JPH03205402A - Lignin glycoside and its use - Google Patents
Lignin glycoside and its useInfo
- Publication number
- JPH03205402A JPH03205402A JP11304890A JP11304890A JPH03205402A JP H03205402 A JPH03205402 A JP H03205402A JP 11304890 A JP11304890 A JP 11304890A JP 11304890 A JP11304890 A JP 11304890A JP H03205402 A JPH03205402 A JP H03205402A
- Authority
- JP
- Japan
- Prior art keywords
- lignin
- glycoside
- polysaccharide
- lignin glycoside
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- 229930182470 glycoside Natural products 0.000 title claims abstract description 47
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Landscapes
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Abstract
Description
【発明の詳細な説明】
[産業]二の利用分野〕
本発明は新規なリグニン配糖体およびその用途に関する
。DETAILED DESCRIPTION OF THE INVENTION [Industry] Second Field of Application] The present invention relates to a novel lignin glycoside and its uses.
〔従来技術・発明が解決しようとする課題]現有の抗癌
剤の殆どは、DNA合戒あるいは細胞分裂を抑制する作
用を持つが、これは正常細胞に対しても同等な作用を示
す。わずかに癌細胞は細胞分裂が速く、正常細胞は遅い
と言う差を利川して、癌細胞に、より多くの障害を与え
ることで治療が或り立っている。正常細胞が受けた障害
+f、副作用として表現され、生体がその副作用にどこ
まで耐えられるかが、癌治療の上で重要なポイントとな
っている。[Prior Art/Problems to be Solved by the Invention] Most of the existing anticancer drugs have the effect of suppressing DNA synthesis or cell division, but they also have the same effect on normal cells. Cancer cells divide slightly faster than normal cells, which is a slower rate, so treatment is possible by giving more damage to cancer cells. Damage to normal cells + f is expressed as side effects, and the extent to which the body can tolerate these side effects is an important point in cancer treatment.
以上の様に、本来の癌治療は癌細胞の生物学、生化学な
どに根ざすべきものであるが、現実に{.よその様な癌
治療法にまで結びついていない。As mentioned above, original cancer treatment should be rooted in the biology and biochemistry of cancer cells, but in reality, {. It has not been linked to other cancer treatments.
さて、癌の原因と言うと発癌物質、放射線およびウイル
スの3つが古くより言われてきた。その内、癌ウイルス
の持つ遺伝情報により細胞が癌化することが明らかにな
り、oncogene. (癌遺伝子)なる言葉が生ま
れた。その後、癌遺伝子は正常細胞にも存在し、それが
ある時スイッチオンされて、細胞が癌化すると言う仮説
が立てられたのである。Well, when it comes to the causes of cancer, three things have long been said to be carcinogens, radiation, and viruses. Among them, it became clear that the genetic information contained in cancer viruses causes cells to become cancerous, and oncogenes. The word (oncogene) was born. Later, it was hypothesized that oncogenes exist in normal cells, and that they are switched on at certain times, causing the cells to become cancerous.
これは、時の流れど共に発展し、今日その大筋は正しか
ったことを誰しも認めるところである。This idea has evolved over time, and today everyone agrees that it was largely correct.
一方、高等動物のゲノムには癌遺伝子となり得るpro
to−oncogeneは50種以上存在し、それらは
正常細胞の増殖や分化に重要な生理機能を果たしている
。それ故、細胞増殖や癌の制御の遺伝子のレベルもしく
は遺伝子産物のレベルでのコントロールの可能性が生ま
れて来た。本発明は癌遺伝子発現の段階を、特異的阻害
剤で抑制する癌治療剤を開発することにある。挿入され
たマウス乳癌ウイルス(ii)ITV)遺伝子の発現が
コルチコイドにより制御されているマウス乳癌細胞を用
い、MMTV遺伝子発現にはクロマチンタンパク質での
脱ポリADP−リボース反応が引金となっていることが
見出されている。即ち、ポリADP−リボースが分解さ
れることにより、その部分のクロマチン構造の局所変化
が、最終的にはRNAボリメラーゼのプロモーターへの
結合と転写促進につながると考えられている。(ジャー
ナル・バイオロジカルケもストリー、258:1537
1 (1983)]。On the other hand, the genomes of higher animals contain pro-oncogenes.
There are more than 50 types of to-oncogenes, and they play important physiological functions in the proliferation and differentiation of normal cells. Therefore, the possibility of controlling cell proliferation and cancer at the gene level or gene product level has arisen. The purpose of the present invention is to develop a cancer therapeutic agent that suppresses the stage of cancer gene expression using a specific inhibitor. Using mouse mammary cancer cells in which the expression of the inserted mouse mammary tumor virus (ii) ITV) gene is regulated by corticoids, the MMTV gene expression is triggered by the depoly ADP-ribose reaction in chromatin proteins. has been found. That is, it is thought that as polyADP-ribose is degraded, local changes in the chromatin structure in that region ultimately lead to binding of RNA polymerase to the promoter and promotion of transcription. (Journal Biology Calke Mostory, 258:1537
1 (1983)].
