JP2004331619A - Histamine release inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a histamine release inhibitor. <P>SOLUTION: This invention provides (1) a histamine release inhibitor containing N-hydroxy-N-methyl-adenosine and (2) the histamine release inhibitor containing the N-hydroxy-N-methyl-adenosine derived from the mycelia of Phellinus linteus. <P>COPYRIGHT: (C)2005,JPO&NCIPI

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]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a histamine release inhibitor comprising N-hydroxy-N-methyl-adenosine.
[0002]
[Prior art]
Conventionally, the following substances have been known as substances having a histamine release inhibitory action.
{Circle around (1)} Tricaffeoylquinic acid, etc. (JP-A-2002-80360)
(2) Tomato extract (JP 2002-80387)
(3) Apigenin and the like (JP-A-2000-86510)
(3) Bark extract of Nikkei, Bayberry, etc. (JP-A-10-287852)
[0003]
[Problems to be solved by the invention]
The inventors of the present invention have conducted intensive studies focusing on the histamine release inhibitory effect of the mycelium of meshimakobu (Phellinus linteus). The present invention was completed by knowing that the action exists.
[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 the mycelium of Mesimakobu is used.
The reason for configuring the present invention as described above is that N-hydroxy-N-methyl-adenosine has not been known to have a histamine release inhibitory effect.
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
[0006]
(1) Extraction (preparation) method of N-hydroxy-N-methyl-adenosine from Meshimakobu (A) Using a large tank (1000 L) of Meshimakobu mycelium, 4.0% glucose as a carbon source, nitrogen source derived from natural products A culture medium containing 0.3% each of yeast extract and polypeptone and 0.05% of KH2PO4 and Na2HPO4, respectively, and inoculated with a mycelium of Mesimachob in a culture medium of an initial medium pH 5.5, was forced to have a 0.22 μm filter. Was passed through the medium, and the cells were cultured at a temperature of 28 ° C. for 45 days.
The mycelium obtained by centrifuging the culture solution was freeze-dried to obtain a dry powder of Mesimakov mycelium.
[0007]
In addition, as for Phellinus linteus used in the present invention, a fruiting body was collected in October 1998 at Suki-mura, Nishimoro-prefecture, Miyazaki Prefecture, and then transformed into a mycelium at IBI Co., Ltd. (IBI) Mushroom Research Institute. The one saved as was used. According to the identification of the fruiting body by Dr. Yasuhisa Abe of the Rot and Disease Laboratory, Department of Forestry and Biology, Forestry Agency, Forestry Agency, Ministry of Agriculture, Forestry and Fisheries of Japan And {circle around (2)} basidiospore morphology, which was identified as Mesimachob.
[0008]
The pre-culture of the test strain was performed by inoculating the mycelium, which had been stored at a low temperature of 5 ° C., into a Potato Dextrose Agar medium (manufactured by Difco) in a Petri dish having an inner diameter of 90 mm, and then inoculated at 25 ° C. in the dark for 15 days. Cultured. The cultured mycelium was cut off 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 Mesimakob mycelium A 10-fold amount of ion-exchanged water was added to the dried powder of the Mesimakob mycelium, a hot water extraction treatment was performed at 100 ° C for 2 hours to remove insolubles, and the insoluble matter was removed. A hot water extract of the body was obtained. This hot water extract was concentrated under reduced pressure at about 70 ° C.
In addition, about 280 g of the concentrate of the hot water extract of meshimakobu 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 the hot water extract of Mesimakobu mycelium with methanol To the hot water extract of Mesimakob was added 3 times the amount of methanol, left to stand for 2 hours, centrifuged, and a methanol-soluble fraction, A methanol-insoluble fraction was obtained.
In addition, 51 g of a methanol-soluble fraction and 226 g of a methanol-insoluble fraction were obtained from 280 g of the concentrated water extract of Mesimakobu.
[0011]
(D) Fractionation of the methanol-soluble fraction with an ion adsorbent (a) An ion adsorbent (Diaion HP-20 (Nippon Rensui), hereinafter also referred to as HP-20) packed in a column is charged with the methanol-soluble fraction. After adsorbing the dissolved 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 a water-eluting fraction and 4.0 g of a methanol-eluting fraction were obtained.
[0012]
(E) Fractionation of methanol-soluble fraction after treatment with ion adsorbent Sephadex LH-20 (manufactured by Pharmacia, hereinafter also referred to as LH-20) column, and the methanol obtained in (C) was subjected to gel filtration chromatography by gel filtration chromatography. 4.0 g of the soluble fraction was fractionated and collected as follows.
Elution of the target component was performed using distilled water, and the whole was fractionated as 8 fractions (Fr1 to Fr8). I took it.)
Table 1 shows the conditions for fractionation by LH-20.
[0013]
[Table 1]

