17, 49 (1987) 17, 49 (1987)
Effect of Rutin on the Isolated Rat Duodenum and Comparison with the Effects of Adrenaline, Isoprenaline and Papaverine.
R u t i n ' i n İzole Sıçan D u o d e n u m u Ü z e r i n e Etkisi v e A d r e n a l i n , İzo-p r e n a l i n , P a İzo-p a v e r i n ile Karşılaştırılması.
O r h a n A L T I N K U R T * Y u s u f Ö Z T Ü R K * *
SUMMARY
T h e experiments were performed b y using isolated r a t d u o d e n u m . R u t i n , a n a t u r a l flavonoid c o m p o u n d , caused relaxations of t h e r a t d u o d e n u m d o s e - d e p e n d e n t l y . T h e r e l a x a n t effect o f r u t i n o n t h e r a t d u o d e n u m was e x a m i n e d in c o m p a r i s o n w i t h t h a t of a d r e n a l i n e , isop r e n a l i n e a n d isop a isop a v e r i n e . I n a d d i t i o n , isop r o isop r a n o l o l , a b e t a a d r e n e r -gic blocking agent, p h e n t o l a m i n e , an a l p h a - a d r e n e r g i c blockingagent a n d nicotinic acid, an a d e n y l cyclase i n h i b i t o r were used as tools to investigate m e c h a n i s m of t h e r e l a x a n t effect of r u t i n on t h e r a t d u o -d e n u m i n vitro.
ÖZET
D e n e y l e r izole sıçan d u o d e n u m u kullanılarak s ü r d ü r ü l d ü . D o ğ a l bir flavonoid bileşiği olan r u t i n d o z a bağımlı b i ç i m d e sıçan d u o d e n u -m u n u gevşetti. R u t i n ' i n sıçan d u o d e n u -m u n d a k i gevşetici etkisi ad-renalin, izoprenalin ve p a p a v e r i n ile karşılaştırmalı olarak incelendi. Ayrıca, r u t i n ' i n sıçan d u o d e n u m u n d a k i gevşetici etkisinin m e k a n i z -masını a r a ş t ı r m a k için b e t a - a d r e n e r j i k bloker olan p r o p r a n o l o l , alfa-adrenerjik bloker olan fentolamin ve bir a d e n i l siklaz i n h i b i t ö r ü olan nikotinik asid de kullanıldı.
Key words: R u t i n , A d r e n a l i n e , Isoprenaline, P a p a v e r i n e , P h e n t o
-l a m i n e , P r o p r a n o -l o -l , Nicotinic acid, S m o o t h musc-le.
R u t i n is found in m a n y plants, p a r t i c u l a r l y extracts from Fagopyrum esculentum, Fagopyrum cymosum, Fagopyrum tataricum
R e d a k s i y o n a verildiği t a r i h : 1 9 . 2 . 1 9 8 7
* D e p t . P h a r m a c o l . , F a c . P h a r m . , U n i v . A n k a r a . * * D e p t . P h a r m a c o l . , F a c . P h a r m . , U n i v . Anatolia.
SO Orhan ALTINKURT, Yusuf ÖZTÜRK
(Polygonaceae), Ruta chalepensis, Ruta graveolens ( R u t a c e a e ) a n d Viola tricolor, Viola lutea splendens (Violaceae). C h e m i c a l l y , it is a flavon glycoside w i t h a s t r u c t u r e of 3,3', 4', 5 , 7 p e n t a h y d r o x y f l a v o n e
-3-rutinoside. It is k n o w n t h a t t h e flavonoid c o m p o u n d s , such as r u t i n , possess v i t a m i n P activity a n d exert their p h a r m a c o l o g i c a l effects by a direct vasoconstriction on t h e capillary bed. Subsequently, these c o m p o u n d s decrease t h e p e r m e a b i l i t y a n d the fragility of t h e vessels
(1). Beside t h e wellknown effects on t h e vessels, r u t i n is a n o n c o m -petitive i n h i b i t o r o f angiotensin I I a n d p r o s t a g l a n d i n E2 o n t h e
guinea-pig ileum (2) Apiin a n d hesperidin w h i c h a r e also k n o w n as flavone glycosides exert similar effects on t h e isolated g u i n e a - pig ileum. T h e studies on t h e guineapig i l e u m showed t h a t hesperidin is a n o n c o m -petitive i n h i b i t o r o f angiotensin I I a n d b r a d y k i n i n . F u r t h e r m o r e , a p i i n is c a p a b l e to a n t a g o n i z e b r a d y k i n i n in a non-competitive m a n n e r (3). T h e a i m of this study is to confirm the r e l a x a n t a c t i o n of r u t i n a n d to a p p r o a c h to its m e c h a n i s m of action on s m o o t h muscle.
