JP2023052160A - Extracted fraction containing polymer condensed tannin obtained from ephedra herb extract or ephedrine alkaloid-free ephedra herb extract as well as manufacturing method and application thereof - Google Patents
Extracted fraction containing polymer condensed tannin obtained from ephedra herb extract or ephedrine alkaloid-free ephedra herb extract as well as manufacturing method and application thereof Download PDFInfo
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- JP2023052160A JP2023052160A JP2022211572A JP2022211572A JP2023052160A JP 2023052160 A JP2023052160 A JP 2023052160A JP 2022211572 A JP2022211572 A JP 2022211572A JP 2022211572 A JP2022211572 A JP 2022211572A JP 2023052160 A JP2023052160 A JP 2023052160A
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- ephedra
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- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 title claims abstract description 15
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Abstract
Description
本発明は、麻黄エキス由来、又はエフェドリンアルカロイド除去麻黄エキス(ephedrine alkaloids-free Ephedra Herb extract: EFE)由来の重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物及びその製造方法、並びに該抽出分画物を含む医薬組成物に関する。本発明の医薬組成物は抗がん薬、抗転移薬、疼痛抑制薬、並びに抗インフルエンザウイルス薬として有用である。 The present invention provides an extract fraction containing polymer condensed tannins derived from Ephedra extract or ephedrine alkaloids-free Ephedra Herb extract (EFE) and having a weight-average molecular weight of 45,000 or more, and a method for producing the same. It relates to a pharmaceutical composition comprising said extract fraction. The pharmaceutical composition of the present invention is useful as an anticancer drug, anti-metastatic drug, pain suppressant, and anti-influenza virus drug.
タンニンは、植物の幹、皮、葉、実等から抽出される天然物で、タンパク質や塩基性化合物等と結合して難溶性にする性質をもつポリフェノールをいう。身近な食品、嗜好品、工業用等に使われる植物に含まれている。タンニンには、酸や酵素が存在するとき加水分解されやすい加水分解型タンニンと、縮合が進む縮合型タンニンがある。加水分解型タンニンは比較的低分子であることも手伝って、生薬等多くの植物材料から単離、構造決定が進み、様々なタイプのものが明らかにされている。一方、縮合型タンニンは明確には同定されていなかった。1989年、Weingesは無色の植物抽出物を酸で加熱した際にアントシア
ニジンを生じる物質をプロアントシアニジンと名づけた。そして構成成分がフラバン 3-
オール又はフラバン 3,4-ジオールで複数の連結したフラボノイド単位からなることを明
らかにした。従来、縮合型タンニンと呼ばれていた多くの果実成分の実体がプロアントシアニジンであることが明らかにされており、プロアントシアニジンに関する研究が多くなされ、プロペラルゴニジン(Propelargonidin)、プロシアニジン(Procyanidin)、プロデルフィニジン(Prodelphinidin)、プロギボルチニジン(Proguibourtinidin)、プロ
フィセチニジン(Profisetinidin)、プロロビネチニジン(Prorobinetinidin)、プロテラカシジン(Proteracacidin)、プロメラカシジン(Promelacacidin)、プロアピゲニニジン(Proapigeninidin)、プロルテオリニジン(Proluteolinidin)等の化学構造及び薬理作用が明らかになっている。すなわち、プロアントシアニジンは、各種植物中に存在する縮合又は重合したタンニンであり、フラバン 3-オール又はフラバン 3,4-ジオールを構成単位として縮重合した化合物群である。
Tannin is a natural product extracted from plant stems, skins, leaves, fruits, etc., and refers to polyphenols that have the property of binding to proteins, basic compounds, etc. to make them insoluble. It is contained in familiar foods, luxury goods, and plants used for industrial purposes. Tannins include hydrolyzable tannins, which are easily hydrolyzed in the presence of acids and enzymes, and condensed tannins, which undergo condensation. Hydrolyzable tannins have relatively low molecular weights, and have been isolated from many plant materials such as crude drugs. Condensed tannins, on the other hand, have not been clearly identified. In 1989, Weinges named proanthocyanidins substances that generate anthocyanidins when colorless plant extracts are heated with acid. and the component is flavan 3-
3,4-diols or flavans were found to consist of multiple linked flavonoid units. It has been clarified that many of the fruit components that were conventionally called condensed tannins are proanthocyanidins, and many studies on proanthocyanidins have been conducted. Delphinidin, Proguibourtinidin, Profisetinidin, Prorobinetinidin, Proteracacidin, Promelacacidin, Proapigeninidin, Prolteolinidin (Proluteolinidin) and other chemical structures and pharmacological actions have been clarified. That is, proanthocyanidins are condensed or polymerized tannins present in various plants, and are a group of compounds condensed with flavan 3-ols or
プロアントシアニジンの化学構造は、一般に、フラバン 3-オール又はフラバン 3,4-ジオールを構成ユニットとして4β位→6位、4β位→8位、4β位→8位・2β位→O→7位等
の結合様式によって重合した二量体以上の重合体である(非特許文献1,2)。結合様式の
違いから数種類に分類されている。その1つは、2つ以上のフラバン 3-オール構造が4β位→8位又は4β位→6位を介して結合しているプロアントシアニジンBタイプ、もう1つはフラバン 3-オール構造が4β位→8位及び2β位→O→7位又は4β位→6位及び2β位→O
→7位の結合が少なくとも1箇所に存在するプロアントシアニジンAタイプである(図1
参照)。
The chemical structure of proanthocyanidins is generally composed of flavan 3-ol or
→ Proanthocyanidin A type with at least one bond at position 7 (Fig. 1
reference).
縮合型タンニンは加水分解型タンニンに比べて分子量の幅が広く、分子量の大きいものでは約20,000に達する(非特許文献3)。 Condensed tannins have a wider range of molecular weights than hydrolyzed tannins, with large molecular weights reaching about 20,000 (Non-Patent Document 3).
プロアントシアニジンは多様な生理活性を示すことが知られており、抗腫瘍、抗炎症、抗老化、抗酸化、抗アレルギー、抗菌、育毛等の活性が報告されているが(非特許文献4,5)、これらの生理活性とプロアントシアニジンの重合度数との構造活性相関に関しては
、全て明確にされているわけではない(特許文献1)。特許文献2では、プロシアニジンA
タイプ及びプロシアニジンBタイプ(カテコールタイプのみ)の単量体単位の結合がC-4、C-8位で生じる構造式が示されている。特許文献3では、プロアントシアニジンAタイプの
重合体、並びにプロアントシアニジンBタイプ(ピロガロールタイプ、カテコールタイプ
、単量体単位の結合がC-4、C-8位で生じる)の縮合体・重合体の肝線維化抑制効果が示されている。特許文献4では、プロアントシアニジンB(ピロガロールタイプ、カテコールタイプ)単量体単位の結合がC-4、C-6、C-8位で生じ、プロアントシアニジンを形成し、こ
れがウレアーゼ阻害作用を有することを報告している。
Proanthocyanidins are known to exhibit various physiological activities, and their activities such as antitumor, anti-inflammatory, anti-aging, antioxidant, anti-allergic, antibacterial, and hair growth have been reported (Non-Patent
Structural formulas are shown in which binding of monomeric units of type and procyanidin B type (catechol type only) occurs at the C-4 and C-8 positions. In
プロアントシアニジンを含む植物には、例えば、リンゴ、ブルーベリー、エルダーベリー、ブドウ、ストロベリー、レッドカーラント、カウベリー、グースベリー、クランベリー、サーモンベリー、ビルベリー、カシス、チェリー、ハクルベリー、ブラックベリー、プラム、ホワートルベリー、ボイセンベリー、マルベリー、ラズベリー、レッドカーラント、ローガンベリー、柿、松、樫、山桃、麦、小麦、大豆、黒大豆、カカオ、小豆、トチ、ピーナッツ、イチョウ葉、緑茶、大黄、麻黄、楊梅皮等が知られている(特許文献5,6
)。
Plants containing proanthocyanidins include, for example, apples, blueberries, elderberries, grapes, strawberries, red currants, cowberries, gooseberries, cranberries, salmonberries, bilberries, cassis, cherries, huckleberries, blackberries, plums, whittles. Berry, boysenberry, mulberry, raspberry, red currant, loganberry, persimmon, pine, oak, yam, barley, wheat, soybean, black soybean, cacao, adzuki bean, horse chestnut, peanut, ginkgo biloba, green tea, rhubarb, mahuang, Yangbai skin etc. are known (
).
麻黄はマオウ科植物Ephedra sinica Stapf、Ephedra intermedia Schrenk et C. A. Meyer又はEphedra equisetina Bunge (Ephedraceae)の地上茎である。麻黄は古来より使
われてきた最重要の生薬である。麻黄は日本薬局方によって総アルカロイド(エフェドリン及びプソイドエフェドリン)を0.7%以上含むと規定され、麻黄の薬理作用のほとんどはエフェドリンアルカロイドに由来すると考えられてきた(非特許文献6)。一方、麻黄の
副作用である動悸、血圧上昇、不眠、排尿障害等は、エフェドリンアルカロイドの中枢神経及び交感神経刺激作用によって生じるが、麻黄の主作用と副作用を分離することは困難であると考えられ、麻黄の副作用を除くために、麻黄からエフェドリンアルカロイドを除去するという発想は、これまでなかった。
Ephedra is the ground stem of Ephedra sinica Stapf, Ephedra intermedia Schrenk et CA Meyer or Ephedra equisetina Bunge (Ephedraceae). Ephedra is the most important crude drug that has been used since ancient times. Ephedra is defined by the Japanese Pharmacopoeia as containing 0.7% or more of total alkaloids (ephedrine and pseudoephedrine), and most of the pharmacological actions of Ephedra have been thought to derive from ephedrine alkaloids (Non-Patent Document 6). On the other hand, side effects of ephedra, such as palpitations, increased blood pressure, insomnia, and dysuria, are caused by the central nervous system and sympathetic nerve stimulating effects of ephedrine alkaloids, but it is considered difficult to separate the main effects and side effects of ephedra. However, the idea of removing ephedrine alkaloids from Ephedra to eliminate the side effects of Ephedra has never been thought of before.
