臺灣博碩士論文加值系統

首先,進行了anthraquinone 類化合物篩選的工作o 在一系列的
anthraquinone-2-carbuxylic acid derivatives 中, N,N-
dimethylaminoethyl anthraquinone-2-carboxylate(3)是抑制兔子腸蠕
動作用最強的化合物o 且其作用是隨劑量反應o此外,化合物3 對小腸蠕動
的抑制作用是屬于可逆性的o 另外也篩選了二十幾個anthraquinone類
化合物,發現emodin,quinalizarin,iso-anthraflavic acid,
anthraflavic acid 和2-hydroxyanthraquinone 是其中抑制腸蠕動活性
最強的一群o 從這些篩選的結果,以結構和抑制腸子蠕動活性的關係而言,
可以歸納出幾點現象:(1)B位置是一個重要的活性位置;(2)具有抑制腸蠕
動活性的anthra-quinone化合物,其取代基團必須是極性的hydroxy,
carboxy. 而且hydroxy的anthra-quinone 化合物其抑制活性遠大於具
carboxy 的同類化合物;(3)hydroxy 雙取代的anthraquinone 化合物,其
生理活性大於單一取代化合物,而且取代基在2,7位置比在2,6位置更有利
於抑制腸子蠕動;(4)1,8位置的hydroxy有益於B位置活性的表現o 在
篩選試驗中,化合物14,19,20,21,22,23,29,30和31有所謂的先興奮後抑制
的作用o由其化合物是19,20,當它們不再採用累加法,而使用各種濃度的單
一劑量投予時,這種現象更是非常明顯o所以這些化合物對小腸的蠕動應該
有雙向的調節作用o 另一個發現就是:化合物32-35對腸子的蠕動有純
粹的刺激作用,而且有劑量相關反應的現象,甚至在實驗中的最高濃度都呈
現有過度刺激而產生痙欒的現象o打破了以往anth-raquinone抑制腸蠕動
的觀念o所以初步認為:硫酸鈉基團本身具有刺激作用,但雙取代的化合物,
刺激腸子蠕動的作用反而降低o而且2,6雙取代的化合物刺激腸蠕動的活
性(IC50:200+-16 nmol/ml)大於2,7雙取代的化合物(IC50:300+-18 nmol/
ml)o 第二部分的實驗是控制條件對作用的影響: 溶媒中,
propylene glycol(PLG),polyethylene glycol 400(PEG400),glycerin和
polyoxyethylene sorbitan monooleate(Tween 80)都對小腸蠕動有抑制
作用,尤其tween80抑制腸蠕動的作用最大o而酒精對小腸的收縮反而具有
強烈的刺激作用o所以使用有機溶媒來助溶必須非常的謹慎o幾乎對腸子蠕
動不影響的溶媒系統有:alcohol:PLG:water(10%:30%:60%);alcohol:PEG
400:water(10%:30%:60%);alcohol:glycerin:water(10%:30%:60%);
alcohol:PEG 400:water(5%:15%:80%)和alcohol:PEG
400:water(10%:20%:70%)等五種,可供參考使用o 另外,配製各種不同
的鹼性溶液,其pH值的變化由7,8,9,10,11到12,經實驗之後,証明0.1 ml
的鹼性溶液對於10ml Tyrode's solution 中的腸子影響不大o但通入高濃
度氧氣者(95%-5%CO2),其活性能維持達11+-1.5 hr,與通入普通空氣者,所
能維持活性的時間為8+-1hr,這種差別是有意義的o 在溶媒和氣體對
活性影響的實驗得知,22,30和31是必須在通入高濃度氧氣和鹼性溶液兩者
併存的環境下,drenergic functio才能發揮最大的活性o只要缺一條件,活
性即降為十分之一左右o這兩條件並存的環境,可能就是使melanosis coli
更嚴重的原因o而且此活性的增加很可能是引起色素性直腸癌的重要因素o
第三部分的實驗是N,N-dimethylaminoethyl anthraquinone-2-
carboxylate(3)作用機轉的探討:經實驗証明,3對小腸蠕動的抑制作用與
交感神經系統的活化無關o因為prazo-sin和propanolol無法影響其抑制作
用o但3抑制小腸的蠕動作用與鈣離子的流動確實有關係,雖然氯化鈣濃度
大於0.72mM時,3無法阻止鈣離子的流入,但在氯化鈣濃度為0.18和0.36mM
時,3確實有阻止鈣離子進入的作用,而使腸子原可被刺激而收縮的作用,明
顯的比對照組低o這種結果顯示3可能干擾了鈣離子所要通過的細胞膜o而
這一觀點被進一步証實,因為3有阻止放射性的鈣同位素湧入腸細胞的作用
o另外腸細胞在KCl和ACh所誘導的特異性鈣同位素湧入腸細胞的作用,依然
受到3的抑制而降低蠕動,而且呈現劑量相關性之現象o因此3作用在鈣離子
通過的細胞膜的重要o在此似乎得到了相當肯定的証明o 當
superoxide dimutase(SOD)和catalase(CAT)存在時,3抑制腸蠕動的作用
有意義的被降低下來o不僅如此,CAT有效的減少3抑制鈣離子湧入腸細胞的
作用o但是,SOD只有在高濃度時才能產生與CAT一樣的作用,於是我們推測3
對兔子小腸的作用與自由基有關,而且似乎比較傾向與H2O2的生成有關o事
實上,也經由實驗証明,濃度為0.0088units/ml的xanthine oxidease(X.