EXPERIMENTAL Materials
A d r e n a l i n e ( E p i n e p h r i n e b i t a r t a r a t e , S i g m a ) , A t r o p i n e s u l p h a t e (Sigma), I s o p r e n a l i n e s u l p h a t e (Ilsan), Nicotinic acid ( J . T . Baker), P a p a v e r i n e h y d r o c h l o r i d e (Sigma), P h e n t o l a m i n e m e t h a n s u l p h o n a t e ( C i b a ) , P r o p r a n o l o l h y d r o c h l o r i d e ( D o ğ u ) , R u t i n ( D i l m e n ) . All dilutions, except of p a p a v e r i n e , were p r e p a r e d w i t h fresh K r e b s ' solution. P a p a v e r i n e was dissolved in saline.
Methods
Isolated rat duodenum
T h e isolated r a t d u o d e n u m was p r e p a r e d a c c o r d i n g t o t h e m e t -h o d described b y H O R T O N (4) w i t -h m i n o r modifications. T -h e proxi-m a l 2 cproxi-m of d u o d e n u proxi-m froproxi-m rats weighing 150-200 g was r e proxi-m o v e d after killing a n d k e p t i n a t r o p i n i z e d (0.143 m M ) K r e b s ' solution a t 6 ° C for two hours. In this w a y s p o n t a n e o u s m o v e m e n t s were d i m i n i -shed. T h e n , t h e d u o d e n u m was suspended i n a 1 0 - m l o r g a n b a t h filled w i t h a t r o p i n i z e d K r e b s ' solution a n d a e r a t e d w i t h a m i x t u r e of
95 p e r cent O2 a n d 5 p e r c e n t C O2 .
T h e relaxations o f r a t d u o d e n u m were r e c o r d e d w i t h a n isotonic t r a n s d u c e r ( U g o Basile, N o . 7006) c o n n e c t e d to a r e c o r d i n g
micro-d y n a m o m e t e r ( U g o Basile, N o . 7050). T h e l o a micro-d o n t h e tissue w a s 1.00 g . T h e r e l a x a t i o n s w e r e m a g n i f i e d 8-folds.
T h e s u s p e n d e d d u o d e n u m w a s a l l o w e d t o e q u i l i b r a t e for 6 0 m i n . D u r i n g this p e r i o d d u o d e n u m w a s w a s h e d o u t every 1 5 m i n . After this i n i t i a l i n c u b a t i o n , d o s e - r e s p o n s e r e l a t i o n s h i p s w e r e o b t a i n e d for a n a g o n i s t u s i n g t w o n o n - c u m u l a t i v e d o s e - r e s p o n s e p r o c e d u r e i n all e x p e r i m e n t s . T h e agonists w e r e a d r e n a l i n e , i s o p r e n a l i n e a n d p a p a -v e r i n e . R u t i n was also used a s agonist i n this p r e p a r a t i o n . I n e a c h e x p e r i m e n t , only o n e c o n c e n t r a t i o n o f a n a n t a g o n i s t w a s tested. T h e d r u g s used a s a n t a g o n i s t s w e r e p r o p r a n o l o l , p h e n t o l a m i n e a n d n i c o -tinic a c i d . T h e isolated r a t d u o d e n u m w a s i n c u b a t e d w i t h these d r u g s for 5 m i n a n d t h e n s a m e dose-response p r o c e d u r e w e r e r e p e a t e d i n t h e p r e s e n c e of these d r u g s .
