A study of the cardiac glucosides with special emphasis on oleandrin

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A STUDY OF THE CARDIAC GLUCOSID1S WITH SPECIAL EMPHASIS ON OLEANDRIN

A T h e sis P r e s e n te d to th e F a c u lty o f th e C o lle g e o f Pharmacy The U n i v e r s i t y of S o u th e rn C a l i f o r n i a

In P a r t i a l F u lfillm e n t of th e R equirem ents f o r th e Degree M aster of S c ie n c e

by M o rris W olfred June 1943

UMI Number: EP63458

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ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

This thesis, written by

under the direction of h%3.. Faculty Committee, a n d a p p r o v e d by a l l its m e m b e r s , has been presented to and accepted by the Council on Graduate S tu d y and Research in partial fulfill­ m e n t of th e r e q u i r e m e n t s f o r the d e g r e e o f

MASTER OF SCIENCE

Dean

Secretary

Date.13.BRUARY,1942

Faculty Committee

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/> Chairman

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TABLE OF CONTENTS

CHAPTER I.

PAGE

INTRODUCTION.............................................................................. E a rly u s e s o f g l u c o s i d e s

...........................

1 1

F u n c tio n perfo rm ed by th e g l u c o s i d e s i n th e

II.

p l a n t .................................................................................

1

The t o x i c i t y o f O le a n d rin .............................................

2

COMPOSITION.............................................................................

5

E m p iric a l form ula

..........................................................

Methods o f h y d r o l y s i s

III.

IV.

V.

V I.

3

..................................................

3

Comparison t o d i g i t a l i s g lu c o s id e s .......................

4

The a l k a l o i d N e r iin e ......................................................

4

D e te rm in a tio n o f chem ical s t r u c t u r e

...................

5

PROCEDURES USED FOR EXTRACTION.....................................

6

W ater s o l u b l e s a l t s o f th e g lu c o s id e .....................

6

COLLECTION AND PREPARATION ..............................................

9

Enzymatic e f f e c t ...............................................................

10

Tem perature e f f e c t ...................................

10

EXTRACTION OF THE DRUG.......................................................

12

Methods u s e d ........................................................................

12

ASSAY OF THE D R U G ................................................................

15

Chick h e a r t m e t h o d ..........................................................

15

R e s u l t s .................................................................................

17

G r a p h s .....................................................................................

19

iii

CHAPTER V II .

PAGE

CONCLUSIONS............................................................................

22

F i g u r e s .................................................................................

23

REFERENCES..........................................................................................

26

CHAPTER I

INTRODUCTION Nerium O le a n d e r, a n a t i v e p l a n t o f A s ia , h a s been in tr o d u c e d as an o rn a m e n ta l shru b i n th e U n ite d S t a t e s and many o f th e warmer c o u n t r i e s o f th e w o rld .

I n th e n o r th e r n

p a r t o f t h e U n ite d S t a t e s i t i s sometimes grown a s a f l o w e r ­ in g house p l a n t .

O leand er b e lo n g s t o t h e Apocynaceae f a m ily ,

and was d e s c r ib e d i n th e w r i t i n g s o f H ip p o c ra te s and by a n c i e n t documents which show t h a t th e knowledge o f th e p o iso n o u s p r o p e r t i e s o f g l u c o s i d e s i s v e r y o l d .

P rim itiv e

t r i b e s s in c e days o f o ld , have used such g l u c o s i d e s t o p r e ­ p a re p o iso n a rro w s, and p r i m i t i v e t r i b e s to d a y i n u n c i v i l i z e d p a r t s of th e w orld s t i l l c o n tin u e t h i s p r a c t i c e .

The p o is o n ­

ous p r i n c i p l e s o f O le a n d rin a r e s i m i l a r t o th o s e p r o p e r t i e s o f th e d i g i t a l i s g l u c o s i d e s , and t h e chem ical s t r u c t u r e of t h e c a r d i a c g l u c o s i d e s t h a t have been i n v e s t i g a t e d i n d e t a i l , show t h a t th e y a r e a l l h y d ro x y la c to n e s o f s t e r o l h y d ro ­ c a rb o n s i n which one h y d ro x y l group i s connected w ith a s u g a r m o lecule o r a c h a in o f s e v e r a l s u g a r s . The f u n c t i o n perform ed by th e c a r d i a c g l u c o s i d e s i n p l a n t s i s a problem t h a t has n o t y e t been s o lv e d , b u t s e v e r a l s p e c u l a t i o n s have been ad v a n ced .

I t seems p o s s i b l e t h a t

th e s e s u b s ta n c e s s e rv e a s a guard a g a i n s t verm in and p r o t e c t th e p l a n t a g a i n s t p a r a s i t e s , b u t o b j e c t i o n s may be r a i s e d

2

toward t h e s e t h e o r i e s and th e c a r d i a c g l u c o s id e s c o n s id e re d m e re ly as m e ta b o lic w aste p r o d u c ts .