[0014]
Of the 8 fractions obtained, Fr7 (see FIG. 1) was used to isolate and separate component B by HPLC using an ODS column (a packing material in which an octadecylsilyl group was chemically bonded to a silica gel carrier, 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]
The component B (methanol solution) isolated and fractionated by HPLC is subjected to mass spectrometry (GC-MS), and the molecular weight of the compound relating 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. The spectrum chart 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, in the case of Compound B, signals of hydrogen in the saccharide and aromatic ring regions were observed.
Further, from FIG. 5, the unit number of Compound B was estimated to be 11, and signals of saccharides and carbon in the aromatic ring region were observed.
[0018]
The infrared spectrum of Compound B was measured.
FIG. 6 shows the result.
[0019]
The ultraviolet / visible spectrum (UV) of Compound B (dissolved in ethanol) obtained by HPLC was measured (λmax: 259 nm). FIG. 7 shows the result.
[0020]
From the above results, Compound B was identified as N-hydroxy-N-methyl-adenosine adenosine (the following formula).
[0021]
Embedded image

[0022]
(2) The inhibitory effect of histamine release from mast cells on N-hydroxy-N-methyl-adenosine obtained from Mesimakov mycelium was examined to determine the type I allergic reaction inhibitory effect.
(A) Preparation of test animals and cells (a) Male Wister rats (6 to 8 weeks old) were used as test animals.
(B) Cell preparation method The rat was anesthetized with ether and exsanguinated, and then 15 ml of Tyrode Buffer was injected into the abdominal cavity of the rat to collect the peritoneal fluid. Further, 10 ml of Tyrode Buffer was injected into the peritoneal cavity of the rat, and the peritoneal fluid was further collected and mixed with the peritoneal fluid collected earlier. The intraperitoneal fluid was centrifuged at 800 rpm at 4 ° C. for 10 minutes, and the supernatant was removed and filtered.
Furthermore, the cell suspension to which Tyrode Buffer was added was centrifuged at 800 rpm at 4 ° C. for 10 minutes, and the supernatant was removed to homogenize the cells. The cell suspension was measured with a hemocytometer, adjusted to 8 × 10 6 cells / ml, and used.
[0023]
(B) Experimental method for bioassay and analysis method for histamine (a) Experimental method for bioassay 370 μl of the cell suspension dispensed into 1.2 ml microtubes was pre-incubated in a 37 ° C. water bath.
After 10 minutes, 20 μl of the sample set at each concentration was added.
After 10 minutes, 10 μl (0.5 μg / ml) of compound 48/80 (mast cell stimulating reagent: Wako Pure Chemical Industries), which is an allergy-inducing agent, was added.
After 15 minutes, the reaction was stopped on ice.
Furthermore, in order to remove mast cells, the mixture was centrifuged at 13.000 rpm at 4 ° C. for 15 minutes, and the supernatant was collected.
This supernatant was used as a diazotized sample for HPLC analysis.
[0024]
(B) Preparation of histamine diazotized derivative An equal volume of a 20 mM p-nitroaniline hydrochloride solution and a 200 mM aqueous sodium nitrite solution were well mixed (diazo reagent).
20 μl of the above (histamine) diazotization sample was added to 10 μl of the diazo reagent, and mixed well with a vortex mixer.
Further, 30 μl of a 10% ethanol solution of sodium carbonate was added and mixed well with a vortex mixer.
20 μl of this solution was used for HPLC analysis.
[0025]
(C) Histamine was analyzed by a diazotization method (Specific Determination of Histamine in Fish by High-performance Liquid Chromatography after sci- tem.
Table 3 shows the histamine HPLC analysis conditions (diazo method) in the biological assay.
[0026]
[Table 3]

[0027]
(C) Measurement of Histamine Release Inhibition Effect Table 4 shows the results of the effect of inhibiting histamine release from mast cells at a certain concentration of N-hydroxy-N-methyl-adenosine obtained from the mycelium of Mesimakobu.
As a comparative material, a commercially available histamine inhibitor (trade name: Intal, main component: sodium cromoglycate, maker Fujisawa Pharmaceutical) was used.
The test results are the average values of the results of three tests.
[0028]
[Table 4]

[0029]
The suppression value was based on the following formula.
a formula:
(1-peak integrated value of sample / peak integrated value of control) × 100
[0030]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, it has the effect that a histamine release inhibitor can be obtained using N-hydroxy-N-methyl-adenosine.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing a step of fractionating a hot water extract of a mycelium of Mesimakobu.
FIG. 2 is a diagram showing a fractionation chart of Fr7.
FIG. 3 shows a mass spectrum (GC-MS) of compound B.
FIG. 4 is a chart showing 1HN-MR spectrum of compound B.
FIG. 5 is a chart showing 13CN-MR spectrum of compound B.
FIG. 6 shows an infrared spectrum of compound B.
FIG. 7 is a diagram showing an ultraviolet-visible spectrum (UV) of compound B.

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 the mycelium of Mesimakobu is used.

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