JP4018592B2 - Histamine release inhibitor - Google Patents

Description

【００１４】
得られた８フラクションのうち、Ｆｒ７（図１参照）について、ＯＤＳカラム（シリカゲル担体に、オクタデシルシリル基を化学結合した充填剤、島津製作所製）を用いたＨＰＬＣにより、成分Ｂを単離・分取した。分析条件を表２に、分取チャートを図２に示す。
【００１５】
【表２】

【００１６】
ＨＰＬＣで単離・分取した成分Ｂ（メタノール溶液）を、質量スペクトル（ＧＣ−ＭＳ）にかけて、ピークＢに係る化合物（以下化合物Ｂという）の分子量及びこの化合物Ｂに結合している官能基を推定した。
その結果、化合物Ｂの分子量は、２９７と推定した。スペクトルのチャートを図３に示す。
【００１７】
化合物Ａ（ＤＭＳＯ及び数滴のクロロホルムで溶解させたもの）を、核磁気共鳴（1HN-MR・13CN-MR）にかけた。
その結果を図４（ 1ＨＮ−ＭＲ）及び図５（13ＣＮ−ＭＲ）に示す。
図４から、化合物Ｂは、糖類及び芳香環領域の水素のシグナルが見られた。
又図５から、化合物Ｂの単素数は１１と推定され、糖類及び芳香環領域の炭素のシグナルが見られた。
【００１８】
化合物Ｂについて、赤外スペクトルを測定した。
その結果を図６に示す。
【００１９】
ＨＰＬＣで得られた化合物Ｂ（エタノールに溶解）について、紫外・可視スペクトル（ＵＶ）を測定した（λmax ２５９ｎｍ）。その結果を図７に示す。
【００２０】
以上の結果から、化合物Ｂは、N-hydroxy-N-methyl-adenosineアデノシン（下式）と同定した。
【００２１】
【化１】

【００２２】
（２）メシマコブ菌糸体から得られたN-hydroxy-N-methyl-adenosineについて、マスト細胞からのヒスタミン遊離抑制効果を調べることによって、Ｉ型アレルギー反応抑制効果を調べた。
（Ａ）被験動物及び細胞の調製方法
（ａ）被験動物には、Wister系ラット雄（６〜８週齢）を用いた。
（ｂ）細胞の調製方法
ラットをエーテルで麻酔させ、脱血死させた後、ラットの腹腔内に Tyrode Buffer １５ｍｌを注入し、腹腔内液を回収した。更にラットの腹腔内にTyrode Buffer 10mlを注入し、更に腹腔内液を回収して先に採取した腹腔内液と混合した。この腹腔内液を、８００ｒｐｍ・４℃・１０分間遠心分離を行い、上澄を除去してフィルターをかけた。
更に、Tyrode Buffer を加えた細胞懸濁液を、８００ｒｐｍ・４℃・１０分間遠心分離を行い、上清を除去して、細胞を均一にした。この細胞懸濁液を血球計算盤で測定し、８×１０6 cells ／ｍｌに調製して用いた。
【００２３】
（Ｂ）生物検定の実験方法およびヒスタミンの分析方法
（ａ）生物検定の実験方法
１．２ｍｌのマイクロチューブに分注した、細胞懸濁液３７０μｌを３７℃のウォーターバスでプレインキュベートした。
１０分後、各濃度に設定したサンプル２０μｌを添加した。
更に、１０分後、アレルギー誘発剤であるcompound48 / 80 （マスト細胞刺激用試薬：和光純薬）１０μｌ（０．５μｇ／ｍｌ) を添加した。
１５分後、氷上で反応を停止した。
更に、マスト細胞を除去するため、１３．０００rpm ・４℃・１５分間遠心分離し、上清を回収した。
この上清を、HPLC分析のジアゾ化するサンプルとした。
【００２４】
（ｂ）ヒスタミンジアゾ化誘導体の調製
等量の２０ｍＭ p −ニトロアニリン塩酸溶液と２００ｍＭ 亜硝酸ナトリウム水溶液をよく混合した (ジアゾ試薬) 。
ジアゾ試薬１０μｌに、前記（ヒスタミン）ジアゾ化用サンプル２０μｌを加え、ボルテックスミキサーでよく混合した。
更に、１０％ 炭酸ナトリウムエタノール溶液３０μｌを加え、ボルテックスミキサーでよく混合した。
この溶液の２０μｌをＨＰＬＣ分析に用いた。
【００２５】
（ｃ）ヒスタミンの分析は、ジアゾ化法により行った（Specific Determination of Histamine in Fish by High-performance Liquid Chromatography after Diazi Coupling : Biosci. Biotech. Biochem. 59(7), 1208-1210(1995) ）。
生物検定におけるヒスタミンのＨＰＬＣ分析条件〈ジアゾ法）は、表３のとおりである。
【００２６】
【表３】

