Some Neuropharmacological Effects of the Crude Venom Extract of

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28
East and Central African Journal of Pharmaceutical Sciences
Vol. 10 (2007) 28-33
Some Neuropharmacological Effects of the Crude Venom Extract of Conus musicus in Mice
K. BALAMURGAN*, D. AKALANKA, S. RAJU AND A. SHARMA
Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, 600 802,
Tamil Nadu, India.
This study reports some neuropharmacological effects of the crude venom
extract of Conus musicus (family Conidae) in mice using various
experimental models. The crude venom was found to significantly increase
tail flick reaction time in mice. The effects of the venom on the central
nervous system were studied by observing spontaneous motor activity, gross
behaviour, rota-rod performance and potentiation of pentobarbitone
sleeping time in mice. Preliminary acute toxicity evaluation was also carried
out and the LD50 was found to be 460.23 μg/kg following intraperitoneal
administration. At a dose of 200 μg/kg i.p., the extract produced a reduction
in spontaneous motor activity, altered gross behavior and motor
coordination and prolonged pentobarbitone-sleeping time. A liquid
chromatography mass spectroscopic study has indicated the presence of ωconotoxin in the crude venom extract.
Keywords: Conus musicus, sedation, spontaneous motor activity, gross behavior, motor
coordination
INTRODUCTION
Predatory marine snails of the genus
Conus (family Conidae) with over 500
species may comprise the largest single
genus of marine animals living today.
These species inhabit tropical reef
environments throughout the world.
Depending on their prey preference, cone
snails can be classified into three major
groups. The piscivorous group comprising
among other species Conus striatus and C.
geographus preys upon fish. The group
that feeds on mollusks (molluscivorous)
includes C. textile and C. pennaceus. A
third group, the vermivorous snails, preys
upon Polychaete annelids and includes C.
imperialis and C. vexillum. All cone snails
are venomous predators and have
developed a sophisticated biochemical
arsenal to rapidly immobilize their prey.
Their venoms are complex mixtures of
small, disulfide-bridged polypeptide
toxins (conotoxins) that inhibit the
functioning of ion channels and
neurotransmitter receptors. In addition to
their vital role in prey capture and
*Author to whom correspondence may be addressed.
defence against predators, conotoxins are
useful tools in neuroscience in the
characterization of receptors and receptor
subtypes due to their high binding affinity
and specificity. Conotoxins also offer
great potential as leads in drug
development. Indeed the N-type calcium
channel blocker from Conus magus ωconotoxin is currently undergoing clinical
trials for the treatment of stroke and
chronic pain. It is anticipated that the
discovery of new toxins displaying
characteristically high specificities will
increase our understanding of the
physiology, pharmacology, biochemistry
and structure of the receptors they bind to,
and may provide leads to the development
of new pharmaceuticals [1-7].
MATERIALS AND METHODS
Preparation of crude venom extract:
Specimens of Conus musicus were
collected from Portonova, Chidambaram,
Tamil Nadu, Southern India, dissected and
a crude extract prepared from the venom
duct material as previously described [8].
29 Balamurgan et al.
East Cent. Afr. J. Pharm. Sci. 10 (2007)
Ground dried ducts were extracted with a
30% acetonitrile/water mixture acidified
with 0.1% trifluoroacetic acid, centrifuged
and the supernatants retained. Crude
venom extract was lyophilized and stored
at -20 °C.
Animals: The pharmacological experiments
were conducted using Swiss albino mice
weighing 20-25 g. The mice were
maintained under standard nutritional and
environmental conditions of 50 ± 10% r.h.
and an alternating 12 h light and dark cycle
throughout the experiment. The animals
were used after an acclimatization period of
at least 5 days to the laboratory environment
and provided with standard food pellets and
water ad libitum. The animals were deprived
of food 24 h prior to experimentation. The
animal ethical committee clearance was
obtained from the institution for the present
study.
