Journal of Current Pharmaceutical Research 2010; 01: 8-11
JCPR 2010; 01: 8-11
© 2010 Medipoeia
Received: 13/03/2010
Revised: 24/03/2010
Accepted: 29/03/2010
Anuj k. Agrahari, Mohd.
Khaliquzzama and Sanjaya k. Panda
The Pharmaceutical College,
Department of Pharmacognosy,
Samaleswari Vihar, Tingipali,
Barpali - 768029, Bargarh,
Odisha, India..
Correspondence:
Anuj k. Agrahari
The Pharmaceutical College,
Department of Pharmacognosy,
Samaleswari Vihar, Tingipali,
Barpali - 768029, Bargarh,
Odisha, India.
E-mail:
agraharimanuj@rediffmail.com,
itsanujagrahari@yahoo.co.in
Tel. No.: +91-9861348888
Evaluation of analgesic activity of methanolic
extract of Trapa natans l.var. Bispinosa roxb.
Roots
Anuj k. Agrahari, Mohd. Khaliquzzama and Sanjaya k. Panda
Sathesh B. P. R, Goli Divakar, Manoj K. Jangid and Kapil K. Purohit
ABSTRACT
Objective: The main objective of the present investigation is to evaluate the analgesic activity of
methanolic extract of Trapa natans L.var. bispinosa Roxb. roots on mice.
Materials & Methods: Analgesic activity of the methanolic extract of the T. bispinosa root at a
dose of 200mg/kg & 400mg/kg was evaluated against the standard drug pentazocine at a dose of
30mg/kg. Adult Swiss albino mice of either sex of six numbers in each group was undertaken for
study and evaluated by tail flick & tail immersion method.
Results: The both doses of T. bispinosa roots methanolic extract was found to produce significant
(p < 0.01) analgesic activity. In tail flick method, the extract at 200mg/kg showed significant
activity (P<0.01) after 45 minutes but in tail immersion method, the extract showed significant
activity at all tested dose levels after 30 minutes interval. The results showed significant analgesic
activity against stimuli.
Conclusion: The methanolic extract of Trapa natans L.var. bispinosa Roxb. roots have potential
analgesic activity against stimuli in the tested animals. So, it can be recommened for further
studies.
Keywords: Trapa bispinosa roots, Floating Herb, Methanol Extract, Analgesic activity, Tail flick
method, Tail immersion test, Pentazocine.
1. INTRODUCTION
Trapa natans L.var. bispinosa Roxb. (Trapaceae), is known as „Water Chestnut‟, in
Orissa as „Pani Singada‟, in Sanskrit „Jalaphala‟, occurring in ponds & lakes (The Ayurvedic
Pharmacopoeia, 2003). It is an annual aquatic floating herb introduced from Asia. Floating leaves
rhomboid, 2-6.5cm diameter, broader than long, denticulate, denate, serrate or incised with entire
base, apex acute, red & densly pubescent or villous beneath. Flowers white. Ca. 1.8cm across, not
much raised above and broad with a short conical, often spinous beak in the centre through which
the radicle is protruded, with two spines at two angles, the second pair of spines often wanting
(Saxena et. al., 1995). The kernels are good source of minerals. It contains carbohydrates &
minerals matters, calcium, phosphate, iron, copper, manganese, magnesium, sodium & potassium.
The kernels also contains some vitamins like thiamine, riboflavin, nicotinic acid, Vitamin – C,
Vitamin – A, D-amylase & considerable amount of phosphorylase (The Wealth of India, 2003).
This is used in many Ayurvedic preparation as nutrient, appetizer, astringent, diuretic, aphrodisiac,
cooling, antidiarrhhoeal & tonic, it is also useful in lumbago, sore throat, bilious affections,
bronchitis, fatigues & inflammation. Fruits are also used in making liniments for the cure of
rheumatism, sores & sunburn. It is also said to have cancer-preventing properties. Stem is used in
the form of juice in eye disorders (The Wealth of India, 2003 & Rehman et. al., 2000). But the use
& effect of roots are not reported till date. An extensive search of the literature reveals no reports
on the analgesic activity of the plant. Thus, the plant. Thus, present investigation was planned to
 Journal of Current Pharmaceutical Research 2010; 01: 8-11