O.),確實會有抑制腸蠕動的現象,而且這一現象可被SOD所反轉o總結而
言:3之所以能抑制小腸蠕動主要是干擾了細胞膜鈣離子的流動,而鈣離子
流動所受的干擾與自由基的生成有關o 第四部分的實驗是
quinalizarin(30)作用基轉的探討:經研究發現prazosin(a-bloc-ker)和
propranolol(B-blocker)都會反轉30對腸蠕動的抑制作用o因此我們推
斷30的抑制作用與交感神經系統的活化有關o 而鈣子流動與30抑制腸
蠕動的關係,經過了實驗後,發現:30存在時,加入各種不同濃度的氯化鈣所
刺激腸子蠕動的收縮作用,被明顯的降低o因此可推論:30確實有阻止鈣離
子的流入而明顯地產生抑制蠕動的作用o所以這種結果確實顯示30干擾了
鈣離子流過細胞膜(trans-membrane)的過程o這可能是干擾到KCl作用的結
果,因此我們使用小腸細胞,以放射性的鈣同位素當作指示劑,在KCl的刺激
下進行實驗o這種特異性鈣同位素的湧入依然會受到30的抑制而降低o而且
有劑量相關性的現象o由此可証明30會抑制鈣離子通過細胞膜且與KCl的去
極化有關o SOD的加入有反轉30抑制腸蠕動現象,而且減少了30抑制鈣
離子湧入腸細胞的作用o但catalase的加入不僅沒有反轉30抑制腸蠕動的
作用,反而有促進30的作用,雖然這個實驗結果,沒有表現出有意義的統計
值o對於減少30抑制鈣離子湧入腸細胞的作用也沒有任何有意義的表現o可
是在catalase與SOD的併用後,對於反轉30抑制腸蠕動的程度,當與
catalase和SOD個別投予單劑量的結果比較起來,確實也呈現出有互相抵消
的現象o Quinalizarin的作用機轉經過探討之後,所得的結論是:30抑
制轈蠕動的作用與透過自主神經的傳導有關o而且明顯的有抑制鈣離子通
過細胞膜的現象o但是否因為透過神經系統而干擾鈣離子的通過,有待進一
步的探討o而自由基在此就沒有像3和31這麼重要o 第五部分的實驗是
emodin(31)致效基轉的探討:emodin幾乎是所試驗藥物中活性最強的o其作
用機轉經過prazosin和propranolol試驗之後,確定與交感神經系統的活化
無關o但對於每一個濃度的氯化鈣所刺激腸蠕動的收縮作用,幾乎都明顯的
被emodin抑制下來o在ACh的誘導下,放射性鈣同位素湧入腸細胞的現象依
然受到31的抑制而降低o而且呈相對劑量相關性的現象o由此可証31確有阻
止鈣離子的流入而產生明顯的抑制腸蠕動的作用o 31對小腸蠕動的抑
制作用,可被SOD和catalase有意義的降低下來,而且SOD會減少31抑制鈣離
子湧入腸細胞的作用,但會隨著劑量的增加反而效果下降o亦即SOD在高劑
量時會降低其保護能力o而CAT對31的影響,與SOD對31的影響差不多o但CAT
反轉emodn抑制作用的效果比SOD還大o不僅反轉31對腸蠕動的抑制作用,也
減少了31抑制鈣離子湧入腸細胞的作用o於是讓我們更確認emodin對兔腸
蠕動的抑制作用與自由基有關o而且emodin抑制小腸蠕動的作用似乎比較
傾向與H2O2的生成有關o所以整體而言:emodin對小腸蠕動抑制活性主要是
阻斷細胞膜鈣離子的湧入,而阻斷鈣離子湧入的作用與自由基的生成有關o

The effects of anthraquinone-2-carboxylate and
anthraquinone-2-carboxamideson the motility of isolated rabbit
small intestines was examined. It was found that some possessed
concentration-dependent inhibitory activity. Comparisonof IC50
values revealed a requirement for a polar group, especially a
hydroxygroup at B-1,2,5,8-tetrahydroxy and 3-methyl-1,6,8-
trihydroxyl anthraquinoneinhibited the intestianl motility more
potently than did the lead compound anthraquiaone-2-carboxylic
acid. The potency of quinalizarin and emodin were increased
about 50 times. Four structure-activity relationships were
suggested from the results: (1) a polar group, especially
hydroxy group at the B-position of anthraquinone derivatives is
required for inhibition of motility; (2) thepresence of a polar
group at the 2,7-position increased inhibition of motilitymore
than substitution at the 2,6-position increased inhibition of
motility more than substitution at the 2,6-position; (3)