Acute toxicity test: The mice were divided
into groups of ten and the crude venom
extract of Conus musicus (CMV) was
injected i.p. in doses ranging from 50 to 500
μg/kg. The number of deaths within 24 h of
injection was recorded. The LD50 was
estimated from the graph of percent
mortality against log-dose of the crude
venom using the Miller and Tainter method
[9].
Analgesic activity: To evaluate the central
analgesic effects of the extract the tail flick
test was performed. In this test, the time
taken for a mouse to withdraw its tail when
immersed in water maintained at 55±0.5 °C
was recorded [10]. The first group was
treated with normal saline while groups 2, 3
and 4 received the venom extract at doses of
50 μg/kg, 100 μg/kg and 200 μg/kg i.p.
respectively. The fifth group was treated
with pentazocine (10 mg/kg, i.p.) as a
standard drug.
Spontaneous motor activity (SMA):
Spontaneous motor activity was measured
using an actophotometer (Techno LE3806,
Lucknow, India). Mice were grouped in
sixes and treated with normal saline or the
CMV extracts (50, 100 and 200 μg/kg i.p.)
or diazepam 4 mg/kg i.p. Activity was
automatically recorded 30 min after
treatment. The experiments were repeated at
an interval of 30 min for a total of 60 min.
The results obtained were compared with
those of the control group at each time
interval [11].
Gross behavioral pattern: After intraperitoneal administration of the same test
doses of the CMV extract as in the earlier
experiments to a group of 6 mice, each
animal was observed for gross behavioral
effects. The behavior of the animals was
continuously observed for 3 h after
administration of the CMV extract and then
after every 30 min for the next 3 h. The
study was carried out for 6 h, 12 h and 24 h
[12].
Motor coordination: A rota-rod (Techno
3C, Lucknow, India) biological research
apparatus was used for this test. The
instrument (a horizontal rotation device) was
set at a rate of 16 rpm. Mice were placed on
the rod and those that were able to remain
on the rod for longer than 3 min were
selected for the study. The first group was
treated with normal saline, while groups 2 to
4 received intraperitoneal CMV extract at
doses of 50, 100 and 200 μg/kg. The fifth
group received diazepam 4 mg/kg i.p. Mice
unable to remain on the rod for at least three
min were considered as a positive result and
the time taken to fall was recorded [13].
Pentobarbitone sleeping time: The mice
were divided into 4 groups of six each.
Group 1 received normal saline while
groups 2, 3 and 4 received 50, 100 and 200
μg/kg i.p. respectively, of the CMV extract.
The mice in group 5 received diazepam 4
mg/kg i.p. as the standard drug. The animals
were administered with 40 mg/kg i.p. of
sodium pentobarbitone 30 min later. The
index of hypnotic effect was recorded. For
this purpose, the time lapse between the
administrations of pentobarbitone and the
loss of righting reflex was recorded as the
30 Balamurgan et al.
East Cent. Afr. J. Pharm. Sci. 10 (2007)
onset of sleep. The duration of sleep was
taken as the time between the loss and
recovery of the righting reflex [14].
Statistical analysis: The data obtained was
expressed as mean ± standard error.
Differences in means were estimated by use
of ANOVA followed by Dunnet's post hoc
test. Results were considered significant at
p< 0.05.
RESULTS AND DISCUSSION
Acute toxicity studies: The LD50 of the
CMV extract following i.p. administration
was found to be 460.23 μg/kg. While
conducting the toxicity studies, animals
were observed continuously for any gross
behavioral changes and significant reduction
of
spontaneous
locomotor
activity,
drowsiness and remarkable quietness were
observed [15].
Analgesic activity: Analgesic activity was
investigated by the tail flick test. The
average tail flick latency before and after
treatment in the normal saline treated group
was 3.72  0.31 (Table 1). The CMV extract
treatment induced dose dependent related
changes in tail-withdrawal latencies when
compared to control group. The maximum
analgesic effect was reached 60 min after
administration. A cut-off time of 10 s was
taken as the maximum analgesic response to
avoid injury to the tail due to heat.