Analysis of data and statistics
I n o r d e r t o e v a l u a t e t h e actions o f a g o n i s t a n d a n t a g o n i s t s o n t h e r a t d u o d e n u m , p D2, p D2' a n d p A2 v a l u e s w e r e c a l c u l a t e d i n a m a n n e r consistent w i t h t h e m e t h o d d e s c r i b e d b y A R I E N S a n d V A N R O S S U M (5). All v a l u e s r e p o r t e d r e p r e s e n t t h e results of i n d i v i d u a l e x p e r i m e n t s . T h e dose-response curves o b t a i n e d i n t h e a b s e n c e a n d i n t h e p r e s e n c e o f a n t a g o n i s t s w e r e a n a l y s e d b y m e a n s o f l i n e a r regression p r o c e d u r e (6). R E S U L T S A N D D I S C U S S I O N T h e m a i n p u r p o s e o f this s t u d y w a s t o a p p r o a c h t o t h e m e c h a n i s m of i n h i b i t o r y effect of r u t i n on s m o o t h m u s c l e in v i t r o . T h e p r e v i o u s results o b t a i n e d o n t h e g u i n e a - p i g i l e u m s h o w e d t h a t r u t i n exerts non-specific i n t e r a c t i o n s a n g i o t e n s i n I I a n d p r o s t a g l a n d i n E2 t h r o u g h a c o m m o n m e c h a n i s m (2). A c c o r d i n g to t h e c u r r e n t c o n c e p t s (8), if a d r u g exerts n o n - c o m p e t i t i v e a n d n o n - s p e c i f i c i n t e r a c t i o n s w i t h s o m e agonists t h r o u g h a c o m m o n m e c h a n i s m o n a n isolated o r g a n , i t s h o u l d possibly a c t o n s a m e r e c e p t o r c o u p l e d effector system. T h e r e -fore, r u t i n m a y asct o n such a n effector system i n r e g a r d t o s m o o t h muscle r e l a x a n t a c t i o n . A t this p o i n t , i t s h o u l d b e t h o u g h t w h a t t h e effector system is.
C a t e c h o l a m i n e s m a y c a u s e r e l a x a t i o n s o f s o m e s m o o t h muscles ( 8 - 1 4 ) , s o m e v a s c u l a r s m o o t h muscles (8, 1 5 - 1 7 ) , u t e r u s (8, 1 8 - 2 3 ) . T h e a c t i o n o f c a t e c h o l a m i n e s son these muscles i s m a i n l y d u e t o b e t a
-Orhan ALTINKURT, Yusuf ÖZTÜRK a d r e n o c e p t o r a c t i v a t i o n a n d a b l e t o b e b l o c k e d b y b e t a - a d r e n e r g i c b l o c k i n g a g e n t s ( 1 2 1 4 , 17, 19). Cyclic a d e n o s i n e 3 ' , 5 ' m o n o s p h o s -p h a t e (cyclic A M P ) i s believed t o b e t h e i n t r a c e l l u l a r m e d i a t o r o f t h e effect o f b e t a a d r e n o c e p t o r a c t i v a t i o n i n t h e s m o o t h muscles m e n t i o n e d a b o v e ( 1 2 2 3 ) . O n t h e o t h e r h a n d , a l p h a a d r e n o c e p t o r a c t i -v a t i o n m a y also cause r e l a x a t i o n o f i n t e s t i n a l s m o o t h m u s c l e (9, 1 2 - 1 4 ) . T h i s r e l a x a t i o n is m o s t p r o b a b l y r e s u l t i n g from c a l c i u m efflux. In o u r e x p e r i m e n t s , r u t i n (in a c o n c e n t r a t i o n r a n g e of 5 g / m l t o 160 u g / m l ) c a u s e d r e l a x a t i o n s o f r a t d u o d e n u m d o s e - d e p e n d e n t l y . T h e pD2 v a l u e w h i c h is d i r e c t m e a s u r e for t h e affinity of t h e d r u g s to site of a c t i o n was c a l c u l a t e d for t h e r e l a x a n t a c t i o n s of a d r e n a l i n e , i s o p r e n a l i n e , p a p a v e r i n e a n d r u t i n o n t h e r a t d u o d e n u m b y m e a n s o f A R I E N S a n d V A N R O S S U M ' s p r o c e d u r e (5). T h e r e l a x a n t effect o f r u t i n o n t h e r a t d u o d e n u m w a s e x a m i n e d b y c o m p a r i s o n o f these p D2 v a l u e s ( T a b l e I ) .
