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Indian Journal of Pharmacology 1992
;
24: 12
-
17.
EFFECTS OF SHILAJIT
ON
MEMORY, ANXIETY AND BRAIN
MONOAMINES IN RATS
A.K.JAISWAL
AND S.K.BHATTACHARYA
Neuropharmacology Laboratory, Department of Pharmacology,
institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005
Accepted for publication January 3, 1992.
Summary The effect of Shilajit was investigated for putative nootropic and anxiolytic activity, and its
effect on rat brain monoamines using Charles Foster strain albino rats. Nootropic activity
was assessed by passive avoidance learning and active avoidance learning acquisition and
retention. Anxiolytic activity was evaluated by the elevated plus-maze technique. Rat brain
monoamines and monoamine metaboliteswere estimated
bya
HPLC technique. The results
indicated that Shilajit had significant nootropic and anxiolytic activity. The biochemical
studies indicated that acute treatment with Shilajit had insignificant effects on rat brain
monoamine and monoamine metabolite levels. However, following subacute (5days) treat-
ment, there was decrease in 5-hydroxytryptamine and 5-hydroxyindole acetic acid concentra-
tions and an increase in the levels of dopamine, homovanillic acid and
3.4-dihydroxyphenyl-acetic acid concentrations, with insignificant effects on noradrenaline
and 3-methoxy-4- hydroxyphenylethylene glycol levels. The observed neurochemical effects
induced by Shilajit, indicating a decrease in rat brain 5-hydroxytryptamine turnover, associ-
ated with an increase in dopaminergic activity, helps to explain the observed nootropic and
-
_
anxiolytic effects of the drug.
Key word Shilajit nootropic effect anxiolytic action
Shilajit
is a blackish-brown exudation, of variable
consistency, obtained from steep rocks of differ-
ent formations found in the Himalayas at altitudes
between 1000 to 5000 metres, from Arunachal
Pradesh in the East to Kashmir in the West. It is
also found in Afghanistan, Nepal, Bhutan, Paki-
stan, China, Tibet and U.S.S.R.
(Tien-Shan,
Ural
and Caucasus).
Shilajit,
apart from being used in
diverse clinical conditions in
Ayurveda,
has been
proposed to arrest aging and induce rejuvenation,
and to improve memory, major attributes of
Ayurvedic rasayanas and medha rasayanas’. It
has been proposed that the modern equivalents
of rasyanas and medha rasayanas
are-adaptoge-
nic and nootropic activity,
respectively².
Shilajit,
long regarded as a bitumen (asphalt) or mineral
resin, or as a plant fossil exposed by elevation of
the Himalayas, has now been subjected to
exten-
Correspondence::
S.K.
Bhattacharya
rat brain monoamines and metabolites
sive chemical investigations3 and it has now been
shown to contain significant quantities of organic
compounds, including the bioactive oxygenated
dibenzo-alpha-pyrones, tirucallane triterpenes,
phenolic lipids and small tannoids.
Shilajit,
ob-
tained from different sources, has now been
stan-
dardized on the basis of its major organic
constituents³.
While investigating the putative adaptogenic activ-
ity of
Shi/ajit4,
some behavioural characteristics
emerged which suggested that the drug may have
nootropic and anxiolytic activity. We, therefore,
investigated these two actions of Shilajit, by using
acceptable behavioural paradigms. Changes in
the rat brain monoamines or their metabolite were
also investigated to explain the observed pharma-
cological effects.
Awarded Gufic prize in XXIV Annual conference of Indian Pharmacological Society held at Ahmedabad. December 29-31. 1991
NOOTROPIC AND
ANXIOLYTIC
ACTIVITY OF
SHILAJIT
13
MATERIALSAND METHODS
Standardized Shilajittwas obtained from Prof.
S.Ghosal,
Department of Pharmaceutics, Institute
of Technology, Banaras Hindu University. Freshly
prepared suspension of Shilajit in distilled water
was used and the doses mentioned represent the
dry weight of the drug. Shilajit was administered
60 min before behavioural testing, unless men-
tioned otherwise. All the experiments were done
with one lot of
Shilajit.
Young Charles Foster strain rats (100-150 g), of
both sexes, were used for the pharmacological
investigations, whereas older rats (200-220 g), of
either sex, were used for the neurochemical stud-
ies. The rats were housed in colony cages at an
ambient temperature of
25±2°C
and 4555% rela-
tive humidity, with a 12 hr light/l2 hr dark cycle.
