JPH0578384A - Flavonol derivative - Google Patents

Abstract

PURPOSE: To provide a new compound which has an enhancement effect on nerve growth factor activity and is useful as an Alzheimer type anti-demential agent.

CONSTITUTION: This new derivative is the compound represented by the formula. Also, the preparation of the compound comprises: grinding Gansujidou (a kind of bean produced in China); subjecting the ground material to extraction treatment with an organic solvent such as alcohol or acetone to obtain a liquid extract; removing the solvent from the liquid extract by using distillation to obtain a residue; suspending the residue in water; thereafter, adding an organic solvent such as n-hexane to the suspension; drying the resulting organic layer with anhydrous sodium sulfate; thereafter removing the solvent from the dried organic layer by distillation to obtain an organic solvent-extracted fraction; dissolving this fraction in an organic solvent such as chloroform; and purifying and isolating the extracted material by silica-gel column chromatography or gel filtration using Sephadex (R), to prepare the objective compound.

COPYRIGHT: (C)1993,JPO

Description

Translated fromJapanese

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】本発明は、新規なフラボノール誘
導体に関する、更に詳しくはアルツハイマー型抗痴呆薬
として用いることのできる新規なフラボノール誘導体に
関する。TECHNICAL FIELD The present invention relates to a novel flavonol derivative, and more particularly to a novel flavonol derivative which can be used as an Alzheimer type anti-dementia drug.

【０００２】[0002]

【従来の技術】近年増えつつあるアルツハイマー型痴呆
症は、前脳基底野のコリン作動性神経細胞の選択的な脱
落によって起こるといわれており、更にその神経細胞の
脱落を神経成長因子（以下、ＮＧＦと略称する。）は抑
制することが知られ、ＮＧＦの顕著な作用増強効果を有
する物質の出現が望まれている。2. Description of the Related Art Alzheimer's dementia, which is increasing in number in recent years, is said to be caused by selective loss of cholinergic nerve cells in the basal forebrain, and the loss of these nerve cells is caused by nerve growth factor (hereinafter, referred to as (Abbreviated as NGF)) is known to be suppressed, and the emergence of a substance having a remarkable action-enhancing effect of NGF is desired.

【０００３】[0003]

【発明が解決しようとする課題】本発明の目的は、ＮＧ
Ｆの作用増強効果を有する新規な物質を提供することに
ある。DISCLOSURE OF THE INVENTION The object of the present invention is NG
It is to provide a novel substance having the effect of enhancing the action of F.

【０００４】[0004]

【課題を解決するための手段】本発明者らは、上記目的
の達成のために各種の植物成分について種々検討した結
果、中国甘粛省で採取された甘粛棘豆（Ｏｘｙｔｒｏｐ
ｉｓ ｋａｎｓｕｅｎｓｉｓ）の抽出物にＮＧＦの作用
増強効果を有する新規な物質が存在することを見出し本
発明を完成した。[Means for Solving the Problems] As a result of various studies on various plant components for achieving the above object, the present inventors have found that Gansu spinach (Oxytrop) collected in Gansu Province, China.
The present invention has been completed by finding that a novel substance having an action-enhancing effect of NGF exists in an extract of iskansuensis).

【０００５】本発明はThe present invention is

【化２】で表されるフラボノール誘導体である。[Chemical 2] Is a flavonol derivative represented by.

【０００６】この甘粛棘豆より本発明のフラボノール誘
導体を単離するには、以下の抽出方法によって行う。す
なわち、甘粛棘豆（薬用全草）を粉砕し、アルコール、
アセトンなどの有機溶媒で抽出し、有機溶媒を留去して
得られた残さを水に懸濁した後、ｎ−ヘキサンなどの有
機溶媒を加える。有機層を無水硫酸ナトリウムで乾燥
後、溶媒を留去し、有機溶媒抽出画分を得る。この画分
を再度クロロホルム、メタノールなどの有機溶媒に溶解
し、シリカゲルカラムクロマトグラフィー及びセファデ
ックスを用いたゲル濾過等により、本発明のフラボノー
ル誘導体を精製単離することができる。The flavonol derivative of the present invention is isolated from this Gansu tsubame by the following extraction method. That is, crushed Gansu spinach (medicinal whole plant), alcohol,
After extraction with an organic solvent such as acetone, the organic solvent is distilled off, the obtained residue is suspended in water, and then an organic solvent such as n-hexane is added. After drying the organic layer over anhydrous sodium sulfate, the solvent is distilled off to obtain an organic solvent extraction fraction. The flavonol derivative of the present invention can be purified and isolated by re-dissolving this fraction in an organic solvent such as chloroform and methanol and performing silica gel column chromatography and gel filtration using Sephadex.

