University of NigeriaChudi.P_1988_1579.pdf · Chemical composition of Venoms 2 Enzymes Present in...

University of Nigeria Research Publications

OKEKE, Chudi P.

PG/M.Sc/82/1579

In-vitro And In-vivo Studies On The Antagonism Of Some Toxic Effects Of Echis carinatus Venom By An Alcoholic Extract From Diodia scandens

Pharmaceutical Sciences

Pharmacology and Therapeutics

July, 1988

OF SOME TOXIC EFFECTS OF ECHIS CARMATUS VmOM

BY AN ALCOHOLIC EXTRACT FRON DIODIA SCANDE3TS

A DISSERTATION SUBMITTED TO

THE DEPARTEEN!T OF PHAMACOLOGY AND THEEAP[TTICS

COLLEGE OF MEDICINE

UrlCVERSI!TY OF NIGERIA, EaTTGW CAMPUS

IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR TEE

DEGREE OF

MASTER OF SCIEJWE

IN-VITRO AFTD IN-VIVO STUDIES OH THE ANTAGOITISM

OF SOME TOXIC EFFECTS OF ECRIS CARINATUS VENOM

BY AN ALCOHOLIC EXTRACT FROM D I O D I A SCADENS

Pe C e OKEKE, FeImM.L.Te DEPARTMENT O F PHAREACOLOGY AND TEIERAPEWTICS

COLLEGE OF MEDICINE UNIVERSITY OF NIGERIA, ENUGU CAMPUS

SUPERVISOR :

I certify t h a t this is a n original work

c a r r i e d out b y O k e k e , C h u d i P a u l in t he D e p a r t m e n t

of P h a r m a c o l o g y & Therapuekics, U?,!iiC f o r the ward

of r:iSc deg ree o f the University of ~ i g e r i a .

TABLE OF CONTENTS

CONTENTS

D E D I C A T I O N

ACKNOWLEDGEMENT v i

S U M M A R Y viii

PUBLICATION/COMMUNICATION AT SCIENTIFIC MEETING i

L I T E R A T U R E R E V I E W - SNAKES A N D THEIR VENOMS

S n a k e S p e c i e s A

Chemical composition of Venoms 2

Enzymes P r e s e n t in S n a k e Venoms - 2 Pharmacology of venom

I. Hydrophidae V e n o m s

2 . Elapidae V e n o m s 6

3 . Crotalidae venoms E3

4. V i p e r i d a e V e n o m s

~oxicalogy of S n a k e Venoms

:. Coagulation Disorders

2. Haemorrhage

3 . Myonecrosis

4. Nephrotoxicity

Chemical Neutralization of Snake Venoms

1. E s t r o g e n s

2, H e p a r i n

3 . Compounds c o n t a i n i n g s u l p h u r

S . C h e l a t i n g compounds

5. P r o t a r n i n e S u l p h a -.e 2 2

CONTENTS

PROBLEM I N MANAGEMENT OF SNAKE VENOM POISONING

EXPERIMENTAL STUDIES

I n t r o d u c t i o n

M e t h o d s a n d Materials

SECTION ' A ' : EXTRACTION METHODS

PAGE - 32

SECTION ' B ' : COAGULATION S T U D I E S

T h e e f f e c t of E c h i s C a r i n a t u s Venom on W h o l e Blood C l o t t i n g t i m e 26

T h e e f f e c t of t h e h e r b a l e x t r a c t o n t h e a c c e l e r a t e d w h o l e b l o o d c l o t t i n g t i m e i n d u c e d by venom 27

The e f f e c t of venom o n P r o t h r o m b i n t i m e 28

T h e e f f e c t o f h e r b a l e x t r a c t o n venom a n d b r a i n t h r o m b o p l a s t i n a c t i v i t y 29

I n v e s t i g a t i o n o f the venom t h r o m b o - p l a s t i c a c t i v i t y a n d h e r b a l e x t r a c t a n t i t h r o m b o p l a s t i c a c t i v i t y 31

T h e effect of t h e h e r b a l e x t r a c t o n venom t h r o m b i n activity 3 3

SECTION 'C': ELECTROPHORESTIC STUDIES

E v a l u a t i o n of A n t i p r o t e o l y t i c a c t i o n of t h e h e r b a l e x t r a c t o n v e n o m i s e d p l a s m a e l e c t r o p h o r e t o g s a r n

SECTION 'D' TOXICITY STUDIES

( a ? A c u t e T o x i c i t y S t u d i e s i n Mice 37

(b) E f f e c t o f p r e t r e a t m e n t w i t h h e r b a l e x t r a c t o n m o r t a l i t y due t o venom 3 7

I c ) Toxicity s t u d i e s o f the h e r b a l e x t r a c t i n mice. 3'1

CONTENTS PAGE

SECTION ' E ' : EXPERIMENTS ON THE CARDIOVAS- CULAR SYSTEM.

1. P e r f u s e d r a t h i n d q u a r t e r s p r e p a r a t i o n

20 T h e e f f e c t s o f venom a n d h e r b a l e x t r a c t o n b l o o d p r e s s u r e , ECG, a n d r e s p i r a - t i o n of a n a e s t h e t i z e d c a t .

RESULTS

WEIGHTS OF ETHANOLIC EXTRACTARLE FRACTIONS FROM FRESH MATERIAL

COAGULATION STUDIES

E f f e c t o f Echis c a r i n a t u s venom o n w h o l e b l o o d c l o t t i n g t i m e

T h e a c t i o n o f f r a c t i o n B o f t h e h e r b a l e x t r a c t o n t h e a c c e l e r a t e d c l o t t i n g time of w h o l e b l o o d i n d u c e d b y t h e venom

c o m p a r i s o n of p r o t h o r m b i n t i m e s of p l a s m a t r e a t e d w i t h venom a n d u n t r e a t e d p l a s m a

T h e e f f e c t o f t h e h e r b a l e x t r a c t o n t h e venom a n d b r a i n t h r o r n b o p l a s t i n a c t i v i t y

T h e e f f e c t of t h e h e r b a l e x t r a c t o n t h e c l o t t i n g t i m e of plasma t o w h i c h venom a n d Ca2+ were a d d e d

The e f f e c t of t h e h e r b a l e x t r a c t o n t h e c l o t t i n g t i m e of p l a s m a t o w h i c h venom w a s a d d e d .

ELECTROPHORETIC STUDIES

E v a l u a t i o n of A n t i p r o t e o l y t i c a c t i o n o f t h e h e r b a l e x t r a c t o n v e n o m i s e d p lasma electro- p h o r e t o q r a r n

ACUTE TOXICITY STUDIES

T o x i c i t y s t u d i e s of venom i n mice

E f f e c t o f h e r b a l e x t r a c t 1.5uq/q b o d y w e i g h t o n m o r t a l i t y d g e t o venom

CONTENTS

Toxicity studies of the herbal e x t r a c t in mice

EXPERIMENTS ON THE CARDIOVASCULAR SYSTEM

Perfused rat hindquarters preparation

The effect of venom and herbal e x t r a c t on anaesthetized cat blood pressure ECG and respiration

DISCUSSION

CONCLUSIONS

REFERENCES

DEDICATION

To My C h i l d r e n , Chukwudubem,

Nnamadi, Emeka, Ngozi a n d m y

Wife Doris Okeke

A l m i g h t y God i n H ~ S i n f i n i t e mercies h a s made t h i s

p i e c e o f work a r e a l i t y . T h r o u g h H i s a u t h o r i t y H e made

i t . p o s s i b l e f o r m e t o h a v e the s i n g u l a r p r i v i l e g e t o be

t a u g h t , g u i d e d a n d s u p e r v i s e d b y P r o f e s s o r G.O.

O n u a g u l u c h i , t h e Head , D e p a r t m e n t o f P h a r m a c o l o g y a n d

T h e r a p e u t i c s , C o l l e g e of M e d i c i n e , U n i v e r s i t y o:E N i g e r i a

Teaching H o s p i t a l , Enuqu. H e i n f o r m e d m e o f the u s e o f

D i o d i a s c a n d e n s i n t r a d i t i o n a l m e d i c i n e . H e a l s o

s u p e r v i s e d a l l a s p e c t s of t h i s work, n a m e l y

e x p e r i m e n t a l w o r k , d r a f t s r e a d i n g and the p r o d u c t i o n

o f t h i s f i n a l c o p y . I a m g r e a t l y i n d e b t e d t o h i m for

h i s i n n u m e r a b l e e x c e l l e n t s u g g e s t i o n s a n d i n v a l u a b l e

c o n s t r u c t i v e criticisms.

I w i s h a l s o t o e x p r e s s m y s i n c e r e g r a t i t u d e t o

Dr. J .C. Okafor of t h e E n u g u F o r e s t H e r b a r i u m for

b o t a n i c a l l y i d e n t i f y i n g the p l a n t ( h e r b ) and Mr, S . A .

I g w e , w h o s e a s s i s t a n c e i n t h e e x p e r i m e n t a l work was

i n v a l u a b l e . My t h a n k s a l s o g o t o the Technical S t a f f

o f t h e D e p a r t m e n t o f P h a r m a c o l o g y a n d T h e r a p e u t i c s ,

U.N.T.H., Enugu e s p e c i a l l y t o M r . D.0. Opara a n d

Mrs. C.N. Ike, who s u p p l i e d a l l t h e too l s r e q u i r e d for

t h i s s t u d y .

v i i .

T h e members of s t a f f of t h e Medica l I l l u s t r a t i o n

U n i t U n i v e r s i t y of N i g e r i a , Enugu Campus were v e r y

h e l p f u l w i t h t h e t r a c i n g s , d i a g r a m s , graphs a n d

p h o t o g r a p h s . M r . W i l l i a m O k o of t h e Personnel

D e p a r t m e n t , University of ~ i q e r i a T e a c h i n g Hospital,

E n u q u d i d a l l t h e s t e n o g r a p h i c w o r k .

F i n a l l y , let m e t h a n k my f a m i l y for t h e i r s u p p o r t

t h r o u g h o u t t h e period o f t h i s s t u d y .

V i i i

SUMMARY

T h e m e t h o d of extraction of a p o s s i b l e

a n t i v e n o m f r a c t i o n from D i o d i a s c a n d e n s i s d e s c r i b e d .

T h r e e f r a c t i o n s (A,B,C) were s e p a r a t e d . F r a c t i o n

B w a s the most p l e n t i f u l a n d w a s t h e f r a c t i o n u s e d

i n t h i s s t u d y .

I t w a s f o u n d t h a t f r a c t i o n 3 of t h e e t h a n o l i c

e x t r a c t ( h e r e a f t e r c a l l e d t h e h e r b a l e x t r a c t ) d i d

n o t a f f e c t h u m a n w h o l e k l l o o d c l o t t i n g t i m e , but

i t i n c r e a s e d t o 2.9 m i n the w h o l e blood c l o t t i n g

time r e d u c e d t o -1.8 rnin b y t r e a t m e n t with E c h i s

c a r i n a t u s v e n o m . T h e h e r b a l e x t r a c t was shown t o

p o s s e s s some a n t i t h r o m b o p l a s t i n p r o p e r t y a g a i n s t

b r a i n t h r o m b o p l a s t i n and v e n o m t h r o m b o p l a s t i n

a c t i v i t y , h o w e v e r j . t d i d n o t i n h i b i t the t h r o m b i n -

l i k e a c t i v i t y o f t h e venom. I t i s s u q - i e s t e d t h a t t h e

a n t a q s n i s t i c e f f e c t o f t h e h e r b a l e x t r a c t i s due t o

t h e i n h i b i t i o n of t h r o m b o p l a s t i n , F a c t o r V I I

a c t i v a t i o n of Factor X.

E l e c t r o p h o r e t i c s t u d i e s t h a t t h e v e n o m

c a u s e d c o n s i d e r a b l e loss of a l p h a 1, a l p h s I1 a n d

b e t a g l o b u l i n s a n d com3lete d i s a p p e a r a n c e of t h e

f i b r i n o g e n - b a n d . T h e h e r b a l e x t r a c t r e d u c e d t h e

loss of t h e a l p h a 1, a l p h a 11 a n d be t a g l o b u l i n

b a n d s b u t d i d n o t p r e v e n t t h e loss i n t h e

f i b r i n o g e n band.

T h e h e r b a l e x t r a c t a t 1.5mg/kg a d m i n i s t e r e d

i n t r a p e r i t o n e a l l y i n c r e a s e d t h e LD50 of the v e n o m

i n mice from 2.0mg/kg i n t r a p e r i t o n e a l l y t o 3,2mg/kg

i n t r a p e r i t o n e a l l y . T h e h e r b a l e x t r a c t o n i t s own

c a u s e d n o dea ths e v e n a t 2g/kg i n t r a p e r i t o n e a l l y

I t s LDS0 of 3.3g/kg t h e r e f o r e i n d i c a t e s t h a t t h e

h e r b a l e x t r a c t h a s a w i d e s a f e t y m a r g i n .

I n t h e r a t h i n d q u a r t e r s p r e p a r a t i o n t h e ver .om

i n d u c e d v a s o c o n s t r i c t i o n w h i l e t h e h e r b a l extract

i n d u c e d s l i g h t v a s o d i l a t a t i o n .

O n t h e a n a e s t h e t i z e d cat b l o o d p r e s s u r e , t h e

v e n o m a t 3mg/kg i v . , c a u s e d a p r o f o u n d r i se i n

b lood p r e s s u r e w h i c h d i d n o t f a l l t o t h e base l i n e

u n t i l after a p e r i o d of 4 m i n . A s m a l l rise i n

blood p r e s s u r e , u p t o 20mrnHg w a s i n d u c e d by

t he v e n o m a t 0.5mq/kq. T h e h s r b a l e x t r a c t c a u s e d

s l i g h t f a l l i n b l o o d p r e s s u r e .

Cn t h e ECG o f t h e a n a e s t h e t i z e d c a t , t h e v e n o m

( 0 , 5 r n g / k g ) i . v . p r o d u c e d d e f i n i t e d e p r e s s i o n of the

ST s e g m e n t . k t 3mg/kg j.v. t h e ST d e p r e s s i o n was

more marked a n d was a c c o m p a n i e d by t h e d e p r e s s i o n

of t h e T w a v e s .

T h e change i n the QRS c o m p l e x e s a t the a b o v e

dose l e v e l s was s u g ~ e s t i v e o f e a r l y stages of

v e n t r i c u l a r d y s r r h y t : h m i a m T h e e f f e c t of the

herbal e x t r a c t o n t h e ECG c h a n g e s d u e t o

venom w a s n o t d e t e r m i n e d i n t h i s s t u d y .

T h e venom induced m a r k e d r e s p i r a t i o n

d e p r e s s i o n i n the a n a e s t h ~ t i z e d c a t e v e n

a t 0.5rng/kg i . v . T h e h e r b a l extract

a t 10mg/kq i . v . a n t a q o n i s e d t h e r e s p i r a t o r y

d e p r e s s i o n induced by the venom.

PUBLICATION

O n u a g u l u c h i G. a n d Okeke P.C. P r e l i m i n a r y i n - v i t r o

s t u d i e s o n t h e a n t a g o n i s t i c e f f e c t s o f a n e t h a n o l i c

e x t r a c t from D i o d i a s c a n d e n s o n Echis c a r i n a t u s

venom - induced changes i n the c l o t t i n g of human

blood. P h y t o t h e r a p y R e s e a r c h 1988 ( i n press) .

C o m m u n i c a t i o n A t S c i e n t i f i c M e e t i n q

O n u a g u l u c h i G. a n d Okeke P.C. A n t i v e n o m activity of

a n e x t r a c t f r o m Dioda scandens. Meeting of t h e

West A f r i c a n Society f o r P h a r m a c o l o g y A p r i l (1986) .

.- 1 I

S N A K E S A N D T H E I R VE?ICMS - A REVIEW

Human b e i n g s have t r a d i t i o n a l l y been f e a r f u l of

a n d p u z z l e d b y t h e d e a d l y a c t i o n o f venom from

v e n o m o u s s n a k e s . Symptoms a r i s i n g i n the v i c t i m

r e s u l t f r o m c o m b i n e d e f f e c t s of complex p r o t e i n

c o m p o u n d s i n venom ( T u , 1 9 7 7 ) . TU ( 1 9 7 7 ) a l s o

s h o w e d t h a t v e n o m s c o n s i s t o f a p p r o x i m a t e l y 90%

protein. T h e p r o p o r t i o n s o f t h e d i f f e r e n t

p r o t e i n s u b s t a n c e s a n d t h e i r s p e c i f i c c h a r a c t e r i s t i c s

v a r y among species . H o w e v e r , t h e c loser t h e

p h y l o q e n e t i c r e l a t i o n s h i p o f t h e snake, t h e more

s i m i l a r a r e t h e c o m p o s i t i o n o f t h e v e n o m s a n d t h e i r

p h a r m a c o l o g i c a l p r o p e r t i e s . A c l a s s i f i c a t i o n of

t h e p r o t e i n a ~ d n o n - p r o t e i n c o n s t i t u e n t s of s n a k e

venom was a t t e m p t e d by D e v i (1968) . T h e r e are

more t h a n o n e t o x i c p r i n c i p l e i n a venom; t h e s e

p r i n c i p l e s a c t t o g e t h e r i n p r o d u c i n g p o i s o n i n g .

A b o u t t w o t h o u s a n d d i f f e r e n t t y p e s of snake

e x i s t a n d a p p r o x i m a t e l y 300 a r e known t o be v e n o m o u s .

T h e v e n o m o u s snake c o m p r i s e of f i v e f a m i l i e s .

