing the PLC effective fraction was administered, the swelling of the right footpad was obviously suppressed and the remarkable allergic inflammation suppressing activity was recognized as compared with the control group. Further, it is recognized that the HPLC effective fraction shows an activity equivalent to that of the crude extract at a dose of about 1/50 of that of the crude extract.

Ii) Rat skin tuberculin reaction (IV
Next, the method of Kuriyama et al. (Nippon Pharmacology, Vol. 94, 113-11)
(Page 8, 1980), the effect of the above HPLC effective fraction on the rat skin tuberculin reaction was examined.

First, by the same operation as in Test Example 1, the concentration was 0.5.
A test solution containing 0 mg / ml of HPLC effective fraction was prepared. For control experiments, the crude extract was used at a final concentration of 2
The suspension was prepared in the above aqueous solution of aramid gum containing dimethyl sulfoxide so as to be 5 mg / ml.
Bacillus Calmatte-Guerin (BCG) was suspended in physiological saline to a final concentration of 2.5 mg / ml.
The thing autoclaved at 5 degreeC was prepared. Further, the purified tuberculin was dissolved in physiological saline to prepare 200
What was made into μg / ml was prepared. As test animals, Wistar male mice weighing 180 to 220 g were used.

First, 0.2 ml of the above BCG suspension (B
As a CG, 0.5 mg) was intraperitoneally administered to the test animal.

7 days after the administration of BCG, 2 ml of the above test solution
/ Kg (1.00 mg / k as the effective fraction of HPLC)
g, 50 mg / kg as a crude extract amount) was intraperitoneally administered to a test animal.

Immediately after this administration, 0.1 ml of the above physiological saline containing tuberculin (20 g of purified tuberculin was prepared).
μg) was administered intradermally to the back of test animals to induce a skin tuberculin reaction. At the same time, 0.1 ml of physiological saline alone was intradermally administered to another site on the back as a control.

Twenty-four hours after induction, the erythema diameter (mm) was measured with a caliper and the color difference meter (Minolta CR-20) was used for the sites of tuberculin administration and physiological saline administration.
0) was used to measure the erythema intensity. For each individual, a value obtained by subtracting the value at the physiological saline administration site from the value at the tuberculin administration site was determined, and the product of the respective erythema diameter and erythema intensity values was calculated as the skin erythema index. Then, the tuberculin administration site and the physiological saline administration site were punched out with a punch having a diameter of 16 mm and the weight thereof was measured.
The value at the physiological saline-administered site was subtracted from the value at the tuberculin-administered site, and the obtained value was taken as the skin swelling weight.

As a control for this test, the HP
The dimethyl sulfoxide-containing arabic gum aqueous solution containing no LC effective fraction was used, and otherwise the same as the above-mentioned operation.

This test was carried out using 5 rats each, and the average value of the values obtained for these rats is shown in FIG. 3 and FIG. 4 as the test results.

As is clear from these figures, Example 2
In the group administered with the HPLC effective fraction and the crude extract of
Compared with the control group, erythema and edema due to cutaneous tuberculin reaction were clearly suppressed, and remarkable type IV allergic inflammation suppressive activity was observed. Further, it is recognized that the HPLC effective fraction shows an activity equivalent to that of the crude extract at a dose of about 1/50 of that of the crude extract.

Test Example 3 (Action against Adjuvant Arthritis) The above H against rat adjuvant arthritis was prepared by the following method.
The effect of the PLC effective fraction was investigated.

First, 0.50 m in the same manner as in Test Example 1.
A test solution for HPLC effective fraction having a concentration of g / ml was prepared. For control experiments, the crude extract was added to a final concentration of 25 mg.
/ Ml was prepared by suspending it in the above aqueous solution of arabic gum containing dimethyl sulfoxide. Also,
As test animals, male Wistar Lewis rats weighing 200 to 250 g were used.

Mycobacterium tubercurosis H37RA (Dif. Tuberculosis) suspended in liquid paraffin at a concentration of 0.6% by weight.
0.1 ml of a bacterial solution adjuvant (manufactured by Co.) was administered into the rat right footpad.

Thereafter, the above test solution was applied to rats at 2 ml / k.
g (1.0 mg / kg as HPLC effective fraction amount, 50 mg / kg as crude extract amount) from the day 1 on the day
The drug was administered intraperitoneally every day for 22 days. In addition, the volume of the footpads of the right and left hind limbs of the rat was measured daily for 23 days from the day of adjuvant administration using a Plethysmometer (manufactured by Ugo Basile) to examine changes in these volumes.

As a control for this test, the above HP
Using the dimethyl sulfoxide-containing arabic gum aqueous solution containing no LC effective fraction, the same procedure as in the above was carried out in the other points to examine the change in the volume of the foot pads of the left and right hind limbs.

This test was carried out using 5 rats each, and the volume of the footpads of the left and right hind limbs is shown in FIGS. 5 and 6 as the average of the values obtained for these rats.

As is clear from these figures, Example 2
In the group to which the test solution containing the HPLC effective fraction of was administered, the increase in the volume of the footpads of the right and left hind limbs was obviously suppressed as compared with the control group, and remarkable efficacy against adjuvant arthritis was observed. The HPLC effective fraction is
It is observed that at a dose of about 1/50 of the crude extract, the same activity is shown.