本発明者らは、これまでの研究から、麻黄の非アルカロイド画分に肝細胞増殖因子(HGF)受容体c-Met キナーゼ阻害作用を介した抗がん抗転移作用があることを突き止め、さら
に麻黄の非アルカロイド画分からHerbacetin 配糖体を分離し、非糖部分のHerbacetin にc-Met キナーゼ阻害作用を含むマルチキナーゼ阻害作用があることを明らかにした。その中でも、Herbacetin のTrkA(NGF 受容体)阻害作用は強く、NGF-TrkA シグナル阻害を介した疼痛抑制作用を有することも見出した (特許文献7,8)。これらの結果から、麻黄の薬理作用の一部はエフェドリンアルカロイドに依存せず、したがって麻黄から副作用成分を除去できることが示唆された。そこで麻黄エキスからエフェドリンアルカロイドを除去したEFEを作製したところ(非特許文献7)(特許文献9)、疼痛抑制作用、抗インフルエンザ
ウイルス作用を含むいくつかの薬理作用を有することや(特許文献9)(非特許文献8)、不眠、動悸、興奮等の副作用が消失していることが明らかになった(非特許文献9)。しか
し、麻黄エキスはもとより、EFEは様々な成分を含んでいるため、通常服用可能な嵩が制
限要因となるため、より有効性が期待される高力価の投与ができなかった。
The present inventors have found from previous studies that the non-alkaloid fraction of mahuang has an anti-cancer anti-metastatic effect mediated by hepatocyte growth factor (HGF) receptor c-Met kinase inhibitory action. We isolated Herbacetin glycosides from the non-alkaloid fraction of Ma Huang, and clarified that the non-sugar portion of Herbacetin has multikinase inhibitory activity, including c-Met kinase inhibitory activity. Among them, it was found that Herbacetin has a strong TrkA (NGF receptor) inhibitory action and has a pain suppressing action via NGF-TrkA signal inhibition (
一方、麻黄由来の低分子のプロアントシアニジンAタイプあるいはプロアントシアニジ
ンBタイプ(ピロガロールタイプ、カテコールタイプ)の重合したプロアントシアニジン
は既に報告されている(非特許文献10,11,12)。さらに最近、麻黄の70%アセトン抽出液
から、プロアントシアニジンAタイプ及びプロアントシアニジンBタイプの両方が重合したオリゴマーが単離された(非特許文献13)。
On the other hand, polymerized proanthocyanidins of low-molecular-weight proanthocyanidin A type or proanthocyanidin B type (pyrogallol type, catechol type) derived from Ephedra have already been reported (Non-Patent
麻黄由来のプロアントシアニジン含有液を、分画分子量が5000以下の限外濾過膜及び/又は食塩排除率が30%以下の逆浸透膜で処理した後、分画分子量が500~5000の画分を分取することを特徴とするプロアントシアニジンの製造法が知られている(特許文献6)。 After treating the Ephedra-derived proanthocyanidin-containing liquid with an ultrafiltration membrane with a cutoff molecular weight of 5000 or less and/or a reverse osmosis membrane with a salt rejection rate of 30% or less, the fraction with a cutoff molecular weight of 500 to 5000 is A method for producing proanthocyanidins characterized by fractionation is known (Patent Document 6).
しかしこれまでに、重量平均分子量が20,000を超える高分子タンニンに関する報告はない。 However, to date, there have been no reports of high-molecular-weight tannins with a weight-average molecular weight exceeding 20,000.
麻黄エキスからエフェドリンアルカロイドを除去したEFEの製造方法が確立され、c-Met
キナーゼ阻害作用を介する抗がん作用・抗転移作用、疼痛抑制作用、及び抗インフルエ
ンザウイルス作用を有し、エフェドリンアルカロイドによる副作用を排除した医薬品が提
供できるようになった。しかし、麻黄エキスはもとより、EFEは様々な成分を含んでいる
ため、通常服用可能な嵩が制限要因となるため、より有効性が期待される高力価の投与ができなかった。麻黄エキスあるいはEFE中のc-Met キナーゼ阻害作用を介する抗がん作用
・抗転移作用、疼痛抑制作用、及び抗インフルエンザウイルス作用を有する活性成分を精査した結果、麻黄の有効性成分の1つとしてHerbacetin 配糖体が見出されているが、薬効が高くないことから、医薬品として最適でなかった。
A method for producing EFE by removing ephedrine alkaloids from mahuang extract was established, and c-Met
It has become possible to provide pharmaceuticals that have anti-cancer, anti-metastatic, pain-suppressing, and anti-influenza virus effects through kinase inhibitory action, and eliminate the side effects of ephedrine alkaloids. However, since EFE contains various ingredients, not to mention Ephedra extract, the bulk that can be taken normally is a limiting factor, and high-potency administration, which is expected to be more effective, could not be administered. As a result of careful examination of active ingredients in mahuang extract or EFE that have anti-cancer, anti-metastatic, pain-suppressing, and anti-influenza virus activities mediated by c-Met kinase inhibitory action, it was found to be one of the effective ingredients of mahuang. Herbacetin glycosides have been found, but they were not optimal as pharmaceuticals because they are not highly efficacious.
本発明は、麻黄エキスあるいはEFEから、c-Met キナーゼ阻害作用を介する抗がん作用
・抗転移作用、疼痛抑制作用、及び抗インフルエンザウイルス作用を持つ新規活性成分を明らかにし、その成分の製造方法を確立することにより、高力価の投与が可能で、かつ、活性成分以外の成分による副作用発現リスクが排除された医薬品を提供することを目的とする。さらに、本発明に関する研究過程で、活性成分の新規効能効果(c-Met発現量の低
下作用及び、上皮成長因子受容体(EGFR)リン酸化阻害作用とEGFR発現量の低下作用を介する抗がん作用)を見出したので、本活性成分の用途として提供することを目的とする。
The present invention clarifies a novel active ingredient having anticancer activity, anti-metastatic activity, pain suppression activity, and anti-influenza virus activity mediated by c-Met kinase inhibitory action from mahuang extract or EFE, and a method for producing the ingredient. By establishing this, the purpose is to provide a drug that can be administered with a high potency and eliminates the risk of side effects due to ingredients other than the active ingredient. Furthermore, in the course of research related to the present invention, new efficacy effects of active ingredients (c-Met expression level lowering action and epidermal growth factor receptor (EGFR) phosphorylation inhibitory action and EGFR expression level lowering action) Action) was discovered, and the object is to provide it as a use of the present active ingredient.
本発明者らは、上記課題を解決するために鋭意研究した結果、麻黄エキスあるいはEFE
を水/n-ブタノールで抽出した水可溶部を、芳香族系合成吸着剤Diaion HP-20カラムを用いて水、20%~100%メタノール(MeOH)で溶出させて分画し、20%メタノール以上の溶出部について分析したところ、この化学構造がプロシアニジンAタイプ、及びプロシアニジンBタイプ(ピロガロールタイプ及びカテコールタイプ)のC-4、C-6、C-8位で重合する重量
平均分子量45,000以上の高分子であることを見出した。
The present inventors have made intensive studies to solve the above problems, and found that Ephedra extract or EFE
was extracted with water/n-butanol, and the water-soluble portion was fractionated by eluting with water and 20% to 100% methanol (MeOH) using a synthetic aromatic adsorbent Diaion HP-20 column to obtain 20% Analysis of the portion eluted with more than methanol revealed that this chemical structure polymerized at the C-4, C-6, and C-8 positions of procyanidin A type and procyanidin B type (pyrogallol type and catechol type) with a weight average molecular weight of 45, 000 or more macromolecules.
さらに、得られる成分群の構造を明らかにし、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物にc-Metキナーゼ阻害作用、c-Metリン酸化阻害作用、c-Met
発現量の低下作用を介する抗がん・抗転移作用、疼痛抑制作用、EGFRのリン酸化阻害作用及びEGFR発現量の低下作用を介する抗がん作用、並びに抗インフルエンザウイルス作用が存在することを発見して、本願発明を完成した。
Furthermore, we clarified the structure of the obtained component group, and found that the extract fraction containing high molecular weight condensed tannins with a weight average molecular weight of 45,000 or more has c-Met kinase inhibitory activity, c-Met phosphorylation inhibitory activity, and c-Met inhibitory activity.
Discovered that it has anti-cancer/anti-metastatic effect, pain suppression effect, anti-cancer effect and anti-influenza virus effect through inhibition of EGFR phosphorylation and reduction of EGFR expression level. As a result, the present invention was completed.
すなわち、本願発明は以下のものを提供するものである。
[請求項1]
麻黄エキス由来、又はエフェドリンアルカロイド除去麻黄エキス(EFE)由来の重量平
均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物であって、前記重量平均分子量45,000以上の高分子縮合型タンニンを30%以上含む抽出分画物。
[請求項2]
高分子縮合型タンニンが、エクステンションユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールが主にプロアントシアニジンBタイプで縮合し、その一部にプロアントシアニジンAタイプの縮合型タンニンユニットを含み、さらにターミ
ナルユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールを
主に含み、ピロガロールタイプとカテコールタイプを約5:1で含む、請求項1記載の抽出
分画物。
[請求項3]
麻黄エキス由来、又はエフェドリンアルカロイド除去麻黄エキス(EFE)の水溶性画分
をカラムクロマトグラフィーによって、水とメタノール又はエタノールから選択されるアルコールとの混合溶媒を用いて、アルコール濃度を高くしながら順次溶出し、得られる各分画の分子量を測定して、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を得ることからなる、請求項1記載の抽出分画物の製造方法。
[請求項4]
請求項1又は2に記載の抽出分画物を含む医薬組成物。
[請求項5]
疼痛抑制薬である、請求項4に記載の医薬組成物。
[請求項6]
c-Metを発現するがんに対する抗がん・抗転移薬である、請求項4に記載の医薬組成
物。
[請求項7]
EGFRを発現するがんに対する抗がん薬である、請求項4に記載の医薬組成物。
「請求項8」
抗インフルエンザウイルス薬である、請求項4に記載の医薬組成物。
[請求項9]
請求項1に記載の抽出分画物を用いる、高分子縮合型タンニンを活性成分として含有する医薬組成物の含量測定法。
That is, the present invention provides the following.
[Claim 1]
An extract fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more derived from mahuang extract or derived from ephedrine alkaloid-removed mahuang extract (EFE), wherein 30 Extracted fraction containing more than %.
[Claim 2]
Polymer condensed tannins, as extension units, are condensed mainly with proanthocyanidin B type catechol-type or pyrogallol-type flavan 3-ols, part of which contains proanthocyanidin A-type condensed tannin units, and
[Claim 3]
Water-soluble fractions derived from Ephedra extract or Ephedrine alkaloid-removed Ephedra extract (EFE) are eluted by column chromatography using a mixed solvent of water and an alcohol selected from methanol or ethanol while increasing the alcohol concentration. 2. The method for producing an extract fraction according to
[Claim 4]
A pharmaceutical composition comprising the extract fraction according to
[Claim 5]
5. The pharmaceutical composition according to
[Claim 6]
5. The pharmaceutical composition according to
[Claim 7]
5. The pharmaceutical composition according to
"Claim 8"
5. The pharmaceutical composition according to
[Claim 9]
A method for measuring the content of a pharmaceutical composition containing polymer condensed tannin as an active ingredient, using the extract fraction according to
本発明により、麻黄エキス由来、又はEFE由来の重量平均分子量45,000以上の高分子縮
合型タンニンを含む抽出分画物を有効成分として含む医薬組成物を提供することが可能となり、疼痛抑制作用、c-Metを発現するがんの抗がん・抗転移薬、EGFRを発現するがんの
抗がん薬、及び抗インフルエンザウイルス作用を有する高力価で投与可能な医薬品を提供することができる。本発明の抽出分画物は、高分子画分(重量平均分子量45,000以上)
のプロアントシアニジンAタイプ及びプロアントシアニジンBタイプの重合したプロアントシアニジン(縮合型タンニン、高分子ポリフェノール)である。
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a pharmaceutical composition containing, as an active ingredient, an extract fraction containing a polycondensed tannin derived from mahuang extract or derived from EFE and having a weight average molecular weight of 45,000 or more. It is possible to provide anti-cancer/anti-metastatic drugs for cancers expressing -Met, anti-cancer drugs for cancers expressing EGFR, and pharmaceuticals with anti-influenza virus activity that can be administered with high potency. The extract fraction of the present invention is a high molecular weight fraction (weight average molecular weight of 45,000 or more)
proanthocyanidin A type and proanthocyanidin B type polymerized proanthocyanidins (condensed tannins, high molecular weight polyphenols).
本発明によって、高力価の投与が可能で、かつ、活性成分以外の成分による副作用発現リスクが排除された医薬品を提供することが可能となる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a drug that can be administered with a high potency and that eliminates the risk of developing side effects due to ingredients other than the active ingredient.