そこで、発明者はポリADP−リボースの分解を阻止す
れば、癌遺伝子が活性化されなくなることが予想された
ため、ADP−リボースの分解に関与する酵素であるポ
リ(ADP−リボース)グリコヒドロラーゼをヒト胎盤
より分離精製し、本酵素に対し阻害作用をもつ化合物を
検討した結果、幾つかの天然化合物に強い阻害活性を見
出した。Therefore, the inventor predicted that if the decomposition of polyADP-ribose was prevented, the oncogene would not be activated, so the inventors introduced poly(ADP-ribose) glycohydrolase, an enzyme involved in the decomposition of ADP-ribose, into humans. As a result of separating and purifying the placenta and examining compounds that have an inhibitory effect on this enzyme, we found that several natural compounds have strong inhibitory activity.
また、この化合物にはllf!瘍壊死因子(TNF)の
もつ細胞殺傷作用と細胞分化誘導作用を増強する全く新
しい活性があることを見出し、この増強効果がTNF受
容体への”FNFの結合親和性を高めることに起因する
ことも突き止めた。さらに、この化合物にはマクロファ
ージからのTNF、インターロイキンlの産生を誘導ず
る作用があることを見出した。そして、さらに検討を進
め、ポリ(ADP−リホース)グリコヒトロラーゼ明害
、及びTNFをはしめとするサイトカインの誘導および
それらの作用増強効果に基づく抗癌作用を有する医薬と
して、使用に耐え得る新規化合物を見出し、本発明を完
威した。Also, this compound has llf! We discovered that tumor necrosis factor (TNF) has a completely new activity that enhances the cell-killing and cell differentiation-inducing effects, and that this enhancement effect is due to increasing the binding affinity of FNF to the TNF receptor. Furthermore, they discovered that this compound has the effect of inducing the production of TNF and interleukin l from macrophages.Then, they further investigated the effects of poly(ADP-rehose) glycohydrolase, The present invention has been completed by discovering a new compound that can be used as a drug having anticancer effects based on the induction of cytokines, including TNF, and their action-enhancing effects.
即ち、本発明は次の要旨を有するものである。 That is, the present invention has the following gist.
■ 以下に示す性質を有するリグニン配糖体(i)リグ
ニンおよび多糖類が結合
(i])分子量は60000−140000(山)リグ
ニンと多wM類の結合比は1:1〜201(分子比)
(iv)多糖類はウロン酸60〜70%、中性1!30
〜40%で構或されている。■ Lignin glycosides with the following properties (i) Lignin and polysaccharides are bound (i]) Molecular weight is 60,000-140,000 (mountain) The binding ratio of lignin and multi-wMs is 1:1-201 (molecular ratio) (iv) Polysaccharide is 60-70% uronic acid, 1.30% neutral
~40%.
■ リグニン配糖体を主成分とするポリ (ADP−−
リボース)グリコヒドロラーゼ阻害剤。■ Poly(ADP--) whose main component is lignin glycoside
ribose) glycohydrolase inhibitor.
■ リグニン配糖体を主成分とするサイト力イン、すな
わち腫瘍壊死因子(TNF)作用増強剤。■ Cytolycoin, which is mainly composed of lignin glycosides, is a tumor necrosis factor (TNF) action enhancer.
■ リグニン配糖体を主或分とするサイトカイン(TN
F,IL−1)の産生誘導剤。■ Cytokine (TN) whose main component is lignin glycoside
F, IL-1) production inducer.
■ リグニン配糖体を主或分とする(TNFなどのサイ
ト力インとの併用による)癌免疫療法剤。■ Cancer immunotherapeutic agents that contain lignin glycosides as the main component (in combination with cytotoxic agents such as TNF).
本発明のリグニン配糖体は、リグニンとIlt (多m
l)との結合体である。リグニンと糖(多vMilQ)
との結合はエーテル結合による。その結合比は、リグニ
ン:構或糖の重量比で1:3〜5程度が例示される。ま
た、リグニンと多糖体との分子比で1〜20:1程度が
例示される。The lignin glycoside of the present invention comprises lignin and Ilt (polymer
It is a conjugate with l). Lignin and sugars (polyvMilQ)
The bond with is through an ether bond. The binding ratio is exemplified to be about 1:3 to 5 in terms of weight ratio of lignin to structural sugar. Further, the molecular ratio of lignin to polysaccharide is exemplified by about 1 to 20:1.