Experiments were conducted under these ambi-
ent conditions between 0900 and 1400 hours. The
rats were fed standard pellet diet (Hind Lever) and
tap water was given through drinking bottles.
Food, but not water, was withheld 16 hr before
experimentation.
The following behavioural paradigms were used:
a. Passive avoidance learning and retention
The method used was essentially the same as
described
earlier5-7.
Briefly, the rat was placed on
the elevated platform situated in the centre of the
floor of the passive avoidance test box and the
latency to step down was recorded. Immediately
after stepping down, the rat received electric
shock (0.5
mA)
of 3
sec
duration through the grid
floor, and was then returned to its home cage. On
the following day (24 hr retention interval) the rat
was once again placed on the platform and the
latency to step down was recorded. Electric
shock was not given on day 2. If the rat remained
on the platform for the 5 min test duration, it was
assigned a maximum score of 300 sec. Latency
to step down was again assessed a week later on
day 9 in order to evaluate the retention of passive
avoidance learning. The results have been ex-
pressed as retention scores after 24 hr or one week
for each rat, by calculating the ‘inflexion ratio’ by
the formula:
b.
Active avoidance learning and retention
lnflexion ratio =
(LI
-
LI$/LJ
where
Lo
= initial step down latency in
sec
and
LI
= step down latency after 24 hr or 1 week, in
sec.
The method used was essentially the same as
described earlier’, using a Sidman jumping box
(Techno,
Lucknow). All the rats used were trained
upto 100% learning criterion of active avoidance
response. During the training period, each rat was
placed in one of the two chambers of the Sidman
box, and after 5
sec
the buzzer (conditioned stimu-
lus, CS) was sounded for 2
sec,
followed by an
electric shock (unconditioned stimulus, UCS;
3Ov,
0.5
sec)
through the grid floor. Thereafter, a rest
pause of 180
sec
was allowed. If the rat jumped
within the CS duration
to the
unelectrified safe box,
so as to avoid the UCS, it was allowed to rest there
for 30 sec. However, if the rat did not show the
avoidance response, it was removed from the
shock chamber after 180
sec
and was initiated for
the next trial. The rats were given 10 trials daily
until they reached the criterion of 100% active
avoidance response. The rats were subjected to a
repeat test after an interval of 15 days in order to
assess the retention of the previously learned ac-
tive avoidance response.
c. Elevated Plus-maze test
This test was used to evaluate anxiolytic activity.g
The plus-maze consisted of two opposite open
arms, 50 X 10 cm, crossed with two closed arms
of the same dimensions with walls 40 cm high. The
arms were connected with a central square, 10 X
14
A.K.JAISWAL AND
S.K.BHATTACHARYA
10 cm. to give the apparatus a plus-sign appear-
ance. The maze was elevated 70 cm above the
floor in a dimly lit room. Rats were individually
placed in the central square facing an enclosed
arm. The time spent by the rat, during the next 5
min, on the open and closed arms was recorded.
An arm entry was defined when all four limbs were
on the arm.
Estimation of rat brain monoamines and
monoamine metabolites
Whole brain concen-
trations of
5-hydroxytryptamine
(5-HT),
5-
hydroxyindole acetic acid (5-HIAA), dopamine
(DA),homovanillic
acid
(HVA),
3,4-dihydroxyphe
nyl acetic acid (DOPAC), noradrenaline (NA) and
3-methoxy-4-hydroxyphenylethylene
glycol
(MHPG) levels were assessed by a high pressure
liquid chromatographic (HPLC) technique”.
RESULTS
a. Passive avoidance learning and retention
The results summarized in Table 1 indicate that
Shilajit
had a dose-related facilitatory effect on the
retention of passive avoidance learning, though
the effect was statistically significant for the higher
dose (50
mg/kg,
p.o.)
at both retention intervals of
24 hr and 1 week.
b. Active avoidance learning and retention
The results summarized in Table 2 indicate that
neither of the doses (25 and 50
mg/kg,
p.o.)
of
Shilajit had any significant effect on the acquisition
of active avoidance learning. However,
Shilajit
had a dose-related facilitatory effect on the reten-
tion of the previously learnt active avoidance task
when tested after an interval of 15 days. The rats
Table 1. Step down latencies, represented as inflexion ratios, following pretreatment with Shilajit, in the passive avoidance test in
rats (Mean ± SEM)
Groups
n
Retention intervals
24hr 1 Week
Control
Shilajit
(25
mg/kg)
Shilajit
(50
mg/kg)
8
3.0 ± 1.7
4.0
±
1.5
8
2.2 ± 1.4
4.8
±
1.8
7
12.4 ±
4.1”*
9.0
±
l.ga
a
p <0.05,
*
p
<
0.05 as compared to control and Shiljit (25mg/kg) groups respectively (unpaired Student’s
't'
test).