【０００７】以上の精製法によって得られたフラボノー
ル誘導体の理化学的性質は、次の通りである。 （１）外観：淡黄色粉末 （２）融点：１５５〜１５８℃ （３）元素分析値；Ｃ_３８Ｈ_５２Ｏ_１２として、 計算値：Ｃ；６５．１３％，Ｈ；７．４８％ 実測値：Ｃ；６４．８８％，Ｈ；７．３０％The physicochemical properties of the flavonol derivative obtained by the above purification method are as follows. (1) Appearance: pale yellow powder (2) Melting point: 155 to 158 ° C. (3) Elemental analysis value; as C₃₈ H₅₂ O₁₂ , calculated value: C; 65.13%, H; 7.48% measured value : C; 64.88%, H; 7.30%

【０００８】（４）Ｅｌマススペクトル ｍ／ｚ ３００（アグリコン）^＋ （５）ＦＡＢマススペクトル ｍ／ｚ ７２３（Ｍ＋Ｎａ）^＋ ｍ／ｚ ７３９（Ｍ＋Ｋ）^＋ （６）分子式：Ｃ_３８Ｈ_５２Ｏ_１２ （７）分子量：７００(4) El mass spectrum m / z 300 (aglycone)⁺ (5) FAB mass spectrum m / z 723 (M + Na)⁺ m / z 739 (M + K)⁺ (6) Molecular formula: C₃₈H₅₂O₁₂ (7) Molecular weight: 700

【０００９】（８）ＵＶ吸収スペクトル： メタノール溶液で測定した結果、 λ_Ｍａｘ＝ ２６６ｎｍ ２９８ｎｍ（ｓｈ） ３４９ｎｍ ナトリウムメチラートを添加した結果、 λ_Ｍａｘ＝ ２６０ｎｍ ２６５ｎｍ ３９８ｎｍ 塩化アルミニウムを添加した結果、 λ_Ｍａｘ＝ ２７６ｎｍ ３０３ｎｍ ３５５ｎｍ ３９８ｎｍ 塩化アルミニウム＋塩酸を添加した結果、 λ_Ｍａｘ＝ ２７６ｎｍ ３０３ｎｍ ３４９ｎｍ ３９８ｎｍ 酢酸ナトリウムを添加した結果、 λ_Ｍａｘ＝ ２６６ｎｍ ２９８ｎｍ（ｓｈ） ３４８ｎｍ 酢酸ナトリウム＋ホウ酸を添加した結果、 λ_Ｍａｘ＝ ２６６ｎｍ ２９８ｎｍ（ｓｈ） ３４８ｎｍ(8) UV absorption spectrum: As a result of measurement with a methanol solution, λ_Max = 266 nm 298 nm (sh) 349 nm As a result of adding sodium methylate, λ_Max = 260 nm 265 nm 398 nm As a result of adding aluminum chloride, λ_Max = 276 nm 303 nm 355 nm 398 nm As a result of adding aluminum chloride + hydrochloric acid, λ_Max = 276 nm 303 nm 349 nm 398 nm As a result of adding sodium acetate, λ_Max = 266 nm 298 nm (sh) 348 nm As a result of adding sodium acetate + boric acid, λ_Max = 266 nm 298nm (sh) 348nm