F u rth erm o re i t i s

p o s s i b l e t h a t t h e p l a n t u s e s th e s o l u b l e g l u c o s i d a l form s in o r d e r t o t r a n s p o r t s p a r i n g l y s o lu b l e s t e r o l l a c t o n e s from one p a r t o f th e p l a n t t o a n o t h e r , o r th e p l a n t may s t o r e s u g a rs o r s t e r o l s i n th e form of such g l u c o s i d e s . The d i s t r i b u t i o n o f th e c a r d i a c g lu c o s id e s i n th e p l a n t v a r i e s from ty p e to ty p e and d e te rm in e s which p a r t of th e p l a n t i s used a s th e raw m a t e r i a l f o r t h e i r i s o l a t i o n . I n th e c a s e o f O lean d er, th e l e a v e s a r e u t i l i z e d ; i n S tro p h a n th u s , th e s e e d s ; i n S q u i l l , th e b u lb s ; i n C onvall a r i a , m a in ly th e f l o w e r s ; and Ouabaio, th e wood and th e bark. ^ F or c e n t u r i e s th e O leander p l a n t has been known t o be p o iso n o u s, t h e l e a v e s b e in g h ig h ly t o x i c b o th in th e g re e n and d ry c o n d i t i o n .

C a t t l e , h o r s e s , sh ee p , and c h ic k e n s have

been p o iso n ed by e a t i n g th e l e a v e s .

A few c a s e s of p o is o n ­

in g have been r e p o r t e d among humans, and o t h e r s r e l a t e t h a t c o n t a c t w ith th e le a v e s a l s o produced a d e r m i t i t i s i n c e r ­ ta in in d iv id u a ls .

Symptoms of p o is o n in g a r e —n a u s e a ,

v o m itin g , c o l i c , d i z z i n e s s , d ro w s in e s s , d e c re a se d p u ls e r a t e , i r r e g u l a r h e a r t a c t i o n , marked m y d ria s e s , bloody d i a r r h e a , u n c o n s c io u s n e s s , r e s p i r a t o r y p a r a l y s i s and f i n a l l y d e a t h .^

CHAPTER I I

COMPOSITION Under e a r l y i n v e s t i g a t i o n th e a c t i v e p r i n c i p l e s o f Nerium O leander was th o u g h t t o be an a l k a l o i d , b u t f u r t h e r r e s e a r c h showed t h a t a g lu c o s id e c a l l e d O le a n d rin , o b ta in e d from th e l e a v e s , was th e main c o n s t i t u e n t .

O le a n d rin

r o t a t e s th e p la n e of p o l a r i z e d l i g h t t o th e l e f t and has a m o le c u la r w e ig h t r e p r e s e n te d by th e form ula

It

i s r e s i s t a n t t o d i l u t e a c id s and i s slo w ly s p l i t i n t o an ag lu co n c a l l e d O le a n d rig e n in .

O le a n d rig e n in t o which p r e ­

v i o u s l y th e form u la CggH^gC^was a s c r i b e d was shown t o have th e co m p o sitio n

and was o b ta in e d a l s o by t r e a t i n g

g i t o x i g e n i n w i t h a c e t i c an h y d rid e i n p y r i d i n e a t 0° C.

When

O lea n d rin was b o i l e d w ith HC1 i n an aqueous s o l u t i o n of m ethyl a l c o h o l , i t y ie l d e d o l e a n d r ig e n in p l u s o le a n d r o s e . The c o n s t i t u t i o n of o le a n d r ig e n in and O le a n d rin was e s t a b ­ l i s h e d in th e fo llo w in g m anner.

O le a n d rig e n in y i e l d e d on

o x id a tio n w ith CrO^ i n g l a c i a l a c e t i c a c id o le a n d rig e n o n e w ith a m e ltin g p o i n t 250 - 2° C. which gave on t r e a tm e n t w ith c o n c e n tr a te d HC1 d ia n h y d ro o le a n d rig e n o n e , w hich was i d e n t i c a l w ith d i g i ta lg e n o n e a s pro v en by th e mixed m e ltin g p o i n t and th e a b s o r p t i o n c u r v e . 3

These r e s u l t s showed t h a t o l e a n d r i ­

g e n in c o n ta in e d a f r e e h y d ro x y l group a t th e t h i r d carbon which was o x id iz e d t o a k eto n e g ro u p , and th e a c e t y l r e s t was

4

a t th e s i x t e e n t h ca rb o n .

O le a n d rin c o n ta in e d t h e r e f o r e th e

s u g a r o le a n d ro s e a t t a c h e d to th e t h i r d ca rb o n .

0 / e an J r fj

G30H46°9 * H2° O le a n d rin

en

/

------G23H34°5 + G7H14°5 G ito x ig e n in D i g i t a l o s e

As h as been p ro v en , O le a n d rin i s v e ry c l o s e l y r e l a t e d to th e d i g i t a l i s g ro u p in g s .

A ccording t o Windaus, on h y d r o l ­

y s i s of O le a n d rin , t h i s g lu c o s id e d i f f e r s from d i g i ta l i n u m verum

on ly by th e l o s s of a g lu c o s e m o le c u le .