本発明は一態様として、麻黄エキス由来、又はエフェドリンアルカロイド除去麻黄エキス(EFE)由来の重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を
提供する。
As one aspect, the present invention provides an extract fraction containing polymer condensed tannins having a weight-average molecular weight of 45,000 or more derived from an Ephedra extract or derived from an ephedrine alkaloid-removed Ephedra extract (EFE).
本発明の抽出分画物を得るための原料として用いられる麻黄エキスは、日本薬局方の麻黄の熱水抽出物である。または、麻黄エキスからエフェドリンアルカロイドを除去したEFEである(特許文献9)。EFEは、麻黄エキスから陽イオン交換クロマトグラフィーによっ
てエフェドリンを除去し、濃縮乾燥を経ることによって得ることができる。後述する実施例に示すように、麻黄エキスを原料として用いた場合も、EFEを原料として用いた場合も
、同様に本発明の抽出分画物を得ることができる。
The Ephedra extract used as a raw material for obtaining the extract fraction of the present invention is a hot water extract of Ephedra in the Japanese Pharmacopoeia. Alternatively, it is an EFE obtained by removing the ephedrine alkaloids from the mahuang extract (Patent Document 9). EFE can be obtained by removing ephedrine from mahuang extract by cation exchange chromatography, followed by concentration and drying. As shown in the examples described later, the extraction fraction of the present invention can be obtained in the same manner when Ephedra extract is used as a raw material and when EFE is used as a raw material.
本発明の抽出分画物は、重量平均分子量45,000以上の高分子縮合型タンニンを含むことを特徴とする。重量平均分子量はGPC用標準ポリスチレンを標準物質として換算した重量
平均分子量である。本発明の抽出分画物は、重量平均分子量45,000以上の、プロアント
シアニジンAタイプ及びプロアントシアニジンBタイプの重合したプロアントシアニジン(縮合型タンニン、高分子ポリフェノール)である。
The extract fraction of the present invention is characterized by containing a polymer condensed tannin having a weight average molecular weight of 45,000 or more. The weight average molecular weight is the weight average molecular weight converted using standard polystyrene for GPC as a standard substance. The extract fraction of the present invention is polymerized proanthocyanidins of proanthocyanidin A type and proanthocyanidin B type (condensed tannins, high molecular weight polyphenols) having a weight average molecular weight of 45,000 or more.
本発明の抽出分画物は、重量平均分子量45,000以上の高分子縮合型タンニンを30%以上
、より好ましくは40%以上含む。
The extract fraction of the present invention contains 30% or more, more preferably 40% or more, of polymer condensed tannins having a weight average molecular weight of 45,000 or more.
本発明の抽出分画物に含まれる高分子縮合型タンニンは、エクステンションユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールが主にプロアント
シアニジンBタイプで縮合し、その一部にプロアントシアニジンAタイプの縮合型タンニンユニットを含み、さらにターミナルユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールを主に含み、ピロガロールタイプとカテコールタイプを約5:1で含む(図1参照)。
In the polymer condensed tannin contained in the extract fraction of the present invention, as an extension unit, catechol-type or pyrogallol-type flavan 3-ol is mainly condensed with proanthocyanidin B type, and a part thereof is proanthocyanidin A type Furthermore, as a terminal unit, it mainly contains catechol-type or pyrogallol-type flavan 3-ols, and contains pyrogallol-type and catechol-type at a ratio of about 5:1 (see FIG. 1).
本発明は別の一態様として、麻黄エキス由来、又はエフェドリンアルカロイド除去麻黄エキス(EFE)の水溶性画分をカラムクロマトグラフィーによって、水とメタノール又は
エタノールから選択されるアルコールとの混合溶媒を用いて、アルコール濃度を高くしながら順次溶出し、得られる各分画の分子量を測定して、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を得ることからなる、本発明の抽出分画物の製造方法を提供する。
As another aspect of the present invention, a water-soluble fraction derived from Ephedra extract or Ephedrine alkaloid-removed Ephedra extract (EFE) is subjected to column chromatography using a mixed solvent of water and an alcohol selected from methanol or ethanol. , The extraction of the present invention, which consists of sequentially eluting while increasing the alcohol concentration, measuring the molecular weight of each fraction obtained, and obtaining an extracted fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more. A method for producing a fraction is provided.
具体的には、麻黄エキス由来、又はエフェドリンアルカロイド除去麻黄エキス(EFE)
を水に溶解したものを、極性の低い有機溶媒から極性の高い有機溶媒を用いて順次分配し、得られる水エキスをカラムクロマトグラフィーによって、水とメタノール又はエタノールから選択されるアルコールとの混合溶媒を用いて、アルコール濃度を高くしながら順次溶出し、得られる各分画の分子量を測定して、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を得る。
Specifically, Ephedra extract-derived or Ephedrine alkaloid-removed Ephedra extract (EFE)
is dissolved in water, sequentially distributed using an organic solvent with low polarity to an organic solvent with high polarity, and the resulting water extract is analyzed by column chromatography with a mixed solvent of water and an alcohol selected from methanol or ethanol. was sequentially eluted with increasing alcohol concentration, and the molecular weight of each resulting fraction was measured to obtain an extracted fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more.
極性の低い有機溶媒の例としては、ヘキサン、ジエチルエーテル、酢酸エチル等が挙げられ、極性の高い有機溶媒の例としては、n-ブタノール、エタノール、メタノール、水等が挙げられる。分画に用いるカラムクロマトグラフィーでは、Diaion HP-20等のスチレン-ジビニルベンゼン系合成吸着剤あるいはその他の芳香族系合成吸着剤を用いることができる。各分画の分子量測定は、粘度法、ゲル浸透クロマトグラフィー(GPC)、質量分析
法、浸透圧法等を用いることができる。本願実施例では、GPC用標準ポリスチレンを標準
物質とするGPCにより重量平均分子量を求めた。
Examples of organic solvents with low polarity include hexane, diethyl ether, and ethyl acetate, and examples of organic solvents with high polarity include n-butanol, ethanol, methanol, water, and the like. In column chromatography used for fractionation, styrene-divinylbenzene synthetic adsorbents such as Diaion HP-20 or other aromatic synthetic adsorbents can be used. For measuring the molecular weight of each fraction, viscosity method, gel permeation chromatography (GPC), mass spectrometry, osmotic pressure method and the like can be used. In the examples of the present application, the weight-average molecular weight was determined by GPC using standard polystyrene for GPC as a standard substance.
具体的には、麻黄エキス又はEFEを水で溶解したものを、酢酸エチル、水飽和n-ブタノ
ールで順次分配し、酢酸エチルエキス、n-ブタノールエキス、及び水エキスの各分配エキスを得る。得られた各分配エキスのうち、水エキスに活性が認められたので、活性の認められた水エキスについて、さらにDiaion HP-20カラムクロマトグラフィー(5.5 x 40 cm
、三菱化学)を用いて、H2O→20%MeOH→40%MeOH→MeOHの順で順次溶出して分画し、H2O分画物、20%MeOH分画物、40%MeOH分画物、MeOH分画物の各分画物を得る。各分画物の活性を測定した結果、20%MeOH分画物、40%MeOH分画物、MeOH分画物にc-Metキナーゼ阻害作用、
リン酸化阻害作用、発現量の低下作用、がん細胞の運動能抑制作用、EGFRのリン酸化阻害作用、発現量の低下作用、疼痛抑制作用、抗インフルエンザウイルス作用のあることがわかった。また、20%MeOH分画物、40%MeOH分画物、MeOH分画物は、重量平均分子量45,000以上の高分子縮合型タンニンであった。
Specifically, Ephedra huang extract or EFE dissolved in water is sequentially partitioned with ethyl acetate and water-saturated n-butanol to obtain partitioned extracts of ethyl acetate extract, n-butanol extract and water extract. Among the obtained partitioned extracts, the water extract was found to be active.
, Mitsubishi Chemical) was used to fractionate by sequentially eluting with H 2 O → 20% MeOH → 40% MeOH → MeOH in that order, H 2 O fraction, 20% MeOH fraction, 40% MeOH fraction A fraction, a MeOH fraction, are obtained. As a result of measuring the activity of each fraction, 20% MeOH fraction, 40% MeOH fraction, c-Met kinase inhibitory effect on MeOH fraction,
It was found to have phosphorylation inhibitory action, expression level reduction action, cancer cell motility suppression action, EGFR phosphorylation inhibitory action, expression level reduction action, pain suppression action, and anti-influenza virus action. In addition, the 20% MeOH fraction, 40% MeOH fraction and MeOH fraction were polymer condensed tannins with a weight average molecular weight of 45,000 or more.
したがって、本願発明の抽出分画物は、20%MeOH分画物、40%MeOH分画物、MeOH分画物を単独で、又は任意の組合せで用いることにより調製できることが明らかとなった。これらの結果から、効率のよい調製方法としては、上述する水エキスを100%MeOHで抽出して、100%MeOH分画物をさらにSephadex LH-20カラム等にかけて高分子画分を得ることで調製することも可能である。 Therefore, it was clarified that the extract fraction of the present invention can be prepared by using 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction alone or in any combination. Based on these results, as an efficient preparation method, the above water extract is extracted with 100% MeOH, and the 100% MeOH fraction is further applied to a Sephadex LH-20 column, etc. to obtain a high molecular weight fraction. It is also possible to
本発明は別の一態様として、上記高分子縮合型タンニンを含有する抽出分画物を含む医薬組成物を提供する。本発明の医薬組成物は、後述する実施例に示すように、疼痛抑制薬、抗がん・抗転移薬、及び抗インフルエンザウイルス薬として有用である。また、本発明の医薬組成物は、麻黄エキスを原料として用いた場合も、EFEを原料として用いた場合も
、同様に有効な薬理作用を示す。
Another aspect of the present invention provides a pharmaceutical composition containing an extract fraction containing the polymer condensed tannin. The pharmaceutical composition of the present invention is useful as a pain suppressant, an anti-cancer/anti-metastatic drug, and an anti-influenza virus drug, as shown in the examples below. In addition, the pharmaceutical composition of the present invention exhibits a similar effective pharmacological effect whether Ephedra extract is used as a raw material or EFE is used as a raw material.
本発明にかかる疼痛抑制薬、抗がん・抗転移薬、抗インフルエンザウイルス薬は、例えば、錠剤、顆粒剤、細粒剤、カプセル剤等のような経口投与の製剤、経口投与に適した様々な液体製剤、又は注射剤のような非経口投与用製剤とすることが可能である。経口投与が好ましい。 The pain suppressant, anti-cancer/anti-metastatic drug, and anti-influenza virus drug according to the present invention are, for example, formulations for oral administration such as tablets, granules, fine granules, capsules, etc., and various formulations suitable for oral administration. It can be a simple liquid formulation, or a formulation for parenteral administration such as an injection. Oral administration is preferred.
経口投与の製剤の場合、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出
分画物を製剤担体と共に錠剤、顆粒剤、細粒剤、カプセル剤等の形態で製剤化して得られる。
In the case of preparations for oral administration, extract fractions containing polymer condensed tannins with a weight-average molecular weight of 45,000 or more are formulated together with pharmaceutical carriers in the form of tablets, granules, fine granules, capsules, and the like.