T h e Crotalidae c o m p r i s e s i x qenera a n d are f o u n d o n l y

i n N o r t h , C e n t r a l a n d S o u t h A m e r i c a . The V i p e r i d a e

commonly known a s v i p e r i d s o r v i p e r s are f o u n d i n

A f r i c a , E u r o p e a n d A s i a . T h e y a r e n o t f o u n d o r s e e n

i n A m e r i c a n c o n t i n e n t s o r A u s t r a l i a . T h e

E l a p i d a e a r e f o u n d i n F!or th a n d C e n t r a l

A m e r i c a a n d

a l s o i n New G u i n e a and A u s t r a l i a , T h e

a r e sea s n a k e s a n d live f n t r o p i c a l and sub-

t r o p i c a l sea waters bordering the I n d i a n and P a c i f i c

O c e a n s . C o l u b r i d a e has the largest f a m i l y a n d consist

of 250 q e n e r a and o v e r 1000 s p e c i e s , Not a l l o f t h e m a r e

p o i s o n o u s . P o i s o n o u s C o l u b r i d a e i n c l u d e the q e n e r a

Dispholidus and T h o l o t o r n i s , h o t h o f which are f o u n d i n

Africa.

C h e m i c a l C o m ~ o s i t i o n of Venoms

T h e n o n - p r o t e i n c o m p o n e n t s of venom a r e m a i n l y

metals, a m i n o a c i d s and small p e p t i d e s , n u c l e o t i d e s and

r e l a t e d c o m p o u n d s , c a r b o h y d r a t e s , l i p i d s a n d b i o q e n i c

a m i n e s . S n a k e venoms c o n t a i n v a r i o u s m o n o v a l e n t c a t i o n s

a n d a n i o n s w h i c h n e u t r a l i s e the charged p r o t e i n m o l e c u l e s ,

Some d i v a l e n t m e t a l s known as c o - f a c t o r s for b i o l o g i c a l

a n d enzymatic a c t i v i t i e s are also p r e s e n t .

The c o m p o s i t i o n of Naja n a j a a n d v i p e r r u s s e l l i

venoms was s t u d i e d b y Dev i (1968) a n d o t h e r w o r k e r s . Zinc

was f o u n d commonly i n many venoms ( F r e d e r i c k a n d Tu , 1979).

The b i o l o g i c a l s i g n i f i c a n c e o f zinc c a n be s e e n from t h e

fact t h a t some venoms p o s s e s s a n t i c h o l i n e s t e r a s e a c t i v i t y

which r e q u i r e s z k b n ( ~ u m a r et a1 , 1973). Some venom

protases are m e t a l l o p r o t e i n s and c o n t a i n c a l c i u m a n d zinc i o n s .

The c a r b o h y d r a t e componen t s of venoms dre m a i n l y

in the form of g l y c o p r o t e i n s r a t h e r t h a n Ere2 s u g a r s and

a re f o u n d i n many venoms (Basu e t al, 1 9 7 0 ) . T h e total

lipid c o n t e n t of Naj_a - nfia venom i s v e r y s m a l l a n d a c c o u n t s

for only 0.4% of t h e d r y w e i g h t (Kabara and F i s c h e r , 1969).

T h e majpr component i s p h o s p h o l i p i d a n d p h o s p h o t i d y l c h o l i n e .

Because o f t h e s m a l l q u a n t i t y of l i p i d s i n venoms, i t i s

b e l i e v e d t h a t t h e y d o n o t p l a y a major ro le i n venom

a c t i o n .

B i o g e n i c arnines: b r a d y k i n i n , h i s t a m i n e , 5-hydroxy-

t r y p t a m i n e and N: N ' d i m e t h y l ( 1,5 h y d r o x y t r y p t a m i n e )

p r e s e n t i n venoms have been i m p l i c a t e d a s being r e s p o n s i -

b l e for t h e p r o d u c t i o n of p a i n a t t h e s i t e of snake b i t e

which i s more commonly s e e n i n bites due t o t h e v i p e r i d a e

and t h e C r o t a l i d a e t h a n bites due t o t h e E l a p i d a e a n d

Hydroph idae ,

Enzymes P r e s e n t i n S n a k e Venoms

Phospholipase A2 is present i n venoms o f a l l famil ies

o f s n a k e s . A l t h o u g h p h o s p h o l i p a s e A2 f r o m v a r i o u s venoms

e x h i b i t more o r less s i m i l a r enzymatic p r o p e r t i e s they

are n o t i d e n t i c a l i m m u n o l o g i c a l l y . S p e c i f i c a n t i s e r u m

a g a i n s t t h e venom of a p a r t i c u l a r s p e c i e s i n h i b i t s the

p h o s p h o l i p a s e A a c t i v i t y of venoms of c l o s e l y r e l a t e d 2

species, b u t i n most cases fails t o s u p p r e s s t h e phospho-

l i p a s e A a c t i v i t y o f venoms f rom other families (Nair et 2

al., 1975).

phosphotidylcholine, lecthin, are the most common substrates

for phospholipase A2. The hydrolysis of these substrates

can take place in the free substrate form in egg yolk (TU

et al., 1 9 7 0 ) and in serum. This enzyme is known to release

fatty acids from p positions.

Phospholipase A2 of the venom is considered to be

the prime factor for the disruption of the electron trans-

port chain and integrity of mitochondria1 sturcture

(Patruschka et al., 1 9 5 9 ) . Petruschka and co-workers used

heat treated venoms of Agkistrodom piscivorus and Naja naja

as lipolytic agents to study the role of phosphorylation in

a manner identical to that of whole venom. It has been

observed that phospholipase A2 had marked effect on the

muscle and nerves. For example the force of contrf,iction

following stimul.ation of frog sartorious nerve-muscle

preparation was greatly enhanced by phospholipase A (Brazil 2

et al., 1 9 7 3 ) and at low concentration it increased membrane

permeability of the lobster leg axon. At high concentration,

axonal conduction was blocked (Rosenberg and Ehrenpresis,

1 9 6 1 ) . Such increase in membrane permeability and block of

axona.1 conduction were attributed to the activity of

phospholipase. A2 (Condrea and Rosenberg, 1 9 6 8 ) . However,

there is evidence that enzymes are not responsible for the

major venom reactions (Tu and Passey, 1 9 7 1 ) .

In Aqkistrodom acutus venom, the main toxicity can

be separated from phospholipase A2 activity (Yanq et al.,

1959) . Chiu (1 9 6 0 ) investigated phospholipase A2

activity in six venoms of Formosan snakes and found

no correlation between enzyme activity and venom potency.

It was thought that histamine release by rattle snake

venom (Feldberg and Kellaway, 1937) was due to phospholipase

A present in the snake venom. Phospholipase A2 was proved 2

to be responsible for the convulsive action of snake venom.

For example convulsions were induced by intraventricular

injection of N a j a n a j a venom. Partially purified phos-

pholipase A2 at dose of 2.5mg pzoduced convulsions in rabbit

(Lysz and Rosenberg, 1974) . There are other enzymes present in venoms such as

phosphodiesterase, amino acid oxidase, acetylcholines-

terase, proteolytic enzymes, arginine ester hydrolase and

other esterases etc.

FHARMACOLOGY OF VENOMS

1. H y d r o p h i d a e ( sea s n a k e ' venoms c o n t a i n p o t e n t n e u r o -

t o x i n s t h a t are among the most t o x i c s u b s t a n c e s i n t h e

w o r l d . Because o f t h e h i g h l y t o x i c n a t u r e o f t h e v e n o m s ,

sea s n a k e p o i s o n i n g , s h o u l d n o t be r e g a r d e d lightly

( H a l s t e a d , 7 9 7 0 ) . Sea! s n a k e venoms a c t a t t h e

n e u r o m u s c u l a r j u n c t i o n . F r o m the c l i n i c a l observation

of m y o q l u b i n u r i a i n human v i c t i m s , i t w a s c o n c l u d e d

t h a t sea s n a k e venoms are m y o t o x i c , A c u t e tubular

n e c r o s i s w a s a l s o r e p o r t e d b y ~ i t p r i ja e t a l . ( 2 9 7 3 ) .

2 , E l a p i d a e Venom

T h e c u r a r e - l i k e a c t i o n of c o b r a venom a n d its

t o x i n s i s w e l l known ( v i c k e t a l . , 1965 ) . T h e venom

blocks t r a n s m i s s i o n a t t h e n e u r o m u s c u l a r j u n c t i o n . I t

d o e s n o t a f f e c t t r a n s m i s s i o n w i t h i n t h e a x o n (Schmidt

et al,, 2964) . T h e action of c o b r a neurotoxin d i f f e r s

from t h a t of c u r a r e , h o w e v e r , a n d s t r i c t l y s p e a k i n g i t

s h o u l d n o t be referred t o a s c u r a r i s a t i o n because i t s

m e c h a n i s m of a c t i o n i s n o t one of p u r e c o m p e t i t i o n

( C h e y m o l e t a l . , 1974 ) . Cobra n e u r o t o x i n s b i n d t o

r e c e p t o r s i n the motor e n d - p l a t e a n d t h e y do no t affect

t h e e l ec t r i ca l p r o p e r t i e s of m u s c l e f i b r e s kioocs e t

a l . , 1 9 7 3 ) . ind ding o f n e u r o t o x i n s t o t h e e x t r a j u n c t i o n a l

c h o l i n e r q i c r e c e p t o r s i s v e r y s t r o n g a n d e s s e n t i a l l y

i r r e v e r s i b l e ,

A n o t h e r i n t e r e s t i n g fac t a b o u t C o b r a venom i s t h e

b i n d i n g of two f r a c t i o n s of r e l a t i v e l o w t o x i c i t y wh ich

h a v e a d e p o l a r i z i n g effect on s k e l e t a l m u s c l e membrane.

T h e d e p o l a r i z i n g a c t i o n of t h e venom was n o t a t t r i b u t e d

t o p h o s p h o l i p a s e A p o r protease, b u t r a t h e r t o h e a t

s table basic p r o t e i n s (Earl and E x c e l l , 1 9 7 1 ) .

E a r l and Excel (1972) p r o p o s e d t h a t t h o s e d e p o l a r i -

z i n g f r a c t i o n s produced non-specific i n c r e a s e i n

membrane p e r m e a b i l i t y b y displacing membrane c a l c i u m .

T h e s e f r a c t i o n s are i d e n t i c a l with c a r d i o t o x i n s a n d

c a r d i o t o x i n s a r e t h e n e x t mos t t o x i c components .

Onuagu luch i (1960) d e s c r i b e d a case o f cobra

e n v e n o m a t i o n w h i c h c a u s e d damage t o t h e c e n t r a l n e r v o u s

s y s t e m i n t h e f o r m of a n a c u t e a s c e n d i n g s p i n a l p a r a l y s i s .

T h e p a t i e n t was a 12 year o l d g i r l who w a s a d m i t t e d

to t h e G e n e r a l H o s p i t a l Wukari w i t h a 7 d a y h i s t o r y

of s n a k e b i t e , o v e r t h e left foot .

A p a r t from the b l o c k a d e a t t h e n e u r o m u s c u l a r

j u n c t i o n and t h e a u t o n o m i c g a n g l i o n , and a s c e n d i n g

s p i n a l p a r a l y s i s , cobra venom h a s b e e n f o u n d t o have

effect on t h e r e s p i r a t o r y sys t em, Lee a n d P a n g ( 1 9 6 1 )

i n v e s t i g a t e d t h e c a u s e of r e s p i r a t o r y f a i l u r e i n

d o g s a n d c a t s b y r e c o r d i n g t h e r e s p i r a t o r y d i s c h a r g e

down the p h r e n i c n e r v e a n d t h e a c t i o n p o t e n t i a l s i n

t h e i n t e r c o s t a l a n d d i a p h r a g m a t i c m u s c l e s . T h e p h r e n i c

d i s c h a r g e w a s f o u n d e v e n a f t e r c o m p l e t e p a r a l y s i s of

t h e r e s p i r a t o r y m u s c l e s , if t h e a n i m a l s w e r e k e p t

a l i v e b y a r t i f i c i a l v e n t i l a t i o n , T h e d i s c h a r g e w a s

i n t e n s i f i e d a n d p r o l o n g e d if a s p h y x i a d e v e l o p e d . T h e

c o n t i n u e d a c t i v i t y of t h e central r e s p i r a t o r y mechan i sm

w a s i n d i c a t e d b y t h e p r e s e n c e of t h e H e r i n g - B r e u e r

r e f l e x . L e e a n d Pang t h e r e f o r e c o n c l u d e d t h a t r e s p i r a t o r y

f a i l u r e c a u s e d by Naja n a j a venom w a s i n d e e d p e r i p h e r a l

i n o r i g i n d u e t o m u s c l e p a r a l y s i s .

T h e effect o f Naja naja a t r o x venom on b l o o d

p r e s s u r e w a s i n v e s t i g a t e d b y Pang (1952). He c o n c l u d e d

from experiments on r a b b i t s t h a t f a l l i n b l o o d p r e s s u r e

o n e n v e n o m a t i o n i s o f p e r i p h e r a l o r i g i n a n d i s m a i n l y

due t o v a s o d i l a t a t i o n i n s p l a n c h i c area and sometimes

i n the s k i n and t h e m u s c l e s . He a l s o c o n c l u d e d t h a t

the rise i n b l o o d p r e s s u r e a f t e r a d m i n i s t r a t i o n of

t h e venom a n d j u s t b e f o r e d e a t h i s d u e t o s t i m u l a t i o n

of the v a s o m o t o r centre by a s p h y x i a .

3. B66oms of C r o t a l i d a e : C r o t a l i d s , P i t V i p e r s

C r o t a l i d a e venoms c o n t a i n a r a t h e r l a r g e number

of p h a r m a c o l o g i c a l l y a n d b i o c h e m i c a l l y a c t i v e p r o t e i n s .

The Brazillian rattle snakc c ~ n t a i n s n e u r o t o x i n s and also

t o x i n s w h i c h i n d u c e i n t r a v a s c u l a r h a e m o l y s i s . Some c a n

c a u s e a n i m m e d i a t e a n d t r a n s i t o r y f a l l i n blood p r e s s u r e

( B r a z i l et a l . , 1 9 6 7 ) . T h e p r i n c i p a l t o x i n s p r e s e n t i n

C r o t a l i d a e v e n o m s a r e c r o t o x i n a n d crotarninp. T h e n e u r o t o x i c

a c t i o n of c r o t o x i n i s of p e r i p h e r a l origin and i s very

s i m i l a r t o the e f f e c t e l i c i t e d by c u r a r e which m a n i f e s t s a s

p a r a l y s i s and f l a c c i d i t y of s k e l e t a l m u s c l e . A decrease i n

s e n s i t i v i t y of the m o t o r - e n d - p l a t e t o the d e p o l a r i z i n g

a c t i o n of a c e t y l c h o l i n e i s t h e m a i n cause of c r o t o x i n n e u r o -

m u s c u l a r blockade, I n human e n v e n o m a t i o n b y t h e

E r a z i l l i a n r a t t l e s n a k e s , r e n a l l e s i o n s were observed i n

a u t o p s y an43 b i o p s y tissues ( A m o r i n anci Nel lo, 1954 ) .

4. V i p e r i d a e Venoms

T h e many species of v i p e r i d a e h a v e a c c o u n t e d f o r t h e

d i v e r ~ i t y i n t h e t o x i c i t y a n d p h a r m a c o l o g y o f their v e n o m s ,

T h e s e i n c l u d e n e u r o t o x i c i t y a n d p a r a l y s i s , c i r c u l a t o r y

f a i l u r e , h a e m o r r h a q e s and i n t r a v a s c u l a r c l o t t i n g .

T h e a n t i c o a g u l a n t a c t i o n o f v i p e r i n e s n a k e venom

h a s been d o c u m e n t e d , t h e mechan i sm of t h i s a n t i c o a g u l a n t

effect i s n o t c e r t a i n (Okonkwo a n d O n u a g u l u c h i , 1977).

Af i b r i n o g e n a e m i a i s u s u a l l y a f r e q u e n t c h a r a c t e r i s t i c o f

v i p e r i n e p o i s o n i n g ( Okonkwo a n d O n u a g u l u c h i , 1977;

Warrell et a l . , 1977). I t h a s been s u g g s s t e d t h a t d i s s e -

m i n a t e d i n t r a v a s c u l a r c o a g u l a t i o n ( D I C ) c a u s e d the

d e p l e t i o n of f i b r i n o g e n (Weiss, P h i l l i p s , H o e w e l l ,

P h i l l C h r i s t y a n d N i t t i , 1973). However , Okonkwo a n d

O n u a g u l u c h i ( 1 9 7 7 ) from p h a r m a c o l o g i c a l a n d b i o c h e m i c a l

s t u d i e s of - P u f f A d d e r venom, s u g g e s t e d t h a t t h e m o s t

a p p r o p r i a t e e x p l a n a t i o n was t h a t t h e a f i b r i n o g e n a e m i a was

due t o f i b r i n o g e n o l y t i c a c t i v i t y o f t h e venom. D v i l a n s k y

a n d B r a i n (1973) were also u n a b l e t o d e m o n s t r a t e d i s s e m i -

n a t e d i n t r a v a s c u l a r c o a g u l a t i o n i n a p a t i e n t b i t t e n by

E c h i s c o l e r a t a . I n d e e d OkonEwo a n d O n u a g u l u c h i ( 1977)

showed t h a t the n o n - c l o t t i n g o f t h e blood i n d u c e d by

p u f f a d d e r venom was d u e t o t h e p r o d u c t i o n of h e p a r i n o i d

s u b s t a n c e s i n t h e systemic c i r c u l a t i o n of t h e v ic t im.