The g lu c o s id e G ito x in (from d i g i t a l i s ) h y d ro ly z e s p r a c t i c a l l y i d e n t i c a l t o O le a n d rin . °41H64°14 * 3H2° G ito x in

G23H34 + 3G6H12°4 G ito x ig e n in D ig ito x o se

G i t a l i n (a n o th e r g lu c o s id e from d i g i t a l i s ) can a l s o be con­ v e r t e d by d e h y d r a tio n i n t o g i t o x i g e n i n h y d r a t e . 3 M ention i s a l s o made i n t h e l i t e r a t u r e of an a l k a l o i d which was found i n O leander by Schm iedeberg. s u b s ta n c e N e r i i n e .

He named t h i s

N e r iin e e x i s t s i n t h e form of a y e llo w ­

i s h w h ite powder, v e ry b i t t e r , nonhydroscopic and e x tre m e ly s o l u b l e i n HgO, a lc o h o l and a c e to n e , and i n s o l u b le i n e t h e r , c h lo ro fo rm and b e n z in e .

In c o n t a c t w ith H2S04 one drop of

t h i s s o l u t i o n of N e r iin e l/lOOO c o n c e n t r a t i o n g iv e s an orange y e llo w c o l o r a t i o n which s lo w ly d e v e lo p s i n t o a r o s e l i k e c o l o r t h a t may he o b ta in e d im m ed iately by o x i d a t i o n , u s in g th e v a p o rs o f n i t r o g e n o x id e and brom ine.

The s e n s i ­

b i l i t y of t h i s r e a c t i o n i s common t o th e s tr o p h a n th u s f a m ily and o u a b a in e .

The a c t i o n i s so s e n s i t i v e t h a t i t can be

d e t e c te d i n 1/2000 c o n c e n t r a t i o n of a m illig r a m of N e r iin e so lu tio n .

The c o l o r a c t i o n i s due t o th e d i f f e r e n c e i n

g lu c o s e p o ly m e r iz a tio n . The g lu c o s id e (O le a n d rin

has th e fo llo w in g

p h y s i c a l p r o p e r t i e s : w h ite n e e d le s from 50 p e r c e n t a l c o h o l ; p r a c t i c a l l y i n s o l u b l e i n w a te r and m ethyl a l c o h o l ; and s o l u b l e i n a l c o h o l , e t h e r and c h lo ro fo rm .

I t a l s o h as a

m e ltin g p o i n t o f 249° C - 250° C and { ° c )D - 4 5 . 9 5 . 4

CHAPTER I I I

PROCEDURES USED FOR EXTRACTION P ro c e s s e s f o r th e p ro d u c tio n of g l u c o s i d e s from O leander l e a v e s , which have an a c t i o n on th e h e a r t , have o f t e n been d e s c r i b e d .

The g e n e r a l p ro c e d u re i s as f o llo w s :

th e aqueous o r aqueous a l c o h o l i c e x t r a c t s r e q u ir e d in ob­ t a i n i n g th e s u b s ta n c e h av in g an a c t i o n on th e h e a r t a r e p u r i f i e d w ith l e a d a c e t a t e and th e f i l t r a t e s a r e f u r t h e r t r e a t e d t o o b t a in g l u c o s i d e s i n s o l i d form .

The y i e l d s

g r e a t l y v a r y and i n th e most f a v o r a b le c a se amount to about .15 p e r c e n t of th e l e a v e s u s e d . S u b s ta n c e s h av in g an a c t i o n on t h e h e a r t may be ob­ t a i n e d from O leander le a v e s by e x t r a c t i o n w ith a s u i t a b l e so lv e n t.

The aqueous a l c o h o l i c e x t r a c t s a r e o b ta in e d by

th e u s u a l m ethods from th e d r i e d o r f r e s h l e a v e s ; s u i t a b l e s o l v e n t s a r e u sed e s p e c i a l l y C h lo rin a te d A l i p h a t i c h y d ro ­ ca rb o n s such a s CHClg, CC14 , or m ethylene c h l o r i d e .

From

th e e x t r a c t of t h e s e s o l v e n t s , th e e f f e c t i v e s u b s ta n c e s a r e p r e c i p i t a t e d by a d d i t i o n o f s a t u r a t e d o rg a n ic h y d ro c arb o n s, such a s b e n z in e , p e tro le u m e t h e r , c y c lo h e x a n e , o c ta n e , nonane, and d ec an e, t h e s e b e in g most s a t i s f a c t o r y .

A fte r

t h i s s e p a r a t i o n th e e f f e c t i v e s u b s ta n c e may a g a in be d i s ­ s o lv e d and r e p r e c i p i t a t e d .