T a b l e I . Affinity constants ( p D2) a n d slopes o f d o s e - r e s p o n s e curves ( m ) o f a d r e n a l i n e , i s o p r e n a l i n e , p a p a v e r i n e a n d r u t i n o n t h e isolated r a t d u o d e n u m . R e l a x a n t A g e n t p D2 ± S E M m ± S E M A d r e n a l i n e I s o p r e n a l i n e P a p a v e r i n e R u t i n 6 . 8 7 3 ± 0 . 0 0 8 8 . 2 4 2 ± 0 . 0 0 4 7 . 4 9 8 ± 0 . 0 0 1 4 . 6 7 1 ± 0 . 0 0 1 7 0 . 9 0 4 ± 0 . 2 8 8 7 1 . 4 1 8 ± 0 . 3 6 0 7 2 . 8 7 9 ± 0 . 2 8 2 3 5 . 2 0 7 ± 0 . 0 5 2 S E M : S t a n d a r d e r r o r o f m e a n ( n = 1 2 for e a c h r e l a x a n t a g e n t ) P h e n t o l a m i n e , a l p h a - a d r e n e r g i c b l o c k i n g a g e n t , i n h i b i t e d t h e r e l a x a t i o n s elicited by all agonists, n o n - c o m p e t i t i v e l y . It is likely t h a t these i n h i b i t i o n s a r e n o t d u e t o a l p h a - a d r e n e r g i c b l o c k a d e , b e c a u s e t h e c o n c e n t r a t i o n o f p h e n t o l a m i n e w h i c h w a s tested i n this s t u d y i s h i g h e r t h a n t h e c o n c e n t r a t i o n r e q u i r e d for a l p h a - a d r e n e r g i c b l o c k a d e . C o n f i r m a t i v e l y , i t i s w e l l - k n o w n t h a t p a p a v e r i n e does n o t possess a n y a d r e n e r g i c activity a n d its effect on s m o o t h m u s c l e is also n o t d u e to c a t e c h o l a m i n e release. H o w e v e r , t h e r e l a x a t i o n o f r a t d u o d e n u m elicited b y p a p a v e r i n e w e r e i n h i b i t e d b y p h e n t o l a m i n e . A c c o r d i n g t o o u r u n p u b l i s h e d results, p h e n t o l a m i n e causes a n i n h i b i t i o n o f c a l c i u m -i n d u c e d c o n t r a c t -i o n o f K + - d e p o l a r -i z e d r a t d u o d e n u m (24). T h e r e f o r e i t likely t h a t t h e i n h i b i t o r y effect o f p h e n t o l a m i n e o n t h e r a t d u o d e n u m a g a i n s t a d r e n a l i n e , i s o p r e n a l i n e , p a p a v e r i n e a n d r u t i n a c c u r s a s a c o n s e q u e n c e of this c a l c i u m a n t a g o n i s t i c a c t i o n .
P r o p r a n o l o l , b e t a a d r e n e r g i c b l o c k i n g a g e n t i n h i b i t e d t h e r e l a x -a t i o n s elicited b y -a d r e n -a l i n e -a n d i s o p r e n -a l i n e , n o n - c o m p e t i t i v e l y , b u t d i d n o t i n h i b i t r u t i n a n d p a p a v e r i n e r e l a x a t i o n s . T h i s f i n d i n g c o n f i r m s t h e results o b t a i n e d b y A N T O N I O (11). S e e i n g t h a t r u t i n - i n d u c e d r a t d u o d e n u m r e l a x a t i o n s w e r e n o t i n h i b i t e d b y p r o p r a n o l o l , i t m i g h t b e t h o u g h t t h a t t h e r e l a x a n t effect o f r u t i n o n s m o o t h m u s c l e does n o t o c c u r as a result of c a t e c h o l a m i n e relaease or c a t e c h o l a m m e - l i k e a c t i o n . A n o t h e r i n t e r s t i n g p o i n t was t h a t , n i c o t i n i c acid i s a b l e t o i n h i b i t a d r e n a l i n e - a n d i s o p r e n a l i n e - i n d u c e d r e l a x a t i o n s o f r a t d u o d e n u m . T h e s e i n h i b i t i o n s a r e c o m p e t i t i v e i n n a t u r e . I t i s k n o w n t h a t n i c o t i n i c acid is an i n h i b i t o r of a d e n y l cyclase (25). T h e r e f o r e , these c o m p e t i t i v e i n h i b i t i o n s s h o u l d n o t be a surprise, since b e t a - a d r e n o c e p t o r s a r e c o u p l e d b y a d e n y l cyclase. I