T h e venom of Echis c a r i n a t u s c o n t a i n s p r o t h r o m b i n

a c t i v a t i n g p r i n c i p l e w h i c h c o n v e r t s p r o t h r o m b i n i j t o a n

ac t ive enzyme t h r o m b i n ( K o r n a l i k e , 1963). However ,

K o r n a l i k e (1963) o b s e r v e d t h a t the t h r o m b i n a c t i v i t y

a c h i e v e d a s s u c h was lower t h a n t h a t o b t a i n e d w i t h t i s s u e

t h r o m b o p l a s t i n . When t h i s p r o t hrornbin a c t i v a t i n g p r i n -

c i p l e i s i n j e c t e d i n t o ra ts , t h e t h r o m b i n time i s p r o l o n g e d

f o r s e v e r a l d a y s . T h e i n c o a g u l a b i l i t y of b l o o d i s d u e t o

c o n s u m p t i o n of p r o t h r o m b i n by t h e p r o t h r o m b i n a c t i v a t i n g

p r i n c i p l e o f t h e venom. T h e p r o t h r o m b i n a c t i v a t i n g

p r i n c i p l e c a n be s e p a r a t e d f r o m t h e f r a c t i o n w i t h

haemorrhagic, c a e s i n o l y t i c a n d f i b r i n o g e n o l y t i c a c t i v i t i e s

( S c h i e k e t al . , 1972).

Tu (1977) a l so o b s e r v e d t h a t v i p e r i d a e venom h a v e been

known t o p r e c i p i t a t e b l o o d i n - c o a g u l a b i l i t y t h r o u g h various

m e c h a n i s m s . F o r e x a m p l e t h r o m b o p l a s t i n ( f ac to r 111) i s a

b l o o d c o a g u l a t i o n f a c t o r f o u n d i n t h e e x t r i n s i c system,

a n y venom c o m p o n e n t e i t h e r a c t i v a t i n g o r i n h i b i t i n g

t h r o m b o p l a s t i n would h a v e p r o n o u n c e d effect on b l o o d

c o a g u l a t i o n .

Some w o r k e r s a l s o o b s e r v e d t h a t t h e c o a g u a a n t a c t i o n

of v i p e r i d a e venom w a s d u e t o a c t i o n on c e r t a i n c o a g u l a t i o n

f a c t o r s . F o r i n s t a n c e , M a c f a r l a n e a n d B a r n e t t (1934)

o b s e r v e d t h a t R u s s e l l q s v i p e r venom e n h a n c e d b l o o d co-

a g u l a t i o n a n d b e l i e v e d i t t o be d u e t o t h e a c t i v a t i o n o f

S t u a r t f a c t o r ( F a c t o r X) p r e s e n t i n p l a s m a b y t h e venom

enzyme, A l s o a c o a g u l a n t enzyme p a r t i a l l y p u r i f i e d from

t h e venom of B i t i s q a b o n i c a ( M a r s h a n d W h a l e r , 1974)

releases f i b r i n o p e p t i d e s A a n d B from f i b r i n o g e n a n d

b r i n g s a b o u t b l o o d c o a g u l a t i o n .

Venom of V i p e r a r u s s e l l i c o n t a i n s a n a c t i v a e o r o f

factor V ( P r o a c c e l e r i n ) a n d t h e r e f o r e c a n be u s e d f o r t h e

a s s a y o f f a c t o r V ( P o o l a n d R o b i n s o n , 1971) R a p p o r t e t

a l . ( 1 9 6 6 ) f o u n d t h a t t h e u s e o f c r u d e venom g a v e

u n r e l i a b l e r e s u l t s . F a c t o r V ( P r o a c c e l e r i n ) t r e a t e d w i t h

R u s s e l l l s v i p e r venom i s n o t able b y i t se l f t o c o n v e r t

p r o t h r o m b i n t o t h r o m b i n a n d c a n n o t t h e r e f o r e be r e g a r d e d

as t h e f i n a l p r o t h r o m b i n c o n v e r t i n g p r i n c i p l e , T h e

p r o t h r o m b i n c o n v e r t i n g p r i n c i p l e c o n t a i n i n g a c t i v a t e d

f a c t o r V, S t u a r t f a c t o r , p h o s p h o l i p i d s a n d caCf i s able

t o g e n e r a t e t h r o m b i n f r o m p r o t h r o m b i n more r e a d i l y J w h e n

f a c t o r V h a s b e e n t r e a t e d w i t h venom t h a n when i t i s

u n t r e a t e d a n d t h e r e f o r e t h r o u g h t h i s mechan i sm t h e v i p e r

venom e h h a n c e c o a g u l a t i o n .

C a s e s of B i t i s A r i e n t a n s e n v e n o m a t i o n i n human b e i n g s

i n d i c a t e t h a t a n t i h a e r n o p h i l i c factor l e v e l s i n c r e a s e s i g n i -

f i c a n t l y f o r t h e f i r s t f e w d a y s , ( P h i l l i p s et a l , , 1 9 7 3 ) .

T h e a n t i h a e m o p h i l i c f a c t o r is p r e s e n t i n the m i d d l e p h a s e s

of i n t r i n s i c b l o o d c o a g u l a t i o n sys t em. T o g e t h e r w i t h

c a l c i u m i o n , c h r i s t m a s f a c t o r and p h o s p h o l i p i d s a n t i -

h a e m o p h i l i c f a c t o r a c t i v a t e s S t u a r t f a c t o r and w h o l e

p r o c e s s of c o a g u l a t i o n i s finally consuma ted .

V i p e r i d a e venom hafe a l s o been known t o h a v e e f f e c t

on b l o o d p r e s s u r e a n d r e s p i r a t i o n . F o r example t h e c r u d e

venom of V i p e r a ammodytes and two b a s i c f r a c t i o n s , induced

r a p i d d e c r e a s e i n a r t e r i a l b l o o d p r e s s u r e , and r e s p i r a t o r y

f a i l u r e in r a t (Tu, 1977) b u t t h e y had n o effect on r a t

d i a p h r a g m - p h r e n i c n e r v e p r e p a r a t i o n e v e n a f t e r p r o l o n g e d

s t i m u l a t i o n . T h u s t h e r e s p i r a t o r y d e p r e s s i o n may be due

t o d e p r e s s i o n of the r e s p i r a t o r y c e n t r e a n d n o t d u e t o

paralysis of t h e r e s p i r a t o r y muscles. By f r a c t i o n a t i o n

o f Vipera ammodytes venom N o v a l e et a l . (1973) o b t a i n e d

t h r e e f r a c t i o n s r e s p o n s i b l e f o r t h e d e p r e s s i o n of t h e

a r t e r i a l blood p r e s s u r e . They attributed t h i s a c t i o n t o

t h e effect o f p h o s p h o l i p a s e A2.

TOXICOLOGY OF SNAKE VENOMS

Snake b i t e s have c o n t i n u e d t o be r e s p o n s i b l e f o r many

d e a t h s i n many t r o p i c a l c o u n t r i e s . T h e s e r i o u s n e s s o f

t h i s p r o b l e m i s best a p p r e c i a t e d i n t h e r u r a l a r e a s , w h e r e

f ac i l i t i e s f o r i m m e d i a t e t r e a t m e n t are n o t a v a i l a b l e .

S n a k e venoms c o n t a i n d i r e c t and i n d i r e c t l y t i c f a c t o r s ,

Direct l y t i c factor i t s e l f c a n h a e m o l y s e r e d ce l l s s l o w l y ,

however i t s l y t i c a c t i v i t y c a n be g r e a t l y a c c e l e r a t e d by

t h e a d d i t i o n of p h o s p h o t i d y l c h o l i n e (Roy, 1955) . T h e

i n d i r e c t l y t i c f a c t o r has been i d e n t i f i e d as p h o s p h o l i p a s e

h (Tu , 1 9 7 7 ) . Venoms o f E l a p i d a e u s u a l l y c o n t a i n b o t h d i r e c t 2

a n d i n d i r e c t l y t i c f a c t o r s (De Veries e t a l , , 1962) . Haemo-

g l o b i n u r i a i s o n e of t h e c l i n i c a l symptoms o f A u s t r a l i a n

ELap idae p o i s o n i n g (Tu 1 9 7 7 ) . Cobra venom a l s o shows t h i s

h a e m o l y t i c a c t i v i t y . Chopra and Roy ( 1 9 3 6 ) o b s e r v e d t h a t

R u s s e l l ' s v i p e r venom haernolysed human a n d g u i n e a p i g r e d

ce l l s b u t n o t s h e e p r e d cells.

Coawlation Diso~ders

The Russell's viper on envonomation causes coagulation

of blood, This action, as already mentioned, results

from the activation of Stuart factor, The arvin (ancord)

poison from aakistrodom rhodostoma depletes the fibrinogen

level by conversion of prothrombin to thrombin which in

turn converts fibrinogen to fibrin, This fibrinogen

depletion results in retarded blood coagulation and the

victim may die from severe haemorrhage. Snake venom exert

effects on platelet, in particular they cause platelet

aggregation. The venom of B i t i s nasiconis increases

platelet adhesiveness (Nackay et al, , 1970). Crotalus

atrox venom is also followed by aggregation of p l a t e l e t s

and haemostasis (Ownby et al., 1974): the lumen of

capillaries being completely occluded by aggregates of

platelets, It was also observed that viperine snake venom

caused a decrease in platelet count, (Warrell et al., 1977).

In crotalus mber ruber and puff adder bites low platelet

counts were observed (Lyons, 1971)~ These observations

suggest that venom has direct destructive effect on

platelets in vivo, This was confirmed by injecting

venom into rats which resulted in decrease in platelet

count (Takahashi et al., 1970).

H a e m o r r h a g e

V a r i o u s h a e m o r r h a g i c m a n i f e s t a t i o n s a r e common i n v i p e r i n e

b i t e s , T h e s e i n c l u d e haemorrhagic o o z i n g from the site of

t h e s n a k e b i t e s a n d m i l d d e g r e e of h a e m o p t y s i s . H a e m a t u r i a

a n d c i r c u l a t o r y c o l l a p s e were c o n s i d e r e d t h e m a j o r p r o b l e m s

i n v i p e r i n e e n v e n o m a t i o n ( O n u a g u l u c h i , 19601. T h e bites o f

v i p e r i d a e ( p u f f a d d e r , R u s s e l l ' s v i p e r a n d r a t t l e s n a k e s )

c a u s e p e r s i s t e n t p a i n a n d s e v e r e l o c a l r e a c t i o n s a t t h e site

o f t h e bite. If: f a t a l , s u c h p a t i e n t s die some t w o t o s i x

d a y s later. Severe a n d e x t e n s i v e b l e e d i n g into the s k i n a n d

e l s e w h e r e a re s e e n on p o s t m o r t e m e x a m i n a t i o n ( S e a t o n , 1 9 5 9 ) .

H a e m o r r h a g e a s s o c i a t e d w i t h local t i s s u e damage of t e n f o l l o w

p o i s o n i n g b y c r o t a l i d a e a n d v i p e r i d a e . I n severe p o i s o n i n g .

h a e m o r r h a g e c a n be o b s e r v e d i n many i n t e r n a l organs (Tu and

Homan, 1970) .

M o s t crotalidae venoms p r o d u c e h a e m o r r h a g e and m y o n e c r o s i s

s i m u l t a n e o u s l y u p o n e n v e n o m a t i o n (Tu a n d Homan, 1970) . Some

v i c t i m s s u r v i v e , b u t s u s t a i n p e r m a n e n t damage t o t i s s u e s ,

such as muscles, c a r t i l a g e a n d t e n d o n s ( E m e r y a n d R u s s e l ,

1963) .

N e p h r o t o x i c i t y

S n a k e p o i s o n i n g can c a u s e a c u t e t u b u l a r n e c r o s i s i n human

v i c t i m s . C l i n i c a l c a s e s of r e n a l f a i l u r e have b e e n r e p o r t e d

from e n v e n o m a t i o n b y sea s n a k e q ( M a r s d e n and R e i d , 1951).

Tu ( 1 9 7 7 ) r e p o r t e d t h a t f e w s t u d i e s on c r o t a l i d a e - venom

effect on t h e k i d n e y h a v e been made. H e however r e p o r t e d

t h a t t h e r e w a s a s i g n i f i c a n t ? n c r e a s e i n u r i n a r y a l k a l i n e

p h o s p h a t a s e and l e u c i n e a m i n o - p e p t i d e s e a c t i v a t i o n i n ra ts

t r e a t e d with Agki s t rodo rn p i s c i v o r u s venom. T h e increase

i n e n z y m a t i c a c t i v i t y s u g g e s t e d t h a t t h e k i d n e y was damaged

b y t h e venom. C r o t o x i n c a u s e s k i d n e y damage when i n j e c t e d

i n t r a v e n o u s l y i n t o d o g s (Mohamed e t a l . , 1 9 5 6 ) . N e p h r o t o x i c

a c t i o n c a n a l so be i n d u c e d by v i p e r i d a e venoms. Sank et

al . ( 1 9 7 4 ) repor ted t h a t E c h i s carinatus venom p roduced -- l e s i o n s i n k i d n e y . o f r a b b i t s and monkeys.

T h e most p o t e n t component i n Elapidae venoms i s

n e u r o t o x i n and t h e n e x t mos t p o t e n t i s c a r d i o t o x i n .

However c r u d e venom c o n t a i n s more t h a n t h e s e two c o m p o n e n t s

a n d many E l a p i d a e venoms c a u s e k i d n e y damage. A lethal

dose of Naja n a j a venom c a u s e d f o c a l t u b u l a r d e g e n e r a t i v e - l e s i o n s w i t h i n t a c t g l o m e r u l i (Moharned et a l . , 1 9 7 5 ) . The

t u b u l a r membrane w a s a l s o i n t a c t a n d t h e b r u s h b o r d e r o f

the p r o x i m a l c o n v o l u t e d t u b u l e s was d e s t r o y e d o n l y i n t h e

m a r k e d l y degenerated t u b u l e s . ~acuolGa+lon and s h e d d i n g

of t h e e p i t h e l i a l l i n i n g of t h e cells of t h e loop of H e n l e a n d

d i s t a l c o n v u l t e d t u b u l e s were howeve r o b s e r v e d ( T U , 1977).

Envenomat ion by t h e sea s n a k e - L e t i c a n d a semifasciata

d i d n o t result i n a n y u l t r a s t r u c t u r a l modifications of

t h e p r o x i m a l t u b u l e ( S c h m i d t et a l . , 1976). However ,

i n t r a c e l l u l a r o e d e m a a n d s e l e c t i v e s w e l l i n g of o r g a n e l l e s ,

e n d o s p l a s m i c r e t i c u l u m a n d m i t o c h o n d r i a were n o t e d i n

most of the v i s c e r a l epithelial cel ls .

CHEMICAL NEUTRALIZATION CF SNAKE VENOMS

A t p r e s e n t the most effective t r e a t m e n t f o r s n a k e b i t e is

h o r s e a n t i s e r u m t o snake venom ( ~ n t i v e n i n ) w h i c h i s u s u a l l y

p r o d u c e d by i n j e c t i n g very s m a l l a m o u n t s o f venom o v e r a

l o n g p e r i o d . B e c a u s e of the h i g h t o x i c i t y and severe l oca l

t i s s u e damage i n d u c e d by most s n a k e venoms o n l y small

a m o u n t s of venom c a n be a d m i n i s t e r e d t o t h e h o r s e . However,

t h e p r o d u c t i o n of t h e a n t i s e r u m with h i g h t i t r e i s a very

l o n g and t e d i o u s p r o c e d u r e . T h e approach is r a t h e r cumbersome

and t h e r e f o r e a n o t h e r procedure f o r p r o d u c i n g a n a n t i s e r u m

w i t h h i g h t i t r e i s r e q u i r e d . T h i s h a s b e e n achieved b y

m a k i n g t o x o i d s f r o m snake venoms t h a t still retain t h e i r

a n t i g e n i c i t y . S u c h t o x o i d s a r e i n j e c t e d i n t o horses which

s t i m u l a t e a n t i b o d y p r o d u c t i o n . The h o r s e s a r e bled a n d the

serum c o l l e c t e d a s a n t i s e r u m after appropriate t r e a t m e n t .

E s t r o g e n s

The a n t i h a e m o r r h a g i c a c t i o n of e s t r o g e n s has l o n q b e e n

r e c o g n i s e d , b u t t h e b a s i s for t h i s a c t i o n i s still n o t

clear (Rona, 1963). E s t r i o l d i s o d i u m s u c c i n a t e h a s b e e n

u s e d t o s t o p the h a e m o r r h a q e i n d u c e d by cobra venom because

of its p r o t e c t i v e e f f e c t o n blood v e s s e l s . I t a p p a r e n t l y

s t r e n g t h e n s t h e p e r i v a s c u l a r c o n n e c t i v e tissue b y c a u s i n g

a c h a n g e i n t h e a c i d m u c o p o l y s a c c a r i d e s , s o t h a t t h e s o l - g e l

e q u i l i b r i u m s h i f t s t o w a r d s t h e more s o l i d g e l state.

Hepa r in

H e p a r i n i s a s u l p h a t e - c o n t a i n i n g m u c o p o l y s a c c a r i d e t h a t

acts as a n a n t i c o a g u l a n t b y p r e v e n t i n g t h e a c t i v a t i o n of

c h r i s t m a s f a c t o r ( p l a s m a t h r o m b o p l a s t i c c o m p o n e n t ) . It

a l s o ac t s w i t h p l a sma c o f a c t o r t o i n h i b i t t h e a c t i o n o f

t h r o m b i n t h u s p r e v e n t i n g c o a g u l a t i o n of b l o o d . H e p a r i n i s

h i g h l y a c i d i c and i s known t c form an i r r e v e r s i b l e complex

w i t h p r o t a r n i n e a h i g h l y basic p r o t e i n . B h a r g a r a e t a l .

( 1 9 7 0 ) h a v e shown t h a t h e p a r i n f o r m s a n i n a c t i v e complex

w i t h hae rno r rhag ic oomponent of cobra venom, t h e r e b y i n h i b i t -

i n y t h e h a e m o r r h a g i c a c t i v i t y o f t h i s venom. Bon ta e t a l .

