JP3602574B2 - Method for producing inflammation-inhibiting substance - Google Patents

Description

【００２９】
結果から明らかなように、インドメタシンを投与した群では、コントロールと比べて漏出色素量に差は認められないが、実施例１で得られた炎症抑制物質を含有する供試液を投与した群では、コントロールと比べて、ラット４時間異種ＰＣＡ反応部の皮膚に漏出する色素量が大幅に減少した。すなわち、本発明により得られた炎症抑制物質はアレルギー性炎症抑制活性を有する。
【００３０】
（試験例２）毒性試験
以下の方法で実施例１で得られた炎症抑制物質の毒性を調べた。
まず、炎症抑制物質を、最終濃度が４０ｍｇ／５ｍｌになるように、上記ジメチルスルホキサイド含有アラビアゴム水溶液に懸濁し、供試液とした。被験動物としては体重２５〜３０ｇのＩＣＲ雄性マウスを用いた。
上記供試液をマウスに５ｍｌ／ｋｇ（試験例１の有効量の５０倍に相当）を腹腔内投与した。
その結果、毒性症状は特に認められず、また供試液投与２週間後の死亡率は０％であった。生存した被験動物の剖検においても、何ら異常は認められなかった。この結果からも明らかなように、実施例で得られた炎症抑制物質は有効量の５０倍量で毒性を示さなかった。なお試験は５匹のマウスを用いて行った。
【００３１】
（比較例１）
従来法、すなわち培養液に硫酸アンモニウムを添加し、その沈殿物からの抽出物を、ＯＤＳカラムクロマトグラフィー及び再結晶化により炎症抑制物質を精製する方法で炎症抑制物質を単離し、１週間あたりに単離できる炎症抑制物質量を、本発明の方法を用いた場合と比較した。結果を表２に示す。
【００３２】
【表２】

