JP3571747B2 - Method for producing inflammation-inhibiting substance - Google Patents

Description

実施例１と比較例１の結果を示す表１から明らかなように、放線菌Ｓ．ノビリスを培養する際、Ｄ−セリンを含有しない澱粉・アンモニウム培地を用いた場合では、培養開始後４日目でＳＥＫ−１００５の生産量がピークとなり、その生産量は、１４６．３μｇ／ｌであった。しかし、同培地にＤ−セリンを０．０１重量％添加した場合には、生産量のピークは４日目と同じであったが、その生産量は２６０．２μｇ／ｌであった。すなわちＤ−セリンを添加することによって、約２倍の量のＳＥＫ−１００５生産を達成できることがわかる。
【００２８】
比較例２
培地として、Ｄ−セリンを含有しない澱粉・アンモニウム培地（蒸留水１００ｍｌ中に、可溶性澱粉を１ｇ、Ｋ_２ ＨＰＯ_４ を０．０５ｇ、ＮＨ_４ Ｃｌを０．０５ｇを含む）を用いる以外は、実施例２と全く同じ方法で放線菌Ｓ．ノビリスを培養し、培養時間とＳＥＫ−１００５生産量との関係を調べた。その結果を実施例２の結果とともに表２に示す。また、培養開始から３日後の培養液を酢酸エチルで抽出処理し、この抽出液のＨＰＬＣ分析を行った。この分析チャートを図３に示す。
【００２９】
【表２】