(1970) studied t h e damaging effect of c o b r a venom on t h e

pu lmonary r n i c r o c i r c u l a t i o n and t h e i n a c t i v a t i o n of the

v a s c u l o t o x i n of the venom by h e p a r i n . The p r o t e c t i v e

e f f e c t o f h e p a r i n on t h e v e s s e l s s u p p o r t s t h e v i e w t h a t

t h e h e p a r i n - p r e c i p i t a b l e , s t r o n g l y b a s i c v a ~ c u l o t o x i c

f a c t o r of t h e venom i s n o t i d e n t i c a l w i t h t h e main l e t h a l

( n e u r o t o x i c ) component . Hepa r in a l s o p r e v e n t e d pu lmonary

haemor rhage and p r o l o n g e d s u r v i v a l time when a d m i n i s t e r e d

l o c a l l y p r i o r t o t h e i n j e c t i o n o f t h e venom.

L u n g s of mice p r e t r e a t e d w i t h h e p a r i n 10mg/kg i n t r a -

t h o r a c i c a l l y p r i o r t o t h e i n j e c t i o n of t h e venom of Naja

n a j a were f o u n d a t p o s t mortem t o be free f r o m h a e m o r r h a g e .

T h e i n c r e a s e i n l u n g w e i g h t d u e t o N a ja n a j a venom was

p r e v e n t e d a n d s u r v i v a l time of t h e a n i m a l s was p r o l o n g e d ,

However , d e a t h was n o t p r e ~ e n t e d by h e p a r i n i z a t i o n of t h e

venom.

D e c r e a s e d v a s c u l a r p e r m e a b i l i t y a f t e r h e p a r i n h a s been

d e s c r i b e d (Nazarov a n d P e t r i s c h e r , 1968) t h o u g h t h e e x a c t

mechan i sm i s p o o r l y u n d e r s t o o d , However , (Bonta et a l , ,

1970) p o i n t e d o u t i n t h e i r e x p e r i m e n t t h a t w h e t h e r h e p a r i n

p r o t e c t i o n o f t h e v e s s e l s i s d u e t o i t s i n f l u e n c e on the

m u c o p o l y s a a c h a r i d e i s s t i l l a q u e s t i o n for f u r t h e r

e x p e r i m e n t a l s t u d i e s , T h e e f f e c t o n m u c o p o l y s a a c h a r i d e

c o m p o s i t i o n of c o n n e c t i v e t i s s u e h a s b e e n d i s c u s s e d i n a

review by G a s e p a r ( 1965). E v i d e n c e of a direct effec t of

h e p a r i n o n t h e g r o u n d s u b s t a n c e of p e r i v a s c u l a r c o n n e c t i v e

t i s s u e , i s n o t y e t agailabae, t h o u g h i t h a s b e e n s u g g e s t e d

by G a s t p a r (1965) t h a t h e p a r i n may p o s s i b l y i n c r e a s e the

p o l y m e r i z a t i o n of t h e a c i d m u c o p o l y s a c c h a r i d e s t h r o u g h

i n c o r p o r a t i o n i n t o t h e i n t r a c e l l u l a r g r o u n d s u b s t a n c e s ,

Such a n e f f e c t of h e p a r i n may r e s u l t i n a n i n c r e a s e d

r e s i s t a n c e of t h e vessel permeability.

Compounds c o n t a i n i n q S u l p h u r

O f t h e t h i o l c o m p o u n d s , d i h y d r o t h i o t i c a c i d seems t o be

t h e m o s t e f f e c t i v e i n preventing h a e r n o r r h a g e a n d d e a t h

when m i x e d w i t h h a b u venom b e f o r e i n j e c t i n g i n t o a n i m a l s .

T h e a c t i v i t y o f t h e h a b u venom r e s i d e s i n t h e S-S bond.

H e n c e , a l o w m o l e c u l a r w e i g h t compound c o n t d i n i n l ; ari SW

r a d i c a l c o u l d s u b s t i t u t e f o r t h e S-S b o n d a n d t h u s

i n a c t i v a t e t h e venom ( K u r i h a r a a n d S h i b a t a , 1971). On t h e

o t h e r h a n d , t h i o l c o m p o u n d s m i g h t p r e v e n t t h e i n h i b i t o r y E.

a c t i o n of h a b u venom o n t h e a c t i v i t y of s u c c i n i c d e h y d r o -

g e n a s e ( S H e n z y m e ) , However , t h i o l c o m p o u n d s are g e n e r a l l y

h i g h l y l i m i t e d a s a n t i v e n o m . F o r e x a m p l e glutathione d i d

r e d u c e t h e Gethalbky.of h a b u venom in v i t r o , b u t d i d n o t

r e d u c e h a e m o r r h a y e i n d u c e d by C r o t a l u s s p e c i e s venoms.

C y s t e i n e , t h i o g l y c o l a t e a n d t h i o u r e a d i d n o t p r e v e n t

h a e m o r r h a g e d u e t o s n a k e venom.

C h e l a t i n g c o m p o u n d s

Venoms p r o t e a s e a c t i v i t i e s a n d h a e m o r r h a g i c a c t i v i t y c a n

be removed b y a d d i n g EDTA a p o w c r f u l c h e l a t i n y a g e n t t o

v e n o m s ( F r i e n d e r i c h a n d T u , 1 9 7 1 ) , T h e e f f e c t i v e n e s s o f

c h e l a t i n g compound E.D,T,A. i s d u e t o r e m o v a l o f metals

from venom p r o t e i n s . T h e s t u d y of (Tu et a l . , 1 9 7 0 ) a n d

(Ownby et a l . , 1975) showed t h a t s e v e r a l of c h e l a t i n g a g e n t s

t e s t e d p r e v e n t e d h a e m o r r h a g e i n v i t r o b u t n o t i n v i v o .

Protamf n e S u l p h a t e

Okonkwo and Onuaguluchf (1977) recommended t h a t c o a g u l a -

t i o n defect which fo l low v i p e r i n e bites can be overcame

by r e p l a c i n g t h e depleted p l a s m a p r o t e i n s e s p e c i a l l y

f i b r i n o g e n a n d by g i v i n g p r o t a r n i n e s u l p h a t e t o antagonise

t h e h e p a r i n o i d s u b s t a n c e l i b e r a t e d by the venom a n d elso

t o protect f i b r i n o g e n from further l y s i s by t h e venr-.

Prob lems I n Manaqement o f Snake Venom P o i s o n i n q

It would appear f rom t h e l i s t o f s u b s t a n c e s u t i l i z e d in

a n t a g o n i s i n g t h e effects of snake venom that t he re is

no t much choice I n t h e s e l e c t i o n of a g e n t s for therapy.

Even the ava i l ab l e agents have l i m i t a t i o n s . For i n s t a n c e ,

a n t i v e n i n being a foreign p r o t e i n i s known t o p r o d u c e

s e r i o u s a n a p h y l a c t i c r e a c t i o n s i n i n d i v i d u a l s h y p e r s e n s i t i v e

t o horse serum. The s p e c i f i c i t y of the m o n o v a l e n t a n t i v e n i n

i s a f u r t h e r l i m i t a t i o n , so t h a t the use of such a n t i v e n i n

r e q u i r e s invariably the i d e n t i f i c a t i o n o f the t y p e of

species of t h e o f f e n d i n g snake, P o l y v a l e n t antivenin

does n o t h a v e t h i s t y p e of l i q i t a t i o n but may be less

e f f e c t i v e . Heparin is only effective i n cobra e n v e n o m a t i o n

but o n l y against t h e v a s c u l o t o x i c f a c t o r . C h e l s t i n g a g e n t s

are o n l y e f f e c t i v e a s i n v i t r o agents. Therefore, they

a r e a t best o n l y useful tools i n experimental p r o c e d u r e s .

- L J - EXPERIMENTAL S T U D I E S

I N T R O D U C T I O N

In the N i g e r i a n e n v i r o n m e n t , v i p e r i n e bites are

v e r y common a n d h a v e c a u s e d v e r y many d e a t h s . H o w e v e r , I

t h i s d o e s not mean t h a t o u r h e r b a l i s t s d o n o t t reat

s n a k e b i t e s w i t h some s u c c e s s . L o c a l h e r b s s u c h a s

~ i o d i a s c a n d e n s h a v e a p p a r e n t l y been u s e d s u c c e s s f u l l y

t o treat s n a k e b i t e s . P l a t e l ( a a n d b) are p h o t o g r a p h s

of D i o d i a s c a n d e n s s h o w i n g t h e stem, leaves a n d f l o w e r s ,

T h e f l o w e r s are w h i t e a n d are a g g r e ~ a t e d i n l a r g e

n u m b e r s a t t h e n o d e s a n d a t the e n d s o f t h e b r a n c h e s .

D i o d i a s c a n d e n s i s a member of t h e ~ u b i a c i ' a e a n d i s a

c r e e p i n g o r c l i m b i n g h e r b a c i o u s p l a n t , which g r o w s

l u x i r i a n t l y d u r i n g the r a i n y s e a s o n but withers s o m e w h a t

i n t h e d r y s e a s o n . T h e h e r b used i n t h i s study was

collected from Achi i n Oji River L o c a l G o v e r n m e n t Area

i n ~ n a m b r a State i n t h e m o n t h o f A u g u s t 1984.

H o w e v e r , some were planted i n P r o f . G. o n u a q u l u c h i * ~

g a r d e n a t E n u g u i n order to have a r e a d y s o u r c e of the

h e r b whenever the n e e d a r i ses .

I n t r e a t i n g the s n a k e bite victim the h e r b a l i s t

uses b o t h w a t e r e x t r a c t and a t h i c k paste o f the g r o u n d -

u p h e r b . T h e a rea of t h e bite i s s c a r i f i e d w i t h c l e a n

r a z o r b l a d e a n d a thick paste o f t h e a r cund -up h e r b i s

a p p l i e d o v e r t h e wound. A s e g m e n t of t h e s t e m i s

f a s t e n e d a l i t t l e a b o v e t h e w o u n d , i n the belief t h a t

i t c o u l d c a u s e t h e venom t o g r a v i t a t e and remain

b e l o w the wound, Some of t h e h e r b i s t h e n c h o p p e d u p

a n d b o i l e d f o r a b o u t o n e h o u r i n a p o t of w a t e r and t h e

p a t i e n t i s g i v e n a c u p ( a b o u t 200ml) o f t h e i n f u s i o n

t o d r i n k . H e c o n t i n u e s t o t a k e t h i s q u a n t i t y o f t h e

infusion 3 o r 4 t i m e s d a i l y f o r a n o t h e r 3 d a y s o r more

u n t i l a c u r e i s e s t a b l i s h e d .

T h i s s t u d y i n v e s t i g a t e d t h e e f f e c t s of a n ethanolic

e x t r a c t from D i o d i a s c a n d e n s o n some effects of t h e

venom f r o m E c h i s c a r i n a t u s .

S E C T I O N A: EXTRACTION METHODS

T h e h e r b w a s c o l l e c t e d f r o m t h e f i e l d s and s t o r e d

i n a refirgerator o v e r n i g h t . I t was t h e n choped u p ,

w e i g h e d a n d e x t r a c t e d i n a s o x h l e t a p p a r a t u s with

b o i l i n g e t h a n o l f o r 1 2 h o u r s . C l a r i f i c a t i o n of the

c r u d e t o t a l e x t r a c t w a s a c h i e v e d by h o m o g e n i s i n g i t

i n 50% a q u e o u s e t h a n o l and p a s s i n g t h e s o l u t i o n through

a c o l u m n of activated c h a r c o a l . T h e f i l t r a t e t h u s

o b t a i n e d w a s c o l o u r l e s s , C r y s t a l s d e p o s i z ~ d i n t h e

f i l t r a t e o n s t a n d i n g . H o w e v e r , more c r y s t a l s were

h a r v e s t e d when t h e f i l t r a t e was k e p t c o v e r e d i n the

r e f r i g e r a t o r . T h e c r y s t a l s were r e m o v e d ( f r a c t i o n A )

by f i l t r a t i o n u s i n g a medium s i z e d w h a t m a n n u m b e r 542

f i l t e r p a p e r a n d a i r dired. T h e m o t h e r liquor w a s

0 e v a p o r a t e d t o d r y n e s s a t 40 C, T h e d a r k residue

o b t a i n e d ( f r a c t i o n B) w a s collected a n d d r i e d i n a

d e s i c a t o r .

F i n a l l y t h e a c t i v a t e d c h a r c o a l c o l u m n w a s eluted

w i t h a q u e o u s e t h a n o l c o n t a i n i n g 0.05% glacial acet ic

a c i d . Distil l a t i o n of t h i s e l u a t e yielded a b r o w n

r e s i d u e ( f r a c t i o n C ) w h i c h was d r i e d a t 4 0 " ~ a n d

p r e s e r v e d i n t h e d e s i c a t o r . T h e c o n t a i n e r s w e r e

w e i g h e d d a i l y u n t i l c o n s t a n t w e i g h t s were a c h i e v e d .

T h e w e i g h t s of t h e f r a c t i o n s B a n d C o b t a i n e d from t h e

p l a n t were t h e n d e t e r m i n e d a n d p e r c e n t a g e yields w e r e

c a l c u l a t e d ,

SECTION £3: COAGIJLATIOM STUDIYS

E x p e r i m e n t la - r h ~ e f f e c t of E c h i s C a r i n a t u s ?r~\nom o n

' J h o l e Rlood C l o t t i n n T i m e

F r e l i m i n a r y s t u d y w a s c a r r i e d out followin? t h e observation

t h a t m o s t v i p e r s e x e r t their l e t h a l effect by p r o d u c i n g

i n t e r n a l h a e m o r r h a q e ( O n u a q u l u c h i , 1960). I t was t h e r e f o r e

d e c i d e d t o i n v e s t i g a t e how E c h i s c a r i n a t u s venom p r o d u c e d

( d e r a n g e m e n t i n b l o o d c o a q u l a t i o n . The assay method was

s i m i l a r to t h a t of Lee a n d W h i t e ( 1 P 3 1 1 , e x c e p t - t h a t 2 m l of

blood were u s e d i n s t e a d o f 1 m l of b lood u s ~ d by L e e a n d

W h i t e . S p e c i m e n s were c o l l e c t e d from k e n a d ~ l l t v o l u n t e e r s

c o n s i s t i n g of t h r e e females a n d s e v e n males. Two m w s of ten

t es t t u b e s w e r e a r r a n g e d s s against f o u r test tubes f o r e a c h

s p e c i m e n i n t h e o r i g i n a l method. I n o t h e r words each specime

h a d t w o test t u b e s . T h i s a r r a n G e m e n t w a s necessary f ~ r t h e

economy of m a t e r i a l s . T h e t u b e s were k e p t for 5 rnin. i n a

0 w a t e r b a t h m a i n t a i n e d at 37 C. A stop watch was started as

soon as v e n o u s blood e n t e r e d t h e s y r i n g e .

O.?ml ( 2 0 0 u g ) o f Imu/rnl venom solution was d e l i v e r e d i n t o

each o f the second row t e s t t u b e s previously before blood

a d d i t i o n . T h e tubes were i n c o n s t a n t rotation b ~ r t e v e r y

m i n u t e each t u b e w a s r e m o v e d from i t s s t a n d a n d was t i l t e d

t h r o u g h a n a n g l e greater t h a n 90". NO spilling of blood

indicated that clot t i n c naa occurred.

Exper iment Ib - The Effect of The H e r b a 1 , E x t r a c t on The

A c c e l e r a t e d Whole Blood C l o t t i n g T i m e

Induced by Venom

A s a r e s u l t of Exper iment l ( a ) the effect of t h e herbal

e x t r a c t on t h e d e c r e a s e d whole b lood c l o t t i n g time f o l l o w i n g

a d d i t i o n of venom was inves t iga ted . In this study t h e

method of L e e and W h i t e (1932) was again adopted , Samples

were c o l l e c t e d f r o m f o u r adult v o l u n t e e r s . Irnl p o r t i o n of

each sample was d e l i v e r e d i n t o each of the test t u b e s

marked A B C D. T h e test t u b e s were k e p t i n a water b a t h

a t 3 7 ' ~ for 10 m i n before the blood samples were delivered

i n t o them. T h e quantity of herbal e x t r a c t o r venom u s e d was

O.1ml of lmg/ml s o l u t i o n , The t o t a l volume i n e a c h test tube

was k e p t constant a t 1.2ml as shown i n t h e scheme below,

Blood ( m l )

S a l i n e ( m l )

HE (lrng/ml)

Venom ( lmg/ m l

T U B E S

5 x p c s i m e n t 3Ia - Effec t of venom on prothrombin t i m e

Quick's One Stage p r o t h r o m b i n time assay technique was

a d o p t e d . T h i s invo lved t h e r ~ c a l c i f i c a t i o n of c i t r a t e d

plasma i n t o w h i c h was added t i s s u e t h r o m b a p l a s t i n and

the c l o t t i n o time e s t i m a t e d . T r i s o d i u r n ci t r a t c d p lasma

from t e n a d u l t volunteers was used. Two rows of ten

t es t t u b e s were a r r anged and k e p t warm i n water b a t h

main t a ined a t 37O~.

O . l r n l p l a s ~ a f rom esch samplc was d e l i v e r e d into each test

t u b e . O.1ml b r a i n thromboplastin was a l s o delivered i n t o

all the tuhcs. To the tubes i n t h ~ first r o w 0.01rnl no rma l

saline (9aR1) was added t o m a k c u p f o r t h c addition of 0.02

ml of lmg/rnl venom solution added t o tubes i n t h e s econd

row. T h e ccntents in a l l the tubes vtre t h e n recalcified

w i t h O . 1 r n l 0.025PI CaC12 k e p t warm at 37O~. The time

t a k e n for t h e devclcpment of f i b r i n clot was noted .

P = Plasma; B = Brain thromboplastin; S = S a l i n e

C = C a l c i u m c h l o r i d e and V S= Venom.

E x p e r i m e n t IIb - T h e E f f e c t of t h e H e r b a l Extract o n t h e Venom and B r a i n T h r o m b o p l a s t i n

A c t i v i t y

T r i s o d i u m c i t r a t e d p l a s m a f r o m t w o a d u l t v o l u n t e e r s

(Eu and B O ) w a s u s e d . T h e tes t w a s done i n d u p l i c a t e .