【００３３】
上記の結果から、本発明によると、同一時間あたりに従来法の４倍量の炎症抑制物質を単離することが可能となった。
【００３４】
【発明の効果】
本発明の方法は上述のとおりであり、これによると、活性を損なうことなく、効率的に短時間で炎症抑制物質を大量に精製できる。[0001]
[Industrial application fields]
The present invention relates to a method for producing an anti-inflammatory substance effective for both allergic and non-allergic inflammatory reactions.
[0002]
[Prior art]
Inflammatory reactions are broadly divided into allergic inflammation and non-allergic inflammation. Allergy is a kind of pathological symptom in which an immune reaction that occurs upon a second contact with a certain antigen appears excessively or inappropriately by an individual, depending on the type of antibody involved, type I, type II , Type III and type IV reactions.
Among these four types, allergic inflammatory reactions involved in type III reaction (immune complex reaction: Arthus reaction) and type IV reaction (cellular immune reaction: delayed hypersensitivity reaction) are like rheumatoid arthritis. It plays an important role in the development of various inflammatory diseases such as autoimmune diseases, as well as asthma, hepatitis, nephritis, and dermatitis.
[0003]
By the way, various antibiotics have been conventionally 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 ever been found in actinomycete culture filtrate.
In addition, aspirin (Nikkei Science, 3:70, 1991) and indomethacin (Ther. Res., 3: 1057, 1985), which are conventional anti-inflammatory agents, have a problem that their inhibitory action against allergic inflammation is extremely weak. is there.
[0004]
Therefore, as a result of searching for substances having an allergic inflammation inhibitory activity using actinomycetes, allergenicity was found in a crude extract of Streptomyces nobilis (hereinafter referred to as “S. nobilis”) culture solution. Confirmed remarkable inhibitory action and safety against inflammatory reaction, and succeeded in finding a very useful composition having no problem as an anti-inflammatory agent (Japanese Patent Laid-Open No. 5-25053). It proposes to isolate allergic inflammatory response inhibitors using chromatography. Further, an ammonium sulfate is added to the culture broth, and the extract obtained by extracting the precipitate at that time with a solvent is used to efficiently purify the anti-inflammatory substance using ODS column chromatography and recrystallization from the solvent. It also proposes a method.
[0005]
However, in the conventional purification method, since the amount of impurities in the precipitate due to ammonium sulfate is very large, the extraction operation becomes complicated. Therefore, in order to secure a large amount of inflammation-inhibiting substances, purification must be performed in multiple steps, which requires a great deal of time.
[0006]
[Problems to be solved by the invention]
The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a method for purifying a large amount of an inflammation-inhibiting substance efficiently in a short time.
[0007]
[Means for Solving the Problems]
The inventors have described that actinomycetes S. cerevisiae. The cell extract obtained by extracting the cell of Nobilis with at least one solvent selected from the group consisting of dichloromethane, ethyl acetate and acetone was subjected to ODS column chromatography, and the polarity was methanol: acetonitrile = 1: From the fraction eluted with methanol between an aqueous solution containing 85% of 1 mixed solvent, it was found that the inflammation-inhibiting substance can be obtained as a single product by purifying it using recrystallization from the solvent. The present invention has been completed based on the findings.
[0008]
In the present invention, the actinomycete S. Nobilis can be obtained from a public preservation institution, for example, bacteria such as a preserved bacterium (JMC4274) from RIKEN (which is also preserved as ATCC 19252 in the United States and CBS 198.65 in the Netherlands).
[0009]
Streptomyces S. Nobilis is cultured using a medium containing appropriate nutrients. In the case of liquid culture, as a medium component, for example, an aqueous solution containing at least one saccharide such as glucose, proteins such as peptone and malt extract, vitamins, nucleic acids, amino acids, complex carbohydrates and the like is preferably used. . As a typical medium, for example, a starch / ammonium-based liquid medium (containing soluble starch, K ₂ HPO ₄ , NH ₄ Cl) may be mentioned.
The pH of the liquid medium is preferably in the range of 2-9, and the culture temperature is preferably 15-42 ° C. The culture time for liquid culture is preferably 1 to 14 days.
[0010]
The culture broth thus obtained was filtered and actinomycetes S. cerevisiae. Obtain nobilis cells. The bacterial cells are extracted with a solvent to obtain a bacterial cell extract.
In the extraction treatment, the cells may be brought into contact with the solvent as they are, but it is preferable to use them in advance by grinding with a mortar or the like, or by pulverizing with a homogenizer, an ultrasonic generator or the like.
[0011]
The solvent used in the extraction process, dichloromethane, is limited to at least one selected from the group consisting of ethyl acetate and acetone.
[0012]
In the above extraction treatment, the ratio of the cells and the solvent is not particularly limited, but 10 to 20 ml of the solvent per 1 g (wet weight) of the cells is preferable from the viewpoint of extraction efficiency and operability. Moreover, although extraction time changes with the kind of solvent, extraction temperature, etc., 10 minutes-24 hours are preferable.
During extraction, the liquid is allowed to stand or left with stirring. The extraction operation is preferably repeated multiple times for the same sample.
[0013]
Next, column chromatography for the above bacterial cell extract will be described.
As a column chromatography packing material, ODS (octadecyl silane such as octadecyldimethylchlorosilane) is used, and the packing amount is not particularly limited, but it is 10 to 500 times by weight with respect to the cell extract to be charged. Filling is preferred.
[0014]
When the cell extract is charged to the column, it is first adsorbed to ODS. The amount of ODS at this time is not particularly limited, but is preferably 0.5 to 20 times by weight with respect to the microbial cell extract to be adsorbed. Then, suspend in a small amount of solvent and charge the column.
As an elution solvent for column chromatography, one having a polarity between an aqueous solution containing 85% methanol: acetonitrile = 1: 1 mixed solvent and methanol is used .
[0015]
Next, recrystallization with a solvent will be described.
The solvent used for recrystallization is not particularly limited as long as it can dissolve the inflammation-inhibiting substance in the present invention. For example, methanol, ethanol and the like are preferable.
As a recrystallization method, for example, heat may be applied to dissolve the ODS elution fraction containing the substance in a small amount of solvent, and the solvent may be gradually cooled to cause recrystallization. The ODS elution fraction containing the substance may be dissolved in a high solvent, and a solvent having a low solubility of the substance may be gradually added thereto for recrystallization.
[0016]