For example th e cru d e p ro d u c t

may be d i s s o l v e d i n benzene and s e p a r a te d i n a p u r i f i e d

form by th e a d d i t i o n of one o f t h e above named o rg a n ic l i q u i d s such as cy clohexan e o r b e n z in e . Now i t has been found t h a t th e p r o d u c ts o b ta in e d a c c o rd in g to th e above p ro c e s s may be s e p a r a te d by means of e t h e r i n t o g l u c o s i d e s r e a d i l y s o l u b le i n e t h e r and g l u c o ­ s i d e s i n s o l u b l e in e t h e r , b o th k in d s of components h av in g an a c t i o n on th e h e a r t .

The g l u c o s i d a l f r a c t i o n s s e p a r a te d

by means o f e t h e r a r e r e a l g l u c o s i d e s a s th e y can be broken down by means o f a c i d s i n t o g e n in e s (n o t y e t d e f in e d ) and su g ar.

The p r o d u c tio n o f g l u c o s i d e s r e a d i l y s o lu b l e i n

w a te r and h av in g an a c t i o n on th e h e a r t have alw ays been em phasized; t h e r e f o r e , a g lu c o s id e such a s O le a n d rin which i s i n s o l u b l e i n w a te r and hav in g t h e same a c t i o n i s s u r ­ p risin g . The W inthrop Chem ical Company has a p ro c e s s f o r p r e ­ p a r in g hydrogen e s t e r s (su ch s a l t s b e in g r e a d i l y s o l u b l e i n w a te r) of th e g lu c o s i d e s h a v in g an a c t i o n on th e h e a r t .

They

have found t h a t compounds r e a d i l y s o lu b le in w a te r may be o b ta in e d by c o n v e r tin g c a r d i a c g lu c o s i d e s i n t o d e r i v a t i v e s which a r e s a l t s o f a c id e s t e r s .

These compounds have a d d i ­

t i o n a l ad v a n ta g e t h a t th e y a r e e x c e l l e n t l y t o l e r a t e d .

The

new compounds a r e made by ca u sin g h a l i d e s o r e s t e r a n h y d rid e s of o rg a n ic and in o r g a n ic a c i d s ( e s p e c i a l l y phosphorous o x y c h lo rid e and phosphorous t r i c h l o r i d e ) i n th e p re se n c e of a c id b in d in g s u b s ta n c e s such a s p y r i d i n e o r d i m e t h y l a n i li n e

to r e a c t w ith g lu c o s i d e s and by tr a n s f o r m in g t h e p r o d u c ts o f th e r e a c t i o n i n t o s a l t s . T h e ir method u sed i s a s f o l l o w s : 1 gram O le a n d rin i s d i s s o l v e d i n 10 c c . o f p y r i d i n e and .27 grams o f phosphorus o x y c h lo rid e d i s s o l v e d i n 55 c c . p y r id i n e i s added w h ile c o o lin g .

The whole i s allo w ed t o sta n d f o r some h o u rs f i r s t

w h ile c o o lin g , th e n a t room te m p e ra tu r e , th e n ic e d and a s o l u t i o n o f 1 .2 5 grams of sodium a c e t a t e in 5 c c . o f w a te r i s th e n added and t h e whole i s b ro u g h t t o d ry n e ss a t a low t e m p e r a tu r e .

The r e s i d u e i s d is s o lv e d i n w a te r and th e s o l u ­

t i o n a c i d i f i e d w ith d i l u t e HG1.

The a c id which s e p a r a t e s a t

once i s f i l t e r e d w ith s u c t i o n , washed w ith w a te r and n e u t r a l ­ iz e d w ith NagCOrj s o l u t i o n .

The c l e a r s o l u t i o n i s e v a p o ra te d

to d ry n e s s and th e r e s i d u e mixed w ith OgH^OH, f i l t e r e d and washed a g a in w i t h CgH^OH and w ith e t h e r .

The p r o d u c ts a re

w h ite s u b s ta n c e s r e a d i l y s o lu b l e in w a t e r . 5

CHAPTER IV

COLLECTION AND PREPARATION The l e a v e s f o r t h i s ex p erim en t were p ic k e d from O leander t r e e s growing on t h e campus of th e U n i v e r s i t y or from t h e a d j a c e n t park*

These O leander t r e e s were i n v e s t i ­

g a te d and found t o be of th e same o f f i c i a l s p e c ie s used by many o t h e r i n v e s t i g a t o r s , namely Nerium Oleander*

The t r e e s

however, were c u l t i v a t e d m a in ly f o r th e p u rpo se o f ornamen­ t a t i o n r a t h e r th an f o r t h e i r m e d ic in a l v a l u e , a s t h i s p l a n t grows r e a d i l y i n S o u th e rn C a l i f o r n i a .

The l e a v e s were

p ic k e d d u rin g th e months betw een Septem ber and J a n u a ry , and were o b ta in e d from f u l l y m atu red t r e e s , ra n g in g i n h e ig h t from e i g h t to tw elve f e e t .