E a c h s a m p l e w a s a s s a y e d i n f o u r rows of test t u b e s t w o

i n e a c h row. The test tubes w e r e k e p t i n water b a t h

m a i n t a i n e d a t 37Oc.

The f i r s t row c o n t a i n e d 3 , l m l p l a s m a , O.lml b r a i n

t h r o m b o p l a s t i n , 0 , l l m l n o r m a l s a l i n e (9g /X) a n d O . 1 m l

CaC1 (0 .025M). S e c o n d row c o n t a i n e d 0 . l m l p lasma,

O . l m l brain t h r o m b o p l a s t - i n 0.Olrril ( l O p g ) , h e r b a l e x t r a c t

( l m g / m l ) , 0 , l m l n o r m a l s a l i n e ( 9 q 1 / 1 ) a n d O, l rn l ,

Row t h r e e c o n t a i n e d 0.1111 p l a s m a , U , O l m l ( 1 0 p g ) venom

( m q / m l ) , 0.2ml n o r m a l s a l i n e ( 9 g / l ) a n d O.lml.

(0.025M) cac12.

F o u r t h r o w c o n t a i n e d O . l m l p l a s m a , 0,lml b r a i n

t h r o r n b o p l a s t i n , h e r b a l extract O . O l m l ( 1 0 p g ) Img /ml ,

O , O ? m l (1Opg) venom l m q / m l , U.09ml n o r m a l s a l i n e ( 9q /1 )

a n d O . 1 m l CaCl ( 0 . 0 2 5 ~ ) . T h e t e c h n i q u e u s e d f o r t h i s 2

assay w a s t h e s a m e a s t h e q u i c k ' s One S t a g e P r o t h r o m S i n

t i m e a s s a y . T h e d e v e l o p m e n t of fibrin c l o t marked t h e

e n d p o i n t a n d t h e r e f o r e the t i m e t a k e n f o r the

f o r m a t i o n o f t h i s f i b r i n w a s n o t e d a s p r o t h r o m b i n time.

E x p e r i m e n t I I C - I n v e s t i g a t i o n of the Venom T h r o n S o ~ l a s t i c k c t i v i t j t a n d Eerba: E X ~ K - ~ C ' ;,ntl thrornbo-

~ l a s t i c A C ,:vi t v

E x p e r i m e n t IIb was s l i g h t l y d e f i c i e n t i n i t s p l a n n i n g

b e c a u s e w e c o u l d n o t separate t h e a n t a g o n i s t effect of

t h e h e r b a l e x t r a c t o n t h e b r a i n t h r o m b o p l a s t i n a n d o n

t h e t h r o m b o p l a s t i c a c t i v j - t y o f t h e venom, E x p e r i m e n t I I C

was t h e r e f o r e d e s i g n e d s p e c i f i c a l l y to i n v e s t i g a t e t h e

effect of t h e h e r b a l e x t r a c t o n t h e t h r o m b o p l a s t i c

a c t i v i t y o f t h e venom. Two r o w s each o f 4 t e s t tubes

were se t up. T h e f i r s t row o f 4 test t u b e s were u s e d

in a s s a y i n g t h r o m b o p l a s t i c a c t i v i t y o f t h e venom.

E a c h tube c o n t a i n e d 0 , l m l p l a s m a , 0 , O l m l ( l o p g ) venom

I rng/ml , 0 .01ml n o r m a l s a l i n e ( 9 g / ~ ) a n d O , l r n l

CaC12 0.025M. T h e s e c o n d row o f f o u r test tubes w e r e used

i n a s s a y i n g the a n tithromboplas tic a c t i v i t y of t h e h e r b a l

e x t r a c t i n t h e p r e s e n c e of t h e venom,

E a c h t u b e c o n t a i n e d U - l m l o f p l a s m a , 0.01ml (10)aq) venom

l m g / m l , 0 ,01ml ( 1 0 p g ) h e r b a l e x t r a c t Irng/ml and 0 , I r r l

CaC12 0.025M. Venom i n t h i s e x p e r i m e n t r e p l a c e d the

brain t h r o m b o p l a s t i n . F o r m a t i o n of fibrin clot m a r k e d

the e n d p o i n t after recslcification of t h e m i x t u r e . T h e

t i m e t a k e n f o r t h i s f i b r i n formation w a s noted a s t h e

p r o t h r o m b i n t i m e .

P = Plasma; V = Venom;

S = S a l i n e ; C - Calcium Chloride; H = Herbal extr3ct.

ROWS 1

C o n ten ts

Experiment IIIa -

If t h e venom

To Demonstrate Possible Thrombin A c t i v i t y of t h e Venom and the Effect of the H e r b a l

Extract on t h i s A c t i v i t y

has th rombin a c t i v i t y i t w i l l c l o t

citrated plasma i n absence of c a l c i u m i o n s . This thrombin-

l i k e a c t i v i t y of t h e venom was i n v e s t i g a t e d by conducting

a series of t h r o m b i n assays. The p r o c e d u r e adapted was

e s s e n t i a l l y t h a t used by Dacie and Lewis ( 2 9 7 5 ) , who added

0,lml thrombin s o l u t i o n t o 0,21111 c i t r a t e d plasma dnd t h e t i m e

taken by the mixture to c l o t was regarded as t h e thrombin

t i m e , The same p r o c e d u r e was a d o p t e d except t h a t varying

amaunts of t h e Herbal extract were added t o 0 ,Zml c i t r a t e d

plasma g i v i n g f i n a l d i l u t i o n s of 1:3142, 1: 1100 and 1:275

before adding a fixed dose of 0.02mg ( 2 0 u g ) of l rng/ml venom

by us ing 0.02m1, Specimens were collected from t h r e e adult

v o l u n t e e r s marked A, B, C, Each specimen was assayed i n

q u a d r u p l i c a t e against a p a r t i c u l a r d rug a n t i v e n o m c o n c e n t r a -

t i o n . Thus four test t u b e s of 75 x 12mm w e r e arranged i n

a s t r a i g h t l i n e in a water bath m a i n t a i n e d a t 3 7 O ~ . 0.2.1

of t h e c i t r a t e d plasma was d e l i v e r e d i n t o each of the tubes,

his was f o l l o w e d by the a d d i t i o n of 0.07ml I m g / m l ( 7 0 ~ 9 1 ,

herbal extract and 0,13ml normal saline (9g/L) for t h e first

set of four t u b e s , The same p r o c e d u r e was adopted for t h e

second set of f o u r tubes using 0,2ml l m g l m l h e r b a l extract

( 2 0 0 p g ) and O . 1 m l 8 m q / m l (800ug) h e r b a l e x t r

normal s a l i n e were used for t h e t h i r d set of

Finally 0,02ml I m g / m l venom were added into all t h e tubes

in the three sets. The time t a k e n f o r t h e formation of

f i b r i n clot was n o t e d as t h e thrombin t i m e .

P = Plasma; V = Venom; H = Herbal

extract; S = S a l i n e , Variable amounts

of H and S were used: H = 0.07- 0 - 2 m l

and S E 0 - 0.13m1.

SECTICN C E l e c t r o ~ h o r e s i s S t u d i e s

E v a l u a t i o n o f An tiproteolytic A c t i o n o f T h e Herbal E x t r 3 c t

O n Venornised P l a s m a E l e c t r o ~ 9 o r e t o a r a m - -

he o b s e r v a t i o n by ~ ~ ( 1 9 7 7 ) 'and Okonkwo and O n u a q u l u c h i ( 1 9 7 7 )

that viperine venom i s p r o t e o l y t i c n e c e s s i t a t e d a n

i n v e s t i a a t i o n i n t o the p o s s i h l e a n t i p r o t e o l y t i c activity of t h e

h e r b a l extract, o n t h e v a r i o u s p r o t e i n b a n d s of k p a r i n i s e d

p lasma treated w i t h venom e l e c t r o p h o r e s e d on cellulose

a c e t a t e s t r i p . H e p a r i n i s e d p l a s m a ( 1 5 i u / r n l ) f rom t h r e e a d u l t s

was u s e d , T h e f o l l o w i n q p a t t e r n of e x p e r i m e n t were arranged

for e a c h plasma s p e c i m e n ,

I 1 T u b e s M a t e r i a l s

I Flas rna 1 m l + 0.21~11 normal s a l i n e I I1

I P l a s m a I m l + 0:lrnl venom ( I rnq/ml ) + O . l m l 1 n o r m a l s a l i n e

T h u s t h e c o n t e n t s of e a c h tube was s e p a r a t e l y e l e c t r o p h o r e s e d

1 I F l a s m a I m l + O . ? r n l herbal extract ( T m g / m l )

O.'ml n o r m a l s a l i n e

o n a celluloce e c e t a t e s t r i p .

T h e t!m e l e c t r o p h o r e t i c t a n k chambers were f i l l e d with

B1a.rma 1 m l + C ,;ml h e r b a l extract ( lrnq/rnl) + 0.1 venom ( I m g / m l )

5 0 0 m l each of t h e b a r b i t o n e b u f f e r s o l u t i o n pH 8.6 c o n t a i n i n q

3.129 d i e t h y l b a r b i t u r i c a c i d and 17.lg s o d i u m d i e t h y l b a r b i t u r a t e ,

T h e c e l l u l o s e a c e t a t e s t r i p w a s cut t o s i z e (4.5x5cm) and was

s o a k e d i n t h e buffer s o l u t i o n for 5 m i n .

T h e r e a f t e r t h e soaked s t r i p was d r i e d b e t w e e n two

medium sized Whatman Number 30 f i l t e r p a p e r s . It w a s p l a c e d

i n p o s i t i o n across t h e bridge p i e c e s t o t e n s i o n . I t w a s

l o a d e d w i t h 10 m i c s o l i t e r s of t h e s p e c i m e n a p p l i e d a l o n g

a line about 1 c m on the m i d - l i n e d r a w n a c r o s s t h e s t r i p

2 crn from t h e cathodal ed$e. The e l e c t r o p h o r e t i c t a n k was

t h e n covered a n d t h e e lec t r i ca l leads w e r e c o n n e c t e d t o

t h e power pack, u s i n g a constant v o l t a g e of 1 8 0 vo l t s .

T h e run w a s f o r 20 min. T h e strip w a s s t a i n e d with

N i g r o s i n d y e O. lg /L i n 100g/L t r i c h l o r o a c e t i c acid f o r

30 m i n u t e s a n d d e s t a i n e d i n aqueous acetic a c i d ( 5 0 m l / L )

f o r a f u r t h e r 30 m i n u t e s . I t w a s f i n a l l y d r i e d b e t w e e n

f i l t e r p a p e r s a n d left u n d e r w e i g h t e d b l o c k t o a v o i d c u r l -

i n g up. T h e s e p a r a t e d p r o t e i n bands were t h e n i n s p e c t e d .

SZCTIC!?T D: TOXICITY STUDIES

A c u t e Toxicity Studies i n Nice

Acu te t o ~ i c j tp s t u d y 17-7s carried out for the

d e t e m i n ~ t i o n o f t h e LUgO, The followin? v e n o ~ dose?:

qm~rSmnnt v faC c~rried out under ve ry ~ t r i c t septic

161b nrensure/sq. i nch e a u i v a l ~ n t t o 1.lTk~

2 2 pressure/cm (0.045N/m ) for '25min. Yice of e i t h e r sex

welghj ng Setween 2 0 - 3 5 ~ were ussd, They were d i v i d e d

i n t o croups of 10 mice each. Each Fl;ronr, reccjved a

known dose of t h e venom i n t r aae r j tone~lly ?f t e r

~ p i ~ i t . The , ~ n i m n l s wene o b s e r v e d for 24 hours, The

mortality IPS c a l culated . Log dose res-nonse c u ~ . r e

w n s p l o t t e d and t h e LDgQ m s c r l cu l9 ted . .

15 minutes, Yice of e i t h p r sex w e i ~ h i n g betrrrcen

2C - 25g were used. There were five gsoups, each

h a v i n g ten nice . E ~ c h mouse wgs p r e t r e a t e d with

her931 e x t r a c t ( 1 . 5 ~ ~ / g body weight) 3C: mjnutes

before giving a dose of the venom,

(0.5, 1.0, 2.0, 3.0 r n d 4.+g/g body weight). Both

d m g s were administered intraperitoneally after

s t e r i l i z , i n g the s k i n over t h e abdomen with rnethvlated

snirlt. The animls were observed f o s 24 hours, b

The numher o f f i e ~ t h s w a s recorded ~ n d percentage

mortality was calculzted. The p e r c e n t ~ g e mor t a l i t y

in the presence and absence of the herbal extract w a s

plotted a p i n s t log-dose of venom and the log-dose

response curves were cons t ruc ted . The LD,-o of the

venom in the presence and absence of the herba l

extract was determined,

SECTICFI E:

E x p e r i m e n t s o n the I s o l a t e d P e r f u s e d Rat H i n d q u a r t e r s L reparation

T h i s e x p e r i m e n t was e m b a r k e d u p o n i n order t o observe t h e

effects of t h e venom and h e r b a l extract o n t h e p e r i p h e r a l

b l o o d v e s s e l s .

Method:

Rats of ei ther s e x w e i g h i n g between 200-2509 were k i l l e d

by a blow o n t h e hesd. T h e i r t h r o a t s were cut and t h e

ensuing blood allowed t o d r a i n o u t . T h e a b d o m i n a l c a v i t y

w a s opened by means of l o n g i t u d i n a l incision e x t e n d i n g from

t h e sternum t o t h e anus a n d t h e rectum. T h e i n f e r i o r and

superior r n e n s e n t e r i c arteries were d i v i d e d b e t w e e n ligatures.

T h e abdominal viscera were removed and t h i s e x p o s e d the

abdominal aorta which was c a n n u l a t e d . The body w a l l a n d

vertebral c o l u m n were t ransected above t h e p o i n t of cannu-

l a t i on . The a o r t i c c a n n u l a w a s filled with h e p a r i n i s e d

s a l i n e t o p r e v e n t clot formatio? w i t h i n t he vessels. T h e

h i n d q u a r t e r s p r e p a r z t i o n was therefore p l a c e d o n wire mesh

L y i n ? o n a f u n n e l h e l d i n p o s i t i o n by means of tripod stand

an(: t h e o u t f l o t : collectud i n a m e a s u r i n g c y l i n d e r s . This

rnc ld i f i ca t ion was d u e t o t h e faulty T h o r p e d r o p r e c o r d i n g

a s s e m b l y . T h e p r e p a r a t i o n w a s p e r f u s e d with the physiological

f l u i d at constant p r e s s u r e (55cmJ-120) t h r o u g h the abdominal

aorta. ( B u r n , 1952). Also t h e set u p was gassed continuously

with a mixture of 95% oxygen a n d 5 5 carbondioxide w h i c h

k e p t t h e s o l u t i o n a t a pH 7 . 4 ,

T h e vessels ?.:ere p c r f u s c d with oxygene ted !?in<_ er-Locke

solution ( r !aCl , 99, hcL 0.429, C a c L p , 0.249; PJa3CO3 0.59

glucose, 1.Og) made u p to s litre with distilled water at

room t e n p e r ~ t u r c .

T h e rate of f10l.i cf the p c r f u s i n g f l u i d was c o n t r a l l e d by

means of i d j u s t i b l e c l i p , and p e r f u s a t e s were collected

every 10 ninutes u n t i l a steady s t a t e of flow was obtained.

Usually a stead;' fLov I:as obta ined i n 40-50 m i n u t e s a f t e r

w h i c h t h e d ru(2s ~ ' c r e i n j e c t e d .

C o n t r o l experiments ( A ) were ~ e r f orned i n w h i c h the flow

r a t e was s t u d i e d over 20-30 minutes after e q u i l i b r a t i o n a n d

f o l l o w i n g t h e i n j e c t i o n of 0. l m L of the p c r f u s i m f l u i d .

Then t h c effcct of 200 and 4OCug of venom and 100 a n d

200ug oL the herbal e x t r a c t was determincd. T h e volumeof t h e

herbal extract or venom i n j e c t e d was O . 1 n l .

T h e effect of -.renom or herbal evtr .ct w ~ s ~valuctcd a n d

c ~ l c u l a t e d a s increase or decrease over the f l o v rate at

equilibration. The t i r e of o n s c t of m x i m a l activity was

also recorded.

The Ef fec t s of Vcnom a n d Herbal E x t r a c t on Zlood E2ressure,

ECG a n d R e s p i r a t i o n of R n ~ e s t h e t i e e d Cat.

Cannu 1 a t i o n of Trachea

Three ca t s w e i c j h i n c b e t w e e n 1 a n d 3kg were used . T h e y were

3 c h u s t e r I.7yographi.c Table (C.F. Palmer L o n d o n ) a n 4 t h e l e g s

wcr.2 f a s t e n e d t o t h e table by means of s t r o n g ropes. The

n e c k s were s h a v e d w i t h s5a rp razor blade. T h e larynx was

felt a s a lump t h r o u p t h e s k i n a n d below it, a length of - s!,:in was pic!:& up anu c u t open w i t h s h a r p scissors. T h i s

exposed t h e trachea. It was t h u s freed from connective

t i s s u e . Two s t r o n g l i g a t u r e s werc passed r o u n d t h e trachea.

,'. small i n c i s l o n was mide o n t h e trachca a n d a rnetalic

tracheal t u b e c o n n c c t c d tc a tambour t h r o u g h a rubbe r

t u b i n g was i n s e r t e d so t h a t r e s p i r a t o r y effect c o u l d be

recorded on t h ~ ! snolccd papgr , It was secured i n pl;.,ce with

t h ? i n f e r i o r l isature. Hot..:ever t h e s u p e r i o r l i g a t u r e w a s

used l n tfring t o g e t h e r the t r a c h e a l ",3e and trachea f o r

proper a l i g n m e n t .