【００２７】
（Ｃ）ヒスタミン遊離抑制効果の測定
メシマコブ菌糸体から得られたN-hydroxy-N-methyl-adenosineの一定濃度におけるマスト細胞からのヒスタミン遊離の抑制効果の結果を表４に示す。
比較資料として、市販のヒスタミン抑制剤（商品名：インタール，主要成分：クロモグリク酸ナトリウム，メーカー藤沢薬品工業）を用いた。
試験結果は、両者共３回試験した結果の平均値である。
【００２８】
【表４】

【００２９】
抑制値は下記の計算式によった。
計算式：
（１−サンプルのピーク積分値／コントロールのピーク積分値）×１００
【００３０】
【発明の効果】
本願発明によれば、N-hydroxy-N-methyl-adenosineを用いて、ヒスタミン遊離抑制剤を得ることができるという効果を有する。
【図面の簡単な説明】
【図１】 メシマコブ菌糸体の熱水抽出物の分画工程を示す図である。
【図２】 Ｆｒ７の分取チャートを示す図である。
【図３】 化合物Ｂの質量スペクトル（ＧＣ−ＭＳ）を示す図である。
【図４】 化合物Ｂの 1ＨＮ−ＭＲのスペクトルを示す図である。
【図５】 化合物Ｂの13ＣＮ−ＭＲのスペクトルを示す図である。
【図６】 化合物Ｂの赤外スペクトルを示す図である。
【図７】 化合物Ｂの紫外・可視スペクトル（ＵＶ）を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a histamine release inhibitor characterized by containing N-hydroxy-N-methyl-adenosine.
[0002]
[Prior art]
Conventionally, the following substances are known as substances having an inhibitory action on histamine release.
(1) Tricaffeoylquinic acid, etc. (JP 2002-80360)
(2) Tomato extract (JP 2002-80387)
(3) Apigenin and the like (JP 2000-86510)
(3) Bark extracts such as Nikkei and bayberry (Japanese Patent Laid-Open No. 10-287582)
[0003]
[Problems to be solved by the invention]
The inventors conducted extensive research focusing on the histamine release inhibitory action of the mycelium of Phellinus linteus, and found that N-hydroxy-N-methyl-adenosine extracted and isolated from Meshimakobu has a remarkable inhibition of histamine release. Knowing that the action exists, the present invention has been completed.
[0004]
[Means for Solving the Problems]
The present invention is constituted by the following claims (1) and (2).
(1) A histamine release inhibitor comprising N-hydroxy-N-methyl-adenosine.
(2) The histamine release inhibitor according to claim 1, wherein N-hydroxy-N-methyl-adenosine obtained from Meshimakobu mycelium is used.
The reason for constituting the present invention as described above is that N-hydroxy-N-methyl-adenosine has not been known to have a conventional histamine release inhibitory action.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
[0006]
(1) Extraction (preparation) method of N-hydroxy-N-methyl-adenosine from Meshimakobu (A) 4.0% glucose as a carbon source and nitrogen source derived from natural products using a large tank (1000L) of Meshimakobu mycelium Yeast extract and polypeptone 0.3% each, KH2PO4 and Na2HPO4 0.05% each, inoculate the mycelium of Meshimakobu in the culture medium of the initial medium pH 5.5, forcibly 0.22μm filter Aseptic air passed through was passed through the medium and cultured at a temperature of 28 ° C. for 45 days.
The mycelium obtained by centrifuging this culture broth was freeze-dried to obtain a dry powder of Meshimakob mycelium.
[0007]
The Meshima Cob used in the present invention was collected in October 1998 in Sukimura, Nishimoro County, Miyazaki Prefecture, and mycelium was converted to PL-08 strain after being converted into mycelium at IBI Application Mushroom Research Institute. What was stored as was used. This strain has a yellow-brown bristle body peculiar to Mashimakobu fruit body according to the appraisal of Dr. Yasuhisa Abe of Forest Biology Department Forest Microbiology Department of Forest Microbiology Department of Forestry, Forestry Agency, Ministry of Agriculture, Forestry and Fisheries And {circle around (2)} basidiospores, and those identified as Meshimakobu were used.
[0008]
For pre-culture of the test strain, the mycelium that had been cryopreserved at 5 ° C was inoculated into Potato Dextrose Agar medium (Difco) in a petri dish with an inner diameter of 90 mm and surfaced for 15 days in the dark at 25 ° C. Cultured. This cultured mycelium was cut out with a cork borer having an inner diameter of 5 mm (corresponding to a dry mycelium weight of 0.35 mg) and used as the strain of paragraph 0006, (A).
[0009]
(B) Hot water extract of Meshimakob mycelium 10 times the amount of ion-exchanged water is added to the dried powder of Meshimakob mycelium, followed by hot water extraction treatment at 100 ° C. for 2 hours to remove insoluble matter and Meshimakob mycelium. A body hot water extract was obtained. The hot water extract was concentrated under reduced pressure at about 70 ° C.
In addition, about 280 g of the Meshimakobu hot water extract was obtained from 1300 g of the dried mycelium powder of Meshimakobu.
This hot water extract was fractionated by the method shown in FIG.
[0010]
(C) Fractionation of hot water extract of Meshimakob mycelium with methanol Three times the amount of methanol was added to the Meshimakob hot water extract, left to stand for 2 hours, centrifuged, and a methanol soluble fraction, A methanol-insoluble fraction was obtained.
In addition, 51 g of methanol-soluble fraction and 226 g of methanol-insoluble fraction were obtained from 280 g of the Meshimakobu hot water extract concentrate.
[0011]
(D) Fractionation of the methanol soluble fraction with an ion adsorbent After adsorbing the soluble fraction, it was fractionated into a water-eluting fraction and a methanol-eluting fraction.
From 51 g of the methanol-soluble fraction, 43 g of water-eluted fraction and 4.0 g of methanol-eluted fraction were obtained.
[0012]
(E) Fractionation of methanol soluble fraction after ion adsorbent treatment
Using a Sephadex LH-20 (Pharmacia, hereinafter also referred to as LH-20) column, 4.0 g of the methanol-soluble fraction obtained in (C) above was fractionated and fractionated by gel filtration chromatography as follows. I took it.
Elution of the target component was performed using distilled water, and the whole was fractionated into 8 fractions (Fr1 to Fr8) (4.0 g of the methanol soluble fraction was divided into 5 portions each containing 0.5 g. Took.)
Table 1 shows the fractionation conditions with LH-20.
[0013]
[Table 1]