リグニン配糖体の糖部分はウロン酸および中性糖より構
戒される。その組威としてはウロン酸60〜70%、中
性#M30〜40%程度が例示される。The sugar moiety of lignin glycosides is more sensitive than uronic acids and neutral sugars. Examples of its composition strength include 60 to 70% uronic acid and 30 to 40% neutral #M.
中性糖としては、グルコース、ガラクトース、マンノー
ス、アラビノースが挙げられる。その糺y戊としてはグ
ルコース15〜201101 %、ガラクトース25〜
30mol %、マンノース35〜50mol %、ア
ラビノース10=15mol%程度が例示される。Examples of neutral sugars include glucose, galactose, mannose, and arabinose. The main ingredients are glucose 15~201101% and galactose 25~201101%.
Examples include 30 mol %, mannose 35 to 50 mol %, and arabinose 10=15 mol %.
これらの構成糖は全体としてI!鎖構造を取っており、
多糖類を形或する。These constituent sugars as a whole are I! It has a chain structure,
Forms polysaccharides.
リグニン配糖体の分子量はsoooo〜140000程
度であり、多糖類部分の分子量は4万〜10万程度であ
る。またリグニン部分は分子ffi1000〜1000
0程度を有する。The molecular weight of the lignin glycoside is about soooo to 140,000, and the molecular weight of the polysaccharide part is about 40,000 to 100,000. In addition, the lignin part has a molecular ffi of 1000 to 1000.
It has about 0.
本発明リグニン配糖体は、元素的にはC原子35〜45
重量%、H原子1〜10重量%、0原子45〜64重量
%程度が例示される。Elementally, the lignin glycoside of the present invention has 35 to 45 C atoms.
Examples include 1 to 10% by weight of H atoms, and 45 to 64% by weight of 0 atoms.
本発明のリグニン配糖体は以下のように調製される。The lignin glycoside of the present invention is prepared as follows.
出発原料としては茶(葉・枝)、紫根、三豆根、朝鮮人
参、松(かさ・葉)、草みつき(幹)等が挙げられる。Examples of starting materials include tea (leaves/branches), purple root, three bean root, Korean ginseng, pine (cap/leaf), and grass roots (trunk).
原料を各種溶媒(例えば、熱水、エタノール、アセ1・
ン等)で処理する。処理時間は1〜15時間程度である
。処理済み原料をアルカリ性溶液(0.1〜IN水酸化
ナトリウム、アンモニウム等)で抽出する。抽出液をp
H4〜6に調整し、15倍量のエタノールを添加して沈
澱画分を回収する。沈澱画分をゲル濾過で精製して活性
部分を回収する。The raw materials are mixed with various solvents (e.g., hot water, ethanol, acetic acid, etc.).
processing). The processing time is about 1 to 15 hours. Extract the treated raw material with an alkaline solution (0.1-IN sodium hydroxide, ammonium, etc.). The extract is p
Adjust to H4-6, add 15 times the volume of ethanol, and collect the precipitate fraction. The precipitated fraction is purified by gel filtration to recover the active portion.
こうして得られたリグニン配糖体は透析、遠心分離、凍
結乾燥等を施すことができる。The lignin glycoside thus obtained can be subjected to dialysis, centrifugation, freeze drying, etc.
本発明のリグニン配糖体は、ポリ(ADP−リボース)
グリコヒドロラーゼ阻害作用、及びTNFの産生誘導お
よびその作用の増強効果を有し、ヒトを含む啼乳動物(
ヒト、ウマ、イヌ、マウス、モルモノl・、ラット等)
に対してポリ (ADPリボース)グリコヒドロラーゼ
阻害活性、及び゛PNFの産生誘導およびその作用の増
強活性を有し、ボリ(ADP−リボース〉グリコヒド0
ラーゼ阻害剤、及びTNFの産生誘導およびその作用増
強活性剤として悪性腫瘍、ウイルス性感染症の治療、予
防に有用なものである。The lignin glycoside of the present invention is poly(ADP-ribose)
It has a glycohydrolase inhibitory effect, and an effect of inducing TNF production and enhancing its effect, and is effective in mammals including humans (
humans, horses, dogs, mice, mormons, rats, etc.)
It has poly (ADP-ribose) glycohydrolase inhibitory activity against poly(ADP-ribose) glycohydrolase and activity of inducing the production of PNF and enhancing its action.
The compound is useful for the treatment and prevention of malignant tumors and viral infections as a TNFase inhibitor and an activator for inducing TNF production and enhancing its action.
本発明のリグニン配糖体は、経口的または非経口的に投
与される。The lignin glycosides of the present invention are administered orally or parenterally.