製剤担体としては、賦形剤、結合剤、崩壊剤、滑沢剤、及び可塑剤等を使用できる。賦形剤としては、例えば、白糖、塩化ナトリウム、マンニトール、乳糖、ブドウ糖、でんぷん、炭酸カルシウム等を使用できる。結合剤としては、例えば、水、エタノール、プロパノール、ブドウ糖液、デンプン液、ゼラチン液、等を使用できる。崩壊剤としては、例えば、カルボキシメチルセルロースカルシウム、乾燥デンプン、炭酸水素ナトリウム、等を使用できる。滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ酸末、ポリエチレングリコール等を使用できる。可塑剤としては、例えば、グリセリン脂肪酸エステル、ジオクチルフタレート、ジブチルフタレート、ヒマシ油等を使用できる。 Excipients, binders, disintegrants, lubricants, plasticizers and the like can be used as pharmaceutical carriers. Examples of excipients that can be used include sucrose, sodium chloride, mannitol, lactose, glucose, starch, calcium carbonate, and the like. Examples of binders that can be used include water, ethanol, propanol, glucose solution, starch solution, gelatin solution, and the like. Examples of disintegrants that can be used include carboxymethylcellulose calcium, dry starch, sodium bicarbonate, and the like. Examples of lubricants that can be used include purified talc, stearate, boric acid powder, and polyethylene glycol. Examples of plasticizers that can be used include glycerin fatty acid esters, dioctyl phthalate, dibutyl phthalate, and castor oil.
上記の経口投与の製剤化の場合、水溶性高分子及び界面活性剤等の分散剤を使用することも可能である。水溶性高分子としては、例えば、ヒドロキシプロピルセルロース、ポリビニルアルコール、ポリビニルピロリドン等を使用できる。界面活性剤としては、例えば、ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム等を使用できる。 Dispersing agents such as water-soluble polymers and surfactants can be used in the above formulations for oral administration. Examples of water-soluble polymers that can be used include hydroxypropylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone. Examples of surfactants that can be used include sodium lauryl sulfate and magnesium lauryl sulfate.
錠剤を調製するには、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を、上記製剤担体を用いて常法により錠剤とする。顆粒剤又は細粒剤は、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物に上記製剤担体を添加し、流動層造粒、高速攪拌造粒、攪拌流動層造粒、遠心流動造粒、押し出し造粒等で顆粒化することにより調製できる。カプセル剤は、不活性な医薬充填剤又は希釈剤と共に混合して調製し、硬ゼラチンカプセル又は軟カプセルに詰める。 To prepare tablets, the extract fraction containing polymer condensed tannins with a weight-average molecular weight of 45,000 or more is made into tablets by a conventional method using the above formulation carriers. Granules or fine granules are produced by adding the above formulation carriers to the extracted fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more, followed by fluid bed granulation, high-speed stirring granulation, stirring fluid bed granulation, and centrifugation. It can be prepared by granulation by fluid granulation, extrusion granulation, or the like. Capsules are prepared by mixing with an inert pharmaceutical filler or diluent and filled into hard gelatin capsules or soft capsules.
経口液体製剤は、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物と、甘味料(例えば、ショ糖)、保存剤(例えば、メチルパラベン、プロピルパラベン)、着色料、香料等とを混合して調製する。 Oral liquid preparations contain extract fractions containing high molecular weight condensed tannins with a weight average molecular weight of 45,000 or more, sweeteners (e.g. sucrose), preservatives (e.g. methylparaben, propylparaben), colorants, fragrances, etc. Prepared by mixing
注射用製剤は、例えば、液剤、乳濁液、又は懸濁液の形態で調製され、血液に対して等張にされる。液体、乳濁液又は懸濁液の形態の製剤は、例えば、水性媒体、エチルアルコール、プロピレングリコール、エトキシ化イソステアリルアルコール等を用いて調製される。 Injectable formulations are prepared, for example, in the form of solutions, emulsions, or suspensions and are made isotonic with blood. Liquid, emulsion or suspension form formulations are prepared using, for example, aqueous media, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, and the like.
注射用製剤において、当技術分野で通常使用されている添加剤を適宜用いることができ、例えば、等張化剤、安定化剤、緩衝剤、保存剤、キレート剤、抗酸化剤等を使用できる。等張化剤としては、例えば、ブドウ糖、ソルビトール、マンニトール等が挙げられる。安定化剤としては、例えば亜硫酸ナトリウム等が挙げられる。緩衝剤としては、例えば、ホウ酸緩衝剤、リン酸緩衝剤、クエン酸緩衝剤等が挙げられる。保存剤としては、例えば、パラオキシ安息香酸エステル、ベンジルアルコール等が挙げられる。キレート剤としては、例えば、エデト酸ナトリウム、クエン酸ナトリウム等が挙げられる。抗酸化剤としては、例えば、亜硫酸ナトリウム、亜硫酸水素ナトリウム等が挙げられる。 Additives commonly used in the art for injection preparations can be used as appropriate. For example, tonicity agents, stabilizers, buffers, preservatives, chelating agents, antioxidants and the like can be used. . Tonicity agents include, for example, glucose, sorbitol, mannitol and the like. Examples of stabilizers include sodium sulfite and the like. Buffers include, for example, borate buffers, phosphate buffers, citrate buffers and the like. Preservatives include, for example, p-oxybenzoate, benzyl alcohol and the like. Chelating agents include, for example, sodium edetate, sodium citrate and the like. Examples of antioxidants include sodium sulfite, sodium hydrogen sulfite and the like.
本発明の疼痛抑制薬、抗がん・抗転移薬、抗インフルエンザウイルス薬の投与量としては、例えば、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を、一回の投与につき体重1 kgあたり0.5mgから500mg、好ましくは10mgから200 mgの範囲で投与量が選択できる。しかしながら、本発明の医薬組成物はこれらの投与量に制限されるものではない。 The dose of the pain suppressant, anticancer/anti-metastatic drug, and anti-influenza virus drug of the present invention is, for example, a single administration of an extract fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more. The dosage can be selected in the range of 0.5 mg to 500 mg, preferably 10 mg to 200 mg, per 1 kg of body weight. However, the pharmaceutical composition of the present invention is not limited to these doses.
さらに本発明は別の一態様として、本発明の抽出分画物を用いる、高分子縮合型タンニンを活性成分とする医薬組成物の管理方法のひとつとして含量測定法を提供する。具体的
には、本発明で示した高分子縮合型タンニンの分子量の解析に用いたGPC分析を用いて、
含量未知の試料及び高分子縮合型タンニン標準物質について、それぞれ高分子縮合型タンニンのピーク面積を求め、ピーク面積の相対値から試料中の高分子縮合型タンニン含量を算出することができる。また、この含量測定法は、麻黄エキスやEFE中の高分子縮合型タ
ンニン含量を測定することも可能で、高分子縮合型タンニンが活性成分とする薬効、すなわち、疼痛抑制薬、抗がん・抗転移薬、及び抗インフルエンザウイルス薬として製造する際の管理方法のひとつとして用いることができる。
Furthermore, as another aspect of the present invention, there is provided a content measurement method using the extract fraction of the present invention as one of the control methods for a pharmaceutical composition containing polymer condensed tannin as an active ingredient. Specifically, using the GPC analysis used to analyze the molecular weight of the polymer condensed tannin shown in the present invention,
The peak areas of polymer condensed tannins are obtained for each of a sample of unknown content and a standard substance of polymer condensed tannins, and the content of polymer condensed tannins in the sample can be calculated from the relative values of the peak areas. In addition, this content measurement method can also measure the content of polymer condensed tannins in mahuang extract and EFE. It can be used as one of the management methods when manufacturing anti-metastatic drugs and anti-influenza virus drugs.
本発明を実施例によりさらに詳しく説明するが、本発明はこれに限定されない。種々の変更、修飾が当業者には可能であり、これらの変更、修飾も本発明に含まれる。
(実施例1)麻黄エキス又はEFEの分画、分析
麻黄エキス(300 g)を水(5 L)で溶解したものを、n-ヘキサン(4 L)、酢酸エチル
(12 L)、水飽和n-ブタノール(12 L)で順次分配し、n-ヘキサンエキス(92.3 mg)、
酢酸エチルエキス(13.2 g)、n-ブタノールエキス(65.7 g)、水エキス(161.1 g)を
得た。麻黄エキス由来の得られた各分配エキスにつき、c-Metキナーゼ阻害活性、がん細
胞の運動抑制活性を求めた。その結果、n-ブタノール及び水エキスに活性が認められた。
The present invention will be described in more detail by examples, but the present invention is not limited to these. Various changes and modifications are possible for those skilled in the art, and these changes and modifications are also included in the present invention.
(Example 1) Fractionation and analysis of Ephedra extract or EFE Ephedra extract (300 g) was dissolved in water (5 L), n-hexane (4 L), ethyl acetate (12 L), water saturated n -butanol (12 L), n-hexane extract (92.3 mg),
Ethyl acetate extract (13.2 g), n-butanol extract (65.7 g) and water extract (161.1 g) were obtained. The c-Met kinase inhibitory activity and cancer cell motility inhibitory activity were determined for each partitioned extract derived from the Ephedra extract. As a result, n-butanol and water extracts were found to be active.
収量が多く、活性の認められた水エキス(90 g)について、さらにDiaion HP-20カラムクロマトグラフィー(5.5 x 40 cm、三菱化学)を用いて、H2O→10%MeOH→20%MeOH→30%MeOH→40%MeOH→50%MeOH→MeOHの順で順次溶出して分画し、H2O分画物(47.9 g)、10%MeOH分画物(2.5 g)、20%MeOH分画物(2.6 g)、30%MeOH分画物(7.0 g)、40%MeOH分画物
(11.5 g)、50%MeOH分画物(5.1 g)、MeOH分画物(2.6 g)の各分画物を得た。得られ
た各分画物について、c-Metキナーゼ阻害活性、がん細胞の運動抑制活性試験を行った結
果、特に30%MeOH分画物、40%MeOH分画物、次いで50%MeOH分画物に強い活性が認められた
。
The water extract (90 g), which yielded a large amount and was found to be active, was further subjected to a Diaion HP-20 column chromatography (5.5 x 40 cm, Mitsubishi Chemical) for H 2 O → 10% MeOH → 20% MeOH → Fractions were obtained by sequentially eluting with 30% MeOH → 40% MeOH → 50% MeOH → MeOH, and the H 2 O fraction (47.9 g), 10% MeOH fraction (2.5 g), 20% MeOH fraction fraction (2.6 g), 30% MeOH fraction (7.0 g), 40% MeOH fraction (11.5 g), 50% MeOH fraction (5.1 g), MeOH fraction (2.6 g). A fraction was obtained. As a result of conducting c-Met kinase inhibitory activity and cancer cell motility inhibitory activity tests for each of the obtained fractions, 30% MeOH fraction, 40% MeOH fraction, and then 50% MeOH fraction A strong activity was observed in the substance.
そこで、水エキスの各分画物の含有成分について比較する目的で、HPLC分析を行った。HPLC条件は以下の通りである。 Therefore, HPLC analysis was performed for the purpose of comparing the components contained in each fraction of the water extract. HPLC conditions are as follows.
カラム:L-column ODS(2.1 I.D. x 150 mm)(化学物質評価研究機構)、
カラム温度:40℃、
流速:0.3 mL/min、
測定波長:200-400 nm、
移動相:(A)0.1%ギ酸‐蒸留水及び(B)0.1%ギ酸‐アセトニトリル〔濃度勾配条件(B in A):0→30 min(0→50%)、30→35 min(50→85%)、35→40 min(85%)、40→50 min(85→90%)、50→55 min(90→100%)、55→60 min(100%)〕。
Column: L-column ODS (2.1 ID x 150 mm) (National Institute for Chemical Evaluation and Research),
Column temperature: 40°C,
Flow rate: 0.3mL/min,
Measurement wavelength: 200-400 nm,
Mobile phase: (A) 0.1% formic acid-distilled water and (B) 0.1% formic acid-acetonitrile [Concentration gradient conditions (B in A): 0 → 30 min (0 → 50%), 30 → 35 min (50 → 85 %), 35→40 min (85%), 40→50 min (85→90%), 50→55 min (90→100%), 55→60 min (100%)].