The t r e e s c o n ta in e d blossom s

th ro u g h o u t t h i s p e r io d and t h e r e were no s ig n s of d e g e n e ra ­ t i o n o r fungus grow th on t h e le a v e s o r any p a r t o f t h e p l a n t The tim e of day a t which th e le a v e s were p ic k e d did no t m a t e r i a l l y a f f e c t th e y i e l d o f th e g l u c o s i d e ; however, th e y were p ic k e d a t v a r io u s tim e s in th e morning betw een th e h o u rs o f e i g h t A.M. to tw e lv e noon. A s e r i e s o f ex p e rim e n ts were a l s o c a r r i e d o u t t o d e te rm in e th e d i f f e r e n c e in g l u c o s i d a l c o n te n t betw een th e o ld and th e young le a v e s and th e sm a ll and th e l a r g e l e a v e s . R e s u lts showed t h a t a b e t t e r y i e l d may be o b ta in e d from th e s m a l l e r younger l e a v e s .

These r e s u l t s may have been due to

10

e x p e rim e n ta l e x t r a c t i o n m ethods, and u n t i l a more u n ifo rm a s s a y i s e s t a b l i s h e d , one may n o t be c e r t a i n of th e above statem en t.

Many more i n v e s t i g a t i o n s must be c a r r i e d out on

a s ta n d a r d a s s a y method to p ro v e t h e s e r e s u l t s ; however, t h e r e i s a s tr o n g i n d i c a t i o n t h a t th e g l u c o s i d a l c o n te n t v a r i e s betw een s i z e and age o f th e l e a v e s . The le a v e s were chosen f o r t h i s work b ecause e x p e r i ­ m e n ta l e v id e n c e had pro ven t h a t th e y c o n ta in th e h i g h e s t p e rc e n ta g e o f g lu c o s i d e .

They were p ic k e d by hand, s o r t e d

and im m ediately i n v e s t i g a t e d t o p r e v e n t th e d e c o m p o sitio n o f th e g lu c o s id e by enzymes i n th e p l a n t .

The le a v e s were

th e n c o a r s e l y ground and th e e x p r e s s e d sap added to th e ground d ru g .

The grounds were th e n p la c e d i n a p e r c o l a t o r

and th e g l u c o s i d e e x t r a c t e d by methods h e r e i n d e s c r i b e d . The drug need n o t be m a ce rated more th a n t w e n ty - f o u r ho urs w ith th e p ro p e r s o l v e n t a s r e s u l t s have prov en t h a t th e menstruum c o n t a i n s 85 p e r c e n t of th e e x t r a c t e d O le a n d rin g lu c o sid e .

During th e p r o c e d u r e s , n e u t r a l s a l t s were added

to p re v e n t enzym atic a c t i o n from t a k i n g p la c e d u rin g t h e m a c e ra tin g p e r i o d .

Ammonium

s u l f a t e proved to be th e most

s a t i s f a c t o r y n e u t r a l s a l t f o r enzym atic d e s t r u c t i o n and re ta rd a tio n . At tim e s th e l e a v e s were d r i e d im m ediately a f t e r p ic k in g i n d ry a i r c o n t a i n e r s which ranged in te m p e ra tu re from 40 - 60° C. (d ry a i r t h e r m o s ta t was c o n t r o l l e d by

11

a llo w in g com pressed a i r t o p a s s th ro u g h copper h e a te d tu b in g in to a g l a s s c o n t a i n e r f i t t e d w ith e x t e r n a l o u t l e t s which allo w ed f r e e c i r c u l a t i o n th ro u g h o u t th e d ry in g )*

The d ry in g

p e r io d ranged from f o u r t o e i g h t h o u rs , and lo n g e r p e r io d s were av o id ed f o r f e a r o f g l u c o s i d a l d e s t r u c t i o n *

R e s u lts

showed t h a t th e d r i e d le a v e s d id n o t y i e l d th e same q u a n t i t y o f g lu c o s id e a s th e f r e s h o n e s, and t h a t th e y i e l d was con­ t i n u o u s l y lower*

These o b s e r v a tio n s a l s o showed t h a t th e

g lu c o s id e i s e i t h e r a f f e c t e d by enzymes or t h a t h e a t i n g te n d s t o d e s t r o y i t o r m a t e r i a l l y a l t e r i t s p r e p a r a t i o n and p ro p e rtie s*

CHAPTER V

EXTRACTION OF THE DRUG 1.

20 grams o f th e g l u c o s i d e m ix tu re d e s c r i b e d i n

th e p re v io u s c h a p te r i s s t i r r e d f o r one h o u r w ith 200 c c . of e t h e r , th e u n d is s o lv e d p o r t i o n s o f th e m ix tu r e a r e s e p a ­ r a t e d by f i l t e r i n g w ith s u c t i o n , th e e t h e r i s d i s t i l l e d from th e f i l t r a t e , and th e r e s i d u e i s d r i e d and th e n i s ta k e n up i n 200 c c . e t h e r and s m a ll p o r t i o n s o f u n d is s o lv e d s u b s ta n c e s a r e s e p a r a te d by f i l t r a t i o n .

A fte r th e d i s t i l l a ­

t i o n o f e t h e r , t h e r e rem ain s a g l u c o s id e r e a d i l y s o l u b l e i n e th e r. 2.