【００１０】（９）^ｌＨ−ＮＭＲスペクトル：重ジメチ
ルスルフォキシド中、４００ＭＨｚで測定したスペクト
ルを図１に示す。 （１０）^１３Ｃ−ＮＭＲスペクトル：重ジメチルスルフ
ォキシド中、１００ＭＨｚで測定したスペクトルを図２
に示す。 （１１）溶解性：メタノール、エタノール、アセトン、
酢酸エチルエステル及び水に可溶、エチルエーテル、ｎ
−ヘキサン及びベンゼンに難溶。 （１２）呈色反応：陽性：硫酸、ヨウ素、ＦｅＣｌ_３ （１３）塩基性、酸性、中性の区別：酸性 （１４）薄層クロマトグラフィー Ｒｆ値；０．１３ 吸着剤；ＨＰＴＬＣプレート Ｎｏ．１３１２４（メルク社製） 展開溶媒；６０％アセトニトリル(9)^lH-NMR spectrum: heavy dimethy
Spectra measured at 400 MHz in Rusulfoxide
Is shown in FIG. (10)^ThirteenC-NMR spectrum: heavy dimethylsulf
Figure 2 shows the spectrum measured at 100MHz in oxide.
Shown in. (11) Solubility: methanol, ethanol, acetone,
Soluble in ethyl acetate and water, ethyl ether, n
-Slightly soluble in hexane and benzene. (12) Color reaction: positive: sulfuric acid, iodine, FeCl_Three (13) Distinction between basic, acidic and neutral: acidic (14) thin layer chromatography Rf value; 0.13 adsorbent; HPTLC plate No. 13124 (Merck) developing solvent: 60% acetonitrile

【００１１】[0011]

【発明の効果】本発明のフラボノール誘導体は、高いＮ
ＧＦの作用増強効果を有するのでアルツハイマー型抗痴
呆薬として有用である。The flavonol derivative of the present invention has a high N
Since it has the effect of enhancing the action of GF, it is useful as an Alzheimer-type anti-dementia drug.

【００１２】[0012]