[0014]
Among the obtained 8 fractions, Fr7 (see FIG. 1) was obtained by isolating and separating component B by HPLC using an ODS column (a silica gel carrier with a chemically bonded octadecylsilyl group, manufactured by Shimadzu Corporation). I took it. The analysis conditions are shown in Table 2, and the preparative chart is shown in FIG.
[0015]
[Table 2]

[0016]
Component B (methanol solution) isolated and fractionated by HPLC is subjected to mass spectrum (GC-MS), and the molecular weight of the compound related to Peak B (hereinafter referred to as Compound B) and the functional group bonded to Compound B are determined. Estimated.
As a result, the molecular weight of Compound B was estimated to be 297. A chart of the spectrum is shown in FIG.
[0017]
Compound A (dissolved in DMSO and a few drops of chloroform) was subjected to nuclear magnetic resonance (1HN-MR · 13CN-MR).
The results are shown in FIG. 4 (1HN-MR) and FIG. 5 (13CN-MR).
From FIG. 4, compound B showed hydrogen signals in the saccharide and aromatic ring regions.
Further, from FIG. 5, the simple prime number of Compound B was estimated to be 11, and carbon signals in the saccharide and aromatic ring regions were observed.
[0018]
For compound B, an infrared spectrum was measured.
The result is shown in FIG.
[0019]
Compound B (dissolved in ethanol) obtained by HPLC was measured for ultraviolet and visible spectrum (UV) (λmax 259 nm). The result is shown in FIG.
[0020]
From the above results, Compound B was identified as N-hydroxy-N-methyl-adenosine adenosine (the following formula).
[0021]
[Chemical 1]