Spontaneous motor activity: The CMV
extract produced a significant decrease in
spontaneous motor activity in mice. This
effect was dose dependent and was observed
within 30 min of drug administration,
persisting for 60 min (Table 2).
Motor coordination: The results of motor
coordination test are presented in Table 3.
The CMV extract elicited a marked
reduction in motor coordination rendering
the mice unable to hold onto the rotating
rod. This effect was dose dependent with the
reaction time decreasing with dose.
Pentobarbitone induced sleeping time:
Prior administration of CMV extract
significantly increased pentobarbitoneinduced sleeping time in mice. Table 4
shows sleeping time in mice treated with
pentobarbitone with or without extract.
The average sleeping time was found to
be 40.48 ± 1.92 min in mice treated with
pentobarbitone alone. Prior administration of the CMV extract significantly
altered the onset and increased the
duration of action of pentobarbitoneinduced sleeping time. The maximum
duration of sleep was observed at a dose
of 200 μg/kg of CMV extract, 92.95 ±
1.54 min [16].
Gross behavior pattern: A significant
difference in response was observed when
the animals that received the CMV extract
were compared with the control (Table 5). In
CMV treated mice, there was strong
respiratory depression, severe writhing,
tremors, convulsions and hind limb paralysis
in the initial 3 h after drug administration,
which became mild in subsequent 3 h
followed by no above responses at the end
of the 24 h of the study. But there was a
mild to strong stimulation of salivary
secretions with increased sense of touch and
sound perceptions. No signs of diarrhea and
mortality were observed in the study [17].
The results presented heretofore report some
pharmacological activities of the crude
venom extract of Conus musicus in mice.
Results indicated that the CMV extracts
significantly increased tail flick response.
The tail flick response is usually considered
suitable for testing central analgesic activity.
The CMV was found to produce alteration
in general behavior pattern, significant
reduction of spontaneous motor activity,
changes in gross behavior pattern, motor
coordination
and
prolongation
of
pentobarbitone-induced sleeping time. The
present findings suggest that CMV
possesses CNS-depressant effects.
31 Balamurgan et al.
East Cent. Afr. J. Pharm. Sci. 10 (2007)
Table 1: Effect of Conus musicus crude venom on tail flick response in mice
Dose
(mg/kg)
Mean
reaction
time (min)
0.2 ml
3.72 ± 0.31
CMV extract
0.05
3.79 ± 0.31
CMV extract
0.1
4.00 ± 0.26
CMV extract
0.2
3.67 ± 0.33
Pentazocine
10
3.83 ± 0.31
Drug
Normal
saline
Mean reaction time after
administration of drug
15 min
30 min
60 min
3.50 ±
3.39 ± 0.26
3.73 ± 0.19
0.34
6.50 ±
7.16 ± 0.31
8.00 ± 0.37
0.34
7.00 ±
8.30 ± 0.21
9.00 ± 0.26
0.26
8.17 ±
9.00 ± 0.26
9.67 ± 0.21
0.31
7.17 ±
8.50 ± 0.22
9.30 ± 0.21
0.31
% increase in
reaction time
after 60 min
2.6
115.05
141.93
159.94
150.00
Values are mean ± SEM, n=6 in each group, CMV = Conus musicus venom. Percentage increase in reaction
time when compared to control is significant at p <0.05.
Table 2: Effect of Conus musicus crude venom extract on spontaneous motor activity
Mean reaction time after administration of
drug
0 min
30 min
Dose
(mg/Kg)
Mean reaction
time (min)
Normal saline
0.2 ml
408.78 ± 5.77
433.50 ± 6.49
418.39 ± 5.16
CMV extract
0.05
404.22 ± 5.09
150.72 ± 6.36
95.13 ± 1.31
CMV extract
0.10
414.70 ± 6.37
85.25 ± 1.21
60.49 ± 1.44
CMV extract
0.20
421.72 ± 6.15
40.55 ± 1.28
33.40 ± 1.06
Diazepam
4.00
419.99 ± 5.14
33.8 ± 1.91
19.07 ± 0.57
Drug
Values are mean ± SEM, n = 6 in each group, CMV = Conus musicus venom. Significantly different at p<0.05.