その結果、H2O分画物及び10%MeOH分画物以外の画分に縮合型タンニンオリゴマーに特徴的なブロードピークが観察された(図2)。 As a result, broad peaks characteristic of condensed tannin oligomers were observed in fractions other than the H 2 O fraction and the 10% MeOH fraction (Fig. 2).
また、各分画物について13C-NMRによる構造解析を行った。NMRの条件は、測定溶媒としてMeOH-d4:D2O(1:1)を用い、装置はBrucker AVANCE500 (Bruker BioSpin)を用いた。その結果、H2O分画物及び10%MeOH分画物以外の画分にフラバン 3-オール骨格に対応するシグ
ナルが共通して観察され、活性に寄与している画分は縮合型タンニン(プロアントシアニジン)オリゴマー画分であることが確認された。強い活性を示した30%MeOH分画物、40%MeOH分画物、50%MeOH分画物は特に強いシグナルを認めた。また、いずれの分画物も、スペ
クトル解析に基づき、プロアントシアニジンAタイプを含む構造を有すること、さらにカ
テコールタイプのシグナル強度が小さいことから、ピロガロールタイプが多いことが示唆された(図3)。
Structural analysis was also performed on each fraction by 13 C-NMR. As for the NMR conditions, MeOH-d 4 :D 2 O (1:1) was used as a measurement solvent, and Bruker AVANCE500 (Bruker BioSpin) was used as an apparatus. As a result, a signal corresponding to the flavan 3-ol skeleton was commonly observed in fractions other than the H 2 O fraction and the 10% MeOH fraction, and the fraction contributing to the activity was condensed tannin ( proanthocyanidin) oligomer fraction. A particularly strong signal was observed for the 30% MeOH fraction, 40% MeOH fraction, and 50% MeOH fraction, which showed strong activity. In addition, based on spectral analysis, all fractions had structures containing proanthocyanidin A type, and the signal intensity of catechol type was low, suggesting that pyrogallol type was abundant (Fig. 3).
同様に、EFE(400 g)を水(5 L)で溶解したものを、酢酸エチル(10 L)、水飽和n-
ブタノール(10 L)で順次分配し、酢酸エチルエキス(7.3 g)、n-ブタノールエキス(85.0 g)、水エキス(311.0 g)を得た。EFE由来の得られた各分配エキスにつき、疼痛試
験を行った。その結果、水エキスに活性が認められた。活性の認められた水エキス(290 g)について、さらにDiaion HP-20カラムクロマトグラフィー(5.5 x 40 cm、三菱化学)を用いて、H2O→20%MeOH→40%MeOH→MeOHの順で順次溶出して分画し、H2O分画物(190.6 g)、20%MeOH分画物(35.9 g)、40%MeOH分画物(41.7 g)、MeOH分画物(13.9 g)の各
分画物を得た。得られた各分画物について、疼痛試験を行った結果、20%MeOH分画物、40%MeOH分画物,MeOH分画物に活性が認められた。
Similarly, EFE (400 g) was dissolved in water (5 L), ethyl acetate (10 L), water saturated n-
It was partitioned sequentially with butanol (10 L) to obtain ethyl acetate extract (7.3 g), n-butanol extract (85.0 g) and water extract (311.0 g). A pain test was performed on each resulting partitioned extract from EFE. As a result, activity was recognized in the water extract. The active water extract (290 g) was further analyzed using Diaion HP-20 column chromatography (5.5 x 40 cm, Mitsubishi Chemical) in the order of H 2 O → 20% MeOH → 40% MeOH → MeOH. Sequentially eluted and fractionated, H 2 O fraction (190.6 g), 20% MeOH fraction (35.9 g), 40% MeOH fraction (41.7 g), MeOH fraction (13.9 g). Each fraction was obtained. A pain test was performed on each of the obtained fractions, and activity was observed in 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction.
そこで、水エキスの各分画物の含有成分について比較する目的で、同様にHPLC分析を行った。その結果、活性の認められた20%MeOH分画物、40%MeOH分画物,MeOH分画物に縮合型タンニンオリゴマーに特徴的なブロードピークが観察された(図4)。 Therefore, the same HPLC analysis was performed for the purpose of comparing the components contained in each fraction of the water extract. As a result, broad peaks characteristic of condensed tannin oligomers were observed in 20% MeOH fractions, 40% MeOH fractions, and MeOH fractions in which activity was recognized (Fig. 4).
また、各分画物について13C-NMRによる構造解析を行った結果、活性の認められた20%MeOH分画物、40%MeOH分画物,MeOH分画物にフラバン 3-オール骨格に対応するシグナルが共通して観察され、麻黄エキス由来の活性画分と同様の縮合型タンニン(プロアントシアニジン)オリゴマー画分であることが確認された(図5)。 Structural analysis of each fraction by 13 C-NMR revealed activity in 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction corresponding to flavan 3-ol skeleton. A signal was commonly observed, confirming that it was a condensed tannin (proanthocyanidin) oligomer fraction similar to the Ephedra extract-derived active fraction (Fig. 5).
なお、本実施例におけるc-Metキナーゼ阻害活性は実施例5及び7に記載の方法、がん細
胞の運動抑制活性は実施例6及び8に記載の方法、疼痛試験は実施例9に記載の方法に準じ
て行った。
(実施例2)活性画分の分子量分析
活性に寄与している画分が縮合型タンニン(プロアントシアニジン)オリゴマー画分であることが確認されたため、さらに活性画分を限外濾過により分子量による分画を行った。麻黄エキス由来の水エキス-40%MeOH分画物の20%MeOH不溶部を、Vivaspin 500 (GE Healthcare: 3、5、10、30、50、100 KDa)を用いて、11400 x g、10分間遠心分離し、7画分〔<3 KDa (収量約10%)、3~5 KDa(約4%)、5~10 KDa(約5%)、10~30 KDa(約6%)、30
~50 KDa(約2%)、50~100 KDa(約3%)、>100 KDa(約70%)〕を得た。各画分について、c-Metキナーゼ阻害作用の試験を行った結果、>100 KDaの高分子画分(麻黄エキス高分
子画分)に活性が認められた。この高分子画分について13C-NMRによる構造解析を行った
結果、同様の縮合型タンニン(プロアントシアニジン)オリゴマー画分であることが確認された。なお、水エキス-30%MeOH分画物の>100 KDaの高分子画分についても13C-NMR(測
定溶媒としてacetone-d6:D2O(1:1))による構造解析を行った結果、同様のスペクトルが
観察された(図6)。
In this example, the c-Met kinase inhibitory activity was determined by the method described in Examples 5 and 7, the cancer cell motility inhibitory activity was determined by the method described in Examples 6 and 8, and the pain test was performed by the method described in Example 9. I followed the method.
(Example 2) Molecular weight analysis of active fraction Since it was confirmed that the fraction contributing to the activity was the condensed tannin (proanthocyanidin) oligomer fraction, the active fraction was further subjected to molecular weight separation by ultrafiltration. did the painting. The 20% MeOH-insoluble part of the water extract-40% MeOH fraction derived from mahuang extract was centrifuged at 11400 xg for 10 minutes using Vivaspin 500 (GE Healthcare: 3, 5, 10, 30, 50, 100 KDa). 7 fractions [<3 KDa (approximately 10% yield), 3-5 KDa (approximately 4%), 5-10 KDa (approximately 5%), 10-30 KDa (approximately 6%), 30
~50 KDa (~2%), 50-100 KDa (~3%), >100 KDa (~70%)]. Each fraction was tested for c-Met kinase inhibitory activity, and as a result, activity was observed in the high-molecular-weight fraction of >100 KDa (Mahuang extract high-molecular-weight fraction). Structural analysis of this polymer fraction by 13 C-NMR confirmed that it was a similar condensed tannin (proanthocyanidin) oligomer fraction. Structural analysis was also performed on the >100 KDa polymer fraction of the water extract-30% MeOH fraction by 13 C-NMR (acetone-d 6 :D 2 O (1:1) as the measurement solvent). As a result, similar spectra were observed (Fig. 6).
活性画分(麻黄エキス高分子画分、EFE由来水エキス-20%MeOH、40%MeOH、MeOH分画物)についてGPC分析を行った。GPC用標準ポリスチレンを標準物質として、以下の条件で分析した。 GPC analysis was performed on the active fractions (Ephedra extract high-molecular weight fraction, EFE-derived water extract-20% MeOH, 40% MeOH, MeOH fractions). Using standard polystyrene for GPC as a standard substance, analysis was performed under the following conditions.
カラム:TSK-gel Super AW4000(6.0 I.D. x 150 mm)(東ソー)、
カラム温度:30℃、
流速:0.3 mL/min、
測定波長:280 nm、
移動相:N,N-ジメチルホルムアミド+3M ギ酸アンモニウム(0.5%)
分析した結果、いずれの分画物においても、保持時間7.5~8.5分にピークトップを示す高分子領域のピークが観察された(図7)。Chromato-PRO-GPCを使用してデータ処理を行い、各分画物の重量平均分子量を算出した結果、麻黄エキス高分子画分:94372、20%MeOH分画物:113279、40%MeOH分画物:59042、MeOH分画物:45004(GPC用標準ポリスチレンを
標準物質として換算した重量平均分子量で、いずれも3回測定した値の平均値)であった
。
(実施例3)部分構造の解析
活性画分の一つである麻黄エキス由来水エキス-40%MeOH分画物について、フロログルシノール分解を行った。その結果、catechin、epicatechin、gallocatechin、epicatechin-(4→2)-phloroglucinol、gallocatechin-(4→2)-phloroglucinol、prodelphinidin A-type-(4′→2)-phloroglucinolを分解生成物として確認した(図8)。
Column: TSK-gel Super AW4000 (6.0 ID x 150 mm) (Tosoh),
Column temperature: 30°C,
Flow rate: 0.3mL/min,
Measurement wavelength: 280 nm,
Mobile phase: N,N-dimethylformamide + 3M ammonium formate (0.5%)
As a result of the analysis, a high-molecular region peak showing a peak top at a retention time of 7.5 to 8.5 minutes was observed in all fractions (Fig. 7). Data processing was performed using Chromato-PRO-GPC, and the weight average molecular weight of each fraction was calculated. substance: 59042, MeOH fraction: 45004 (weight-average molecular weight converted using standard polystyrene for GPC as a standard substance, average value of values measured three times).
(Example 3) Analysis of partial structure A water extract-40% MeOH fraction derived from Ephedra extract, which is one of the active fractions, was subjected to phloroglucinol decomposition. As a result, catechin, epicatechin, gallocatechin, epicatechin-(4→2)-phloroglucinol, gallocatechin-(4→2)-phloroglucinol, prodelphinidin A-type-(4′→2)-phloroglucinol were confirmed as degradation products ( Figure 8).