n fact, i t h a s b e e n suggested t h a t b e t a -a d r e n o c e p t o r s -a r e i d e n t i c -a l w i t h -a d e n y l cycl-ase (26). H o w e v e r , u n d e r t h e l i g h t of r e c e n t studies, it seems likely t h a t t h e suggestion is o u t of fashion (27). O n t h e o t h e r h a n d , n i c o t i n i c acid d i d n o t affect t h e r e l a x a n t effect o f r u t i n o n t h e r a t d u o d e n u m a s well a s p a p a v e r i n e . T h i s f i n d i n g s t r o n g l y suggests t h a t t h e i n h i b i t o r y effect o f r u t i n o n s m o o t h m u s c l e i s n o t o w i n g t o a d e n y l cyclase a c t i v a t i o n . T a b l e I I shows t h e p D2' a n d p A2 v a l u e s r e p r e s e n t i n g t h e i n t e r a c t i o n s o f a d r e -n a l i -n e , i s o p r e -n a l i -n e , p a p a v e r i -n e a -n d r u t i -n w i t h p h e -n t o l a m i -n e , p r a n o l o l a n d n i c o t i n i c acid o n t h e r a t d u o d e n u m . C o n f i r m a t i v e l y , i t h a s b e e n o b s e r v e d t h a t t h e f l a v o n o i d c o m p o u n d s i n h i b i t p h o s p h o d i e s t e r a s e ( P D E ) w h i c h i s k n o w n a s cyclic A M P -d e g r a -d i n g e n z y m e (28). S i m i l a r l y , p a p a v e r i n e w h i c h i s w e l l - k n o w n P D E i n h i b i t o r causes s m o o t h m u s c l e r e l a x a t i o n (29). T h e r e f o r e , i t m i g h t b e p r o p o s e d t h a t r u t i n - i n d u c e d r e l a x a t i o n i s r e l a t e d w i t h P D E i n h i b i t i o n . H o w e v e r , t h e slope of r u t i n c a l c u l a t e d from its dose-res-p o n s e curves is m u c h less t h e n those of a d r e n a l i n e , i s o dose-res-p r e n a l i n e a n d p a p a v e r i n e ( T a b l e I ) suggesting t h e difference i n t h e m e c h a n i s m o f a c t i o n of r u t i n . T h e r e f o r e , it w o u l d be of i n t e r e s t to i n v e s t i g a t e possible i n t e r f e r e n c e o f c a l c i u m ions a n d c a l m o d u l i n w h i c h a r e i m p o r t a n t fac-tors i n t h e s m o o t h m u s c l e r e g u l a t i o n .
51 O r h a n A L T I N K U R T , Yusuf Ö Z T Ü R K
T a b l e . I I . Effects of p r o p r a n o l o l , p h c n t o l a m i n e a n d nicotinic acid on a d r e n a l i n e - , isopre-n a l i isopre-n e - , p a p a v e r i isopre-n e - a isopre-n d r u t i isopre-n - i isopre-n d u c e d relaxatioisopre-ns of the isolated r a t d u o d e isopre-n u m iisopre-n vitro.
P h e n t o l a m i n e p D '2 ± S E M P r o p r a n o l o l p D '2 ± S E M Nie. Acid. p A , ± S E M R e l a x a n t Agent p D 'P h e n t o l a m i n e 2 ± S E M P r o p r a n o l o l p D '2 ± S E M Nie. Acid. p A , ± S E M A d r e n a l i n e 4 . 0 2 5 ± 0 . 0 6 4 5.141 ± 0 . 0 1 3 3.852 ± 0 . 0 8 9 Isoprenaline 4 . 0 3 1 ± 0 . 0 4 2 4 . 9 1 5 ± 0 . 0 5 8 3 . 8 5 7 ± 0 . 1 2 1 P a p a v e r i n e 4 . 0 6 9 ± 0 . 1 0 3 2 . 6 5 0 0 . 8 4 8 R u t i n 3.902 ± 0 . 0 4 3 2 . 7 7 6 0 . 8 6 7
p D '2 : Non-competitive antagonist affinity constant
p A '2 : C o m p e t i t i v e antagonist affinity constant
S E M : S t a n d a r d e r r o r of m e a n ( n = 5 for each set of experiment)
L I T E R A T U R
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4- H o r t o n , E., H u m a n u r i n a r y kinin excretion, Br. J. Pharmacol., 14, 125-132 (1959). 5 A r i e n s , E.J., V a n R o s s u m , J . M . , p D x , p A x a n d p D ; x values i n analysis o f p h a r m a
-c o d y n a m i -c s , Ar-ch. Int. Pharma-codyn. Ther., 110, 275-299 (1957).