addition of an 1,8-dihydroxy group at the B-position; (4)
substitution with a methyl or ethoxycarbonyl group destroyed
bioactivity. But carboxylic acid group at the B-position
would maked the intestine motilityto biphasic regulation
obviously. It was said that the stimulating effect was atlower
concentration and the inhibitory effect was appeared at higher
dose. In fact, 2-hydroxyanthraquinone, anthraflavic acid,
isoanthraflavic acid, quinalizarin,emodin and rhein had the
smaller stimulating effect at lower dose. One the other hand,
some derivatives stimulated the small intestine motility.For
instance, sodium anthraquinone-2-sulfonate made the action from
inhibition tostimulation (IC50 90 nmol/ml). Thus, we could
conclude as the tranditional view that anthraquinone derivatives
may be termed "stimulant" or "inhibitor" forintestinal motility
mainly depending on the substitued group. The selection of
solvent is very important for dissolving testing compounds.But
propylene glycol (PLG), polyethylene glycol 400, glycerin and
polyoxyethylene sorbitan monooleated (Tween 80) all have
inhibited the spontaneous contraction ofrabbit isolsted small
intestine. Tween 80 had the greatest inhibitory effect
especially.In contrast, alcohol stimulated the contraction of
small intestine. So it was very cautious for dissolving drug
with organic solvent in screening test. In these solventsystem,
it was found that five solvent systems which hardly had no
effect on motilityof small intestine. They were alcohol: PLG:
water (10%: 30%: 60%), alcohol: PEG 400:water (5%: 15%: 80%) and
alcohol: PEG 400: water (10%: 20%: 70%). Spontaneous
motility of small intestine was not affected by the alkaline
solution and their pH value were adjusted from 7, 8, 9, 10, 11
to 12. But high concentrationoxygen (95%O2 -5%CO2) could prolong
the vitro life of intestine tissue compaired with general air
(20%O2-80%N2). In this study with treating compound 3, the IC50
was 80+-10nmol/ml inhigh concentration oxygen (95%O2-5%CO2), but
120+-10 nmol/ml in air. So theactivity of 3 was elevated by high
concentration oxygen. In other cases, the activity of 22,30 and
31 were increased about 10 times by high concentration oxygen
more than general air. So the composition of bubbling air is
very impor-tant for biological activity of 22,30 and 31.