よって、エクステンションユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールが主にBタイプで縮合し、その一部にAタイプの縮合型タンニンユニ
ットを含み、さらにターミナルユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールを主に含むことが明らかとなった。また、ピロガロールタイプと
カテコールタイプのおおよそ存在比をチオール分解したユニットをHPLC分析で算出した結果、おおよそ5:1であった。(図1参照)
(実施例4)EFE由来の抽出分画物中の高分子縮合型タンニンの含有量
縮合型タンニンオリゴマー画分は、Sephadex LH-20カラムクロマトグラフィーで含水アセトン(アセトン-水(7:3))溶出により分画される報告がある(非特許文献14)。本方法に従い、70%アセトン(アセトン-水(7:3))溶出部を高分子縮合型タンニン画分とし
、活性画分(EFE由来水エキス-20%MeOH、40%MeOH、MeOH分画物)中の高分子縮合型タンニン量を見積もった。その結果、20%MeOH分画物に約32%、40%MeOH分画物に約45%、MeOH分画物に約43%であった。
Therefore, as extension units, catechol-type or pyrogallol-type flavan 3-ols are mainly condensed in B-type, and some of them contain A-type condensed-type tannin units. It was found to contain mainly flavan 3-ols. In addition, as a result of calculating the approximate abundance ratio of the pyrogallol type and the catechol type by HPLC analysis of the thiol-decomposed units, it was approximately 5:1. (See Figure 1)
(Example 4) Content of high-molecular-weight condensed tannin in EFE-derived extract fraction There is a report of fractionation by elution (Non-Patent Document 14). According to this method, the 70% acetone (acetone-water (7:3)) elution part is the polymer condensed tannin fraction, and the active fraction (EFE-derived water extract-20% MeOH, 40% MeOH, MeOH fraction ) was estimated. The results were about 32% for the 20% MeOH fraction, about 45% for the 40% MeOH fraction, and about 43% for the MeOH fraction.
(実施例5) 麻黄エキス分配エキスのc-Metキナーゼ阻害作用
麻黄エキスを水で溶解したものをn-ヘキサン、酢酸エチル、水飽和n-ブタノールで順次分配し、得られた各分画物(n-ヘキサンエキス、酢酸エチルエキス、n-ブタノールエキス、水エキス)のc-Metキナーゼ阻害作用をc-Metのキナーゼドメインの組換えタンパク質と合成基質ペプチドを用いたインビトロアッセイにより調べた。
(Example 5) c-Met kinase inhibitory action of partitioned Ephedra extract Ephedra extract dissolved in water was sequentially partitioned with n-hexane, ethyl acetate, and water-saturated n-butanol, and each fraction obtained ( n-hexane extract, ethyl acetate extract, n-butanol extract, and water extract) were investigated by an in vitro assay using a recombinant protein of the kinase domain of c-Met and a synthetic substrate peptide.
アッセイ条件は、以下の通りである。40 mM Tris-HCl pH7.5、2 mM DTT、7 mM MgCl2、10 μM ATP に4 nM 組換えc-Metキナーゼドメイン、0.2 μg/mL Poly (Glu4,Tyr1)及び各分画を5 μg/mLとなる溶液を調製し、室温で1時間インキュベートした。反応停止後、ADP-Glo試薬を用いてADP量に依存したルミノール発光強度をルミノメーター (パーキンエル
マー社、EnSpire)で測定し、c-Metキナーゼ活性を算出した。その結果、n-ブタノールエ
キス及び水エキスにキナーゼ阻害作用があることが明らかになった(図9)。
The assay conditions are as follows. 4 nM recombinant c-Met kinase domain in 40 mM Tris-HCl pH 7.5, 2 mM DTT, 7 mM MgCl 2 , 10 μM ATP, 0.2 μg/mL Poly (Glu4, Tyr1) and 5 μg/ml of each fraction. A mL solution was prepared and incubated for 1 hour at room temperature. After stopping the reaction, the ADP-Glo reagent was used to measure the ADP amount-dependent luminol emission intensity with a luminometer (PerkinElmer, EnSpire) to calculate the c-Met kinase activity. As a result, it was revealed that the n-butanol extract and the water extract have a kinase inhibitory effect (Fig. 9).
(実施例6) 麻黄エキスの分配エキスのがん細胞の運動能阻害作用
麻黄エキスを水に溶解したものをn-ヘキサン、酢酸エチル、水飽和n-ブタノールで順次分配し、得られた各分画物(n-ヘキサンエキス、酢酸エチルエキス、n-ブタノールエキス、水エキス)、がん細胞の運動能阻害作用を、トランスウエルを用いた試験により評価した。アッセイには、c-Metを発現しており、HGFによって運動能が誘導される高転移性ヒト乳がん細胞株MDA-MB-231細胞を用いた。
(Example 6) Cancer cell motility inhibitory effect of partitioned extract of Ephedra extract The extracts (n-hexane extract, ethyl acetate extract, n-butanol extract, water extract) and cancer cell motility inhibitory effects were evaluated by transwell tests. The assay used MDA-MB-231 cells, a highly metastatic human breast cancer cell line that expresses c-Met and whose motility is induced by HGF.
アッセイ条件は、以下の通りである。MDA-MB-231細胞(5x104細胞/ウエル)をVehicle (2.5%EtOH-DMEM)あるいは、25 μg/mLの各分配エキスを溶解した2.5%EtOH-DMEMに懸濁し
、上部ウエルに添加した。下部ウエルはコラーゲンコートした24ウエルプレートを用いた。下部ウエルには、50 ng/mL HGF-DMEMを600 μL添加した。24時間培養後に、下部ウエルに移動した細胞数を顕微鏡下に測定して、運動能の指標とした。その結果、n-ブタノールエキス、水エキスにがん細胞の運動能抑制作用があることが明らかになった(図10)。(実施例7)水エキスのDiaion HP-20カラム分画物のc-Metキナーゼ阻害作用
実施例9の疼痛試験の結果から、経口投与により薬理作用を示す分配エキスは水エキス
であり、n-ブタノールエキスの経口投与は薬理作用を示さないことがわかった。そこで、麻黄エキスの分配で得られた水エキスをDiaion HP-20カラムに添加し、H2O、10%MeOH、20%MeOH、30%MeOH、40%MeOH、50%MeOH、MeOHで順次溶出した分画物を得た。各分画物についてc-Metのキナーゼドメインの組換えタンパク質と合成基質ペプチドを用いたインビトロ
アッセイにより調べた。
The assay conditions are as follows. MDA-MB-231 cells (5×10 4 cells/well) were suspended in Vehicle (2.5% EtOH-DMEM) or 2.5% EtOH-DMEM in which 25 μg/mL of each partitioned extract was dissolved, and added to the upper wells. A collagen-coated 24-well plate was used for the lower wells. 600 μL of 50 ng/mL HGF-DMEM was added to the lower wells. After culturing for 24 hours, the number of cells that migrated to the lower well was measured under a microscope and used as an index of motility. As a result, it was revealed that the n-butanol extract and the water extract have an inhibitory effect on the motility of cancer cells (Fig. 10). (Example 7) c-Met kinase inhibitory effect of Diaion HP-20 column fraction of water extract
From the results of the pain test in Example 9, it was found that the distributed extract showing pharmacological action by oral administration was the water extract, and the n-butanol extract did not show pharmacological action by oral administration. Therefore, the water extract obtained by partitioning the Ephedra extract was added to a Diaion HP-20 column and eluted sequentially with H 2 O, 10%MeOH, 20%MeOH, 30%MeOH, 40%MeOH, 50%MeOH, and MeOH. A fine fraction was obtained. Each fraction was examined by an in vitro assay using a recombinant protein of the c-Met kinase domain and a synthetic substrate peptide.
アッセイ条件は、以下の通りである。40 mM Tris-HCl pH7.5、2 mM DTT、7 mM MgCl2、10 μM ATP に4 nM 組換えc-Metキナーゼドメイン、0.2 μg/mL Poly (Glu4,Tyr1)及び各分画を5 μg/mLとなる溶液を調製し、室温で1時間インキュベートした。反応停止後、ADP-Glo試薬を用いてADP量に依存したルミノール発光強度をルミノメーター (パーキンエル
マー社、EnSpire)で測定し、c-Metキナーゼ活性を算出した。その結果、Diaion HP-20カ
ラムの20%MeOH分画物、30%MeOH分画物、40%MeOH分画物、50%MeOH分画物及びMeOH分画物にc-Metキナーゼ阻害作用があることが明らかになった(図11)。
(実施例8)水エキスのDiaion HP-20カラム分画物のがん細胞の運動能抑制作用
実施例9の疼痛試験の結果から、経口投与により薬理作用を示す分配エキスは水エキスであり、n-ブタノールエキスの経口投与は薬理作用を示さないことがわかった。そこで、麻黄エキスの分配で得られた水エキスをDiaion HP-20カラムに添加し、H2O、10%MeOH、20%MeOH、30%MeOH、40%MeOH、50%MeOH、MeOHで順次溶出した画分を得た。各画分についてHGFによって誘導されるがん細胞の運動能の抑制作用を解析した。
The assay conditions are as follows. 4 nM recombinant c-Met kinase domain in 40 mM Tris-HCl pH 7.5, 2 mM DTT, 7 mM MgCl 2 , 10 μM ATP, 0.2 μg/mL Poly (Glu4, Tyr1) and 5 μg/ml of each fraction. A mL solution was prepared and incubated for 1 hour at room temperature. After stopping the reaction, the ADP-Glo reagent was used to measure the ADP amount-dependent luminol emission intensity with a luminometer (PerkinElmer, EnSpire) to calculate the c-Met kinase activity. As a result, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, 50% MeOH fraction and MeOH fraction of Diaion HP-20 column have c-Met kinase inhibitory activity. (Fig. 11).
(Example 8) Diaion HP-20 column fraction of water extract inhibits motility of cancer cells
From the results of the pain test in Example 9, it was found that the distributed extract showing pharmacological action by oral administration was the water extract, and the n-butanol extract did not show pharmacological action by oral administration. Therefore, the water extract obtained by partitioning the Ephedra extract was added to a Diaion HP-20 column and eluted sequentially with H 2 O, 10%MeOH, 20%MeOH, 30%MeOH, 40%MeOH, 50%MeOH, and MeOH. A fraction was obtained. Each fraction was analyzed for its inhibitory effect on cancer cell motility induced by HGF.
アッセイ条件は、以下の通りである。MDA-MB-231細胞(5x104細胞/ウエル)をVehicle (DMEM)あるいは、10 μg/mLの各画分を溶解したDMEMに懸濁し、上部ウエルに添加した。
下部ウエルはコラーゲンコートした24ウエルプレートを用いた。下部ウエルには、50 ng/mL HGF-DMEMを600 μL添加した。24時間培養後に、下部ウエルに移動した細胞数を顕微鏡下に測定して、運動能の指標とした。その結果、30%MeOH分画物、40%MeOH分画物、及び、50%MeOH分画物は、HGFにより誘導される運動能を強く抑制することが明らかになった(図12)。
(実施例9)マウスを用いたホルマリン誘発疼痛に対する抑制作用(ホルマリン試験)
EFEの各分配エキス及び水エキスのDiaion HP-20カラム分画物について、ホルマリン誘
発疼痛に対する抑制作用を調べた。
The assay conditions are as follows. MDA-MB-231 cells (5×10 4 cells/well) were suspended in Vehicle (DMEM) or DMEM in which 10 μg/mL of each fraction was dissolved, and added to the upper wells.
A collagen-coated 24-well plate was used for the lower wells. 600 μL of 50 ng/mL HGF-DMEM was added to the lower wells. After culturing for 24 hours, the number of cells that migrated to the lower well was measured under a microscope and used as an index of motility. As a result, 30% MeOH fraction, 40% MeOH fraction, and 50% MeOH fraction strongly inhibited HGF-induced motility (Fig. 12).