In order to formulate the anti-inflammatory substance obtained by the present invention, it is usually made into the form of a pharmaceutical composition together with a pharmaceutical carrier. As the carrier, those usually used for adjusting a drug according to the dosage form are used, for example, a filler, a disintegrant, an extender, a binder, a coloring agent, a flavoring agent, a pH adjusting agent, a solubilizing agent. Agents, suspending agents, buffers, stabilizers, preservatives, texture agents, surface activators, lubricants, excipients and the like.
In addition, by selecting an appropriate solvent, the obtained anti-inflammatory substance can be used as it is as an external solution.
[0017]
The dosage unit form of the anti-inflammatory agent formulated from the anti-inflammatory substance obtained by the present invention includes, for example, tablets, pills, drinking liquids, powders, suspensions, emulsions, granules, extracts in addition to the above-mentioned external liquids. Agent, fine granules, syrup, soaking agent, decoction, eye drop, troche, poultice, liniment, lotion, eye ointment, plaster, capsule, suppository, injection (liquid, suspension, etc.) ), Patches, ointments and the like.
The amount of the anti-inflammatory substance to be contained in the anti-inflammatory agent is not particularly limited and is appropriately selected over a wide range, but is preferably 0.01 to 50% by weight in the anti-inflammatory agent.
[0018]
The anti-inflammatory agent obtained from the anti-inflammatory substance in the present invention is administered by a method according to various forms when used. For example, in the case of the above-mentioned liquid preparation for external use, it is applied directly to the required site such as the skin or mucous membrane, and in the case of tablets, pills, liquid drinks, suspensions, emulsions, granules and capsules, it is orally applied. In the case of injections, it is administered intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally, in the case of suppositories, it is administered intrarectally, and in the case of patches and ointments, it is applied or applied. The
[0019]
The dose of the inflammation inhibitor is appropriately selected depending on the purpose of use, symptoms, etc., but is usually about 0.001 to 50 mg / kg as an inflammation inhibitor in the present invention per day. Of course, the above-mentioned pharmaceutical composition may be administered 3 to 4 times per day.
[0020]
【Example】
The present invention will now be described with reference to examples.
(Example 1)
Actinomycetes purchased from RIKEN Nobilis (JCM4274) was shaken and cultured for about 48 hours in 250 ml of a starch / ammonium medium supplemented with 0.2% yeast extract (previously cultured), and then 12 ml of the same medium was inoculated with 240 ml of the precultured bacterial solution. Cultured (seed culture) with aeration and agitation for 24 hours. Furthermore, the whole amount of seed culture was inoculated into 700 L 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), and 1 to 10 at about 30 ° C. The culture was aerated and agitated for a day. The culture was filtered and about 900 g of actinomycetes S. cerevisiae in wet weight. Nobilis cells were obtained.
[0021]
After adding 1 L of dichloromethane to 100 g (wet weight) of the cells and irradiating ultrasonic waves for 30 minutes, 4 L of dichloromethane was further added and stirred at room temperature for 1 hour. After filtration, the extract was dried using an evaporator to obtain 8 g of a cell extract.
[0022]
Next, 8 g of the above bacterial cell extract was adsorbed on 25 g of ODS (octadecyldimethylchlorosilane) and suspended in a small amount of solvent (acetonitrile: methanol: water = 7: 7: 6), an ODS column (diameter 32 mm, It was charged and eluted in a length of 760 mm, a packed ODS carrier amount of 275 g, and an in-column solvent acetonitrile: methanol: water = 7: 7: 6).
The elution solvent was acetonitrile: methanol: water = 1 L of acetonitrile: methanol: water = 1.2 L of acetonitrile: methanol: water = 8: 8: 4, acetonitrile: methanol: water = 200 ml of 9: 9: 2, acetonitrile: Methanol: water = 19: 19: 2 was flowed in the order of 20 ml × 50 and methanol in the order of 500 ml.
[0023]
Next, among the elution fractions with the above-mentioned elution solvent of acetonitrile: methanol: water = 19: 19: 2, the 5th to 15th fractions were combined and dried using an evaporator to obtain 106 mg of eluate. Obtained. This was dissolved in an appropriate amount of methanol, a small amount of water was added, and the mixture was allowed to stand at 4 ° C. for 1 day for crystallization. This operation was repeated several times to obtain 50 mg of an inflammation suppressing substance.
[0024]
(Test Example 1) Action on type III allergic reaction i) Preparation of rabbit anti-ovalbumin serum According to the method of Eda et al. (Nippon Pharmacology, 66: 237, 1970), rabbit anti-ovalbumin was prepared by the following method. Serum was prepared. That is, an antigen solution consisting of an equal volume mixed emulsion of a 2 mg / ml solution of ovalbumin (manufactured by Sigma) dissolved in physiological saline and complete Freund's adjuvant (manufactured by Difco) was prepared. Each 0.5 ml of this antigen solution was injected 4 times a week into the left and right gluteal muscles of a New Zealand white male rabbit weighing approximately 3 kg. Seven days after the final injection, blood was collected from the carotid artery, and only the serum was isolated and obtained as rabbit anti-ovalbumin serum. The titer of this antiserum rat 4 hour heterogeneous passive skin anaphylaxis (4 hour heterologous PCA) reaction was 1:32.
[0025]
ii) Action on rat 4-hour heterologous PCA reaction (type III allergic skin reaction) Dimethyl sulfoxide in 5% by weight aqueous solution of gum arabic so that the inflammation inhibitory substance obtained in Example 1 was 0.4 mg / ml. Was suspended in a solution containing 5% by weight. For control experiments, indomethacin was suspended in the above dimethyl sulfoxide-containing gum arabic aqueous solution so as to be 1.0 mg / ml. The suspension thus obtained was used as a test solution. As a control, a dimethyl sulfoxide-containing aqueous gum arabic solution containing no inflammation inhibitor was used as a test solution. As test animals, Wistar male rats weighing 120 to 200 g were used.
[0026]
First, the above test solution was intraperitoneally administered to rats at 2 ml / kg (0.8 mg / kg as the amount of inflammation-inhibiting substance and 2 mg / kg as the amount of indomethacin). Then, 18 to 22 hours later, 0.05 ml of an injection solution obtained by diluting the rabbit anti-ovalbumin serum 4 times with physiological saline was injected into the back skin, and the rats were sensitized with the antiserum.
Further, 4 hours later, 0.5 wt% Evans blue physiological saline containing 2 mg / ml ovalbumin as a corresponding antigen was intravenously injected at 2.5 ml / kg to induce a heterologous PCA reaction for 4 hours.
[0027]
The leaking dye at the site that caused the intradermal reaction was extracted and quantified according to the method of Harada et al. (J. Pharm. Pharmacol., 23: 218, 1971). That is, the animal was sacrificed 1 hour after the antigen injection, and the skin of the heterogeneous PCA reaction part was minced for 4 hours. For 24 hours to extract the leaking dye. The dye extracted in this manner was colorimetrically determined at 620 nm to determine the amount of leaked dye, and this was expressed as the amount of leaked dye (μg) per site (site) injected with rabbit anti-ovalbumin serum. The test was performed using 5 mice, and the amount of leaked pigment (site / μg) was shown as the average value. The results are shown in Table 1.
[0028]
[Table 1]