表２および図３から明らかなように、放線菌Ｓ．ノビリスを培養する際、Ｄ−セリンを含有しない澱粉・アンモニウム培地を用いた場合では、培養開始後３日目でＳＥＫ−１００５の生産量がピークとなり、その生産量は２３．６μｇ／ｌであった。しかし、同培地にＤ−セリンを０．０１重量％添加した場合には、生産量のピークは３日目であったが、その生産量は７７７．４μｇ／ｌであった。すなわち、後者は前者と比較して、約３２倍の量のＳＥＫ−１００５生産を達成できることがわかる。
【００３０】
【発明の効果】
本発明によれば、放線菌ストレプトマイセス・ノビリスをＤ−セリンを含有する澱粉・アンモニウム培地で培養することにより、炎症抑制物質を効率的に生産することができる。
【図面の簡単な説明】
【図１】実施例１において、ＨＰＬＣに供したＳＥＫ−１００５の量とそのピーク面積の関係およびその回帰直線を示すグラフである。
【図２】実施例２において、培養開始から３日後の培養液を酢酸エチルで抽出処理し、この抽出液のＨＰＬＣ分析を行って得られた分析チャートである。
【図３】比較例２において、培養開始から３日後の培養液を酢酸エチルで抽出処理し、この抽出液のＨＰＬＣ分析を行って得られた分析チャートである。[0001]
[Industrial application fields]
The present invention produces allergic and non-allergic anti-inflammatory substances (hereinafter abbreviated as “SEK-1005”) by culturing actinomycetes Streptomyces nobilis (hereinafter abbreviated as “S. nobilis”). Regarding the method.
[0002]
[Prior art]
Inflammatory reactions are broadly divided into allergic inflammation and non-allergic inflammation. Allergy is a type of pathological condition in which an immune reaction that occurs upon a second contact with an antigen appears excessively or inappropriately by an individual, due to differences in the nature of the antibodies involved, type I, Classified into 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 self-affected by rheumatoid arthritis. It has become clear that it plays an important role in the development of immune diseases, as well as various inflammatory diseases such 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 been found so far in actinomycete culture filtrate.
[0004]
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. There is.
[0005]
The present inventors have used S. as a raw material. As a result of searching for substances that have an allergic inflammation inhibitory effect using Nobilis, extracts extracted from the culture broth or its dried solids with organic solvents markedly suppress allergic and non-allergic inflammatory reactions After confirming that it has an action and safety, it was found that there is no problem as an anti-inflammatory agent (Japanese Patent Laid-Open No. 5-25053), and further a purification method was established.
[0006]
[Problems to be solved by the invention]
However, S.A. In the starch / ammonium medium (consisting of soluble starch, K ₂ HPO ₄ and NH ₄ Cl) used for cultivation of Nobilis, the low productivity of SEK-1005 has been a problem.
[0007]
The object of the present invention is that S. An object of the present invention is to provide a method for improving the medium used for cultivating Nobilis and obtaining SEK-1005 more efficiently.
[0008]
[Means for Solving the Problems]
The present inventors cultivated Streptomyces nobilis in a medium obtained by adding D-serine to a conventional starch / ammonium medium, thereby producing productivity of the inflammation inhibitory substance SEK-1005. Has been found to be significantly improved, and the present invention has been completed based on this finding.
[0009]
Actinomycetes that produce anti-inflammatory substances Nobilis can be obtained from public preservation institutions, for example, bacteria such as a preserved bacterium from RIKEN (JCM 4274) (which is also preserved as ATCC 19252 in the United States and CBS 198.65 in the Netherlands).
[0010]
In general, actinomycetes Nobilis is cultured using a medium containing the following nutrients. That is, in the case of a liquid medium, an aqueous solution containing one or more kinds of sugars such as glucose, proteins such as peptone and malt extract, vitamins, nucleic acids, amino acids, complex carbohydrates and the like as components thereof. Used.
[0011]
In the method of the present invention, the medium for producing SEK-1005 is a starch / ammonium medium containing D-serine. A typical example of the starch / ammonium medium is a liquid medium containing soluble starch, dipotassium hydrogen phosphate (K ₂ HPO ₄ ) and ammonium chloride (NH ₄ Cl). D-serine is a kind of amino acid, and a commercially available product can be used.
[0012]
The concentration of soluble starch in the medium is preferably 0.01 to 5.0% by weight, the concentration of K ₂ HPO ₄ is preferably 0.01 to 5.0% by weight, and the concentration of NH ₄ Cl is preferably 0.01. -5.0 wt%. The concentration of D-serine in the medium is preferably 0.001 to 1.0% by weight.
[0013]
The pH of the liquid medium is preferably in the range of 2-9, and the culture temperature is preferably 15-42 ° C. The preferred culture time for liquid culture is 1 to 14 days. In the case of solid culture, the above liquid culture medium further containing agar is used, but the culture conditions for solid culture are almost the same as those for liquid culture.
[0014]
Thus, S.M. After cultivating Nobilis, the product is recovered by conventional means such as solvent extraction or ODS column chromatography.
[0015]
In order to formulate SEK-1005 obtained by the method of the present invention into an anti-inflammatory agent, it is usually in the form of a pharmaceutical composition together with a pharmaceutical carrier. As the carrier, there are used diluents or excipients such as fillers, disintegrants, extenders, binders, moisturizers, surfactants, lubricants and the like that are usually used to prepare drugs according to the dosage form. Illustrated. In addition, by selecting an appropriate solvent, the obtained solvent extract or its concentrate can be used as it is as an external solution.
[0016]
Examples of the dosage unit form of the anti-inflammatory agent formulated using SEK-1005 include tablets, pills, drinking liquids, powders, suspensions, emulsions, granules, capsules, in addition to the above-mentioned external liquids. Examples are suppositories, injections (solutions, suspensions, etc.), ointments and the like.
[0017]
The amount of SEK-1005 to be contained in the inflammation inhibitor is not particularly limited and is appropriately selected over a wide range, but is preferably in the range of 0.1 to 50% by weight in the inflammation inhibitor.
[0018]