【実施例】以下、実施例および試験例を挙げて本発明を
具体的に説明する。 実施例 （１） 中国甘粛省で採取された甘粛棘豆３ｋｇを粉砕
し、９５％エタノール１２Ｌで３回加熱抽出し濾過後溶
媒を留去し、エタノール抽出物１６５ｇを得た。 （２） このエタノール抽出物８０ｇを水１Ｌに懸濁
し、等量のｎ−ヘキサンで４回抽出した。ｎ−ヘキサン
層を無水硫酸ナトリウムで乾燥後、溶媒を留去し、ｎ−
ヘキサン抽出画分１２．２ｇを得た。 （３） ｎ−ヘキサン抽出画分１２．２ｇをｎ−ヘキサ
ン２０ｍｌに溶解し、ｎ−ヘキサンで調製したシリカゲ
ルを充填した５００ｍｌのカラムに吸着させた。ｎ−ヘ
キサン−酢酸エチルエステル混合溶媒（９０：１０〜７
０：３０）で溶出される区分を除き、次いで、クロロホ
ルム−メタノール（５０：５０）の混合溶媒５００ｍｌ
及びメタノール５００ｍｌで溶出される区分を合わせ濃
縮乾固し、褐色のシロップ状物質２．９ｇを得た。 （４） （３）で得られたシロップ状物質２．９ｇを、
クロロホルム１０ｍｌに溶解し、クロロホルムで調製し
たシリカゲルを充填した１３０ｍｌのカラムに吸着させ
た。クロロホルム−メタノール混合溶媒をメタノールの
濃度を徐々に上げながら（０〜１００％）溶出し、活性
画分を合わせ濃縮乾固し、褐色のシロップ状物質２５０
ｍｇを得た。 （５） （４）で得られたシロップ状物質２５０ｍｇを
ｎ−ヘキサン−ジクロロメタン−エタノール（５：５：
１）の混合溶媒に溶解し、上記混合溶媒で調製したセフ
ァデックスＬＨ−２０を充填した３００ｍｌのカラムを
用いてゲル濾過を行った。活性画分を合わせ濃縮乾固
し、褐色粉末６．０ｍｇを得た。 （６） （５）で得られた褐色粉末６．０ｍｇをアセト
ンに溶解し、上記溶媒で調製したセファデックスＬＨ−
２０を充填した１００ｍｌのカラムを用いてゲル濾過を
行った。活性画分を合わせ濃縮乾固し、本発明のフラボ
ノール誘導体の淡黄色粉末４．１ｍｇを得た。EXAMPLES The present invention will be specifically described below with reference to examples and test examples. Example (1) 3 kg of Gansu azuki bean collected in Gansu Province, China was crushed, heated and extracted 3 times with 12 L of 95% ethanol, filtered and the solvent was distilled off to obtain 165 g of an ethanol extract. (2) 80 g of this ethanol extract was suspended in 1 L of water and extracted 4 times with an equal amount of n-hexane. The n-hexane layer was dried over anhydrous sodium sulfate, the solvent was distilled off, and n-hexane was added.
12.2 g of a hexane extracted fraction was obtained. (3) 12.2 g of the n-hexane extracted fraction was dissolved in 20 ml of n-hexane and adsorbed on a 500 ml column filled with silica gel prepared with n-hexane. n-hexane-acetic acid ethyl ester mixed solvent (90: 10-7
The fraction eluted at 0:30) was removed, and then 500 ml of a mixed solvent of chloroform-methanol (50:50) was added.
And the fractions eluted with 500 ml of methanol were combined and concentrated to dryness to obtain 2.9 g of a brown syrupy substance. (4) 2.9 g of the syrup-like substance obtained in (3)
It was dissolved in 10 ml of chloroform and adsorbed on a 130 ml column filled with silica gel prepared with chloroform. The chloroform-methanol mixed solvent was eluted while gradually increasing the concentration of methanol (0 to 100%), and the active fractions were combined and concentrated to dryness to give a brown syrup-like substance 250.
mg was obtained. (5) 250 mg of the syrup-like substance obtained in (4) was added to n-hexane-dichloromethane-ethanol (5: 5:
Gel filtration was performed using a 300 ml column dissolved in the mixed solvent of 1) and packed with Sephadex LH-20 prepared with the above mixed solvent. The active fractions were combined and concentrated to dryness to obtain 6.0 mg of a brown powder. (6) Sephadex LH- prepared by dissolving 6.0 mg of the brown powder obtained in (5) in acetone and using the above solvent.
Gel filtration was performed using a 100 ml column filled with 20. The active fractions were combined and concentrated to dryness to obtain 4.1 mg of a pale yellow powder of the flavonol derivative of the present invention.

