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http://ijps.aizeonpublishers.net/content/2013/6/ijps381-385.pdf
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Vol. 3, No. 6 (2013): 381-385
Research Article
Open Access
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Evaluation of antitussive activity of Lycopus
europaeus on cough reflex induced by different cough
induced models in mice
Abdul Aziz1*, Imran Ahmad Khan2, Aisha Perveen3, Shahnaz Agha4, Shaukat Hussain
Munawar5 and Zahid Manzoor6
1,2 Faculty of Pharmacy, Bahauddin Zakariya University, Multan , Pakistan
3 Department of Pharmacology National Institute of Unani Medicine (NIUM) Kottigepalya, Bangalore, Karnataka, India
4 Department of Chemistry, Govt. Degree College Chungi No.6 Multan.
5,6 Faculty of Veterinary, University of Agriculture, Faisalabad, Pakistan
* Corresponding author: Abdul Aziz; e-mail: a_azizph@yahoo.com
ABSTRACT
The present study was carried out to elucidate the potential of, methanol extract of Lycopus europaeus plant on
antitussive activity in albino mice. The methanol extract (yield 12.5% w/w with respected to dry powdered plant
material) was selected for all experimental procedure. Antitussive activity of the methanolic extract of Lycopus
europaeus (Le.cr) was investigated for its effect on a cough model induced by sulphur dioxide gas and
Ammonium liquor induced cough in mice. Results were revealed that the Le.cr was found to produce significant
antitussive activity (P<0.001) when compared with control, codeine phosphate and Dextramethorphane in a
dose dependent manner. High dose of Le.cr of (500 mg/kg) showed maximum inhibition of cough by 61.21% and
56.63% induced by both inducers. It concludes that Le.cr possessed remarkable antitussive effect, which
provides pharmacological evidence in support of folklore-claim of Lycopus europaeus as an antitussive agent.
Keywords: Lycopus europaeus, methanolic extract; Antitussive activity, codeine phosphate and
Dextramethorphane.
INTRODUCTION
Lycopus europaeus is also known as Bugleweed, bitter
bugle, water horehound. L. virginicus: Paul's betony and
water bugle. Lycopus europaeus is a herbaceous
perennial mint that grows in wet habitats. The leaves
are toothed, and the small white flowers surround the
square stem at the leaf axils in dense clusters. The plant
has little odor; the European species has a bitter taste,
while the American species is not bitter. The whole
herb is used medicinally.
Scientists have played their important for the
evaluationof traditional uses of Lycopus europaeus on
different animals. For example Extracts of L. europaeus
administered to healthy rats reduced the weight of the
thyroid, decreased thyroid hormone activity, and
increased absorption and storage of iodine. The extract
retarded goiter formation in propylthiouracil-treated
rats. All animals treated with the extract demonstrated
reduced metabolism [1]. Other studies in rats have
shown inhibition of serum thyrotropic hormone and
thyroxine after oral administration [2]. Cardiac signs of
hyperthyroidism were reduced in an experiment in rats
treated with L. europaeus extract [3].
Freeze-dried extracts of bugleweed and other related
plants showed a dose-dependent inhibition of bovine
thyroid-stimulating hormone (TSH) binding to human
thyroid membranes, with simultaneous inhibition of
TSH-stimulated adenyl cyclase activity [4;
5]. Formation of covalent adducts with TSH amino acid
residues was postulated; however, the evidence for this
is not conclusive [6]. Traditionally Lycopus europaeus is
being used as astringent, antitussive and sedative
purposes, [7-9]. So the following study is being done to
evaluate the antitussive activities of Lycopus europaeus
in different cough induced models in mice.
MATERIALS AND METHODS
Collection of plant and Preparation of crude extract
Received:
23
October
2013
Accepted:
29
October
201
3
Online:
02
November
201
3
382
http://ijps.aizeonpublishers.net/content/2013/6/ijps381-385.pdf
The plant was collected from the tropical regions of
Pakistan and was identified by a taxonomist in Al-
Manara college of Pharmacy. The plant material was
made free from soil and other adulterants and
vegetative debris. The dried plant material was grinded
to coarse powder with the help of a special herbal
grinder. The powdered plant material (1 kg) was
subjected to maceration in 70% aqueous-methanol in
amber coloured bottle at room temperature for 7 days
with occasional vigorous shaking at room temperature
and keeping the extract in the dark room. The filtrate
was obtained by passing the mixture through a muslin
cloth and then through a Whatman qualitative grade 1
filter paper. The filtrate was evaporated on a rotary
evaporator attached to a vacuum pump at 37ºC under
reduced pressure to thick paste like consistency. And
then the extract obtained was stored at -4°C in air tight
jars.