find out the therapeutic level of methanolic extract of Trapa natans
L.var. bispinosa Roxb. roots in analgesic activity.
2. MATERIALS AND METHODS
Trapa natans L.var.bispinosa Roxb. roots were collected
from the lake of village “Bhaisatara” in Raipur district of
Chhattisgarh. The plant material was authenticated from Botanical
Survey of India, Howrah, Kolkata (No. CNH/ I – I (5)/ 2009/ Tech.
II / 35). Few authentic samples were preserved in department of
Pharmacognosy, The Pharmaceutical College, Barpali for future
reference.
After authentication, fresh plant material was collected in
bulk, washed under running tap water to remove adhering material,
dried under shade and pulverized in a mechanical grinder. The
coarse powder was pass through sieve no. 40 and taken for further
studies.
Preparation of Extract
For the preparation of extract 100gm of dried coarse
powdered roots were charged in to the soxhlet‟s apparatus (hot
extraction) and extracted successively with petroleum ether (600 -
800C), chloroform, ethyl acetate & methanol, in order of their
increasing polarity.
The successive methanolic extract (deep brown colour)
was filtered & dried under reduced pressure to get a solid mass free
from the solvent. The yield was 5.9% with respect to dry starting
material with characteristic odour & greasy consistency. The dried
extract was dissolved in solution of 2% gum acacia in distilled
water (vehicle) for the evaluation of analgesic activity.
Table 1. Analgesic activity of Trapa bispinosa Roxb. by Tail Flick Method
Animals Used
Adult albino rats weighing between 150 – 200gm and
Adult swiss albino mice weighing between 25 – 30gms of either
sex were used for the studies. The animals were maintained under
normal laboratory condition & kept in standard polypropylene
cages at room temperature of 300±20 and 60 to 65% relative
humidity and provided with standard diet & water ad libitum.
The experimental protocols were approved by institutional
Animal Ethical Committee & a written permission from in house
ethical committee has been taken to carry out (Reference no.
PCB/AEC/04/09 on dated 23rd April, 2009) and complete this
study.
Toxicity Study
The selected adult albino rats were used to determine the
dose. The animals were divided in to eight groups of six in each.
The animals were fasted overnight prior to the acute experimental
procedure. The karber‟s method (Ghosh et. al., 2005 & Kale et. al.,
2005) was used to determine the dose; gum acacia (2% w/v) was
used as vehicle to suspend the extracts and administered
intraperitoneally. The control group received 2ml/kg of the vehicle
intraperitoneally. The other group received the extract as test drug
in one of the following doses – 100, 200, 400, 800, 1000, 2000 and
3000mg/kg in a similar manner. Immediately after dosing, the
animals were observed continuously for first four hours for
behavioral changes and for mortality at the end of 24hrs, 48hrs and
72hrs respectively. The toxicity study showed that the methanolic
extract of drug at a minimum dose of 200mg/kg onwards shows the
reaction in experimental animals. However, no mortality was
reported even after 72hours. This indicates that the methanolic
extract is safe up to a single dose of 3g/kg body weight.

Basal Reaction Reaction Time (Sec)

Group Treatment Dose
Time (Sec)
15 min 30 min 45 min 60 min

I 2% w/v Gum acacia 2ml/kg 2.50 ± 0.33 2.80 ± 0.21 2.90 ± 0.21 3.10 ± 0.19 2.60 ± 0.15

II Pentazocine 10mg/kg 2.60 ± 0.28 4.10 ± 0.41 * 5.50 ± 0.39 ** 6.93 ± 0.72** 8.26 ± 0.59**

III Test Drug – I 200mg/kg 2.80 ± 0.23 3.70 ± 0.24* 4.60 ± 0.20 * 5.70 ± 0.54** 7.26 ± 0.49**

IV Test Drug - II 400mg/kg 2.40 ± 0.25 3.90 ± 0.33* 5.00 ± 0.23** 6.20 ± 0.43** 7.60 ± 0.56**

Result expressed as mean ± SEM from six observations; ** indicates P < 0.01 & * indicates P < 0.05

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 Journal of Current Pharmaceutical Research 2010; 01: 8-11

Table 2. Analgesic activity of Trapa bispinosa Roxb. by Tail immersion Method

Basal Reaction Reaction Time (Sec)

Group Treatment Dose Time (Sec)
15 min 30 min 45 min 60 min

I 2% w/v Gum acacia 2ml/kg 2.00 ± 0.33 2.40 ± 0.19 2.54 ± 0.19 2.60 ± 0.19 2.16 ± 0.15