リグニン配糖体は、それ自体または製薬]二許容される
キャリアどの塾薬製剤の形で投与される。The lignin glycosides are administered in the form of a pharmaceutical formulation, either by itself or with a pharmaceutically acceptable carrier.
当該製剤は、自体既知の方法によって調製される。The formulation is prepared by methods known per se.
剤型どしては、錠剤、カプセル剤、散剤、坐剤、注射剤
等が例示される。Examples of dosage forms include tablets, capsules, powders, suppositories, and injections.
リグニン配糖体は、例えば、経口投与の場合、通常0.
1 〜]. O O mg/kg体重程度を] IE
I I ffj+または数回にわたって投与されるが、
年齢、体重、および/または処置すべき病状の重度や治
療に対する反応によりその投与量は変わりうる。For example, when administered orally, lignin glycosides usually have a concentration of 0.
1 ~]. O O mg/kg body weight] IE
I I ffj+ or administered over several times,
The dosage may vary depending on age, weight, and/or the severity of the condition being treated and response to treatment.
毒性実験
本発明のリグニン配糖体のマウスに対する毒性は、いず
れも経口投与でLD5o値が100■/kg以上であり
、投与量にくらべてLD,。値が極めて大きく、安全域
の広い化合物である。Toxicity Experiment Regarding the toxicity of the lignin glycosides of the present invention to mice, the LD5o value was 100 μ/kg or more when administered orally, and the LD5o value was lower than the administered dose. It is a compound with an extremely high value and a wide safety margin.
〔実施例]
実施例1
以下の処理に{1ずこどによってリグニン配糖体を抽出
した。[Example] Example 1 Lignin glycosides were extracted by the following process.
例
松かさ
↓去ソK抽」旦 煮沸時間吐松かさの量、また?
■8
エタノール抽出した松かさを
?■ユ■
抽出液に等量のエタノールを
↓遣(I訟城
透析後の溶液を凍結乾燥にて
実施例2
?グニン配糖体の糖部分、グリコン(glycone)
及び非糖部分、アグリコン(aglycone)の構造
の特徴を検討するために、本配糖体をメタノリシス(メ
タノールー塩酸分解)、あるいは亜塩素酸塩(NaCl
O■)法によりグリコンとアグリコンに分離して分析を
行った。その結果は次の通りである。Example pine cone ↓ leaving water boiling time and amount of pine cone?
■8 Pine cones extracted with ethanol? ■Yu■ Add an equal amount of ethanol to the extract (I) Freeze-dry the solution after dialysis Example 2 - Sugar moiety of gunin glycoside, glycone
In order to investigate the structural characteristics of the non-sugar moiety and aglycone, this glycoside was subjected to methanolysis (methanol-hydrochloric acid decomposition) or chlorite (NaCl).
The glycone and aglycone were separated and analyzed using the O■) method. The results are as follows.
分子I:セファロース(Sepharose)CL−4
Bゲル濾過法により60000〜iooooo
糖組或1 (%) (全重量%)ウロ
ン酸 63,4 48.8中性糖
36,6 28.2グリコンの2/3がウ
ロン酸であるという特徴を持つ。Molecule I: Sepharose CL-4
B Gel filtration method: 60,000 to iooooo Sugar composition 1 (%) (Total weight %) Uronic acid 63.4 48.8 Neutral sugar
36,6 28.2 It has the characteristic that two-thirds of the glycon is uronic acid.
中性糖の組威” (mol %)グルコース
182
ガラクトース 27.4
マンノース 40.2
アラビノース 13.8
フコース O
中性糖としてはグルコース、ガラクトース、マンノース
、アラビノースを含むがフコースは含まない。Neutral sugar composition (mol %) glucose
182 Galactose 27.4 Mannose 40.2 Arabinose 13.8 Fucose O Neutral sugars include glucose, galactose, mannose, and arabinose, but do not include fucose.
a)ウロン酸はカルバゾール法、中性糖はフェノール硫
酸法で定量した。a) Uronic acid was determined by the carbazole method, and neutral sugars were determined by the phenol-sulfuric acid method.
b)中性糖の組或はメタノリシスで生威するメチルグリ
コシドをトリメチル化した後ガスクロマトグラフィーで
分析した。b) Neutral sugar composition or methyl glycoside produced by methanolysis was trimethylated and analyzed by gas chromatography.
分子量:セファロース(Sepharose ) C
L − 6 Bゲル濾過法により4000±2000
赤外吸収分析(IR)
IRはKBrディスクにより測定した。Molecular weight: Sepharose C
4000±2000 by L-6B gel filtration method Infrared absorption analysis (IR) IR was measured using a KBr disk.