【００１３】試験例 （ＮＧＦ作用増強効果） （検体）実施例で得られた淡黄色粉末をメタノールに溶
解し、１ｍｇ／ｍｌ〜３ｍｇ／ｍｌとした。 （試験細胞）ＰＣ−１２細胞 ラット褐色細胞腫（ＮＧ
Ｆ応答細胞） （使用した培地）１０％熱非働化（５６℃、３０分処
理）牛胎児血清（ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕ
ｍ，ＦＢＳ，ギブコ社）、５％熱非働化馬血清（ｈｏｒ
ｓｅ ｓｅｒｕｍ，ＨＳ，ギブコ社）、５０Ｕ／ｍｌペ
ニシリン、５０μｇ／ｍｌストレプトマイシンを含有す
るダルベッコ改良型イーグル培地（Ｄｕｌｂｅｃｃｏ’
ｓｍｏｄｉｆｉｅｄ Ｅａｇｌｅ ｍｅｄｉｕｍ．ＤＭ
ＥＭ，高グルコース含有，ギブコ社） （試験方法）ＰＣ−１２細胞を、上記培地にて、２×１
０^４ｃｅｌｌｓ／ｍｌに調製し、コラーゲンコート２４
孔プレート（２ｃｍ^２，コーニング社）へ、０．５ｍｌ
／ｗｅｌｌずつまき、３７℃、５％ＣＯ_２で培養した。
２４時間後、プレート下部に付着した細胞を残して、
０．５ｎｇ／ｍｌＮＧＦ（シグマ社、５０ｎｇ／ｍｌの
０．１％牛血清アルブミン含有リン酸緩衝生理食塩水溶
液を１％）と各種濃度の検体１％を含む上記培地０．３
ｍｌ／ｗｅｌｌと交換した。さらに４８時間培養後、細
胞の突起伸長を、顕微鏡下に観察した。観察結果を細胞
の突起長で４種類に分類し、形態変化のない細胞を０
点、突起の伸長を伴わず形態変化を起こした細胞を１
点、細胞体の直径以内の突起を持つ細胞を２点、細胞体
の直径以上の突起を持つ細胞を３点とし、１００細胞の
合計点を突起伸長活性とし、３視野を平均した。 （結果）結果を表１に示す。Test Example (NGF action enhancing effect) (Sample) The pale yellow powder obtained in the example was dissolved in methanol to give 1 mg / ml to 3 mg / ml. (Test cells) PC-12 cells Rat pheochromocytoma (NG
F-responsive cells) (Used medium) 10% heat-inactivated (56 ° C., 30 minutes treatment) fetal bovine serum
m, FBS, Gibco), 5% heat inactivated horse serum (hor
Se serum, HS, Gibco), 50 U / ml penicillin, 50 μg / ml streptomycin in Dulbecco's modified Eagle medium (Dulbecco ').
modified Eagle medium. DM
(EM, high glucose content, Gibco) (Test method) PC-12 cells in the above medium at 2 x 1
Prepared to 0⁴ cells / ml and coated with collagen 24
0.5 ml into a perforated plate (2 cm² , Corning)
/ Well, and the cells were cultured at 37 ° C. and 5% CO₂ .
After 24 hours, leaving the cells attached to the bottom of the plate,
0.3 ng of the above medium containing 0.5 ng / ml NGF (Sigma, 1 ng of 50 ng / ml 0.1% bovine serum albumin-containing phosphate buffered saline solution) and 1% of various concentrations of the specimen
Replaced with ml / well. After culturing for a further 48 hours, cell neurite extension was observed under a microscope. The observation results are classified into four types according to the cell protrusion length, and cells with no morphological change are classified as 0.
Cells that have undergone morphological changes without elongation of dots and protrusions
Points, cells having protrusions within the diameter of the cell body were 2 points, cells having protrusions larger than the diameter of the cell body were 3 points, and the total point of 100 cells was the protrusion extension activity, and 3 visual fields were averaged. (Results) The results are shown in Table 1.

【００１４】[0014]

【表１】[Table 1]

【図面の簡単な説明】[Brief description of drawings]

【図１】重ジメチルスルフォキシド中、４００ＭＨｚで
測定したフラボノール誘導体の^１Ｈ−ＮＭＲスペクトル
を示す。FIG. 1 shows the¹ H-NMR spectrum of a flavonol derivative measured at 400 MHz in heavy dimethyl sulfoxide.

【図２】重ジメチルスルフォキシド中、１００ＭＨｚで
測定したフラボノール誘導体の^１３Ｃ−ＭＮＲスペクト
ルを示す。FIG. 2 shows a¹³ C-MNR spectrum of a flavonol derivative measured in deuterated dimethyl sulfoxide at 100 MHz.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 花田 和紀 東京都豊島区高田３丁目24番１号 大正製 薬株式会社内 (72)発明者 楊 峻山 中華人民共和国 北京市宣武区先農壇街１ 号 中国医学科学院葯物研究所内 (72)発明者 王 玉蘭 中華人民共和国 北京市宣武区先農壇街１ 号 中国医学科学院葯物研究所内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuki Hanada 3-24-1 Takada, Toshima-ku, Tokyo Within Taisho Pharmaceutical Co., Ltd. No. 1 Inside the Chinese Institute of Medical Science Anther Research Institute (72) Inventor Wang Yulan No. 1 Xuantan Street, Xuanwu District, Beijing, China No. 1 Inside the Chinese Academy of Medical Science Anther Institute

Claims (1)

Translated fromJapanese

【特許請求の範囲】[Claims]【請求項１】 【化１】で表されるフラボノール誘導体Claims: Flavonol derivative represented by