An a l c o h o l i c aqueous e x t r a c t of O leander le a v e s i s

c o n c e n tr a te d n e a r l y to d r y n e s s .

The s t i c k y r e s id u e i s

t r e a t e d w ith a c e to n e and th e u n d is s o lv e d s u b s ta n c e i s s e p a ­ r a t e d by f i l t e r i n g w ith s u c t i o n .

The a c e to n e s o l u t i o n i s

th e n d r i e d u nder reduced p r e s s u r e and th e r e s i d u e i s mixed s e v e r a l tim e s w ith e t h e r .

The s u b s ta n c e i n s o l u b le i n e t h e r i s

d i s t i l l e d from th e c l e a r e t h e r e a l s o l u t i o n .

The r e s i d u e i s

a g a in d is s o l v e d in e t h e r , f i l t e r e d u n t i l c l e a r and th e e t h e r a l e x t r a c t d i s t i l l e d o f f once more. 3.

A s a t u r a t e d e x t r a c t o f c h lo ro fo rm i s c o n c e n tr a te d

t o o n e - f o u r t h i t s volume by d i s t i l l a t i o n .

The m ix tu r e i s

th e n t r e a t e d w ith 2 .5 N HC1 and allo w ed to s ta n d i n c o ld s o l u t i o n f o r tw e n ty - f o u r h o u r s .

I t i s th e n f i l t r a t e d i n to

13

a 50 p e r c e n t e t h y l a l c o h o l i c s o l u t i o n and th e f i l t r a t e e v a p o ra te d to d r y n e s s .

G lu co sid es a r e e x h a u s te d from

r e s id u e w ith e t h e r , and a g a in c r y s t a l l i z e d o u t from 50 p e r cent a lc o h o lic s o lu tio n . 4.

An aqueous a l c o h o l i c e x t r a c t i s made from th e

l e a v e s and th e t a n n i n s a r e p r e c i p i t a t e d u s in g l e a d sub­ a c e ta te .

The e x c e s s le a d i s th e n e l i m in a t e d u s in g sodium

o x a la te .

I t i s th e n e x t r a c t e d w ith t h e u se of a c e to n e

which e x h a u s ts th e crude g l u c o s i d e s .

A n e u t r a l s a l t such a s

MgSO^, o r b e t t e r , ammonium s u l f a t e , p r e c i p i t a t e s o u t th e g lu c o sid e s.

The g l u c o s i d e s a r e th e n f i l t e r e d and s e p a r a t e d

by d i s s o l v in g i n aqueous a l c o h o l i c s o l u t i o n .

They a r e th e n

e x t r a c t e d from a l c o h o l i c s o l u t i o n s by th e u se of c h lo ro fo rm . E x p erim en tal p r o g r e s s in th e e x t r a c t i o n of th e g lu c o ­ s id e may be t r a c e d w ith th e use of th e m icro sco p e i n i d e n t i f y ­ ing th e p u r i f i e d c r y s t a l s , a s th e c r y s t a l l i n e s t r u c t u r e of th e O le a n d rin g lu c o s id e i s v e r y u n iq u e and q u i t e d i s t i n g u i s h e d when i n pu re s t a t e . V ario u s co m b in atio n s o f s o l v e n t s were u se d , b u t had t o be abandoned, due to low c o n s i s te n c y in th e p e rc e n ta g e o f th e y i e l d s .

The above methods proved m ost s a t i s f a c t o r y and

th e b e s t y i e l d s were o b ta in e d w ith th e s e c o n d i t i o n s and re a g e n ts. At tim e s , f r a c t i o n a l c r y s t a l l i z a t i o n was u n d e rta k e n in o b t a i n i n g th e g lu c o s id e i n p u re form .

T his method was

14

c a r r i e d o u t i n th e g e n e r a l p ro c e d u re u n d er p u r i f i c a t i o n , and a l l of th e drug used i n th e a s s a y was p u r i f i e d by t h i s m ethod.

CHAPTER V I

ASSAY OF DRUG Embryonic c h ic k h e a r t s o f f o r t y - e i g h t h o u rs d e v e lo p ­ ment were d i s s e c t e d o u t and p la c e d i n d ro p s o f Tyrode s o l u t i o n c o n t a i n i n g th e d e s i r e d c o n c e n t r a t i o n of th e drug t o be s t u d i e d .

The Tyrode s o l u t i o n had th e fo llo w in g

p e r c e n ta g e co m p o sitio n : N a C l ........................................................... 18.00 KC1

. . .

...............................................02

CaClg

.................................................................02

MgClg • 6 H g O .....................................................01 NaH2P04

......................................................... 005

N aH C O jj......................................... a p p ro x . .028 T his g iv e s a pH o f 7 . 4 .

The s o l u t i o n was p la c e d i n th e w e ll

o f a s l i d e on th e s t a g e o f a m icrosco pe which was mounted i n a chamber m a in ta in e d a t 38 - 39° C.