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(1980),
8- V o g i n , E.E., B a k e r , W.W., Dichloroisoproterenol a n d the b e t a - a d r e n e r g i c receptor,
Am. J. Pharm., 133, 314-326 R ( 1 9 6 1 ) .
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YAYIM K O Ş U L L A R I
1- Bu dergide aşağıda belirtilen k o ş u l l a r a u y a n ve bilimsel d a n ı ş m a n l a r ı n
yayımla-nabilir nitelikte b u l d u ğ u , d a h a ö n c e hiçbir y e r d e y a y ı m l a n m a m ı ş o l a n orijinal m a k a l e l e r yayımlanır.
2 Metinler, şekiller h a r i ç 10 d a k t i l o sayfasını geçmemelidir. Y a z ı l a r ı n dil k u r a l l a -r ı n a u y g u n l u ğ u n d a n y a z a -r l a -r s o -r u m l u d u -r .
3- M e t i n l e r 3 n ü s h a o l a r a k A-4 f o r m a t t a (21.0 x 29.7 c m ) k a ğ ı d ı n b i r y ü z ü n e n o r m a l p u n t o l u d a k t i l o ile 2 aralıklı o l a r a k yazılmalı, k a ğ ı d ı n alt ve üst k e n a r ı n d a n 2 c m , sol ke-n a r ı ke-n d a ke-n 3 cm ve sağ k e ke-n a r ı ke-n d a ke-n 1.5 cm b o ş l u k bırakılmalıdır. Klişesi y a p ı l a c a k grafik, ş e m a , f o r m ü l gibi şekiller aydınger k a ğ ı d ı n a çini m ü r e k k e b i ile çizilmeli; fotoğraflar p a r -lak k a ğ ı d a k o n t r a s l ı o l a r a k basılmalıdır. H e r şeklin a r k a s ı n a y a z a r a d ı , şekil n o . su ve kü-ç ü l t m e o r a n ı yazılmalı ve 2., 3. n ü s h a l a r ikü-çin şekillerin fotokopisi eklenmelidir.
4- Yazı plânı aşağıdaki şekilde o l m a l ı d ı r :
M a k a l e başlığı, m a k a l e n i n y a b a n c ı dilde başlığı, y a z a r a d ı (yazar adları), T ü r k ç e özet y a b a n c ı dilde özet, a n a h t a r kelimeler, giriş, denel kısım, s o n u ç ve t a r t ı ş m a , teşekkür, lite-r a t ü lite-r .
Özetler t ü r k ç e ve y a b a n c ı dilde en ç o k 100'er kelimeden oluşmalıdır.
Giriş b ö l ü m ü en ç o k 2 sayfa ve başlıksız o l a r a k yazılmalıdır. M a t e r y a l ve M e t o d alt başlık-lar h a l i n d e , D e n e l k ı s ı m d a b u l u n m a l ı d ı r .
S o n u ç v e T a r t ı ş m a kısmı bulguları d a içermelidir.
Literatürler, m a k a l e d e p a r a n t e z içindeki n u m a r a l a r l a belirtilmeli v e m a k a l e s o n u n d a b u n u m a r a l a r a g ö r e sıralanmalıdır.
L i t e r a t ü r l e r : Yazarın Soyadı (siyah), a d ı n ı n ilk harfi (siyah), m a k a l e adı, derginin adı (ita-lik) veya k i t a b ı n a d ı , cilt no (siyah) sayı ( p a r a n t e z içinde), sayfa n u m a r a s ı ve yıl ( p a r a n t e z içinde) o l a r a k yazılmalıdır.
5- Tashihler y a z a r t a r a f ı n d a n yapılacaktır. 6- Y a z a r a 20 ayrı baskı verilir.