その結果、3500〜3700cm−’範囲に3400
crV’にピークをもつ吸収が検出された。この吸収は
フェノール性水酸基の存在を示す。1600cm付近の
吸収は芳香族二重結合を示す。1700cm付近にカル
ポニル基の吸収がないことよりタンニンに見られるよう
なエステル結合はなく、リグニンに見られるようなエー
テル結合で重合している化合物であることを示す。As a result, 3400 in the 3500-3700 cm-' range
Absorption with a peak at crV' was detected. This absorption indicates the presence of phenolic hydroxyl groups. Absorption near 1600 cm indicates an aromatic double bond. The absence of carbonyl group absorption near 1700 cm indicates that the compound does not have ester bonds as seen in tannins, but is polymerized with ether bonds as seen in lignin.
指紋領域も含めて全体のスベクI・ラムはリグニン(ア
ルカリ)と極めて類似している。The entire subek I. rum, including the fingerprint region, is very similar to lignin (alkali).
以上のIRスペクトルの結果から本配糖体のアグリコン
はタンニン様化合物ではなく、リグニン様化合物である
と結論される。From the above IR spectrum results, it is concluded that the aglycone of this glycoside is not a tannin-like compound but a lignin-like compound.
紫外吸収(UV)分析
280nmに最大吸収値、260nmに最小吸収値をも
つ。Ultraviolet absorption (UV) analysis: Maximum absorption value at 280 nm and minimum absorption value at 260 nm.
280/260=1.02
リグニンも同様のUVスペク1・ルをもつ280/26
0=1.03
UVスペクトルにより芳香族(おそらくフェノール)の
存在を示す。280/260=1.02 Lignin also has a similar UV spectrum of 280/26
0=1.03 UV spectrum indicates the presence of aromatics (probably phenols).
電子スピン共鳴(ESR)分析
リグニンと同様にg=2.004EsRシグナルが検出
されることより安定なフリーラジカルを有する構造体を
含むことを示す。なお、タンニンにはこの様なシグナル
は見られない。Electron spin resonance (ESR) analysis shows that a g=2.004 EsR signal, similar to that of lignin, is detected, indicating that the structure contains stable free radicals. Note that no such signal is observed for tannins.
(リグニン配糖体の分析〕
リグニン配糖体全体どしての特徴
元素分析 (weight%)
C 3 9. 8 3
H 4.41
0 5 5. 7 3
N 0. 0 3以下
S O
窒素、硫黄を含有しないことより蛋白、硫酸基を含まず
セフ70−ス(Sepharose ) C I−−4
Bゲル濾過法による分子量は約11万である。(Analysis of lignin glycosides) Characteristic elemental analysis of the entire lignin glycosides (weight%) C 3 9. 8 3 H 4.41 0 5 5. 7 3 N 0. 0 3 or less S O Nitrogen, Since it does not contain sulfur, it does not contain proteins or sulfate groups.Sepharose CI-4
The molecular weight determined by the B gel filtration method is approximately 110,000.
リグニンと糖の結合比は重量比で約1=4である。The binding ratio of lignin and sugar is approximately 1=4 by weight.
従って本配糖体は、糖部分(グリコン)としてウロン酸
を全重量の約50%も含有するという特徴をもっている
。中性糖どしてはグルコース、ガラク1・−ス、マンノ
ース、アラビノースを含む。Therefore, this glycoside has the characteristic that it contains about 50% of the total weight of uronic acid as a sugar moiety (glycone). Neutral sugars include glucose, galactose, mannose, and arabinose.
非糖部分(アグリコン)はリグニンより或るという特徴
を有する。The non-sugar moiety (aglycone) has certain characteristics from lignin.
本配糖体は糖の還元末端ラクトール水酸基がリグニンの
アルコール性またはフェノール性水酸基と脱水縮合して
エーテル状に結合した○−配糖体である。また、リグニ
ンと糖の結合比が重量比で約l:4であり、特に、酸性
糖のウロン酸を多く含有する、分子量約l1万のリグニ
ン配糖体(アグリコンの特徴で分類した場合)である。This glycoside is a ○-glycoside in which the reducing end lactol hydroxyl group of sugar is dehydrated and condensed with the alcoholic or phenolic hydroxyl group of lignin to form an ether-like bond. In addition, the binding ratio of lignin and sugar is approximately 1:4 by weight, and in particular, it is a lignin glycoside (when classified by aglycone characteristics) with a molecular weight of approximately 110,000 liters that contains a large amount of uronic acid, which is an acidic sugar. be.
木配糖体の推定構造モデルは図面に示す通りである。The estimated structural model of xylem glycosides is as shown in the drawing.
試験例l.