Table 3: Effect of Conus musicus crude venom extract on motor coordination
Dose
(mg/Kg)
Mean
reaction time
(min)
Normal saline
0.2 ml
217.50 ± 4.17
226.50 ± 4.83
201.36 ± 6.09
CMV extract
0.05
212.50 ± 7.32
128.30 ± 4.10
145.27 ± 4.15
CMV extract
0.10
217.17 ± 6.05
90.21 ± 2.71
76.50 ± 2.51
CMV extract
0.20
214.06 ± 7.89
78.17 ± 3.68
65.67 ± 2.04
Diazepam
4.00
215.99 ± 5.14
33.8 ± 1.91
19.07 ± 0.57
Drug
Mean reaction time after administration
of drug
0 min
30 min
Values are mean ± SEM; n = 6 in each group, CMV = Conus musicus venom. Significantly different at p<0.05.
32 Balamurgan et al.
East Cent. Afr. J. Pharm. Sci. 10 (2007)
Table 4: Effect of Conus musicus crude venom extract on pentobarbitone induced sleeping
time
Treatment
Dose (mg/kg)
Onset of sleep (min)
Duration of sleep (min)
Pentobarbitone
40.0
3.01  0.17
40.48  1.92
CMV extract
0.05
2.92  0.18
60.79  1.04
CMV extract
0.10
1.86  0.15
75.78  1.32
CMV extract
0.20
1.51  0.11
92.95  1.54
Diazepam
4.00
2.02  0.31
95.48 1.27
Values are mean ± SEM; n = 6 in each group, CMV = Conus musicus venom. Duration of sleeping when
compared to control is significantly different at p<0.05.
Table 5: Effect of Conus musicus crude venom extract on gross behavioral pattern
Gross activity
Time (h)
3
3½
4
4½
5
5½
6
12
24
Respiratory
depression
++
++
+
+
+
+
-
-
-
Writhing
++
++
+
+
+
-
-
-
-
Tremors
++
++
+
+
+
+
-
-
-
Convulsions
++
++
+
-
-
-
-
-
-
Hind limb
paralysis
+
+
++
++
++
++
++
+
-
Sense of touch
and sound
High
High
High
High
-
Low
-
-
-
Salivation
High
High
-
-
-
-
-
-
-
Diarrhea
-
-
-
-
-
-
-
-
-
Mortality
-
-
-
-
-
-
-
-
-
+ = Mild effect, ++ = Strong effect, - = No Effect.
The CMV extract significantly reduced
spontaneous motor activity. This activity is a
measure of the level of excitability of the
CNS and the observed decrease may be
closely related to sedation resulting from
depression of the central nervous system.
Previous studies have related prolongation
of barbital hypnosis to pentobarbital
metabolic inhibition or action on the CNS
centers involved in the regulation of sleep. It
is generally accepted that the sedative effect
of drugs can be evaluated by measurement
of spontaneous motor activity and
pentobarbitone-induced sleeping time in the
laboratory animal model [18]. These results
corroborate previous reports that the
33 Balamurgan et al.
East Cent. Afr. J. Pharm. Sci. 10 (2007)
enhancement of barbital hypnosis is a good
index of CNS depressant activity. Results of
the gross behavior test (Table 5) further
support the sedative effect of the extract and
its possible application in anxiety
management.
Present findings of analgesic activity are
similar to those reported for pentazocine
[19]. It has been reported that ω-conotoxin
has potent sedative activity when tested in
similar models and also inhibits spontaneous
motor activity in mice [20]. Therefore, the
ω-conotoxin content of the crude venom
extract may be partially responsible for the
observed pharmacological effects. Further
studies should be carried out to establish the
mechanism of CNS depressant action of the
CMV extract.
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