Table 2. Effect of Shilajit on active avoidance learning and relearning in rats (Mean
±
SEM)
Groups
Control
Shilajit
(25
mg/kg)
Active avoidance tests
Learning
(acquisition) Relearning
Trials
Shocked trials
Trials
Shocked trials
41.2
±
2.3
13.5
±
1.4
40.0
±
0.6 4.4
±.
0.6
41.2
±
3.5
12.9
±
2.0a 24.3
±
3.7 2.3
±
0.6a
Shilajit
(50
mg/kg)
7
41.4 ± 4.6 10.4 ± 1.3 18.6
±
4.0b
0.9 ±
0.4b’C
a
p
<
0.05 and
b
p
<
0.01 as compared to control; c p < 0.05 as compared to Shilajit (25 mg/kg) group (unpaired Student’s
't'
test).
--
_
f
t
i’
\ 2.
c
i
NOOTROPIC
AND ANXIOLYTIC ACTIVITY OF SHILAJIT
15
pretreated with Shilajit require significantly less
trials and shocked trials to re-learn the problem,
as compared to untreated controls.
c. Elevated plus-maze test
arms. However, the effect was not dose-related,
with maximal effect being induced by the lower
dose (10
mg/kg,p.o.)
used. The results obtained
were comparable with those induced by the stan-
dard anxiolytic agent, diazepam (1
mg/kg,
p.o.).
The results summarized in Table 3 indicate that
Shilajit exhibited an anxiolytic activity as
evi-
d. Rat brain monoamines and monoamine
metabolites
denced
by significantly greater time spent by the
The results, as summarized in Table
4,
using two
rats on the open arm as compared to the closed doses (25 and 50 mg/kg,
p.o.)
of Shilajit at two
Table 3. Anti-anxiety effect of Shilajit in the elevated plus-maze test in rats (Mean
±
SEM
)
Groups
n
Time spent in enclosed
arms (sec),
Control
6
205.1 ± 12.6
Time spent in open
arms (sec),
94.8
±
12.6
Shilajit
(10
mg/kg)
6
143.4
±
9.2
b
156.5
±
9.2’
Shilajit
(25
mg/kg)
5
160.0
±
15.48
140.0
±
15.4a
Shilajit
(50
mg/kg)
6
145.9
±
20.3’ 154.1
±
20.3=
Diazepam
(1
mg/kg)
5
171.2
±
8.6*
a
p < 0.05,
’
p
<
0.01 and
c
p
<
0.001 as compared with control (unpaired Student’s
't'
test)
128.9
±
8.6a
Table 4
Effect of Shilajit, following acute and subacute treatments, on rat brain monoamines and
monoamine metabolites
(ng/g,
Mean
±
SEM)
Monoamine
Metabolites Vehicle
(8)
Shilajit (50 mg/kg)
(5)
Shilajit (25 mg/kg
x 5 days) (6) Shilajit (50 mg/kg
x 5 days) (6)
5-HT
186.4
±
9.8 202.0
±
8.8
151.0
±
6.Sa 132.1
±
5.9’
5-HIAA
246.2
±
6.6 232.5
±
5.9
206.5
±
6.gb
185.0
±7.4’
Dopamine
1268.2±t
32.4 1292.2
±
62.6
1433.25
49.Ba
1558.4
±39.6’
HVA
136.2”
8.1
126.6
±
5.6
151.3 ±
6.2 169.9
±
7.6a
DOPAC
172.9
±
6.3
156.4
±
9.6
183.3
±
5.0a
196.1
±
5.3b
Noradrenaline 606.4
±
9.9
596.1
±
8.4 634.2
±
7.6 669.3
±
8.4’
MHPG
96.3 ±
4.8 92.0
±
5.6
107.8
±
5.7 129.8
±
7.4a
a
p
<
0.05,
b
p
<
0.01 and
c
p
<O.OOl
as compared with vehicle treated control. (Unpaired Students
‘t’
test
)
Figures in parenthesis indicate the number of animals.