ポリ(ADP−リボース)グリコヒ1・゛ロラーゼに対
する阻害効果
アンセイ用ハソファ−(0.01%ウシ血清アルブミン
−10mMメルカプI・エタノール−5 0 mMカリ
ウム・リン酸、p H 7. 0 )に、″H−(AD
Pリボース) n+I5 を加え、その27μCに被
験物貿4−3よびヒト胎盤より調製した核由来、ポリ(
ADP−リボース)グリコヒドロラーゼン容液を加えて
全量30μ℃とした後、37゜Cにて1時間インキユヘ
ーションした。その後、DE81濾紙に反応液を吸収さ
せ、水、エタノール、アセ1・ンで濾紙を洗浄した後、
それを乾燥させ、液体ジンチレl5
一ションカウンターにて、未反応基t 3H−( AD
P−−リボース)を測定し、本酵素に対する試験物質の
阻害作用を検討した。その結果を示したのが表1であり
、用いた被験物質の全てが、用量依存的にポリ(ADP
−リボース)グリコヒトロラーゼを阻害した。Test example l. Hasofafor (0.01% bovine serum albumin-10mM mercap I ethanol-50mM potassium phosphate, pH 7.0) for assaying the inhibitory effect on poly(ADP-ribose)glycohyperolase, H-(AD
P-ribose) n+I5 was added, and to the 27 μC was added poly(
ADP-ribose) glycohydrolazene solution was added to bring the total volume to 30 .mu.C, followed by incubation at 37.degree. C. for 1 hour. After that, the reaction solution was absorbed into DE81 filter paper, and the filter paper was washed with water, ethanol, and acetic acid.
It was dried, and the unreacted group t3H-(AD
P--ribose) was measured to examine the inhibitory effect of the test substance on this enzyme. Table 1 shows the results, and all of the test substances used showed poly(ADP) in a dose-dependent manner.
-ribose) glycohydrolase.
表1
リグニン配糖体のポリ(ADP−リボース)グルコヒド
ロラーゼ阻害活性
試験例2
遺伝子発現に対する阻害効果
本試験で用いた遺伝子発現系は、#a質コルチコイIJ
感受性遺伝子である、マウス乳癌ウイルス(MMTV)
遺伝子を持つ、マウス乳癌細胞である341株を使用し
た。本細胞は、糖質コルチコイド存在下において、35
SRNAと、さらにスプライシングを受けた24SRN
Aの2種類を発現する。この発現はenv部分のcDN
Aを用いることにより検出することが出来る。そこで、
341株に被験物質を30μg/成となる様に加え、3
7゜C、30分間インキユベーションし、次に、その系
に10−’Mとなる様にデキサメタゾンを添加し、さら
に1時間インキエヘーションした。その後、3IlI細
胞を集め、高分子RNAをグアニジン−塩酸法で抽出、
60゜Cで5分間処理( 2 0 mMMl)PS,
pll7.0.5mM 酢酸ナトリウム、1 mME
DTA)後、1.2%アガロース・ゲル(同バノファ−
)Cこて、電気泳動(40V.16h)を行った。その
後、ニトロセルロースへ1・ランスファーし、32[)
才−TV11N^(envに特異的なcDN八)をハイ
ブリダイズし、X線フィルムによるオートラジオダラム
を作威した。その後、オートラジオダラムより35Sお
よび24SRNAのバンドの濃度をデンシトメーターで
測定することにより、RNA発現量を測定し、被験物質
の無添加の場合と比較して、RNA発現抑制割合を算出
した。その結果を示したのが表2であり、用いた被験物
質はMMTV遺伝子発現抑制作用を示した。Table 1 Poly(ADP-ribose) glucohydrolase inhibitory activity of lignin glycoside Test Example 2 Inhibitory effect on gene expression The gene expression system used in this test was
Mouse mammary tumor virus (MMTV), a susceptibility gene
The 341 mouse breast cancer cell line containing the gene was used. In the presence of glucocorticoids, this cell
SRNA and further spliced 24SRN
It expresses two types of A. This expression is based on the cDNA of the env part.
It can be detected by using A. Therefore,
The test substance was added to 341 strains at a concentration of 30 μg/form, and 3
After incubation at 7°C for 30 minutes, dexamethasone was added to the system at 10-'M and incubation was continued for an additional hour. After that, the 3IlI cells were collected, and high molecular RNA was extracted using the guanidine-hydrochloric acid method.