C a n n u l a t i o n of .-. t h e --. Carotid ..A- Artery

Two l i g a t u r e s (3/0) were p a s s e d r o u n l t h e cleared l e n g t h

of t h ~ c4: . rokid a r t e ry . T h e t h r e a d f u r t h e r e s t from the

heart was tied and a s t h e a r t e r y d i s t e n d e d with blood a

bulldog clip was p l a c e d a t t h e end n e a r e s t to the h e a r t ,

The a r t e r i ~ l c a n n t l l a (1,Scm)was connected to t h e mecury

manometer t h r o w 3 5 a three way s t o p cock w i t h a s y r i n g e f i l l e d

w i t h h e p a r i n i z ~ d s a l i n e . T h e c o n n x t i o n between t h e three

way s t o p cock and t h e manometer was clcscd, T h e a i r bubbles

i n the a r t e r i a l c a n n u l a were f l u s h e d out with h e p s r i n i s e d

s a l i n e a n c t h - t a p a d j u s t e d t o close off t h e a r t e r i a l c a n n u l a .

A small incision was made on t h e artery between t h e t i e d

ligature and bulldog c l i p . T h e c a n n u l a was t h e n inserted and

tied i n t o p l a c e , The pressure i n the m a n o m e t e r was raised

by m e a n s 05 t he syringe t o t h e e x p e c t e d blood pressure of

t h e a n i m a l , T h e stop cock was a d j u s t e d so t h a t the syringe

was closed o f f and a r t e r i a l c a n n u l a c o n n e c t e d t o t h e r x o r d i n g

device. The bulldog clip was t h e n removed, There was a

l i t t l e flow of blood o u t of t h e a n i m a l i n t o the c a n n u l a .

C a n n u l a t i o n 05 the F e r m o r a l Vein

F u r was c l e a r e d a l o n g t h e i n g u i n a l region close t o t h e abdo-

minal wall. T h e s k i n w a s d i s s e c t e d and the femoral v e i n

exposed. The p o r t i o n between t h e j u n c t i o n with saphenous

vein and i n g u i n a l l i g a m e n t was very c a r e f u l l y c l e a r e d free

of c o n n e c t i v e t i s s u e t h r o u g h blunt d i s s e c t i o n making sure

t h a t no blood vessels were damaged a n d b r a n c h e s thereof were

t i e d o f f . On exposing a reasonable l e n g t h of t h e v e i n , t h e

blood f l o w was o c c l u d e d wich a b u l l d o g clip p l a c e d a t a

p o i n t , n e a r e s t t o t h e h e a r t . Two threads were c a r e f u l l y

p a s s e d r o u n d t h e v e i n h a l f t i e d , The vein w a s distended by

p u s h i n q b l o o d t o w a r d s t h e b u l l d o g c l i p . The inferior thread

was firmly tied and held taut s o as to s t r e t c h t h e vein

slightly. i i small i n c i s l o n was made with a s h a r p p o i n t e d

scissors no t more t h a n h a l f vay t h r o u g h t h e vein b u t very

close t o t h e i n f e r i o r t h r ead . X fine plastic c a n n u l a of

approximately ( 0 . S m m ) f i l l e d with h e p a r i n i s e d s a l i n e m s

inserted a n d c o n n e c t e d t o a three way s t o p cock v h i c h a l l o w e d

t h e i n j e c t i o n of drugs. The s u p e r i o r t h r e a d was t i e d a r o u n d

the cannula t o k e e p i t a l i g n e d c o r r e c t l y .

Connec tLon of The A n i m a l t o Z l ~ c t r a c z r d i o q r a p h i c Machine

The a n i m a l w a s t h e n c o n n e c t e d =o a n electrocardiographic

machine (Narco -Biosys t e rns P h y s i o g r a p h Nic-111) by means of

p i n electrodes i n s e r t e d s u S c u t a n e o u s l y i n t o thc r i s h t fore-

limb and left h i n d l i m b . ECG r e c o r d s were o b t a i n e d o n Lead

I1 c h a n n e l of t h e ZCG machine. A l l ECG r e c o r d i n g s were

o b t a i n e d a t a p3?er s p e e d of 2.5cm per second .

R e c o r d i n q Devicr-s Used

Changes in blood p r e s s u r e cf t h e animal were recorded w i t h a

mercury manometer cf s i m p l e YJVt tube of a S o u t 5mm bore. I t

gave r e a d i n g by g i v i n g t h e p u l s a t i o n . It w a s c v n n e c t c d t o a

writing ~ P V T which wrote o n smoked paper o n a Z r o d i e .';tarling

k y ~ z o g r a p h i c d r c j r o t a t i n g a t 16rnm/min. ;;lso t3c tarnjcur

writing lever was made t o write c n the smoked p..: er ac. t h e

b Tla?-,e 1 (a and b) : Photographs or" Dioda scandens showing

f l o w e r s znd f 01ia~ye.

WEIGEiTS OF ETHAT?CI,IC lXiiTRJ::.CTAE3LE F'KT;4CTIC.!JS

5 RC'PI F R E X f: : TEE1 AL, (Dl O C I A X A T Z ~ E Z ~ S )

Tah le 1 s h o w s the g j e l d s ?TOT- the ethanolic extract

of t h e herb. F r a c t i o n A v a s i n so lub le in. w t e r w?ile

E r a c t i o n s 3 ?nd C were w ~ t e r s o l i ~ b l e . . I t r ~ c t i o n A B w o s

most n 1 ~ r l t . i f u l c o n s t i t 1 1 t j . n ~ 3.474 o f t h e w e i e t of the

f r e s h herb.

Yields from the ethanolic extract of the herb which wei~hed 2 1 6 ~

Percentage

Fractions

Weights PI

SECTIOIB B COAGULATIOR STUDIES

(a) Effect of Echis Carinatus Venom on Whole Blood Clottina Time

The average clotting time of whole blood of 4.9

min was reduced to average 2.1 min by venom at

1: 10,000 dilution. (~fi.01)

Table 2 gives the details of the effect of the venom

in respect of t h e blaod from the ten donors.

TABLE 2

The Effect of Echis Carinatus Venom on Whole Blood C l o t t i a ~ Time

I Clotting Time ( M i d

Donors A Whole Blood

J C O 6.08

S A I 1 4.0

Tota l 1 49.3

mole Blood + Venom (1 :?O,OOO)

A , - 3

(b) The Action of Fraction B of the Herbal Extract on the

Accelerated clot tin^ Time o f Whole Blood Induced by

the venom

The herbal extract a t 1:10,000 d i lu t ion , had no e f fec t

on the c lo t t ing time of whole blood, However a t

1:10,000 d i lu t ion , it increased the c lo t t ing t i m e of

whole blood t o which venom a t (1:10,000) di lu t ion was

added, from the average of 1.8 min t o 2-9 min. This

was however not s t a t i s t i c a l l y significant ( ~ b . 2 )

Table 3 gives the d e t a i l s of the e f fec t of the

herbal extract i n respect of c lo t t ing time of whole

blood from t h e three donors i n the presence and

absence of venom.

The Action of F rac t ion B of the Herbal Extrac t on The Accelerated Clo t t ing Time o? h o l e Blood Induced by t he 7enom and on the c l o t t j na t:me of normal. b l o o d

Donor Blood

Clotting Time I n Min

Whole Blood I Whole Blood

+ Herbal Whole Blood + Venom (I :10,000)

Whole Blood + Herbal Ex- t r ac t (1: 10,000) + Venom (1:10,000)

( c ) Comparison of Prothrombin T i m e s of Plasma T r e a t e d with Venom and Untreated Plasma

The avers-ge prothrombin t ime o f 15.6 sec was

r educed t o 5.7 sec f o l l o w i n g the a d d i t i o n o f

venom a t 1 :3O,OOO dilution (PL0.01). I n o t h e r words

t h e pro thrombin t ime fo l l owing t r e a t m e n t w i t h venom,

was 3776 o f the normal pro thrombin time. Table 4

shows t h e d e t a i l s of e f f e c t o f t h e venom in respec t

o f 10 donor plasma samples.

TAELE 4

Comparison o f Prothrombin T i m e s o f Plasma Trea ted w i t h Venom and Untrezted Plasma

E U S O

Prothrombin Time (Sec )

A Plasma

Plasma + Venom ( 1 :3O,OOO)

Mean 4 SEM 15.620.6 5.7+0.6* 37.02480

( d ) The E f f e c t of the Herbal Extract o n the Venom a n d

G r a i n T h r o m b o p l a s t i n a c t i v i t y

The normal p r o t h r o m b i n time of 11.25 sec w a s i n c r e a s e d

to 13.25 sec f o l l o w i n g treatment with the h e r b 1

e x t r a c t (1:40,000). T h i s result s u g g e s t s t h a t the

herbal e x t r a c t i n h i b i t s the a c t i v i t y of brain thrombo-

p l a s t i n . Ho~iever, when the p r o t h r o m b i n t i m was done

with t h e a d d i t i o n of -,-enom (1:40,000) i n s t e a d of b r a i n

t h r o m b o p l a s t i n t h e t i m e was 20.8 sec indicating t h a t t h e

venom has t h r o m b o p l a s t i n a c t i v i t y . When venom

( l : 4 O , O O O ) and brain thsornboplas t in were used t o g e t h e r

and t h e n incubated with herbal e x t r a c t (1:40,000),

prothrombin time was 17.5 sec.

TABLE 5

The E f f e c t o f t h e Herbal Ex t rac t on Venom and Brain Thromborslast in activity. EU ~ . n d EC, r e f e r the d o n o r s

TYPE OF TEST

Thromboplastin + Ca++

I1 Plasma+Brain Thromboplastin + herba l e x t r a c t 1 : 4O,OOO+Ca++

I V Plasma + E r a i n Thromboplas t in + herba l Extract ( 1 :4O,OOO) + Venom

(1:40,000) + Ca++ i

(el The Effect of Herbal E x t r a c t ( H E ) On t h e C l o t t i n g

Time of Plasma ( P I t o which Venom ( V ) and Calcium

(ca9+) Were Added

E x p e r i m e n t T?b was s l i g h t l y d e f i c i e n t i n its p l a n n i n g

b e c a u s e w e c o u l d n o t s e p a r a t e t h e a n t i t h r o r n b o p l a s t i n

effect o f t h e h e r b a l extract o n b r a i n t h r o m b o p l a s t i n

and on t h e venom t h r o m b o p l a s t i n activity. E x p e r i m e n t

S I C was t h e r e f o r e d e s i g n e d s p e c i f i c a l l y t o i n v e s t i g a t e

t h e effect of the h e r b a l extract on the t h r o m b o p l a s t i n

a c t i v i t y of t h e venom. The a v e r a g e c l o t t i n c j time of t h e

v e n o m - t r e a t e d plasma a f t e r the r e c a l c i f i c a t i o n was 16.75

see. This was i n c r e a s e d t o 19-75 secs f o l l o w i n g the

addition of t h e h e r b a l extract (1:22,000), Table 6 gives

the d e t a i l s i n r e s p e c t of t h e t w o samples used.

TABLE 6

The Effect of Herbal E x t r a c t ( H E ) on t h e C l o t t i n q Time of

Plasma (PI t o which Venon ( V ) and C a l c i u m ( ~ a + + ) Were Added

Plasma C l o t t i n g T i m e ( S e c ) I

Donor 2+ P+V( I: 22,000) HE(^: 22,000) I

Tota l I Time I Average

(f) The Effect of Herbal E x t r a c t (HE) on t h e C l o t t i n q T i m e

of Plasma (PI t o w h i c h Venom ( V ) was added

Plasma separated from b l o o d collected with a n t i c o -

a g u l a n t r e m a i n s f l u i d almost i n d e f i n i t e l y . However,

when venom a lone a t 1:10,000 d i l u t i o n was a d d e d t o

t h e p l a s m a i t clotted i n 18.5 - 2 3 secs, P r e t r e a t m e n t

w i t h he rba l e x t r a c t even a t 1:250 h a d l i t t l e o r nb

effect on t h e c l o t t i n g time of t h e plasma ("thrombin

t i m e r r ) treated w i t h venem, Table 7 shows t h e d e t a i l s

05 effect of va r ious d i l u t i o n s of t h e h e r b a l e x t r a c t

on t h e c l o t t i n g time of plasma t r e a t e d w i t h v a r i o u s

c o n c e n t r a t i o n s of venom.

TABLE 7

The Effect of Herbal Extract ( H E ) On t h e C l o t t i n g Time of Plasma ( P ) to w h i c h Venon (Vl w a s added

Procedure

P + V P + V

+HE (1:314a

P l a s m a C l o t t i n g t i m e ( S e c , )

20 19.5 18 18 18 18 18 18

Total Sec.

75.5 72

Average Sec 2 SEM

18.8+0,5 18.070.0 - ,

SECTICN C: ELECTROFHORETIC STUDIES

P l a t e I1 i s e l e c t r o p h o r e t o g r a m of ( a ) Unt r ea t ed

plasma ( P I ; ( b ) Plasma t r e a t e d with venom (P+V)

(01: 10,000) ; (c) Plasma i n c u b a t e d w i t h h e r b a l extract

( 1 :10 ,000 ) , and to w h i c h venom (1 :10 ,000) w a s added

(P+H+V). I t showed t h a t the venom caused a p p r e c i a b l e

l o s s o f alpha 1, alpha 11, beta g l o b u l i n bands and

comple te loss of the f i b r i n o g e n band. There w a s

c o n s i d e r a b l e p r o t e c t i o n of a l p h a 1, a l p h a 11 and

b e t a g l o b u l i n bands f r c m p r o t e o l y t i c a c t i ~ n of the

venom following incubation o f the plasma with t h e

herbal extract. The f i b r i n o g e n band w a s no t

p r o t e c t e d ( P + H + V )

P l a t e 11. The e f fec t of the herbal extract on the venom induced changes i n the electrophoretic pattern of plasma P=Plasma : P+V=Pl asma+Venom: P+A+V=Plasma+Herbal extract+ venom

SECTION D: TOXICITY STUDIES IN MICE

The mice t r e a t e d with 4ug/g or higher dose, almost

i m m e d i a t e l y became v e r y quiet a n d soon c o n v u l s e d and

d i e d w i t h i n f i v e m i n u t e s of receiving the venom.

Table 8 shows t h e p e r c e n t a g e mortality due t o t h e venom.

TABLE 8

Percentage Mortality Due t o Various Doses o f Venom

Dose of Venom (ug/g body weight)

5 M o r t a l i t y

(b) Effect of Herbal E x t r a c t 1.5ug/g Body w e i q h t on

M o r t a l i t v Due t o Venom

T a b l e 9 s h o w s t h e p e r c e n t a g e m o r t a l i t y d u e t o v a r i o u s doses

of t h e venom alone a n d a f t e r p r e t r e a t m e n t with h e r b a l

e x t r a c t (1.5rng/kq i p ) . T h e venom a t ( 3 m g / k g i p ) caused 80%

m o r t a l i t y , h o w e v e r p r e t r e a t m e n t w i t h t h e h e r b a l e x t r a c t

(1.5mg/kg ip) a t t h e same venom dose level p r o d u c e d 70%

p r o t e c t i o n . S t a t i s t i c a l analysis showed the p r o t e c t i o n t o

be s i g n i f i c a n t ( P - / 0 . 0 2 ) .

T h e LDS0 of t h e venom was 2mg/kg i p b u t t h i s increased

t o 3.2mg/kg i p , f o l l o w i n g p r e t r e a t m e n t with t h e herbal

e x t r a c t ( f i g , I). A l t h o u g h t h e m o r t a l i t y a t 4mg/kg w a s

100% in presence a n d absence of t h e herbal e x t r a c t , i t was

n o t e d t h a t w h i l s t d e a t h s o c c u r r e d w i t h i n 5 m i n i n all a n i m a l

n o t p r e t r e a t e d w i t h t h e h e r b a l e x t r a c t n o mouse p r e t r e a t e d

with t h e h e r b a l extract died i n u n d e r 4 hours.

TABLE 9

E f f e c t of Rerbal Extract (1,5u,q/~ Body W e i ~ h t ) on ;1; i . i o r tn l i t :

Dose o f Venom ug/g Body \h1eight Venom

Alone Venom + Extract

F i g :I Log dose r e s p o n s e curves of the venom following pretreatment w i t h herbal extract ( I. 5ug/g).

( c ) T o x i c i t y s t u d i e s of Herbal ~ x t r a c t i n Piice

The herba l e x t r a c t at 2g /kg i p produced no d e ~ t h s .

t-!ow~vc.r, at 39/kg i p t h e h e r S a 1 e x t r a c t caused

d e a t h s in 30% of t h e mice. T h e LDSn "8s 3.3g/kg

(see Fig. 2) .

T a b l e 10 g i v e s t h e percentaqe m o r t a l i t y d u e to

various doses of t h e h e r b a l e x t r a c t .

TABLE 10

76 M o r t a l i t y in Mice from vari0u.s Doses o f the h e r b a l Extract

Dose of Herbal Extract ug/g Body i ' l e i ~ h t

3.2 3.4 3.6 3.8 Log ~ O S C ~ ~ ! ~ )

Pig. 2: Log dose r e s p n z e s showing$ m o r t a l i t y from various doses o f t h e herb21 extract

.- . - . . - -..--

SECTION E: EXPERIMXYTS ON THE CARDIOV!:SCULAR SYSTEN

( a ) Rat Hindquarters P r e p a r a t i o n

Table 12 (a) shows that venom induced vasoconstriction.

The two doses 200 and 400ug caused a decrease in

volume flow. The average percentage chnnge in

volume f l o w were 14 and 32% respectively, The time

of maximum activity ranged from 10-60 mins.

Table 12(b) shows that the herbal extract produced

vasodilatation. The two doses 100 and 200mg used

caused i n c r e a s e in volume flow; the average percen-

tage change in volume flow w e r e 14.9 and 2176

respectively. Time t o maximum activity w a s found t o

be between 10 and 30 mins.

Fig. 3 shovrs the percentage changes in volume flow

due to the venom and the h e r b a l extract.