16
A.K.JAISWAL AND S.K.BHATTACHARYA
pretreatment time intervals (60 min and 5 days),
indicate that single pretreatment with the drug had
insignificant effects on the rat brain concentrations
of 5-HT and its metabolite
5-HIAA,
DA and its
metabolites HVA and DOPAC, and NA and its
metabolite MHPG. However, 5-day pretreatment
induced decrease in the levels of 5-HT and
5-HIAA,
and increase in the concentrations of DA, HVA and
DOPAC. However, the effect on NA and MHPG
concentrations remained statistically insignificant.
DISCUSSION
Nootropics represent a new class of psychotropic
agents with selective facilitatory effect on integra-
tive functions of the central nervous system, par-
ticularly on intellectual performance, learning
capacity and memory
¹¹.
A number of drugs,
including piracetam, have now been introduced in
therapy to ameliorate cognitive deficits. The pres-
ent studies indicate that
Shilajit,
an Ayurvedic
medha-rasayana, can be regarded as a nootropic
agent in viewof its facilitatory effect on retention
of acquired learning, though it had minimal effect
on the acquisition of active avoidance learning.
Several studies conducted in this laboratory have
indicated that the proposed nootropic agents have
more significant effects on learning and memory,
following induction of memory deficits by under-
nutrition or environmental impoverishment
5-7.
As
such, an extended investigation on the effect of
Shilajit,
under induced conditions of cognitive def-
icits, is required to confirm its putative nootropic
effect.
In the elevated plus-maze test, reduction in open
arm entries, in relation to total arm entries, pro-
vides a measure of fear induced inhibition of ex-
ploratory activity which is attenuated by anxiolytic
agents
‘*‘*.
Thus, the results indicate that
Shilajit
has significant anxiolytic activity, comparable
qualitatively with that induced by diazepam, in
doses lower than that required for nootropic
activity.
The neurochemical basis of learning and memory
remains controversial, despite extensive experi-
mental and clinical studies. Although the role of
the central cholinergic system is fairly well estab-
lished, its deficiency being implicated in memory
deficits, the role of the other neurotransmitter sys-
tems can not be
ignored¹³.
Several studies have
indicated that increase in serotonergic
neu-
rotransmission can interfere with learning acqui-
sition and memory consolidation’4. However, the
role of 5-HT in anxiety is now well established and
it has been conclusively shown that increase in
central serotonergic activity invariably leads to
anxiety, whereas decrease in brain 5-HT activity
results in
anxiolysis.15
Thus, the neurochemical
effects induced
by
Shilajit can explain its nootropic
and anxiolytic actions, particularly the induced
decrease in 5-HT turnover, as indicated by a de-
crease in 5-HT and
5-HIAA
levels. Conversely, the
increase in DA turnover, as evidenced by the in-
duced increase in the levels of DA and its
metab-
olites, HVA and DOPAC, can contribute to the
observed nootropic activity. Piracetam, the clas-
sical nootropic agent, has been reported to aug-
ment rat brain dopaminergic activity’“.
Although some newer pharmacological agents,
like tianeptine, have been reported to exhibit both
anxiolytic and nootropic activity, probably in-
duced by a reduction in central serotonergic func-
tion”’ there is no reason to believe that
improvement in memory is secondary to anxioly-
sis, since benzodiazepines are known to have an
adverse effect on learning and memory”. In the
present investigations also, the doses of Shilajit
exhibiting anxiolytic and nootropic actions, were
at variance.
The present investigations, thus, establish the use
of Shilajit in Ayurveda as rasayana and
medha-
NOOTROPIC
AND ANXIOLYTIC ACTIVITY OF
SHILAJIT
17
t
t
rasayana,
exemplified by its anxiolytic and ACKNOWLEDGEMENT
nootropic actions, respectively. The putative
neu-
rochemical basis for these action has also been
We are thankful
to
Prof.S. Ghosal, Dept.
of
Phar-
provided. However, further investigations, using
more experimental paradigms, are required
be-
fore the nootropic and anxiolytic actions of Shilajit
can be affirmed.
maceutics, of this University, for making available
standardized Shilajit samples.Technical help of
Shri
Hausla
Singh is also thankfully acknowl-
edged.
1.
2.
3.
4.
5.
6.
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