Treated at 60°C for 5 minutes (20 mM Ml) PS,
pll7.0.5mM sodium acetate, 1mM
DTA), then 1.2% agarose gel (vanofar)
) Electrophoresis (40V, 16h) was performed using a C trowel. Then, 1 transfer to nitrocellulose, 32 [)
The DNA-TV11N^ (env-specific cDN8) was hybridized and autoradiodram was generated using X-ray film. Thereafter, the concentration of 35S and 24S RNA bands from the autoradiodrum was measured using a densitometer to measure the amount of RNA expression, and the RNA expression suppression ratio was calculated by comparing with the case without the addition of the test substance. Table 2 shows the results, and the test substance used showed an effect of suppressing MMTV gene expression.
表2
リグニン配糖体の乳癌ウィルス
発現に対ずる作用
(MMTV)遺伝子
デキサメタゾン リグニン配糖体
C10−7M) .C30ug/ml)353
2 4S
バンドの感光度
+
+
十
+
++十干
+
十
++
l9
lは陽性対照、2.3は陰性対照、4は実験群試験例3
.
マウス実験III瘍に対する制癌効果
マウスの腹腔内に、ザルコーマ180腫瘍細胞を1×1
06個移植し、移植後1〜4日間被験物質を腹腔内に連
続投与した。抗腫瘍活性は、生理食塩液投与群との比較
による延命率より求めた。Table 2 Effect of lignin glycosides on breast cancer virus expression (MMTV) gene Dexamethasone Lignin glycoside C10-7M). C30ug/ml) 353
2 4S band photosensitivity + + 10+ ++ 10+ 10++ l9 l is positive control, 2.3 is negative control, 4 is experimental group test example 3
.. Mouse Experiment III Anticancer Effect on Tumors Sarcoma 180 tumor cells were injected into the peritoneal cavity of mice at 1×1.
06 cells were transplanted, and the test substance was continuously administered intraperitoneally for 1 to 4 days after transplantation. Antitumor activity was determined from the survival rate compared with the physiological saline administration group.
腫瘍移植後45日目にて実験終了とした。その結果を示
したのが表3であり、用いた被験物質は制癌作用を示し
た。The experiment was terminated on the 45th day after tumor implantation. Table 3 shows the results, and the test substance used showed anticancer activity.
表3
マウス腫瘍ザルコーマ180!こ対スるりグニン配糖体
の制癌作用
リグニン配糖体
4 0×4
20×4
1 0×4
5×4
1 0 8
2 0 6
120
91
リグニン配糖体は移植日翌日から4日間連続投与した。Table 3 Mouse tumor Sarcoma 180! Anticancer effect of sullignin glycoside Lignin glycoside 4 0×4 20×4 1 0×4 5×4 1 0 8 2 0 6 120 91 Lignin glycoside was administered for 4 consecutive days from the day after transplantation. administered.
試験例4.
マウス線維芽腫細胞L−929に対するTNF作用の増
強効果
TNFのもつ細胞殺傷作用はL−9 2 9細胞のシャ
ーレへの接着性を利用し、生き残った生細胞を染色して
その染色濃度を測定することにより測定することができ
る。そこでL−9 2 9細胞に被験物質を加え、次に
アクチノマイシンDを4μg/dになる様に添加し、さ
らにTNFを加え、37’C,18時間インキユベーシ
ョンする。その後、プレートを生理食塩水で洗い死細胞
を除去する。Test example 4. Enhancement effect of TNF action on mouse fibroblastoma cells L-929 The cell-killing action of TNF utilizes the adhesion of L-929 cells to petri dishes, and the surviving living cells are stained and the staining concentration is measured. It can be measured by Therefore, the test substance is added to L-929 cells, then actinomycin D is added at a concentration of 4 μg/d, TNF is further added, and the cells are incubated at 37'C for 18 hours. The plate is then washed with saline to remove dead cells.
その後、生細胞を0.1%クリスタルバイオレットで染
色し、染色された生細胞を0.5%SDSにより溶解し
、その色素濃度を○D590niで測定し、T N .
F作用に対する増強効果を検討した。Thereafter, the living cells were stained with 0.1% crystal violet, the stained living cells were lysed with 0.5% SDS, and the dye concentration was measured using ○D590ni.
The enhancement effect on F action was investigated.
その結果を示したものが表4であり、用いた被験物質の
全てが、用量依存的にTNFの細胞殺傷作用を増強した
。Table 4 shows the results, and all of the test substances used enhanced the cell killing effect of TNF in a dose-dependent manner.
表4
マウス線維芽細胞L
の増強効果
929に対するTNF作用
リグニン配糖体濃度
(μg/一)
TNFのCD,。濃度
Cg/m支)
増強倍率
(%)
0 0. 0 3 4
1 0 03 0.015
2211 0 0.
