Jordan Journal of Pharmaceutical Sciences, Volume 7, No.

1, 2014

Peppermint Aqueous Extract Counteracts Smooth Muscle Contraction in Rat Ileum

Amjad Abuirmeileh1 *, Abdulrahman Alkhodari1, Amal Alnnjeeli1, Ahmad Talhouni1, Izzeddin Alsalahat1,
and Ahmad Naddaf 1

Faculty of Pharmacy, Al Isra University, Amman, Jordan.

ABSTRACT
Peppermint is widely used for gastrointestinal disorders; however, its pharmacological action on isolated rat
ileum has not yet been evaluated. In this study, the effect of peppermint aqueous extract was investigated for its
ability to reduce rat illial smooth muscle contractility induced by acetylcholine (Ach) and potassium chloride
(KCl). The experiments were conducted using an organ bath apparatus. A section of rat ileum was suspended in
an organ bath containing Tyrodes solution. The tissue was stimulated with either Ach or KCl. The tissue was
kept under 1 g tension at 37C and continuously gassed with O2. Illial smooth muscle contractility was studied in
the absence and presence of peppermint. Peppermint aqueous extract prevented illial smooth muscle contractility
induced by both Ach and KCl. The aqueous extract of peppermint (1g/ml) caused a rightward shift of the Ach
dose response curve and brought about a decrease in the maximum response. This extract also produced a
rightward shift in the KCl dose response curve but without affecting the maximum response. These results
indicate that peppermint aqueous extract counteracts Ach and KCl induced smooth muscle contractility in rat
ileum. The results also suggest that the peppermint effect on Ach induced contractility was non competitive in
nature. The reduction in KCl induced contractility also indicates that peppermint aqueous extract was at least
partially mediated by its effect on illial smooth muscle calcium channels.
Keywords: Peppermint, Rat, Illial Smooth Muscle, Acetylcholine, KCl, Dose Response.

and mechanisms of treatment involve smooth muscles 1, 2.

INTRODUCTION

Treating such disorders will involve medications

As the smooth muscle contractile activity is a major

targeting smooth muscle contractility. There are a good

regulator of functions of the gastrointestinal (GIT)

number of pharmaceutical agents manipulating muscle

system, malfunction of contractility in this system leads

activity that have been derived from plant sources such as

to a host of clinical disorders. Because of this there is a

Papavarine, Anabesine, Cissampeline and Tubocurarine 3.

great deal of interest in smooth muscle physiology and

Several metabolic and physical disorders related to

the mechanisms that manipulate it. Specific disorders of

gastric motility disturbances have been treated by using

GIT smooth muscles include spasm, constipation,

synthetic and herbal medicines. Medicinal plants have

diarrhea and intestinal cramps. Their underlying causes

been used for thousands of years, and more recently there

has been increased interest towards herbal remedies

[email protected], [email protected]
Received on 28/7/2013 and Accepted for Publication on
31/12/2013.

because of their effectiveness and lower cost.

In Jordan, the use of herbal medicine is very

- 88 -

2014 DAR Publishers/The University of Jordan. All Rights Reserved.

Jordan Journal of Pharmaceutical Sciences, Volume 7, No. 1, 2014

common4. 60% of the Jordanian population utilizes

water, the mixture was then filtered and the filtrate was

traditional medicines with around 485 plant species used

used in the conducted experiments. The two different

therapeutically in traditional medicine .

concentrations used (2.5% v/v and 3.75% v/v) were

Peppermint is one of the most common plants used in

representative of the amount in volume of peppermint

Jordan, obtained from the over-ground parts of the

filtrate divided by the final volume of Tyrode's solution

flowering plant Mentba x Piperita L, it contains a large

in the organ bath. The extraction method used here was

amount of essential oil. This oil is characterized by the

based on the common method reported by Jordanian

presence of menthol and monoterpenes which are

traditional medicine providers where they either prepared

mint in this manner, or suggested doing so to patients.

responsible for many of the activities of this herb . It also

7

contains flavonoid compounds and tannins . Peppermint

Although this may differ from common phytochemical

oil has been used traditionally as a carminative agent, in

extraction methods, the goal here was to mimic common

the relief of flatulence and colic pain, and more recently

local practices and investigate their pharmacological

in therapy of the irritable bowel syndrome

8, 9

effectiveness and thus therapeutic value.