(Example 9) Suppressive effect on formalin-induced pain using mice (formalin test)
Diaion HP-20 column fractions of EFE partitioned extracts and water extracts were examined for inhibitory effects on formalin-induced pain.
4週齢の雄性ddYマウスを搬入後、1週間以上馴化したものを1群4~8匹用いた。実験開始日に体重測定を行い、各群でほぼ同じになるようにコントロール群、EFE投与群、各画分
投与群(7群)に分けた。EFE及び各画分は10% DMSO (DMSO:Saline = 1:9)を加え、超音波で溶解した。コントロール群は10%DMSOを経口投与した。EFE群は700 mg/kgEFEを経口投与した。各画分群は、EFE 700 mg/kgに対する各画分の重量収率から算出した量を経口投与し
た。各試料投与6時間後に2.5%ホルマリン溶液10μLを左後足底部に皮内投与した。投与
後速やかにマウスを円柱アクリルケージ(高さ20 cm x 直径10 cm)に入れ、45分間ビデ
オ撮影した。疼痛関連行動は投与直後から5分後までに生じる第1相と10分後から45分後
まで生じる第2相を対象とし、処置した足を舐める行動時間を計測した。統計学的解析はコントロール群と被験物質投与群の疼痛行動時間をDunnett’s testsにて行い、有意水準は5%とした。
4-week-old male ddY mice were brought in and acclimatized for 1 week or longer, and 4-8 mice per group were used. On the day of the start of the experiment, the animals were weighed and divided into a control group, an EFE-administered group, and each fraction-administered group (7 groups) so that each group had approximately the same weight. 10% DMSO (DMSO:Saline=1:9) was added to EFE and each fraction, and dissolved by ultrasonication. The control group was orally administered with 10% DMSO. The EFE group was orally administered 700 mg/kg EFE. Each fraction group was orally administered in an amount calculated from the weight yield of each fraction with respect to
結果を図13に示す。第1相については、試料投与群は疼痛関連行動に影響を与えなか
ったことから、侵害受容性疼痛に対する疼痛抑制作用はないものと考えられた。一方、第2相については、EFE群、水エキス群、20%MeOH分画物群、40%MeOH分画物群、MeOH分画物群において、コントロール群よりも疼痛関連行動が減少し、炎症性疼痛に対する抑制作用を有することがわかった。水エキス群はEFE群よりも少ない投与量で強い疼痛抑制作用を示
したことから、疼痛抑制作用は水エキスに濃縮していること、すなわち、比活性が格段に高くなっていることが明らかになった。一方、in vitro の活性評価で薬効のあったn-ブ
タノールエキスは、in vivoでは薬効を示さなかった。
The results are shown in FIG. As for the first phase, the sample-administered group did not affect pain-related behavior, so it was considered that there was no pain-suppressing effect on nociceptive pain. On the other hand, in
次にこの水エキスをDiaionHP-20カラムで分画した。実施例7及び8より、20%MeOH分画物、30%MeOH分画物、40%MeOH分画物、50%MeOH分画物、及び、MeOH分画物にc-Metキナーゼ阻害作用及び運動能抑制作用があることが明らかになった。そこで、動物試験用に各画分の回収量を上げるために、水エキスをDiaionHP-20によって、H2O分画物、20%MeOH分画物
、40%MeOH分画物、MeOH分画物に分離した。その結果、H2O分画物には疼痛抑制作用はなく、20%MeOH分画物、40%MeOH分画物、MeOH分画物に活性があることが明らかになった。以上より、20%MeOH分画物、40%MeOH分画物、MeOH分画物は、炎症性疼痛に対する鎮痛作用を有することがわかった。
(実施例10)c-Metの発現とc-Metチロシンリン酸化、及びEGFRの発現とEGFRチロシンリン酸化に対する作用
EFE由来の水エキスのDiaion HP-20カラム分画物について、c-Met及びEGFRを過剰発現しているヒト非小細胞肺がん由来H1993細胞を用いて、c-Met及びEGFRに対する作用を解析した。
This water extract was then fractionated on a Diaion HP-20 column. From Examples 7 and 8, 20% MeOH fraction, 30% MeOH fraction, 40% MeOH fraction, 50% MeOH fraction, and MeOH fraction showed c-Met kinase inhibitory action and motility. was found to have an inhibitory effect. Therefore, in order to increase the recovery of each fraction for animal testing, the water extract was processed by DiaionHP-20 into H 2 O fraction, 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction. separated into As a result, it was clarified that the H 2 O fraction had no pain-suppressing effect, while the 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction had activity. From the above, it was found that 20% MeOH fraction, 40% MeOH fraction and MeOH fraction have an analgesic effect on inflammatory pain.
(Example 10) Effects on c-Met expression and c-Met tyrosine phosphorylation, and EGFR expression and EGFR tyrosine phosphorylation
Diaion HP-20 column fractions of the EFE-derived water extract were analyzed for effects on c-Met and EGFR using human non-small cell lung cancer-derived H1993 cells overexpressing c-Met and EGFR.
各分画物は10%DMSO-RPMI1640培地で溶解し、2~4 mg/mLの濃度のStock solutionとした後、RPMI培地で希釈して用いた。培地に含まれるDMSOの最終の濃度は0.125%だった。H1993細胞に100 μg/mL EFE、 50μg/mL H2O分画物、25 μg/mL 20%MeOH分画物、25 μg/mL 40%MeOH分画物、あるいは25 μg/mL MeOH分画物を添加して4時間インキュベーションした
後、細胞を溶解し、遠心分離した上澄みを用いて、Western-blottingを行った。結果を図14に示す。
Each fraction was dissolved in 10% DMSO-RPMI1640 medium to obtain a stock solution with a concentration of 2 to 4 mg/mL, and diluted with RPMI medium for use. The final concentration of DMSO in the medium was 0.125%. H1993 cells were treated with 100 μg/mL EFE, 50 μg/mL H 2 O fraction, 25 μg/
なお、H1993細胞は、c-Met及びEGFRを過剰発現しているため、HGFやEGFの刺激がなくても、自己リン酸化により受容体がリン酸化されている。
(1) c-Metに対する作用:
H2O分画物は、c-Metの発現量及びリン酸化に影響を与えなかった。20%MeOH分画物、40%MeOH分画物、MeOH分画物は、c-Met発現量を低下させ、リン酸化を阻害した。これらの作
用は、40%MeOH分画物が最も高く、EFEとほぼ同程度の作用を示した。したがって、c-Met
に対する作用は、H2O分画物にはなく、20%MeOH分画物、40%MeOH分画物、MeOH分画物にあ
ることが明らかになった。これは、(実施例9)の疼痛試験の結果とほぼ一致していた。(2) EGFRに対する作用:
H2O分画物は、EGFRの発現量及びリン酸化に影響を与えなかった。20%MeOH分画物、40%MeOH分画物、MeOH分画物は、EGFRのリン酸化を阻害した。一方、EGFRの発現量は、EFEと40%MeOH分画物によって低下した。しかし、20%MeOH分画物及びMeOH分画物は、EGFRの発現量をやや低下させる程度であった。
Since H1993 cells overexpress c-Met and EGFR, the receptor is phosphorylated by autophosphorylation even without HGF or EGF stimulation.
(1) Action on c-Met:
The H 2 O fraction had no effect on c-Met expression levels and phosphorylation. 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction decreased c-Met expression level and inhibited phosphorylation. These effects were highest in the 40% MeOH fraction, showing almost the same effect as EFE. Therefore, c-Met
20% MeOH fraction, 40% MeOH fraction, and MeOH fraction had no effect on H 2 O fraction. This was substantially consistent with the results of the pain test in (Example 9). (2) Action on EGFR:
The H 2 O fraction had no effect on EGFR expression level and phosphorylation. 20% MeOH fraction, 40% MeOH fraction and MeOH fraction inhibited EGFR phosphorylation. On the other hand, the expression level of EGFR was decreased by EFE and 40% MeOH fractions. However, the 20% MeOH fraction and the MeOH fraction slightly decreased the expression level of EGFR.
これらの結果から、EGFRのリン酸化阻害作用は、H2O分画物にはなく、20%MeOH分画物、40%MeOH分画物、MeOH分画物にあることが明らかになり、実施例9の疼痛試験の結果及び
上記(1)のc-Metに対する作用とほぼ一致していた。一方、EGFRの発現量を低下させる
作用は、40%MeOH分画物にあることがわかった。
These results revealed that 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction, but not H 2 O fraction, had an inhibitory effect on EGFR phosphorylation. The result of the pain test in Example 9 and the action on c-Met in (1) above were almost consistent. On the other hand, it was found that the 40% MeOH fraction had the effect of reducing the expression level of EGFR.
日本のがん死亡原因1位の肺がんにおいて、分子標的治療薬のEGFRチロシンキナーゼ阻
害剤(EGFR-TKI)が著効するタイプは、生存期間が延長したが、数年で耐性を獲得し再燃することが問題となっており、耐性獲得の原因のひとつにc-Met過剰発現が報告されてい
る(矢野聖二、実験医学、29, 203-208, 2011)。c-Met過剰発現が原因のEGFR-TKI耐性の場合、c-Met阻害剤とEGFR-TKIの併用が有効であると考えられているが、c-Met阻害剤は
医薬品化の目途が立っていない。
In lung cancer, the leading cause of cancer death in Japan, the type for which EGFR tyrosine kinase inhibitor (EGFR-TKI), a molecular-targeted drug, is remarkably effective, prolongs survival, but develops resistance in a few years and relapses. c-Met overexpression has been reported as one of the causes of resistance acquisition (Seiji Yano, Experimental Medicine, 29, 203-208, 2011). In the case of EGFR-TKI resistance caused by c-Met overexpression, the combined use of c-Met inhibitors and EGFR-TKIs is considered to be effective, but c-Met inhibitors are yet to be commercialized. do not have.
20%MeOH分画物、40%MeOH分画物、MeOH分画物は、肺がん細胞が過剰発現しているc-Met
及びEGFRのリン酸化を阻害することから、c-Met過剰発現が原因のEGFR-TKI耐性肺がん患
者の治療に有用でありうる。さらに、40%MeOH分画物は、過剰発現しているc-Met及びEGFRのリン酸化阻害作用だけなく、それの発現量を低下させることから、より有効性の高い分子標的治療薬になりうる。
(実施例11)関節炎モデルマウスに対する疼痛抑制作用
実施例10から40%MeOH分画物の薬効が最も高く、実施例9-10より、H2O分画物には薬効がないことが明らかになった。そこで、これらの分画物の関節炎モデルマウスに対する鎮痛効果をin vivoで比較検討した。結果を図15に示す。
20% MeOH fraction, 40% MeOH fraction, and MeOH fraction are c-Met overexpressed by lung cancer cells.
and inhibit EGFR phosphorylation, it may be useful in treating EGFR-TKI-resistant lung cancer patients due to c-Met overexpression. Furthermore, the 40% MeOH fraction not only inhibits the phosphorylation of overexpressed c-Met and EGFR, but also reduces their expression level, so it can be a more effective molecular target therapeutic drug. .
(Example 11) Pain suppression effect on arthritis model mice From Example 10, the 40% MeOH fraction has the highest efficacy, and from Examples 9-10, it is clear that the H 2 O fraction has no efficacy. became. Therefore, the analgesic effects of these fractions on arthritis model mice were compared in vivo. The results are shown in FIG.