Test Example 4 Effect on rat carrageenin footpad edema reaction (non-allergic reaction) The effect of the above HPLC effective fraction on rat carrageenin footpad edema reaction was examined by the following method.

First, 0.50 m in the same manner as in Test Example 1.
A test solution for HPLC effective fraction having a concentration of g / ml was prepared. For control experiments, the crude extract was added to a final concentration of 25 mg.
/ Ml was prepared by suspending it in the above aqueous solution of arabic gum containing dimethyl sulfoxide. Also,
Male Wistar rats weighing 140 to 160 g were used as test animals.

Also, a solution was prepared by dissolving caranigen in physiological saline to 1%.

First, 2 ml / kg of the above test solution was applied to rats.
(1.0 mg / kg as HPLC effective fraction amount, 50 mg / kg as crude extract amount) was intraperitoneally administered.

The volume of the right hind limb of the test animal was measured with a Plethysmometer.
(Ugo-Basile), the above-mentioned 1% carrageenin was injected into the footpad of the right hind leg of the test animal to induce an edema reaction.

The volume of the right hind limb of the test animal was similarly measured 1, 2, 3, 4, 5 hours after the induction.

As a control for this test, the dimethyl sulfoxide-containing arabic gum aqueous solution containing no HPLC effective fraction was used, and the other points were the same as in the above operation, and the volume of the right hind leg of the rat was adjusted. It was measured.

This test was carried out using 5 rats each, and the right posterior posture volume is shown in FIG. 7 as an average value of the values obtained for these rats.

As is clear from this figure, in the group to which the HPLC effective fraction and the crude extract were administered, the swelling of the right hind limb was obviously suppressed and the remarkable anti-inflammatory activity was recognized as compared with the control group. Further, it is recognized that the HPLC effective fraction shows an activity equivalent to that of the crude extract at a dose of about 1/50 of that of the crude extract.

Test Example 5 Toxicity Test The toxicity of the above HPLC effective fraction was examined by the following method.

First, the HPLC effective fraction obtained in Example 2 was suspended in the above aqueous solution of dimethyl sulfoxide-containing acacia gum so that the final concentration was 40 mg / 5 ml. The suspension thus obtained was used as a test solution, and ICR male mice weighing 25 to 30 g were used as test animals.

The above test solution was intraperitoneally administered to mice at 5 ml / kg (corresponding to 40 to 100 times the effective amount of Test Examples 1 to 4). 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 H obtained in Example 2
The PLC effective fraction showed no toxicity at 40 to 100 times the effective amount. This test was carried out using 5 mice.

[0085]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a composition containing an active ingredient which exhibits a remarkable suppressive action on both allergic and non-allergic inflammatory reactions by administration in a small amount.

[Brief description of drawings]

FIG. 1 Liquid containing crude extract (50 mg / kg), HPLC
Effective fraction-containing liquid (1.0 mg / kg: 50 m of crude extract)
g / kg equivalent amount), HPLC and other elution fraction-containing liquids (4
(9 mg / kg: 50 mg / kg equivalent of crude extract) and its control, the graph shows the amount of leaked pigment in the rat 4-hour heterogeneous PCA reaction.

FIG. 2: Crude extract-containing liquid (40 to 50 mg / kg), H
3 is a graph showing the swelling degree of the right footpad in a mouse footpad-delayed edema reaction of a liquid containing a PLC effective fraction (0.8 to 1.0 mg / kg) and its control.

FIG. 3: Liquid containing crude extract (50 mg / kg), HPLC
It is a graph which shows the skin erythema index in the rat skin tuberculin reaction of an effective fraction containing liquid (1.0 mg / kg) and its control.

FIG. 4 Liquid containing crude extract (50 mg / kg), HPLC
It is a graph which shows the skin weight in the rat skin tuberculin reaction of an effective fraction containing liquid (1.0 mg / kg) and its control.

FIG. 5: Liquid containing crude extract (50 mg / kg), HPLC
It is a graph which shows the volume of the right footpad in the effective fraction containing liquid (1.0 mg / kg) and its control adjuvant arthritis.

FIG. 6 Liquid containing crude extract (50 mg / kg), HPLC
It is a graph which shows the volume of the left footpad in the effective fraction containing liquid (1.0 mg / kg) and its control adjuvant arthritis.

FIG. 7: Liquid containing crude extract (50 mg / kg), HPLC
3 is a graph showing the volume of the right footpad in the rat carrageenin footpad edema reaction of the effective fraction-containing solution (1.0 mg / kg) and its control.

Claims (1)

[Claims] 1. A substance extracted with an organic solvent from a culture broth of Streptomyces nobilis or a dried solid of Streptomyces nobilis is subjected to high performance liquid chromatography using a preparative ODS column to obtain an eluting solvent. To water: acetonitrile = 80: 20 to 0: 100
Up to 80 minutes, and then continue to elute with water: acetonitrile = 0: 100. A composition for an anti-inflammatory agent, which is effective for any inflammatory reaction of sex.