The w e ll was co vered

d u rin g th e ex p e rim e n t to p r e v e n t e v a p o r a tio n and th e tim e from immersion o f th e

h e a r t in th e s o lu tio n

u n t i l th e f i r s t

ap p e aran c e o f c a r d i a c

i r r e g u l a r i t i e s wasm easured w ith a

s to p w atc h . A t r i o - v e n t r i c u l a r - b l o c k was, i n th e g r e a t m a j o r i t y of i n s t a n c e s , th e f i r s t r e a c t i o n g i v i n g ev id en ce o f th e a c t i o n o f t h e drug on th e h e a r t .

O c c a s io n ly , however, dropped b e a ts

o r d i a s t o l i c a r r e s t o c c u rre d f i r s t , and i n such c a s e s th e s e

16

were ta k e n as th e end p o i n t . th e end p o i n t i s q u i t e s h a r p .

With c o n tin u o u s o b s e r v a t io n A s e r i e s of c o n c e n t r a t i o n s

were chosen w ith which c a r d i a c i r r e g u l a r i t i e s a p p e a re d on th e a v e ra g e w i t h i n th e ran ge o f t h r e e t o t e n m in u te s . U s u a lly ab o u t f o u r h e a r t s were u se d f o r each o f t h e s i x c o n c e n t r a t i o n s o f th e d ru g .

S to c k s o l u t i o n s o f th e g l u c o ­

s i d e were made up in 95 p e r c e n t a l c o h o l .

C o n tro l e x p e r i ­

m ents showed t h a t Tyrode s o l u t i o n s c o n ta in in g 2 p e r c e n t a l c o h o l had no d i s c e r n i b l e e f f e o t on th e h e a r t s . 7 R eadings (re c o rd e d by th e s to p watch) on c a r d i a c i r ­ r e g u l a r i t i e s were ta k e n a t one m in u te i n t e r v a l s ( o r l e s s a s in th e c a s e o f stopp age o f th e h e a r t ) and re c o rd e d a c c o rd in g t o th e l e n g t h o f th e h e a r t b e a t s a s m easured i n s e c o n d s. D i l u t i o n s o f th e g l u c o s i d e were made up a c c o r d i n g l y — 1 : 5 , 1 :1 0 , 1 :1 0 0 , 1:1000, and 1:2000 c o n c e n t r a t i o n s .

The g lu c o ­

s id e was d i s s o l v e d i n th e l e a s t amount of aqueous a l c o h o l i c s o l u t i o n which in t u r n was made up o f e t h y l a l c o h o l and Tyrode s o l u t i o n . The fo llo w in g t a b l e shows th e e f f e c t of th e concen­ t r a t i o n o f th e g lu c o s id e on th e r e g u l a r movement o f th e embryo c h ic k h e a r t which i s e x p r e s s e d i n tim e , and m easured by a s to p w atch a c c o rd in g t o t h e fre q u e n c y o f t h e h e a r t b e a t .

17

1 :5 CONCENTRATION H e a rt b lo c k a t e:

Seconds

Seconds

Seconds

1.

3.2 5

4 .5

4 .7 5

3 .2 m in u te s

2.

2 .6

3 .2

3 .2

3 .5 m in u te s

3.

3 .0

3 .2

3 .3

3 .1 m in u te s

4.

3 .1

3 .4

------

3 .0 m in u tes

1:1 0 CONCENTRATION 1.

2 .8

3 .0

3 .1

3 .3 m in u te s

2.

2 .6

2 .4

2 .4

4 .5 m in u tes

3.

3 .0 5

2 .9

3 .4

3 .2 m in u te s

4.

2 .6

2 .9

3 .2

3 .3 m in u te s

1:100 CONCENTRATION 1.

3 .0

3 .1

3 .1

3 .6 m in u te s

2.

2 .5

2 .7

2 .9

3 .7 m in u te s

3.

2 .8

3 .0

3 .1

4 .0 m in u te s

4.

2 .4

2 .4

2 .4

18

1:1000 CONCENTRATION Seconds

Seconds

Seconds

H e a rt block: a t end o f

1.

2 .6

2 .7

2 .7

4 .4

m in u te s

2.

2 .9

2 .6

2 .4

3 .2

m in u te s

3.

3 ,1

2 .9

3 .1

3 .6

m in u te s

4.

2 .5

2 .6

2 .8

5 .9

m in u te s

1:2000 CONCENTRATION 1.

3 .0

3 .1

2 .9

7 .0 m in u te s

2.

2 .7

2 .9

2 .9

6 .3 m in u te s

3.

2 .4

1 .9

2.6

6 .5 m in u te s

4.

1 .5

1 .9

1.2

9 .6 m in u te s

i9 «-

h !-

/ty tm

21

The r e s u l t s o f th e a s s a y showed d e f i n i t e l y t h a t th e g l u c o s id e O le a n d rin h as an e f f e c t on th e c h ic k h e a r t s by d e c r e a s in g t h e number o f b e a t s and i n c r e a s i n g t h e s t r e n g t h o f th e b e a t .