実験動物は、雌性Balb/cの9週齢を用いた。マウスを搬入し1週間程度訓化した後、右後足足蹠にComplete Freud’s Adjuvant (CFA) 5 mg/mLを10 μL接種し、7日間飼育したマ
ウスを関節炎モデルマウスとした。CFA接種7日目のマウスは足関節が有意に腫脹し、機械刺激に対する感受性が最も高くなっているからである。10%DMSO溶液に溶解させた390 mg/kg H2O分画物、あるいは、190 mg/kg 40%MeOH分画物を関節炎モデルマウスに経口投与し
た。Control群は、正常マウスにvehicle(10%DMSO溶液)を投与し、Vehicle群は、関節炎モデルマウスにvehicleを投与した。試験薬投与の6時間後に、右足のvon Frey testを行い
、up-down法を用いて50%反応閾値(50% paw withdrawal threshold: 50%PWT)を算出した(S. R. Chapman et al, Journal of Neuroscience Method 53 (1994) 55-63)。統計解
析は、一元配置分散分析のあとTukeyの多重比較検定法で行った。解析ソフトは、Prism 7(GraphPad Software Inc., San Diego, CA, USA)を使用し、統計的に有意水準p<0.05
を有意差ありと判断した。
As experimental animals, 9-week-old female Balb/c mice were used. After the mice were brought in and acclimatized for about one week, 10 μL of Complete Freud's Adjuvant (CFA) 5 mg/mL was inoculated into the right hind footpad, and the mice were raised for 7 days and used as arthritis model mice. This is because mice on
was judged to be significantly different.
40%MeOH分画物の経口投与により、50%PWTがvehicle群と比較して有意に増加し、機械刺激に対する感受性が低下していた。一方、H2O分画物の経口投与では、50%PWTがvehicle群とほぼ同じで、機械刺激に対する感受性が高いままであった。これらの結果から、40%MeOH分画物は、関節炎モデルマウスに対して疼痛抑制作用を示すが、H2O分画物には疼痛抑制作用がないことが明らかになった。
(実施例12)抗インフルエンザウイルス作用
実施例9のホルマリン試験の結果から、EFEの水エキスをDiaion HP-20で分画した、20%MeOH分画物、40%MeOH分画物、MeOH分画物に疼痛抑制作用があることがわかったことから
、これらの画分の抗インフルエンザウイルス作用を測定した。この測定方法は、インフルエンザウイルスの感染によりMDCK細胞が溶解することを利用し、50%の細胞が生存する試
料濃度で抗インフルエンザウイルス作用を評価する。また、細胞毒性ではなく、抗インフルエンザウイルス作用を測定していることを確認するために、細胞生存率が50%となる濃
度も同時に測定した。
Oral administration of 40% MeOH fraction significantly increased 50% PWT and decreased sensitivity to mechanical stimulation compared to the vehicle group. On the other hand, oral administration of the H 2 O fraction resulted in a 50% PWT similar to that in the vehicle group, and remained highly sensitive to mechanical stimuli. From these results, it was revealed that the 40% MeOH fraction exhibits pain-suppressing activity in arthritis model mice, but the H 2 O fraction does not.
(Example 12) Anti-influenza virus action From the results of the formalin test in Example 9, the water extract of EFE was fractionated with Diaion HP-20, 20% MeOH fraction, 40% MeOH fraction, MeOH fraction Anti-influenza virus activity of these fractions was determined, since it was found that the substance had a pain-inhibiting effect. This measurement method utilizes the lysis of MDCK cells by influenza virus infection, and evaluates the anti-influenza virus activity at a sample concentration at which 50% of the cells survive. In addition, in order to confirm that the anti-influenza virus action was being measured, not the cytotoxicity, the concentration at which the cell viability was 50% was also measured at the same time.
各試料について、それぞれ10%FBS-MEMで25μg/mLの溶液に調製し、さらに10段階の2倍
希釈系列を作製し、試料溶液とした。インフルエンザウイルス(A/WSN/33 (H1N1)株)を
含む液を10%FBS-MEMで1000TCID50/mLに希釈し、インフルエンザウイルス液とした。MDCK
細胞を10%FBS-MEMで懸濁し、96穴培養プレートの各ウエルあたり、3x104個/100μLで播種した。24時間培養した後、培養上清を除去し、試料溶液100μLを各ウエルに添加し、さらに100μLのインフルエンザウイルス液もしくは10%FBS-MEMを添加した。72時間培養した後、クリスタルバイオレットで染色し、マイクロプレートリーダーで各ウエルの560nmにお
ける吸光度を測定した。添加した試料濃度と細胞生存率の曲線について4パラメーラーロジスティック解析を行い、インフルエンザウイルス添加群から50%阻害濃度(IC50値)、
非インフルエンザウイルス添加群から50%細胞障害濃度(CC50値)をそれぞれ求めた(表
1)。
For each sample, a 25 μg/mL solution was prepared with 10% FBS-MEM, and a 10-step two-fold dilution series was prepared to prepare a sample solution. A fluid containing influenza virus (A/WSN/33 (H1N1) strain) was diluted with 10% FBS-MEM to 1000 TCID 50 /mL to prepare an influenza virus fluid. MDCK
Cells were suspended in 10% FBS-MEM and seeded at 3×10 4 cells/100 μL per well of a 96-well culture plate. After culturing for 24 hours, the culture supernatant was removed, 100 μL of sample solution was added to each well, and 100 μL of influenza virus solution or 10% FBS-MEM was added. After culturing for 72 hours, the cells were stained with crystal violet and the absorbance of each well at 560 nm was measured using a microplate reader. A 4-parameter logistic analysis was performed on the added sample concentration and cell viability curve, and the 50% inhibitory concentration (IC 50 value) from the influenza virus addition group,
The 50% cytotoxicity concentration (CC 50 value) was determined from the non-influenza virus addition group (Table 1).
各試料のIC50値は、CC50値よりも低濃度であったことから、抗インフルエンザウイルス作用が測定できていることが確認された。また、20%MeOH分画物、40%MeOH分画物、MeOH分画物のIC50値は、EFEのIC50値と同程度であったことから、EFEの抗インフルエンザウイルス作用を示す活性成分は、20%MeOH分画物、40%MeOH分画物、MeOH分画物に存在することが明らかになった。
(実施例13)高分子縮合型タンニン標準物質の調製
実施例1で得た水エキス-40%MeOH分画物について、さらにSephadex LH-20カラムクロマトグラフィー(GE Healthcare)を用いて精製した。カラムを50%MeOHで洗浄後、70%アセ
トンで溶出し、高分子縮合型タンニンを得た。溶出液を濃縮、凍結乾燥した後、約100 mgを精密に量り(100.0 mg)、50%ジメチルホルムアミド溶液10 mLを正確に加え、完全に溶解した。この溶液について、実施例2で示したGPC分析した結果、重量平均分子量は85642で、低分子量域にピークは確認されなかった(図16)。この溶液約1 mLをバイアルに分注し、高分子縮合型タンニン標準物質溶液(10.0 mg/mL)とした。
(実施例14)GPCを用いた高分子縮合型タンニン含量の測定
実施例2で示したGPCを用いた分子量分析法及び実施例13で示した高分子縮合型タン
ニン標準物質を用いて、麻黄エキスの高分子縮合型タンニン含量を測定した。
Since the IC 50 value of each sample was lower than the CC 50 value, it was confirmed that the anti-influenza virus activity could be measured. In addition, the IC50 values of the 20% MeOH fraction, 40% MeOH fraction, and MeOH fraction were comparable to the IC50 value of EFE, indicating that EFE has an anti-influenza virus effect. was found to exist in 20% MeOH, 40% MeOH and MeOH fractions.
(Example 13) Preparation of Polymer Condensed Tannin Standard Substance The water extract-40% MeOH fraction obtained in Example 1 was further purified using Sephadex LH-20 column chromatography (GE Healthcare). After washing the column with 50% MeOH, it was eluted with 70% acetone to obtain polymer condensed tannins. After concentrating and freeze-drying the eluate, about 100 mg was accurately weighed (100.0 mg), and 10 mL of a 50% dimethylformamide solution was accurately added to dissolve completely. As a result of the GPC analysis shown in Example 2 for this solution, the weight average molecular weight was 85642, and no peak was confirmed in the low molecular weight region (Fig. 16). About 1 mL of this solution was dispensed into a vial to obtain a polymeric condensed tannin standard substance solution (10.0 mg/mL).
(Example 14) Measurement of polymer condensed tannin content using GPC was measured for the content of polymeric condensed tannins.
麻黄エキス3ロットについて、それぞれ10.00 mg/mL となるよう50%ジメチルホルムア
ミド溶液に溶解し試料溶液とした。各試料溶液及び高分子縮合型タンニン標準物質溶液(10.0 mg/mL)について、実施例2で示した条件でGPC分析した。試料導入後5~10分の高分子縮合型タンニンのピーク面積について、次式によって試料1 mL中の高分子縮合型タンニン含量を求めた(表2)。
Three lots of Ephedra huang extract were each dissolved in a 50% dimethylformamide solution to give a sample solution of 10.00 mg/mL. GPC analysis was performed under the conditions shown in Example 2 for each sample solution and polymer condensed tannin standard substance solution (10.0 mg/mL). Concerning the peak area of polymer condensed
試料溶液1 mL中の高分子縮合型タンニンの量(mg)= AT/AS x 10.00
AT:試料のピーク面積
AS:標準物質のピーク面積
10.00:標準物質溶液1 mL中の高分子縮合型タンニンの量(mg)
Amount of polymer condensed tannin in 1 mL of sample solution (mg) = A T /A S x 10.00
A T : Sample peak area
A S : Peak area of standard substance
10.00: Amount of polymer condensed tannin in 1 mL of standard solution (mg)
Claims (9)
均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物であって、前記重量平均分子量45,000以上の高分子縮合型タンニンを30%以上含む抽出分画物。 An extract fraction containing polymer condensed tannins with a weight average molecular weight of 45,000 or more derived from mahuang extract or derived from ephedrine alkaloid-removed mahuang extract (EFE), wherein 30 Extracted fraction containing more than %.
ナルユニットとして、カテコールタイプ又はピロガロールタイプのフラバン 3-オールを
主に含み、ピロガロールタイプとカテコールタイプを約5:1で含む、請求項1記載の抽出
分画物。 Polymer condensed tannins, as extension units, are condensed mainly with proanthocyanidin B type catechol-type or pyrogallol-type flavan 3-ols, part of which contains proanthocyanidin A-type condensed tannin units, and terminal 2. The extract fraction according to claim 1, which mainly contains catechol-type or pyrogallol-type flavan 3-ols as units, and pyrogallol-type and catechol-type at a ratio of about 5:1.
をカラムクロマトグラフィーによって、水とメタノール又はエタノールから選択されるアルコールとの混合溶媒を用いて、アルコール濃度を高くしながら順次溶出し、得られる各分画の分子量を測定して、重量平均分子量45,000以上の高分子縮合型タンニンを含む抽出分画物を得ることからなる、請求項1記載の抽出分画物の製造方法。 Water-soluble fractions derived from Ephedra extract or Ephedrine alkaloid-removed Ephedra extract (EFE) are eluted by column chromatography using a mixed solvent of water and an alcohol selected from methanol or ethanol while increasing the alcohol concentration. 2. The method for producing an extract fraction according to claim 1, which comprises measuring the molecular weight of each obtained fraction to obtain an extract fraction containing polymer condensed tannins having a weight average molecular weight of 45,000 or more.
。 5. The pharmaceutical composition according to claim 4, which is an anticancer/anti-metastatic drug against c-Met-expressing cancer.
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