TABLE 11

R a t I I i n d q u a r t e r s P r e p a r a t i o n

C o n t r o l experiment with O.lml o f Rin~er-Locke S o l u t i o n

Val. P' lovr E q u i l i b r i u m

TAELE 12(a)

R a t H indqua r t e r s P r e ~ a r a t i o n : E f f e c t o f 200 and 4OOug venom FR f l o : ' ~ r a t e (mls/lO mini

Negative s i g n s i n d i c a t e reduction in f l o w

Dose ( u g )

br ium

T ime (min) maximum

V o l . flow at

of Venom

Vol. F l o ~ at peak

activity ~Equilli- E f f e c t I

TAELE 12 (b)

Rat H i n d q u a r t e r s P r e p a r a t i o n : E f f e c t o f 100 and 200u. of the h e r b a l extract on f l ~ w rate ( r n l s / l O m in )

Dose I Time (min ( u g > I Plaximum

C/ Activity extrac tl

Val. Flowl V o l . Flow1 Vo1. 7; Change at Equi-

Librium

Average 01 15

Change + SEN at peak I C h a n p E f f e c t I

Herbal extract alone

Fig. 3, Rat H i n d q u a r t e r s p r e p a r a t i o n . Compar ison of effects

of h e r b a l extract and venom on vol. flow. -. . vertical bars = + S E T4 -

T h e E f f e c t s o f Venom a n d Herba l E x t - - - ~ n a e s t h e t i s e d Cat. B l o o d P r e s s u r e , gram a n d R e s p i r a t i o n

T h r e e c a t s were u s e d f o r this s t u d y - F i r s t c a t

r e c e i v e d a h i g h dose of t h e venom i.e. 3mg/kg i.v.

T h i s p r o d u c e d a n i m m e d i a t e s h a r p r i se i n b l o o d

p r e s s u r e . T h e peak w a s o b t a i n e d i n 1 m i n . T h e

pressure r i s i n g f r o m 120-196mmHg. T h e b l o o d p r e s s u r e

s l o w l y d r o p ~ e d to t h e b a s e l i n e l e v e l f 120mrnHg), 4.6min

a f t e r a d m i n i s t e r i n g t h e venom. T h e venom a t t h a t

dose l e v e l p r o d u c e d ECG c h a n g e s w i t h i n 10 sec of i t s

a d m i n i s t r a t i o n . T h e d e p r e s s i o n of t h e ST s e g m e n t was

the major ECG a b n o r m a l i t y . A b o u t 5 m i n a f t e r

a d m i n i s t e r i n g t h e venom, the ST segment d e p r e s s i o n

was accompanied by t h e d e p r e s s i o n of t h e T waves. The

P wsve and QRS c o m p l e x e s were a l s o n o t c l e a r l y

d e m a r c a t e d a n d t h e h e a r t r a t e w a s i n c r e a s e d f r o m a

c o n t r o l l e v e l o f 1 6 8 / m i n t o 1 9 2 / m i n . F iowever , a b o u t

1 0 m i n a f t e r t h e a d m i n i s t r a t i o n of t h e venom, t h e

ECG showed some r e c o v e r y a l t h o u g h the T waves s t i l l

r e m a i n e d i n v e r t e d . T h e h e a r t rate h a d d r o p ~ l e d t o

1 7 4 / m i n . T h e e f f e c t on r e s p i r a t i o n w a s n o t s t u d i e d .

F i g . 4 s h o w s t h e effect of a d r e n a l i n e a n d venom on

t h e b l o o d p r e s s u r e F i g . 5 s h o w s t h e e f f e c t of

the venom o n t h e ECG.

- 76 - T h e t w o r e m a i n i n g c a t s r e c e i v e d a m u c h l o w e r

d o s a g e o f t h e venom (O.Smg/Kg). I n t h e f i r s t c a t

r e c e i v i n g t h i s d o s e o f t h e venom (O.Smg/kg) i.v.

t h e r e w a s a 20mrnHg r i se i n blood p r e s s u r e w h i c h

r e a c h e d i t s p e a k i n 45 sec b u t s l o w l y d r o p p e d t o

c o n t r o l l e v e l a f t e r 2 min. 1 2 rnin a f t e r a d m i n i s t e r i n g

t h e venom, t h e h e r b a l e x t r a c t ( Z O m g / K q ) was t h e n

g i v e n . T h i s h a d n o e f f e c t o n t h e b l o o d p r e s s u r e ,

I1 m i n a f t e r g i v i n g t h e h e r b a l e x t r a c t , the dose of

t h e venom w a s r e p e a t e d , and t h i s p r o d ~ c e d a s l i g h t

f a l l i n b l o o d p r e s s u r e i n s t e a d o f a r ise i n blood

p r e s s u r e s e e n w i t h t h e f i r s t d o s e of t h e venom

b e f o r e t h e the h e r b a l e x t a c t w a s g i v e n . T h e ECG

c h a n g e s d u e t o t h e venom a t t h e dose l e v e l w e r e

a s f o l l o w s : d e p r e s s i o n of ST s e q r n e r t s , t r a n s i e n t

a t r i a l f l u t t e r a n d t h e h e a r t r a t e f a l l f r o m 1 8 0 / m i n

t o 1 6 0 / m i n . T h e r e w a s n o d e p r e s s i o n o f t h e T w a v e s .

T h e h e r b a l e x t r a c t h a d n o effect on t h e ST d e p r e s s i o n

i n d u c e d by t h e v e n o n , b u t i t i n c r e a s e d t h e h e a r t r a te

from 1 6 0 / m i n t o 1 7 6 / m i n .

On r e s p i r a t i o n , t h e venom e v e n a t t h i s l o w d o s e

l e v e l , c a u s e d a r e d u c t i o n i n t h e r a t e of r e s p i r a t i o n

and t h e y w e r e l o n g p e r i o d s of a p n o e a . T h e h e r b a l

e x t r a c t r e d u c e d t h e a p n o e i c p e r i o d s a n d t h e r a t e of

r e s p i r a t i o n rose.

T?e h ~ r h e l extract w 9 s a3le to reduce the apnoea

iducei! 7 3 7 t h e venom, l i ~ s . 6 a, b , c ,chow the

C C ~ ? _ ~ e c t of t h e venoy c?nd '?erb?l e x t r ~ c t on b lood

nressure , ECf gnd r e sp i s ~ t j - o n .

T 4 6 e f f e c t s of t h e venon on the second c a t

r~c~lving this 6ose of venom, were s i m l l q s except t%at

t h e rice in blood pressure wss only 'lQnn-195 insteed of

2ClmmH~;. The a-onopa induced by the lTenom w a s a s

desc~ihed In the first cat, The 3CG chanqes were

sirilar to chqn~es in previous cat r e c ~ i v i n q the

lorlrpr dose o f t h e venom exceqt thrt ST dppression

whj ch apneared about 5 min a f t e r aclministerrinp; the

venom, was not as marked and consistent as in the

p r p v i o ~ s cat. The h e a r t r a te was rerh~ced from

24~/min t o ?20/min. The he rba l extract hovever

r ~ t n r n ~ d the heart r a t e to t h e original 240 ?eats/min.

30 sec . l-----d

control I . . - . -

/ . 30 sec. Venom 0.5 rngIkcj I - 1

Upper panel: BP Recordings Middle panel: ECG Laver panel : Respiratory exc~~r_s'icsns

Fig. 5 , : Thc effnc-k of venorr, ( O . 5 ~ * l ~ / k ~ ) i.~. on blood prclszurc:, 4CG 2nd rc-.-pir2tion o C the m-test'he- t i z e d c n t n ~ e p z r z t i o n .

Herbal extract 10rngkg

Upper panel: BP Recordings

Middle panel: E C G

Lower panel: f?espiratory Excursions

I 30 set. Venom 0.5 mglkg H

30 sec.

Vencrn 05 rngjkg t-i

Upper panel: BP Recording

Middle panel: E C G Lower panel: Respiratory Excursions

DISCUSSIC!!

V a r i o u s h a e m o r r h a a i c m a n i f e s t a t i o n s a r e common i n

v i p e r i n bites (Warre11 e t al., 1 9 7 7 ) . O n u a q u l u c h i

( 1960 ) noted t h a t h a e r n a t u r i a a n d c i r c u l a t o r y c o l l a p s e

w e r e the major p r o b l e m s i n v i p e r i n e n v e n o m a t i o n . T h e

a n t i c o a g u l a n t a c t i o n of v i p e r i n s n a k e v e n o m h a s been

d o c u m e n t e d but t h e m e c h a n i s m of t h i s a n t i c o a g u l a n t

e f f e c t i s n o t c e r t a i n ( O k o n k w o a n d Onuaquluchi, 1977 ) .

However, X o r n a l i k ( 1 9 6 3 ) ; S c h i c k et a1 (1972) o b s e r v e d

t h a t E c h i s c a r i n a t u s v e n o m c o n t a i n e d prothrorrbin

a c t i v a t i n g p r i n c i p l e . ! d a r r e l l e t a l , ( 1 9 7 7 ) a l s o

were o f t h e o p i n i o n t h a t t h e v e n o m caused direct

a c t i v a t i o n of p r o t h r o b i n l e a d i n g t o disseminated

i n t r a v a s c u l a r c o a q u l a t i o n w i t h s e c o n d a r y f i b r i n o l y t i c

proteolysis. C o a q u l a t i o n cascade i s c s m p o s e d o f a

ser ies of l i n k e d p r o t e o l y t i c r e a c t i o n s ( R o s e n b e r g ,

1984). A t each s t a g e o f t h i s m e c h a n i s m p a r e n t

zymoqen i s c o n v e r t e d t o a c o r r e s p o n d i n g s e t i n e

p r o t e a s e w h i c h c a t a l y s e s a s u b s e q u e n t z y r n o g e n - s e r i n e

p r o t e a s e t r a n s i t i o n ( R o s e n b e r g , 1974) . B l o o d

c o a g u l a t i o n f u n d a m e n t a l o b j e c t i v e i s t h e c o n v e r s i o n

of s o l u b l e p lasma p r o t e i n f i b r i n o g e n t o i n s o l u a b l e

f i b r i n , t h r o u g h t h e i n t r i n s i c a n d e x t r i n s i c p a t h w a y ,

( K a r t i n J r . et a l . , 1 9 6 3 ) .

Active XI1

7 : - m t h e ab0?&-.3~a~rarn it wi 11 b e ahz??ived t h a t t he intrinsic ;:-.. t l ~ w a y ' f o r t h e g e n e r a t i o n of p lasma thromboplastfn commences \.!- .", t h e e x p o s u r e of prekallikrein, h i q h m o l w ~ i q h t Kinino- ,s :r?, f a c t o r X I 1 and f a c t o r XI to a n a c t i v a t i n q s u r f a c e ; ; J , . - rhaps collagen in v i v o . T h i s e x p o s u r e make:; f a c t o r xII r.,:~-e liable to proteolysis S y kallikrein. Fac to r XIIa is r . ? c r a t e d by ~ a l l i k r e i n s e t t i n g u p a r e c ip roca l a c t i v a t i o n . :-,.rctor X I I a re leases bradykinin from h i q h molecular w e i q h t .:i.i?;.nogen'and a c t i v a t e s f a c t o r XI to X I a . Cohen et a 1 ( 1 9 6 9 )

7::prved t h a t g l a n d u l a r t i s s l~es i n v a r i o u s orders of

v e r t e b r a t e s and snake venom

r e l e a s i n g pharmacological ly

c o n t a i n enzyme capable of

a c t i v e p e p t i d e s from plasma

g l o b u l i n . These p e p t i d e s termed k i n i n s (b radyk in in ) cause

pa in , oedema, l eukocyte mig ra t i on , hypotension and cont rac-

t i o n of various smooth muscles,

Cohen e t a1 (1969) observed t h a t Ech i s c o l o r a t u s

venom conta ined k i n i n - r e l e a s i n g enzyme ( K a l l i k r e i n ) . From

t h e p r e s e n t s t u d y one can conclude t h a t E c h i s c a r i n a t u s

venom c o n t a i n s th romboplas t in which c l o t s r e c a l c i f i e d

c i t r a t e d plasma (Table 6 ) . The venom a l s o e x h i b i t e d

thrombin- l ike a c t i o n by c l o t t i n g c i t r a t e d plasma wi thout

t h e a d d i t i o n of calcium i o n s and thromboplas t in . E c h i s

c a r i n a t u s venom was shown t o a c c e l e r a t e i n - v i t r o c o a g u l a t i o n

p roces s i n normal a d u l t human blood samples. T h i s t h e r e f o r e

i s i n agreement w i t h t h e obse rva t ion of Dacie and Lewis

(1975) t h a t v a r i o u s snake venoms produce a c c e l e r a t e d blood

c l o t t i n g v i a t h e e x t r i n s i c pathway. Thus t h e average

u n t r e a t e d whole b l o o d c l o t t i n g time of 4.9 min w a s reduced

t o 2.lmin. The a c t i v i t y of the venom was i n h i b i t e d t o a

v e r y l a r g e e x t e n t by p re t r ea tmen t of whole b lood w i t h

h e r b a l e x t r a c t i n which venomised whole b lood c l o t t i n g time

of 1.8 min was increased t o 2.9 min r e p r e s e n t i n g 61%

i n c r e a s e in c l o t t i n g time. Ro~veves t h e h e r b a l ex t rac t had

no e f f e c t on untreated whole blood clotting t ime.

These r e s u l t s t h e r e f o r e i n d i c a t e d t h a t t h e h e r b a l e x t r a c t

o n l y a n t a g o n i s e d venom produced e f f e c t s . I t was t h e r e f o r e

c o n s i d e r e d p e r t i n e n t t o - e l l u c i d a t e a t what p o i n t a l o n g t h e

c o a g u l a t i o n c a s c a d e t h e h e r b a l e x t r a c t e x e r t s i t s a n t i c o a g u l a n t

e f f e c t s . Prothrombin t ime a s s a y c a r r i e d o u t r e c o r d e d a n

a v e r a g e o f 15.6 s e c . 9hen venom w a s added t o plasma,

s u b s e q u e n t prothroiiibin t ime a s s a y r e c o r d e d a n a v e r a g e

p ro th rombin t ime of 5.7 s e c ( T a b l e 4). T h i s showed t h e

a d d i t i v e n a t u r e of t h e venom t h r o m b o p l a s t i n a c t i v i t y t o t h a t

of b r a i n t h r o m b o p l a s t i n . I n a n a t t e m p t t o d e m o n s t r a t e t h e

p o s s i b l e a r i t i t h r o n l b o p l a s t i c a c t i v i t y of t h e h e r b a l e x t r a c t ,

i t was d i s c o v e r e d t h a t t h e h e r b a l e x t r a c t (1 :40 ,000)

i n c r e a s e d t h e p ro th rombin t ime of normal plasma from 11.25

t o 13.25. From Tab le 5 i t can be su rmised t h a t t h e h e r b a l

e x t r a c t a n t a g o n i s e d b o t h t i s s u e and venom t h r o m b o p l a s t i n .

Exper iment S I C was d e s i g n e d t o i n v e s t i g a t e s p e c i f i c a l l y

t h i s p o s s i b l e t h r o m b o p l a s t i c a c t i v i t y of t h e venom.

The h e r b a l e x t r a c t i n c r e a s e d t h e c l o t t i n g t ime of plasma

t o wmich venom and ca2+ had been added from an a v e r a g e

t ime of 16.75 s e c t o 19.75 s e c ( T a b l e 6 ) .

Prom t h e e x t r i n s i c c o a g u l a t i o n diagram (Page 8 4 ) i t would

a p p e b r t h a t h e r b a l e x t r a c t vias a c t i n g a t t h e t h r o m b o p l a s t i n

( T i s s u e e x t r a c t o r venom) p r o c o n v e r t i n ( f a c t o r V I I ) and C a 2-t

l e v e l by p rever l t ing t h e venom l l t h r o m b o p l a s t i n f t from

a c t i v a t i n g p r o c o n v e r t i n ( f a c t o r V I I ) and t h u s c a u s i n g d e l a y

i n c o a g u l a t i o n ( T a b l e s 5 and 6 ) . I n o t h e r words, t h a t t h e

h e r b a l e x t r a c t e x h i b i t s a n t i t h r o m b o p l a s t i c a c t i v i t y . N a r x e l l

e t a l . (1977) c o n s i d e r e d d i r e c t a c t i v a t i o n o f prothrombin

t o be t h e mechanism o f a c t i o n o f E c h i s c a r i n a t u s venom and

a l s o K o r n a l i k (1363) and S c h i e k et a l . (1972) a g r e e d t h a t t h e

mode o f a c t i o n o f E c h i s c a r i n a t u s venom depends on i t s

pro th rombin a c t i v a t i n g p r i n c i p l e . 'There i s t h e r e f o r e no

agreement on t h e mechanism of a c t i o n of E c h i s c a r i n a t u s

venom.

Our s t u d y showed t h a t E c h i s c a r i n a t u s venom c l o t t e d

c i t r a t e d plasma i n t h e p r e s e n c e and absence o f C a 2+

( T a b l e s 5,6,7). The c l o t t i n g o f r e c a l c i f i e d c i t r a t e d plasma

by t h e venom d e p i c t s t h r o m b o p l n s t i c a c t i v i t y . T h i s a l s o

a g r e e s w i t h t h e o b s e r v a t i o n of Dacie and Lewis (1375) t h z t

v a r i o u s snake venoms produce a c c e l e r a t e d b lood c o a g u l ~ ~ . t i o n

v i a t h e e x t r i n s i c pathway. T h e r e f o r e t h e t h r o m b o p l a s t i n of

2+ t h e venom + p r o c o n v e r t i n ( F a c t o r V I I ) and C a g e n e r a t e d

a p r i n c i p l e ( a c t i v e ) f a c t o r ( V I I a ) which caused a c t i v a t i o n

- 88 .. o f t h e S t u a r t f a c t o r ( F a c t o r X ) t o a c t i v e Factor Xa a

se r ine p r o t e a s e . A d i r e c t a c t i v a t i o n o f p r o t h r o r n b i n t o

t h r o m b i n ( w a r r e 1 1 e t a l . , ( 1 9 7 7 ) w i l l a m o u n t t o a b y p a s s

o f S t u a r t f a c t o r ( F a c t o r X ) a c t i v a t i o n w h i c h serves a s

a m e e t i n g p o i n t f o r t h e i n t r i n s i c a n d e x t r i n s i c p a t h w a y s .

