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University of Nigeria Research Publications
OKEKE, Chudi P.
Aut
hor
PG/M.Sc/82/1579
Title
In-vitro And In-vivo Studies On The Antagonism Of Some Toxic Effects Of Echis carinatus Venom By An Alcoholic Extract From Diodia scandens
Facu
lty
Pharmaceutical Sciences
Dep
artm
ent
Pharmacology and Therapeutics
Dat
e
July, 1988
Sign
atur
e
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
PAGE
v
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
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
iv.
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
PAGE
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
2
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 ,
7
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
8
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 .
10
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 ) .
13
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.
14
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).
16
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
18
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
19
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.
21
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
A
1
B
1
C
1
0.2
- -
D L
1
0.1
- 0.1
0. 1
0.1
-
-
0.1
0.1
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
2
C o n ten ts
PVSC
PVHC
I
t t
t t
I1
9 8
t t
I11
11
11
IV
1 )
t*
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
I11
.
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
IV
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
- 42
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
45
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 ~
I
Percentage
0.4
Fractions
A
Weights PI
0.88
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
G C E
D E O
A O A
V C C
F C U
A N 0
I N A
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
Total
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
J C E
[; c
E C
O R C
E U S O
N U
C N O
Prothrombin Time (Sec )
A Plasma
Total
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
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
Donor
A
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.
63
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 .
G8
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.
71
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
6.5
5.2
6.0
3.2
5.7
8.2
5.5
5.6
T ime (min) maximum
V o l . flow at
5.6
4.5
5.4.
2.6
3.7
5.7
3.7
3.8 -
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
85
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.
86
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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