S o r p t i o n from th e s o l u t i o n to t h e h e a r t t a k e s

p la c e r a p i d l y and i n s e v e r a l c a s e s c a r d i a c f i b r i l l a t i o n s were n o t i c e d .

CHAPTER V I I

CONCLUSIONS The g lu c o s id e O le a n d rin i s c l o s e l y r e l a t e d t o th e g l u c o s i d e s o f d i g i t a l i s and h y d ro ly z e s p r a c t i c a l l y i d e n t i c a l to t h e g lu c o s id e G ito x in .

T h is s i m i l a r i t y i n s t r u c t u r e

a c c o u n ts f o r i t s s tr o n g a c t i o n on th e h e a r t . The methods u sed i n t h e e x t r a c t i o n were a co m b in atio n of e x p e rim e n ta l and p a t e n t e d methods and pro ved c o n c l u s i v e l y t h a t th e g lu c o s id e O le a n d rin may be r e a d i l y o b ta in e d i n crude form ; however, a p u re form i s q u i t e d i f f i c u l t t o produce in q u a n tity .

The m ain d i f f i c u l t y in th e e x t r a c t i o n l i e s i n th e

s e p a r a t i o n o f t h e c h l o r o p h y l l from th e g lu c o s id e w ith o u t d e s t r o y i n g th e g l u c o s i d e .

From e x p e rim e n ta l e v id e n c e th e

e t h e r e a l e x t r a c t i o n s gave th e g r e a t e s t y i e l d and produced th e p u r e s t form of t h e g l u c o s i d e . The r e s u l t s o f t h i s a s s a y was t o t e s t th e e f f e c t of th e a c t i o n of th e g l u c o s id e on th e h e a r t r a t h e r th a n t o o b t a i n an a c c u r a t e q u a n t i t a t i v e s t r e n g t h d e t e r m i n a t i o n . These r e s u l t s showed t h a t th e O lea n d rin g lu c o s id e had a s t r o n g e f f e c t on embryo c h ic k h e a r t s which caused i n most i n s t a n c e s c a r d i a c f i b r i l a t i o n s to ta k e p la c e and w hich ended i n a t r i o - v e n t r i c u l a r b lo c k .

The g ra p h s d e f i n i t e l y show th e

r e l a t i o n s h i p between th e c o n c e n t r a t i o n of th e drug and th e h e a r t b e a t a s m easured i n b o th m in u tes and s e c o n d s .

23

ypJUHE 2

' -tv - % ^

WJt^#*?' jfrMWh »

FIGURE 4

X

24

FIGURE 6

FIGURE 7

F ig u r e s 1 , 2 , 3, and 4 r e p r e s e n t ph o to s ta k e n o f th e g lu c o s id e O le a n d rin , which have been c r y s t a l l i z e d o u t o f a 50 p e r c e n t a l c o h o l i c s o l u t i o n .

The p h o to s were t a k e n from

th e same sample o f t h e d ru g , and r e p r e s e n t th e p u r i f i c a t i o n of t h e g l u c o s i d e .

These p h o to s show th e n e e d l e - l i k e s t r u c ­

t u r e of th e c r y s t a l s as t h e y undergo p u r i f i c a t i o n . F ig u re 4 e x h i b i t s th e g l u c o s i d a l d rug i n p u re

form .

F ig u r e s 5, 6, and 7 r e p r e s e n t c r y s t a l s o f t h e osazone of t h e g lu c o s id e O le a n d rin .

These c r y s t a l s were o b ta in e d by

t a k i n g a sample o f th e g l u c o s i d e , add ing p h e n y lh y d ra z in e h y d r o c h lo r id e and a l i t t l e sodium a c e t a t e , and h e a t i n g th e m ix tu re on a w a te r b a t h f o r t h i r t y m in u te s , th e n p l a c i n g a little

of th e p r e c i p i t a t e on a s l i d e

and a llo w in g t o d r y .

F ig u r e s 5 , 6, and 7 show th e osazone u n d er v a r io u s m a g n ific a tio n s.

REFERENCES 1.

S t o l l , A r t h u r , The C ardiac G ly c o s id e s .

2.

M uenscher, P o iso n o u s P l a n t s o f t h e U n ite d S t a t e s .

5.

B eric h te ,

V o l. 70 1547 (1957)

#7.

4.

B e ric h te ,

V o l. 70 1554 (1937)

#7 .

5.

U n ite d S t a t e s P a t e n t 2 ,1 8 9 ,7 2 3 , F e b ru a ry 6 , 1940.

6.

Compt. r e n d . 194,914 (1 9 3 2 ).

7.

J o u r n a l Pharm acology, V o l. 72, 1941.

8.

B r i t i s h P a t e n t 4 9 8 ,3 0 7 , Ja n u a ry 5 , 1939.

9.

Farm, Z hur. A l k a lo i d s i n N eriium O lea n d er, #2, 1937.

10.

Med. K l i n . 33, 1390-5 (1 9 3 7 ).

11.

Compt. r e n d . 194, 1099, 101 (1 9 3 2 ).

12.

K l i n . 15,

1677-80 (1 9 3 6 ).