II Pentazocine 10mg/kg 2.43 ± 0.19 4.10 ± 0.41 * 5.60 ± 0.31 ** 7.00 ± 0.23 ** 8.50 ± 0.21 **

III Test Drug - I 200mg/kg 2.18 ± 0.15 3.58 ± 0.19* 4.60 ± 0.21 ** 6.10 ± 0.23** 7.20 ± 0.57 **

IV Test Drug - II 400mg/kg 2.62 ± 0.36 3.84 ± 0.28 * 4.90 ± 0.43** 6.60 ± 0.42** 7.80 ± 0.45 **

Result expressed as mean ± SEM from six observations; ** indicates P < 0.01 & * indicates P < 0.05

Analgesic Activity (Kulkarni et. al., 1993, Ghosh et. al., 2005 &
Geetha et. al., 2004)
Tail flick method
Before the study, Swiss albino mice were screened for
sensitivity test by placing the tip of the tail on the radiant heat
source. Any animals that held to withdraw its tail in 5 second was
rejected from the study. The selected animals were divided into
four groups of six rats each. Each animals of the groups received
one of the following extract (200mg/kg & 400mg/kg), Pentazocine
(30mg/kg) and 2% w/v of Gum acacia (2ml/kg) in normal saline
intraperitoneally. Analgesia was assessed with a tail flick apparatus
(Analgesiometer). The basal reaction time was measured initially
and another set of four measures were taken as 15, 30, 45 and 60
minutes interval and the reaction of the animals considered as the
post – drug reaction time. A cut-off period of 10sec. was observed
to prevents tissue damage of the tail of the animals. The results are
tabulated in table. no.1.
Tail immersion Test
Prior to analgesic experiments, the animals were screened
for the sensitivity test by immersing the tail of the mice gently in
hot water maintained at 550 C – 55.50 C . The animal immersing
the tail from hot water with in 5 second was selected for the study.
The selected rat was then divided in to four groups of six rats each.
Group III & Group IV received the extract in 2 % w/v Gum acacia
in normal saline intraperitoneally at a dose of 200mg/kg &
400mg/kg respectively. Group II received Pentazocine (30mg/kg)
and Group I received 2% w/v of Gum acacia (2ml/kg) in normal
saline manner. After administration of the drugs, the reaction time
was measured at 0, 15, 30, 45 and 60 minutes. The results are
tabulated in table. no. 2.
Statistical Analysis
The mean value ± SEM was calculated for each
parameter. The results were analyzed statistically by ANOVA is
followed by Dunnet‟s test. The minimum level of significant was
fixed at p < 0.01.The results of experiments by proper statistical
analysis are tabulated in table. No.1 and 2 respectively.
3. RESULTS AND DISCUSSION
In analgesic studies, the extract showed significant
analgesic activity at all tested dose levels. In tail flick method, the
methanolic extract of T. bispinosa root at a dose of 200mg/kg
showed significant activity (5.7 ± 0.549**) (P < 0.01) after 45
minutes whereas at a dose of 400mg/kg showed significant
analgesic activity (5.0 ± 0.235**) after 30 minutes (table. no. 1)
but in tail immersion method, the methanolic extract of T.
bispinosa root showed significant activity after 30 minutes interval
of experiment at all tested dose levels (table no. 2). The results
showed significant analgesic activity against thermal stimuli.
The analgesic studies revealed that the methanolic extract
of T. bispinosa roots exhibited potent analgesic (central analgesic
activity) effect against thermal noxious stimuli (Vogel et. al., 2002)
and also revealed that the extract shows dose dependent analgesic
effect.
4. CONCLUSION
From the above investigation, it is quite apparent that
methanolic extract of Trapa natans L.var. bispinosa Roxb. roots
possesses potent analgesic effect against different stimuli. This is
evidenced by significant increase in the reaction time by stimuli in
different experimental models.

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 Journal of Current Pharmaceutical Research 2010; 01: 8-11
ACKNOWLEDGEMENTS
The Authors are grateful to extend special thanks to Mr.
R.L. Hota, Chairman, G.B of The Pharmaceutical College, Barpali
for his constant encouragement & support throughout the work.
The authors are extend sincere thanks to Mr. N.K. Hota, President
& Mr. S.K.Sahu, Secretary of The Pharmaceutical College, Barpali
for providing all kind of facilities for this work.
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