[0029]
As is clear from the results, in the group administered indomethacin, no difference was observed in the amount of leaked pigment compared to the control, but in the group administered the test solution containing the inflammation inhibitor obtained in Example 1, Compared with the control, the amount of pigment leaking to the skin of the rat 4-hour heterologous PCA reaction site was greatly reduced. That is, the inflammation inhibitory substance obtained by the present invention has allergic inflammation inhibitory activity.
[0030]
(Test Example 2) Toxicity test The toxicity of the inflammation inhibitor obtained in Example 1 was examined by the following method.
First, an anti-inflammatory substance was suspended in the above dimethyl sulfoxide-containing gum arabic aqueous solution so as to have a final concentration of 40 mg / 5 ml to prepare a test solution. As test animals, ICR male mice weighing 25-30 g were used.
The above test solution was intraperitoneally administered to mice at 5 ml / kg (corresponding to 50 times the effective amount of Test Example 1).
As a result, no toxic symptoms were observed, and the mortality rate after 2 weeks of administration of the test solution was 0%. No abnormalities were found in the autopsy of the surviving test animals. As is clear from this result, the inflammation inhibitory substance obtained in the Examples did not show toxicity at an effective amount 50 times. The test was performed using 5 mice.
[0031]
(Comparative Example 1)
The anti-inflammatory substance is isolated by the conventional method, that is, ammonium sulfate is added to the culture solution, and the extract from the precipitate is purified by ODS column chromatography and recrystallization. The amount of inflammation-inhibiting substance that can be released was compared with that using the method of the present invention. The results are shown in Table 2.
[0032]
[Table 2]

[0033]
From the above results, according to the present invention, it was possible to isolate an anti-inflammatory substance 4 times as much as the conventional method per the same time.
[0034]
【The invention's effect】
The method of the present invention is as described above, and according to this method, a large amount of the inflammation-inhibiting substance can be purified efficiently in a short time without impairing the activity.

Claims (1)

ODS column chromatography was performed by extracting cells of Streptomyces nobilis from Streptomyces nobilis with at least one solvent selected from the group consisting of dichloromethane, ethyl acetate and acetone . Inflammation characterized in that it is purified from the fraction eluted with an aqueous solution containing 85% methanol / acetonitrile = 1: 1 mixed solvent between methanol and methanol using recrystallization from the solvent. Method for producing inhibitory substance.