[0022]
(2) About the N-hydroxy-N-methyl-adenosine obtained from the mycelium of Meshimakobu, the inhibitory effect on type I allergic reaction was examined by examining the inhibitory effect on histamine release from mast cells.
(A) Test animal and cell preparation method (a) Male Wister rats (6 to 8 weeks of age) were used as test animals.
(B) Cell preparation method Rats were anesthetized with ether and allowed to exsanguinate, and then 15 ml of Tyrode Buffer was injected into the peritoneal cavity of the rat, and the intraperitoneal fluid was collected. Further, 10 ml of Tyrode Buffer was injected into the abdominal cavity of the rat, and the intraperitoneal fluid was further collected and mixed with the intraperitoneal fluid previously collected. The intraperitoneal fluid was centrifuged at 800 rpm, 4 ° C., 10 minutes, and the supernatant was removed and filtered.
Furthermore, the cell suspension to which Tyrode Buffer was added was centrifuged at 800 rpm, 4 ° C., 10 minutes, and the supernatant was removed to make the cells uniform. This cell suspension was measured with a hemocytometer and prepared to 8 × 10 6 cells / ml.
[0023]
(B) Experimental method of bioassay and analytical method of histamine (a) Experimental method of bioassay 370 μl of cell suspension dispensed into 1.2 ml microtubes was preincubated in a 37 ° C. water bath.
After 10 minutes, 20 μl of sample set to each concentration was added.
Further, after 10 minutes, 10 μl (0.5 μg / ml) of compound 48/80 (mast cell stimulating reagent: Wako Pure Chemicals), which is an allergen, was added.
After 15 minutes, the reaction was stopped on ice.
Furthermore, in order to remove mast cells, it was centrifuged at 13.000 rpm · 4 ° C. for 15 minutes, and the supernatant was collected.
This supernatant was used as a sample to be diazotized by HPLC analysis.
[0024]
(B) Preparation of histamine diazotized derivative An equal amount of 20 mM p-nitroaniline hydrochloric acid solution and 200 mM aqueous sodium nitrite solution were mixed well (diazo reagent).
To 10 μl of the diazo reagent, 20 μl of the (histamine) diazotization sample was added and mixed well with a vortex mixer.
Further, 30 μl of 10% sodium carbonate ethanol solution was added and mixed well with a vortex mixer.
20 μl of this solution was used for HPLC analysis.
[0025]
(C) The analysis of histamine was performed by the diazotization method (Specific Determination of Histamine in Fish by High-performance Liquid Chromatography after Diazi Coupling: Biosci. Biotech. Biochem. 59 (7), 1208-1210 (1995)).
Table 3 shows the HPLC analysis conditions (diazo method) of histamine in the bioassay.
[0026]
[Table 3]

[0027]
(C) Measurement of histamine release inhibitory effect Table 4 shows the results of the inhibitory effect of histamine release from mast cells at a constant concentration of N-hydroxy-N-methyl-adenosine obtained from Mesimacob mycelium.
As a comparative data, a commercially available histamine inhibitor (trade name: intal, main component: sodium cromoglycate, manufacturer Fujisawa Pharmaceutical Co., Ltd.) was used.
The test result is an average value of the results of testing three times for both.
[0028]
[Table 4]

[0029]
The suppression value was based on the following formula.
a formula:
(1-sample peak integral value / control peak integral value) × 100
[0030]
【The invention's effect】
According to the present invention, there is an effect that a histamine release inhibitor can be obtained using N-hydroxy-N-methyl-adenosine.
[Brief description of the drawings]
FIG. 1 is a diagram showing a fractionation process of a hot water extract of Meshimakob mycelium.
FIG. 2 is a diagram showing a sorting chart of Fr7.
FIG. 3 shows a mass spectrum (GC-MS) of Compound B.
FIG. 4 is a diagram showing a 1HN-MR spectrum of compound B.
5 is a diagram showing a 13CN-MR spectrum of compound B. FIG.
FIG. 6 is an infrared spectrum of Compound B.
7 is a diagram showing an ultraviolet / visible spectrum (UV) of Compound B. FIG.

Claims (2)

A histamine release inhibitor comprising N-hydroxy-N-methyl-adenosine. The histamine release inhibitor according to claim 1, wherein N-hydroxy-N-methyl-adenosine obtained from Mesimacob mycelium is used.

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