The anti-inflammatory agent obtained from SEK-1005 is administered by a method according to various forms when used. For example, in the case of the above-mentioned external liquid preparation, it is applied directly to the required site such as the skin or mucous membrane, and in the case of tablets, pills, drinking liquids, suspensions, emulsions, granules and capsules, it is orally administered. In the case of injections, they are administered intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally, in the case of suppositories, intrarectally, and in the case of ointments.
[0019]
The dose of the inflammation inhibitor obtained from SEK-1005 is appropriately selected depending on the purpose of use, symptoms and the like, but is usually in the range of about 0.2 to 50 mg / kg as SEK-1005 per day. Of course, the pharmaceutical composition may be administered 3 to 4 times per day.
[0020]
【Example】
Next, the present invention will be concretely demonstrated with examples of the present invention.
[0021]
Example 1
In two 500 ml Sakaguchi flasks, D-serine (containing 1 g of soluble starch, 0.05 g of K ₂ HPO ₄ and 0.05 g of NH ₄ Cl per 100 ml of distilled water) (Nacalai Tesque) 0.01% by weight was added to prepare 125 ml of liquid medium. Subsequently, actinomycetes S. cerevisiae purchased from RIKEN were added to these media. Nobilis (JCM4274) was inoculated and cultured with shaking at 30 ° C. and 160 rpm using a shaking incubator (TAITEC, Bio-Shaker BR-300L). In order to examine the relationship between the culture time and the amount of SEK-1005 produced in the culture solution, 50 ml was sampled from each flask 48 hours and 96 hours after the start of the culture. Sampling solutions at the same time were collected, and each collected solution was centrifuged (2500 G × 10 min) to obtain a supernatant. 100 ml of this supernatant was placed in a 300 ml separatory funnel, an equal amount of ethyl acetate was added thereto, and the mixture was shaken for 10 minutes. After standing 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 and the above operation was repeated once more. The ethyl acetate phases thus obtained by three operations were collected, and the collected liquid was concentrated to dryness with an evaporator to obtain a crude extract. A solution obtained by dissolving the crude extract in 1 ml of methanol was used as an analytical sample using high performance liquid chromatography (hereinafter abbreviated as “HPLC”).
[0022]
First, in order to quantify SEK-1005 by HPLC, a calibration curve was prepared as follows. SEK-1005 is an HPLC (Hitachi, pump L-6000L-6200, detector L-3000, column oven 655A-) using an ODS column (manufactured by Tosoh Corporation, ODS-80TM, φ4.6MMID × 25.0 CML). 52), when water: acetonitrile: methanol = 6: 7: 7 is used as the eluent under the conditions of a detection wavelength of 210 nm, a temperature of 40 ° C., and a flow rate of 1 ml / min, a retention time of 41 to 45 minutes is shown. I already know. Taking advantage of this, 40 μl of SEK-1005 dissolved in methanol to give 2, 5, 10, 20, 50, 100 μg / ml, that is, 0.08, 0.2, 0.4, 0. 8, 2.0 and 4.0 μg were each subjected to HPLC under the above conditions, and the relationship between the amount of SEK-1005 and the peak area was examined. At this time, it was found that there was a linear relationship in the range of 0.2 to 2.0 μg, and the straight line obtained by linear regression calculation was taken as a SEK-1005 calibration curve. FIG. 1 is a graph showing the relationship between the amount of SEK-1005 subjected to HPLC and its peak area, and its regression line.
[0023]
Next, the above analytical sample was subjected to HPLC under the same conditions, and the content of SEK-1005 was calculated from the obtained SEK-1005 peak area using the above calibration curve.
[0024]
Table 1 shows the relationship between the culture time and the amount of SEK-1005 production.
[0025]
Example 2
In two 500 ml Sakaguchi flasks, yeast extract (DIFCO) contains starch / ammonium medium (1 g of soluble starch, 0.05 g of K ₂ HPO ₄ and 0.05 g of NH ₄ Cl in 100 ml of distilled water). (Manufactured by Yeast Extract) 0.2% by weight was added to prepare 125 ml of liquid medium. Subsequently, actinomycetes S. cerevisiae purchased from RIKEN were added to these media. Nobilis (JCM4274) was inoculated, and cultured with shaking (seed culture) at 30 ° C. and 160 rpm for 96 hours using a shaking incubator (manufactured by TAITEC, Bio-Shaker BR-300L). Furthermore, in a 10 liter jar fermenter (manufactured by Mitsuwa Biosystems, KMJ-10C-FPMIII), 1 g of soluble starch and 0.05 g of K ₂ HPO ₄ in 100 ml of distilled water, 0.01 wt% of D-serine (manufactured by Nacalai Tesque) was added to NH ₄ Cl (containing 0.05 g) to prepare 6 liters of liquid medium. This medium was inoculated with 42 ml of the above seed culture and cultured at 30 ° C. and 300 rpm for 6 days with aeration and agitation. The aeration rate was 2 liters / min, and the pH was maintained at 6.5 using 0.1 N HCl and NaOH. After the start of culture, about 100 ml was sampled from the culture solution every 24 hours in order to quantify SEK-1005 contained in the culture solution over time. Each sampled culture solution was treated in the same manner as in Example 1, and the SEK-1005 content was measured by HPLC analysis. Table 2 shows the relationship between the culture time and the production amount of SEK-1005. Moreover, the culture solution 3 days after the start of the culture was extracted with ethyl acetate, and HPLC analysis of this extract was performed. This analysis chart is shown in FIG.
[0026]
Comparative Example 1
Implemented except that a starch-ammonium medium containing no D-serine (containing 1 g of soluble starch, 0.05 g of K ₂ HPO ₄ and 0.05 g of NH ₄ Cl in 100 ml of distilled water) was used as the medium. In the same manner as in Example 1, Nobilis (JCM4274) was cultured and the relationship between culture time and SEK-1005 production was examined. The results are shown in Table 1 together with the results of Example 1.
[0027]
[Table 1]