0 0 6 2 5 4 B3 0
0. 0 0 4 4 7 7
3100 ’ 0.0074
458試験例5
マウス実験腫瘍に対するTNF併用にょる制癌効果マウ
スの皮下にザルコーマ180腫瘍細胞をIXIO6個移
植し、移植後1〜5日間被験物質およびTNFを連続投
与した。抗腫瘍活性は生理食塩液投与群での比較による
延命率より求めた。Table 4 TNF effect on mouse fibroblast L929 Enhancement effect Lignin glycoside concentration (μg/1) CD of TNF. Concentration Cg/m branch) Enhancement magnification (%) 0 0. 0 3 4
1 0 03 0.015
2211 0 0.
0 0 6 2 5 4 B3 0
0. 0 0 4 4 7 7
3100' 0.0074
458 Test Example 5 Anticancer effect of combined use of TNF against mouse experimental tumor Six Sarcoma 180 tumor cells were subcutaneously transplanted into mice, and the test substance and TNF were continuously administered for 1 to 5 days after the transplant. Antitumor activity was determined from the survival rate compared with the physiological saline administration group.
腫瘍移植後、45日目にて実験終了とした。その結果を
示したのが表5であり、用いた被験物質はT” N F
との併用により強い制癌作用を示した。The experiment was terminated on the 45th day after tumor implantation. Table 5 shows the results, and the test substance used was T”N F
It showed strong anticancer effect when used in combination with
表5
マウスII!II瘍ザルコーマ180に対ずるリグニン
配糖体どTNFの併用による効果
試料 濃度 T/C(■/ kg
) (%)
0
′T’ N F
”FN F +
リグニン配糖体
100
118
40X4 191
20X4 298
10X4 254
5x4 188
リグニン配糖体はTNFとともに移植日翌日から4日間
連日投与した。Table 5 Mouse II! Effect of combined use of lignin glycosides and TNF on II tumor Sarcoma 180 Sample Concentration T/C (■/kg
) (%) 0 'T' N F "FNF + Lignin glycoside 100 118 40X4 191 20X4 298 10X4 254 5x4 188 Lignin glycoside was administered together with TNF every day for 4 days from the day after the transplantation day.
図面は本発明リグニン配媚体の准定構造を示す。 23 1 : 糖 2 : リグニン 24 The drawings show the quasi-defined structure of the lignin aphrodisiac of the present invention. 23 1: sugar 2: lignin 24
Claims (5)
リグニンと多糖類の結合比は1:1〜20:1(分子比
) (iv)多糖類はウロン酸60〜70%、中性糖30〜
40%で構成されている。(1) A lignin glycoside having the following properties. (i) Lignin and polysaccharides are combined (ii) Molecular weight is 60,000 to 140,000 (iii)
The binding ratio between lignin and polysaccharide is 1:1 to 20:1 (molecular ratio) (iv) Polysaccharide has 60 to 70% uronic acid and 30 to 30% neutral sugar.
It is made up of 40%.
ボース)グリコヒドロラーゼ阻害剤。(2) A poly(ADP-ribose) glycohydrolase inhibitor containing lignin glycoside as a main component.
の増強剤。(3) An enhancer of cytokine action whose main component is lignin glycoside.
の誘発剤。(4) An inducer of cytokine production whose main component is lignin glycoside.
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JP1-280398 | 1989-10-28 | ||
JP28039889 | 1989-10-28 | ||
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ID=26452077
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7176188B2 (en) | 2003-05-07 | 2007-02-13 | UniversitéLaval | Method of lethally sensitizing human and animal cells |
US7838046B2 (en) * | 2001-09-26 | 2010-11-23 | Tampa Bay Research Institute | Plant extracts and uses thereof |
US7838052B2 (en) | 2001-09-26 | 2010-11-23 | Tampa Bay Research Institute | Pine cone extracts and uses thereof |
WO2012023301A1 (en) * | 2010-08-17 | 2012-02-23 | キリンホールディングス株式会社 | Novel plant-derived immunostimulating agent |
-
1990
- 1990-04-28 JP JP11304890A patent/JP2782009B2/en not_active Expired - Lifetime
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7838046B2 (en) * | 2001-09-26 | 2010-11-23 | Tampa Bay Research Institute | Plant extracts and uses thereof |
US7838052B2 (en) | 2001-09-26 | 2010-11-23 | Tampa Bay Research Institute | Pine cone extracts and uses thereof |
US7176188B2 (en) | 2003-05-07 | 2007-02-13 | UniversitéLaval | Method of lethally sensitizing human and animal cells |
WO2012023301A1 (en) * | 2010-08-17 | 2012-02-23 | キリンホールディングス株式会社 | Novel plant-derived immunostimulating agent |
JP5766192B2 (en) * | 2010-08-17 | 2015-08-19 | キリン株式会社 | Novel plant-derived immunostimulant |
Also Published As
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