Another study indicated that the activity of 22,30 and 31
dissolved inalkaline solution were 10 folds more than in organic
solvent and they all wereimproved in high concentration oxygen.
So it was said that both high concentra-tion O2 and alkaline
solution were necessary for the most inhibitory activity on
small intestine motility, the activity was decreased obviously
when thecontrolled condition was lack of anyone above. It seems
to be important to melanosis coli which was much related with
colorectal cancer. In screen test of the bioactivities for
anthraquinone derivatives, N,N-dimethylaminoethyl
anthraquinone-2-carboxylate (3) was found to be the most
effective compound in anthraquinone 2-carboxylic acid analogs to
inhibit themotility of rabbit isolated small intestine. Addition
of 3 into organ bathcontaining rabbit intestine produced a
concentration-dependent reduction of spontaneous motility to
have an approximately IC50 value of 80 nmol/ml. Par-ticipation
of sympathetic activation was ruled out by the finding that
prazo-sin and propanolol at the concentrations sufficient to
block adrenergic recep-tors failed to modify the action of 3. In
the presence of superoxide dismutase(SOD) and catalase, this
inhibition was reversed significantly. Mediation of free
radicals in the inhibition by 3 can thus be considered. Also,
inhibitionby 3 was lowed by an increase of extracellular calcium
ions. Radioactive cal-cium influx into the prepared single
muscle cells was attenuated by 3 in a concen-tration-dependent
manner at concentration that required to inhibit the motili-
ties. Catalase reversed this inhibition by 3, both the motility
and the influx of Ca+2, in a way more markedly than that of SOD.
The results suggested that 3can block the influx of calcium ions
to lower the intestinal motility through the mechanisim related
to free radicals. How did quinalizarin(30) inhibit the
contraction of small intestine?Bothprazosin(1 uM) and
propanolol(0.1uM) would diminish the effect of 30. So we
suggested that the inhibitory effect of 30 was mediated by
sympathetic activa-tion. The partial structure of 30 is same as
catechol group of norepinephrineand considered about inhibiting
the spontaneous motility of small intestine. After
incubation with 30, contraction by CaCL2 at several
concentration was reduced obviously. In small intestine cells,
radioactive calcium was used to evaluated spontaneous influx or
specific influx into cell. Theses results indicated that the
processes of Ca influx into cell was interfered with 30actually
because both spontaneous influx and KCl-induced calcium influx
werededucted. SOD could reversed this inhibitory effect of 30 on
motility of smallintestine and radioactive calcium influx,but
catalase did not. Emodin(31) was the most potent compound
in this screening test for inhi-biting motility. And this
inhibitory effect was not related to autonomic n現象:ous
activation. The stimulating effects of CaCl2 at several
concentrations were lowered by emodin. Also,radioactive calcium
influxed into the prepared single cells was attenuated by emodin
in a dose-dependent manner. In presence of SOD and
catalase,the inhibition of 31 was reversed on both motility and
calcium influx. But this action of SOD is lowered to reversed
the effect of 31 at high dose. In conclusion,the present
study suggested that anthraquinone derivativescould modify the
spont臠 motility depending on substitutes on basic structu-re.
For this action of biological products, emodin and N,N-
dimethylaminoethylanthraquinone-2-carboxylate were related with
free radicals generating to bolck calcium influx. In addition,
quinalizarin inhibited intestinal motility through its catechol
group which can activate noradrenergic function.