Figure 1. Aerial parts of Lycopus europaeus
Chemicals
Methanol, ammonium hydroxide, sodium hydrogen
sulfate, sulfuric acid, ammonium chloride, sodium
bicarbonate, Dextramethorphane and codeine
phosphate were used in the antitussive study.
Evaluation of Antitussive Activity
Experimental Animals used
The experiments were carried out in Albino mice of
either sex weighing between 20–30 g obtained from
animal house of Al-Manara college of Pharmacy Multan,
Pakistan were kept in the animal house at 26±2oC in
polyacrylic cages with not more than six animals per
cage and maintained under standard laboratory
conditions with Standard food and water ad libitum.
The animals were used for the experiment after an
acclimatization period of one week before experimental
sessions. Animals were divided into six groups,
containing 6 mice each. The animal experiment was
performed according to the college’s ethical committee
approval.
The control group was treated with saline solution
orally, and the positive control was treated with
codeine phosphate and dextramethorphan. The
remaining groups were treated with the methanolic
extract of Lycopus europeaus (Le.cr) extracts at doses of
250 and 500 mg/Kg body weight.
Sulfur dioxide gas induced cough reflex in mice
The experimental model is shown in Figure 1 where A
is a 500 ml three-necked flask which contains aqueous
saturated sodium hydrogen sulphite solution. By
opening the stop-cock of a burette (B), the concentrated
sulphuric acid was introduced to generate sulphur-
dioxide gas.
Figure 2. Apparatus used in sulfur dioxide gas induced cough
model
A: Saturated NaHSO3 solution in 500ml flask, B: Conc.
H2SO4 in burette, C: Gas Cylinder, D: Water manometer,
E: Dessicator and a, b, c, d, e, f are stop cocks.
The chemical reaction which occurred in flask A is as
follows:
2 NaHSO3 + H2SO4 →2 SO2 + Na2SO4+ 2 H2O
Flask A and gas cylinder C were filled with sulphur
dioxide (SO2) gas. Cocks c and b were opened to elevate
pressure in gas cylinder C, which was recorded by
water manometer D. Stop-cock b was then closed and
stop-cock d was opened slightly until pressure in D (11
mm, i.d.) reached 75 mm H2O, when stop-cock d was
closed. The procedures were conducted in a draught.
Cough response of all the groups are observed (0
minute) by placing the animals in desiccators E. The
cocks c, f and e are opened in order and when the
pressure in D became 0 mm of H2O, all the cocks are
closed immediately.
A certain amount, 5ml sulfur dioxide gas is induced into
the desiccator and this way. After a minute of
introducing the gas, the animal is taken out of the
dedicator and frequency of cough is observed for five
minutes in an un-ended filter funnel with a stethoscope
at the tip in which mice is confined. In the same fashion
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the frequency of cough are observed for all the animal
groups after every 30 minutes. [10].
Ammonium liquor induced cough
Healthy mice were divided into six groups: Control,
Le.Cr (250 and 500 mg/kg) and standard. Briefly, 1 h
after oral administration of the test drug, each mice was
placed in a glass chamber and exposed to 0.3 ml 25%
NH4OH produced by a nebulizer for 45 s. Animal was
monitored during ammonia exposure and cough
frequency was recorded.
Statistical analysis
The results of pharmacological studies were reported
as Mean ± S.E.M. The total variations present in data
were analyzed by using one way analysis of variance
(ANOVA) followed by Dunnett’s Test.
RESULTS AND DISCUSSION
Lycopus europaeus extract in multiple doses showed
good dose‑dependent antitussive activity (Table 1 and
Table 2 ). Methanolic extract (500 mg/kg) exhibited
significant activity i.e 56.63%, but lower dose of it
showed less activity i.e 23.14% inhibition in
Ammonium liquor induced cough. While on the other
hand, Lycopus europaeus Methanolic extract (500
mg/kg) exhibited significant activity i.e 61.21%, but
lower dose of it showed less activity i.e 32.73%
inhibition in Sulfur dioxide gas induced cough.
Antitussive activity of formulations was evaluated by
using method of [11; 12]. A vial containing 2 ml of
500mg/ml solution of sodium hydrogen sulfite in
double distilled water was placed at the base of a
dessicator and covered with wire gauze to serve as a
platform for placement of mice. To the NaHSO3
solution, 0.2 ml of sulphuric acid was added using a
pipette. After 15 seconds, the mice were placed on the
platform in the dessicator and exposed to SO2 for 45sec.
The mice were then removed from the dessicator and
placed in an observation chamber for counting of bouts
of cough for five minutes thereafter. But in laboratory
condition, when the mice were placed on the platform,
in the dessicator and exposed to SO2 for 45 s and then
removed from the dessicator and placed in an
observation chamber for counting of bouts of cough for
five minutes thereafter, it produced too much cough,
even on exposing for 50 sec to SO2 gas caused death.