T h e c l o t t i n g o f c i t r a t e d p l a s m a b y t h e Venom i n t h e

2+ a b s e n c e o f C a (Table 7 ) w o u l d indicate t h a t t h e venom

p o s s e s s e d a t h r o m b i n - l i k e a c t i o n . T h r o m b i n i s known t o

2+ c l o t c i t r a t e d p l a s m a i n a b s e n c e of C a . Also c e r t a i n

s n a k e venoms e.q. M a l a y a n p i t v i p e r venom - a n c o r d

( A r v i n ) c a n c l o t f i b r i n o g e n d i r e c t l y by c l e a v i n g

f i b r i n o p e p t i d e A f r o m m c h a i n of f i b r i n o g e n ( D a c i e a n d

Lewis, 1975). Warrell et a1 . ( 1 9 7 7 ) a l s o o b s e r v e d s e v e r e

f i b r i n o g e n d e p l e t i o n a s o n e of t h e major problems of

e n v e n o m a t i o n f r o m E c h i s c a r i n a t u s , T h i s may be d u e t o

t h r o m b i n - l i k e a c t i o n of t h e venom o n f i b r i n o g e n , This

f i b r i n o g e n d e p l e t i o n i n t e n s i f i e s t h e b l e e d i n g i n c l u d i n g

h a e m a t u r i a w h i c h c h a r a c t e r i s e s v i p e r i n e e n v e n o m a t i o n .

The p r e s e n t s t u d y d e m o n s t r a t e d t h e t h r o m b i n - l i k e a c t i v i t y

of t h e venom. ~ u r p r i s i n g l y , h o w e v e r , t h e h e r b a l e x t r a c t

e v e n a t 1 :250 d i l u t i o n had p r a c t i c a l l y n o effect o n t h e

t h r o m b i n - l i k e a c t i v i t y o f 1:10,000 d i l u t i o n of the venom

( s e e T a b l e 7 ) .

Okonkwo a n d O n u a g u l u c h i ( 2 9 7 7 ) observed e x t e n s i v e

p r o t e o l y s i s o n p l a s m a t r e a t e d w i t h p u f f a d d e r venom,

I n t h i s s t u d y e l e c t r o p h o r e s i s o f p l a s m a t r e a t ed with

venom s h o w e d c o n s i d e r a b l e l o s s of p r o t e i n b a n d s

e s p e c i a l l y a l p h a I , a l p h a 11, a n d b e t a g l o b u l i n s and

c o m p l e t e l o s s of f i b r i n o g e n . T h e same e f f e c t s were

n o t e d i n t h e p r e s e n t s t u d y when h e p a r i n i s e d p l a s m a w a s

t r e a t e d w i t h E c h i s carinatus venom ( P l a t e 11).

P r e t r e a t m e n t - , o f t h e h e p a r i n i s e d p l a s m a w i t h herbal

extract b e f o r e a d d i n g t h e venom d e c r e a s e d t h e p r o t e o l y t i c

a c t i v i t y of t h e venom o n the g l o b u l i n b a n d s , ( a l p h a I ,

a l p h a I1 a n d be ta g l ~ b u : ~ i n s ) . T h e f i b r i n o g e n band was

h o w e v e r n o t p r o t e c t e d ( P l a t e 11). T h i s r e s u l t r e i n f o r c e s

my c o n t e n t i o n t h a t E c h i s Carinatus venom a l s o p o s s e s s e s

a d e f i n i t e t h r o m b i n - l i k e a c t ion. During t h e electro-

p h o r e t i c study i t was a l s o n o t e d t h a t u n l e s s the

e l e c t r o p h o r e s i s w a s commenced almost i m m e d i a t e l y a f t e r

a d d i n g t h e venom t o the h e p a r i n i s e d p l a sma the plasma I s

c l o t t e d i n t h e t e s t t u b e . I n o t h e r w o r d s , h e p a r i n

f a i l e d t o n e u t r a l i s e t h e t h r o m b i n - l i k e a c t i v i t y of t h e

venom. H o w e v e r , (Warrell et a l , 1 9 7 6 ) n o t e d t h a t t h e

t h r o m b i n g e n e r a t e d by E c h i s c a r i n a t u s venom appears t o

be much less s e n s i t i v e t o h e p a r i n t h a n i s p h y s i o l o g i c a l

t h r o m b i n . B u t m e c h a n i s m of a c t i o n o f h e p a r i n i n v o l v e s

t h e f a c i l i t a t i o n of t h e f o r m a t i o n of c o m p l e x e s o f h e p a r i n

c o - f a c t o r ( W - g l o b u l i n ) w i t h each of t h e f o u r a c t i v a t e d

p r o t e a s e s o f t h e c o a g u l a t i o n D r o c e s s n a m e l y a c t i v a t e d

f a c t o r s I X , X , XI a n d XI1 a n d s i m i l a r s t i m u l a t i o n of t h e

r e a c t i o n b e t w e e n p l a s m a a n t i - t h r o m b i n I11 a n d t h r o m b i n - p-. rragt-~-''* '

*nrr(AF-''

(Goodman a n d G i l f m a n , 1 9 7 5 ) . M o r e o v e r i t r e q u i r e s

30-40 times m o r e h e p a r i n to i n h i b i t t h e a c t i o n of

f o r m e d t h r o m b i n (Goodman a n d G i l l m a n , 1975) . T h e r e f o r e

t h e p r e v e n t i o n of t h r o m b i n f o r m a t i o n i s p e r h a p s t h e

h e p a r i n ' s p r i m a r y effect ( J a q u e s 1966; Jick et al.,

1 9 6 8 ) . S i n c e i n t h e p r e s e n t s t u d y , the venom clotted

h e p a r i n i s e d p l a s m a , o n e c a n c o n c l u d e t h a t t h e venom

h y p a s s e d t h e a c t i v a t i o n of p r o t h r o m b i n to t h r o m b i n .

I t i s m o s t l y l i k e l y t h e r e f o r e t h a t t h e venom of Echis

C a r i n a t u s may c o n t a i n t h r o m b i n - l i k e c o m p o n e n t w h i c h

i s d i f f e r e n t from t h e t h r o m b i n i n n o r m a l b l o o d . I t i s

n o t s u r p r i s i n g t h e r e f o r e t o o b s e r v e t h a t i n a

c o n t r o l l e d t r i a l by Warrell e t a l . ( 1 9 7 6 ) a d d i n g

h e p a r i n t o t h e s t a n d a r d Echis a n t i v e n o m t r e a t m e n t

offered n o a d v a n t a g e o v e r t h e u s e o f s t a n d a r d a n t i v e n o m

treatment,

During t h e t o x i c i t y s t u d i e s i t w a s o b s e r v e d t h a t

s o o n after t r e a t i n g mice with v a r y i n g d o s e s of t h e

venom (0.5-6pq/g b o d y w e i g h t ) t h e a n i m a l s became quiet

a n d w i t h d r a w n f r o m l i v e l y a c t i v i t i e s . H o w e v e r , a t

4 p g / g dose l e v e l o r a b o v e , 90-100% o f t h e m c o n v u l s e d

a n d d i e d w i t h i n f i v e m i n u t e s o f r e c e i v i n g the venom.

T h i s d e a t h p a t t e r n a g r e e d w i t h t h e o b s e r v a t i o n o f

b l a r r e l l et a l . ( 1 9 7 7 ) t h a t i n j e c t i o n o f a l e t h a l d o s e o f

E c h i s C a r i n a t u s venom i n t o a n a n i m a l may c a u s e massive

i n t r a v a s c u l a r c l o t t i n g , c o r v u l s i o n a n d d e a t h within few

minutes, a s w o u l d occur when t h e s n a k e s t r i k e s i t s

- 0 1 - natural p r e y . H o w e v e r , e n v e n o m a t i o n of mice t r e a t e d

3 0 m i n u t e s e a r l i e r w i t h a d o s e of h e r b a l e x t r a c t

1 .5Ag/g b o d y w e i g h t d e m o n s t r a t e d a r e m a r k a b l e

p r o t e c t i v e a c t i o n . T h e LD5" of t h e venom was

i n c r e a s e d f r o m 2mg/kg to 3.2mg/kg, I t w a s a l s o

o b s e r v e d t h a t t h e h e r b a l e x t r a c t ( 1.5mg/kg) had no

e f f e c t o n t h e m o r t a l i t y due t o 4mg of venorn/kg body

w e i g h t . A l l a n i m a l s n o t p r e t r e a t e d with t h e herbal

e x t r a c t d i e d w i t h i n 5 min b u t n o d e a t h s occurred

u n t i l a f t e r f o u r h o u r s i n 75 mice p r e t r e a t e d with t h e

e x t r a c t (Table 9 a n d F i g . 1 ) . T h e t o x i c i t y studies

o f t h e h e r b a l e x t r a c t i n mice d e m o n s t r a t e d a h i g h

safety m a r g i n s i n c e t h e LD5" w a s f o u n d t o be 3 . 3 g / k g

b o d y w e i g h t ( F i g . 1 1 ) . A d o s e of 2 g / k g b o d y w e i g h t

p r o d u c e d n o toxic e f f e c t s , T h e f i r s t e v i d e n c e of

t o x i c i t y was n o t i c e d w i t h 3 g / k g b o d y w e i g h t (Table 2 0 ) .

I t i s t h e r e f o r e n o w o n d e r t h e n a t i v e s u s e t h i s h e r b

s u c c e s s f u l l y i n t r e a t i n g s n a k e b i t e s w i t h o u t o v e r t l y

d e l e t e r i o u s s i d e e f f e c t s f r o m t h e h e r b .

Some s n a k e v e n o m s e . g . chis c o l o r a t u s venom are

known t o c o n t a i n p o w e r f u l k a l l i k r e i n s , a group of

p r o t e o l y t i c e n z y m e s of h i gh substrate s p e c i f i c i t ; ~

( C o h e n e t a l . , 1969) . T h e y w e r e c a p a b l e of cleaving t h e

n o n - a p e p t i d e b a d y k i n i n from the p r e c u r s o r k i n i n o g e n ( s 1

i n p l a s m a q g l o b u l i n f r a c t i o n (Cohen et al . , 1969). 2

I n d e e d , e n d o g e n o u s c o n v e r s i o n of k i n i n o g e n t o k i n i n

; b r a d y k i n i n ) i n v o l v e s a cascade o f e n z y m a t i c r e a c t i o n s

i n plasma a n d a l s o h a v i n g o r i g i n i n a c t i v a t i o n o f

Hageman f a c t o r ( F a c t o r X I I ) . T h e k i n i n s ( B r a d y k i n i n s )

a r e p o t e n t v a s o d i l a t o r s a n d t h e r e f o r e may be r e s p o n -

s i b l e f o r t h e h y p o t e n s i o n s e e n i n s e v e r e e n v e n o m a t i o n .

However o u r s t u d y of t h e e f f e c t o f graded d o s e s of

2 0 0 a n d 4 0 0 p g of the venom on p e r f u s e d r a t hind-

q u a r t e r s p r e p a r a t i o n showed t h a t t h e venom d e c r e a s e d

t h e f l o w ra te ( T a b l e 1 2 a a n d Fig.3). T h u s the venom

h a s a d i r e c t v a s o c o n s t r i c t i v e e f f e c t o n the v a s c u l a r

s m o o t h m u s c l e . T h i s v a s o c o n s t r i c t i v e e f f e c t of the

venom o n t h e v a s c u l a t u r e may be r e s p o n s i b l e a t l e a s t

i n p a r t f o r t h e r i se i n b l o o d p r e s s u r e o f u p t o

76 rnm~g i n the a n a e s t h e t i z e d c a t i n d u c e d by i n t r a v e n o u s

a d m i n i s t r a t i o n of E c h i s c a r i n a t u s venom.

Tu ( 1 9 7 7 ) h o w e v e r , o b s e r v e d t h a t i n j e c t i o n o f a l l

s n a k e venoms for e x a m p l e i n r a t s c a u s e d a very r a p i d

f a l l i n a r t e r i a l blood p r e s s u r e with a r e t u r n t o

n o r m a l i n a f e w minu tes . T h i s c o n t r a s t s s h a r p l y with

the r ise i n b l o o d pressure w h i c h was o b s e r v e d i n t h i s

s t u d y , I t i s h o w e v e r o f i n t e r e s t that Novale et a1

( 1 9 7 3 ) b y f r a c t i o n a t i o n o f v i p e r a smmodytes venom

o b t a i n e d t h r e e f r a c t i o n s r e s p o n s i b l e f o r t h e d e p r e s s i o n

of a r t e r i a l b l o o d p r e s s u r e ,

T h e h e r b a l e x t r a c t ( l O m g / k g ) c a u s e d a small f a l l

i n t h e b l o o d p r e s s u r e of the a n a e s t h e t i z e d c a t a n d

pretreatment w i t h t h e h e r b a l e x t r a c t a l s o r e d u c e d t h e

l e v e l of r i se i n b l o o d p r e s s u r e d u e t o t h e venom.

T h e s l i g h t v a s o d i l a t o r y e f f e c t o f t h e h e r b a l e x t r a c t

d e m o n s t r a t e d i n t h e r a t s hide - q u a r t e r s p r e p a r a t i o n

m o s t p r o b a b l y i s r e s p o n s i b l e f o r t h i s e f fect o n b l o o d

p r e s s u r e

T h e v i p e r i n e venom h a v e b e e n shown t o affect t h e

r e s p i r a t i o n i n e x p e r i m e n t a l a n i m a l s . Tu ( 1 9 7 7 ) for

i n s t a n c e showed t h a t the c r u d e venom of v i p e r a a m m o d y t e s

induced r a p i d d e c r e a s e i n a r t e r i a l b l o o d pressure a n d

r e s p i r a t o r y f a i l u r e , T h e r e s u l t s f r o m t h e e x p e r i m e n t s

o n t h e a n a e s t h e t i z e d c a t s h o w e d t h a t t h e venom e v e n a t

0.5mg/kg h a d d e l i t e r i o u s e f f e c t o n t h e r e s p i r a t i o n ,

T h e r e s p i r a t i o n w a s i r r e g u l a r a n d t h e r e w e r e many

a p n o e i c p e r i o d s . T h e h e r b a l e x t r a c t a t 10mg/kg

c o n s i d e r a b l y i m p r o v e d t h e r e s p i r a t i o n i n t h e s e c a t s

t r e a t e d w i t h t h e venom.

T h e ECG c h a n g e s d u e t o t h e venom noted i n t h i s

s t u d y : d e p r e s s i o n of ST s e g m e n t s a n d T w a v e s , r e d u c t i o n

i n t h e voltage of QRS c o m p l e x e s , b r a d y c a r d i a and atrial

d y s r h y t h m i a . F i g . 6abc showed c l e a r l y that the venom

caused myocardial damage. I t w o u l d a p p e a r t h a t the

r e s p i r a t o r y d e p r e s s i o n a n d m y o c a r d i a l damage may be

r e s p o n s i b l e f o r d e a t h s i n a n u m b e r o f s n a k e - b i t e v i c t i m s .

More s t u d i e s a r e c e r t a i n l y r e q u i r e d t o a s c e r t a i n

t h e n a t u r e o f t h e m y o c a r d i a l d a m a g e d u e t o t h e venom a n d

t h e e f f e c t o f t h e h e r b a l e x t r a c t o n t h e v e n o m - i n d u c e d

c h a n g e s o n t h e c a r d i o v a s c u l a r c h a n g e s .

94 CONCLUSION

The in-vivo pharmacological studies of tl

extract of Diodia scandens have been very exci

was interesting to note that the LD50 of the T

increased from 2mg/kg to 3,2mg/kg following pl

the herbal extract (1,5mg/kg), The toxicity !

herbal extract indicated that it had a very hi

margin, since 2g/kg produced no toxic effect r

was found to be 3,3g/kg,

Interestingly, the herbal extract improvt

t o n depression induced by the venom and also

the rise in blood pressure induced by the vent

the ECG abnormalities due to the venom were a]

affected by the herbal extract at lOmg/kg, FI

using much higher doses of the herbal extract

In-vitro coagulation studies showed that the 1

increased the reduced whole blood-clotting ti1

the venom, The venom was found to possess bod

plastin and thrombin-like activity, The herb:

however antagonised the venom thromboplastin :

even at l : 2 5 O dilution it had no effect on thl

activity of the venom,

Electrophoretic studies showed that the :

plasma and herbal extract considerably protec-

alpha I alpha 11 and $ globulins from proteol;

venom but fibrinogen band was not protected.

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75. ' J a m e l l , D - h . , Davidson N., I . ,cD, G r p ~ n W O C * , F-, .?!. . Orrn~?-nd. C.O., Pope I"!. H. W-Zt ik ins , J O B , and F r e n t j c e , C.F:.E. (1977): Poisoring by b i t e s of the saw-sca led or c3net v iper ( E c h i s c2rinrtus) in N i ~ r p r i a . ',?u?rterl;y Journa l of Yedic'ne 46: ?3 - 62,

76. ? /e i ss , B.J., F h i l l i p s , LL., 3 o ~ e w e l 1 , J.S., "hillchristy and U a t t i (I???): Reprr ln thera~y in a n 3 t i e n t b i t t e n by s saw-scaled v i p e r (Echis c ~ r i n ~ t u s ) a snake whose v e n o r a c t i m t e s prot%rorLbin. !ir. J. Eed. 54: 653 - 660.

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