As is apparent from Table 1 showing the results of Example 1 and Comparative Example 1, actinomycetes When cultivating Nobilis, when a starch / ammonium medium containing no D-serine was used, the production amount of SEK-1005 peaked on the fourth day after the start of the cultivation, and the production amount was 146.3 μg / l. there were. However, when 0.01% by weight of D-serine was added to the same medium, the peak production amount was the same as that on the fourth day, but the production amount was 260.2 μg / l. That is, it can be seen that the addition of D-serine can achieve about twice the amount of SEK-1005 production.
[0028]
Comparative Example 2
Implemented except that starch-ammonium medium containing no D-serine (containing 1 g of soluble starch, 0.05 g of K ₂ HPO ₄ and 0.05 g of NH ₄ Cl in 100 ml of distilled water) is used as the medium. Actinomyces S. cerevisiae in exactly the same manner as in Example 2. Nobilis was cultured and the relationship between culture time and SEK-1005 production was examined. The results are shown in Table 2 together with the results of Example 2. Moreover, the culture solution 3 days after the start of the culture was extracted with ethyl acetate, and HPLC analysis of this extract was performed. This analysis chart is shown in FIG.
[0029]
[Table 2]

As is apparent from Table 2 and FIG. When cultivating Nobilis, when a starch / ammonium medium containing no D-serine was used, the production of SEK-1005 peaked on the third day after the start of the cultivation, and the production was 23.6 μg / l. It was. However, when 0.01% by weight of D-serine was added to the same medium, the peak production amount was on the third day, but the production amount was 777.4 μg / l. That is, it can be seen that the latter can achieve about 32 times as much SEK-1005 production as the former.
[0030]
【The invention's effect】
According to the present invention, an anti-inflammatory substance can be efficiently produced by culturing Streptomyces nobilis in a starch / ammonium medium containing D-serine.
[Brief description of the drawings]
1 is a graph showing the relationship between the amount of SEK-1005 subjected to HPLC and its peak area and its regression line in Example 1. FIG.
FIG. 2 is an analysis chart obtained by extracting a culture solution 3 days after the start of culture with ethyl acetate in Example 2 and performing HPLC analysis of the extract.
FIG. 3 is an analysis chart obtained by subjecting a culture solution 3 days after the start of culture to extraction with ethyl acetate in Comparative Example 2 and performing HPLC analysis of the extract.

Claims (1)

A method for producing an inflammation inhibitor, characterized by culturing Streptomyces nobilis in a starch / ammonium medium containing D-serine.

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