So, there was a need to standardize the method
according to the laboratory condition. Concentration of
H2SO4 and NaHSO3 was 0.2 ml and 2 ml respectively
used through all the experiment. For the
standardization of cough induction model according to
the laboratory condition, the mice were exposed to SO2
in different time durations like 5 second to 50 second
and cough was counted respectively. The effect
exhibited by the entire treated group on sulphur-
dioxide induced cough in experimental animals has
been presented in Tables 1 and 2.
Table 1. Effect of methanolic extract of Le.cr on cough
frequency in sulfur dioxide gas induced cough mice
Treatment Dose
(mg/kg)
Number of
cough
Percentage
of
inhibition
Control 10 58.54± --
Le.Cr 250 39.38±1.90* 32.73
Le.Cr 500 22.71±4.12*** 61.21
Codeine phosphate 10 26.30±1.62** 55.18
Codeine phosphate 20 13.80±4.10*** 76.43
Values expressed as mean±SEM (n=6). *P<0.05, **P<0.01, and
***P<0.001 for comparison of treated groups with control
Table 2. Effect of methanolic extract of Le.cr on cough
frequency in Ammonium liquor induced cough mice
Treatment Dose
(mg/kg)
Number of
cough
Percentage
of
inhibition
Control 10 48.54±3.40 --
Le.Cr 250 37.31±2.30* 23.14
Le.Cr 500 21.04±4.10*** 56.63
Dextramethorphane 10 26.52±2.04** 45.34
Dextramethorphane 20 14.31±3.61*** 71.52
Values expressed as mean±SEM (n=6). *P<0.05, **P<0.01, and
***P<0.001 for comparison of treated groups with control
Figure 3. Comparative study of percent inhibition on cough
on treatment with both standards
Antitussive animal models could be designed by
mechanical stimulus, electrical stimulus, and chemical
stimulus. In this experiment, chemicals like ammonium
liquor and sulfur dioxide were used to induce cough.
These models are widely used animal models for
evaluating antitussive activity of a traditionally used
drug. Cough is a normal physiological response to an
irritation of the laryngo-tracheo-bronchial system
caused by mechanical or chemical stimulation. It may
be painful and require suppression by antitussive
drugs.
In animals, coughing has been elicited by mechanical
[13] or chemical irritation [14] and by electrical
stimulation [15] of tracheal mucosa or by nerve
stimulation [16]. Of all these methods, chemical or
mechanical stimulation are more similar to the
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physiological event and also the experimental models
generally used in man.
Figure 4. Percentage of reduction in cough count by
methanolic extract of Le.cr on cough frequency in both cough-
induced models in mice
Figure 5. Comparison of the standard drugs for inhibition of
induced cough.
0
5
10
15
20
25
30
35
40
Le.cr 250mg/kg Le.cr 500mg/kg
SO2 gas iduced cough
Ammonium liquor induced cough
Figure 6. Reduction in cough by Le.cr plant extracts on the
cough induced by both cough inducers in mice. On Y-axis No.
of cough is mentioned while on X-axis, doses of Le.cr is
mentioned
Anti-tussive agents are used mainly to suppress dry
and painful cough. Cough suppressants act to reduce
the urge to cough. Nonmyelinated C-fibres and rapidly
adapting receptors, which have myelinated Aδ-fibres,
appear to be involved in cough. These putative cough
receptors have myelinated afferents and are found
mainly in the larynx and the extrapulmonary airways
[17]. Vagal afferent nerve provide inputs to brainstem
nuclei, primarily the nucleus of the solitary tract (nTS)
that receive inputs from airway cough evoking
afferents and generate cough reflex in body. Centrally
acting antitussives such as codeine phsophate and
dextromethorphane act within the central nervous
system (CNS) at the level of the brain stem by
depolarization or a dulling of the vagus nerve, the
nerves leading from the brain stem and serving the
chest area. Peripheral antitussive drugs act outside the
CNS to inhibit cough by suppressing the responsiveness
of one or more vagal sensory receptors that produce
cough [18].
CONCLUSION
To conclude, our study indicated that the methanol
extract of Le.cr demonstrated significant antitussive
activity and obtained percentage inhibition of cough
reflex is approximately comparable as standard drug.
These effects are the important evidence for the
traditional use of Le.cr in the treatment of cough and
respiratory disorders. While other we are further
evaluating other traditionally used activities of Le.cr
Acknowledgements
I would like to express my sincere thanks to Mr. Abdul
Shakoor who had always been there for help with
technical advice whenever I encountered problems in
the course of my work and guided me all throughout
the term and I would like to thank all those who have
helped me directly or indirectly for my work and whose
names I have unknowingly missed out, in making this
project a worthy endeavour.
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