Peppermint oil has been shown to relax isolated

gastrointestinal smooth muscle and its mechanism of action

Animals

has been studied in several pharmacological preparations. In

Sprague Dawley rats (190 10 g) were purchased

the latter, it has been suggested that peppermint oil

from the animal facility from Jordan Applied University

influenced a reduction in the availability of calcium in

and housed at 20- 24C with access to food and water.

10, 11

. Evidence for the

The rats were deprived of food (not water) for 24 h before

calcium antagonistic properties of peppermint oil were

the experiment. All procedures concerning animals were

demonstrated when menthol and peppermint oil reduced the

carried out according to Jordanian regulations for animal

binding of specific calcium channel ligands in smooth

experimentation and care, and in compliance with the

gastrointestinal smooth muscles

United Kingdom Home Office animal care guidelines.

muscle and also inhibited depolarization-induced Ca uptake

12

into neuronal preparations .

Ileum Preparation

In this study, we have investigated the effect of

On the day of experiment, rats were sacrificed by

peppermint aqueous extract rather than oil on isolated rat

ileum,

where

the

majority

of

Jordanians

cervical displacement and one or two pieces (1.5-2 cm) of

utilize

ileum were dissected out. The piece of ileum was cleaned

peppermint as an aqueous extract or tea.

from its luminal contents by flushing it's lumen gently

MATERIALS AND METHOD

with a stream of Tyrode's solution by using a pipette then

Materials

mounted between two stainless steel hooks in tissue bath

Ach, KCl and all tyrode solution constituents; NaCl,

(40mL) containing Tyrode's solution (37C, bubble of

KCl, CaCl2, NaHCO3, NaH2PO4, MgCl2 and glucose

oxygen and pH 7.4). The lower hook was fixed at the

were obtained from Sigma-Aldrich. Peppermint aqueous

bottom of the tissue bath and the upper one was

extract was prepared by collecting leaves of fresh whole

connected to an isotonic transducer (Harvard Transducer,

peppermint plant (Mentha x Piperita L, 100g) and then

UK). Each tissue was placed under 1 g resting tension

chopping and soaking them in 100 ml boiling distilled

and equilibrated for 60 min prior to the execution of

- 89 -

Peppermint Aqueous

Amjad Abuirmeileh et al

experimental protocols. During this period, the tissue was

using GraphPad Prism software.

washed with Tyrode's solution every 15 min and the

RESULTS

tension was readjusted to 1 g. Ileum contractions were

Ach Dose Response Curve

displayed

Harvard

Figure (1) indicates the contractile effect of different

Oscillograph, (UK). The ileum contractions were induced

concentrations of Ach (5 x 10-10 to 5 x 10-9 M) on isolated

initially by Ach (15mM) or KCl (10 mM) and then dose

rat ileum. As shown, an increase in Ach concentration

response curves were established. Further dose response

caused increasing illial smooth muscle contraction and a

curves were obtained after the crude peppermint aqueous

maximum response was observed at a concentration of 5

extract was added to the tissue bath.

x 10-9 M. Ileum contraction to Ach was computed based

and

recorded

on

Universal

on the percentage and maximal effect induced by

Measurements and Statistical Analysis:

concentration of 5 x 10-9 M which was considered to be

All data are expressed as mean standard error of the

100% response and other responses were observed as

mean (SEM). Results were analyzed using non linear

percentages of this maximal response. Log EC50 value for

regression (curve fit) with an extra sum-of squares F test

Ach was -8.818.

comparison method for logEC50 and hillslope values

Figure 1. Dose response curve achieved by Ach on contraction of isolated rat ileum.
Each point represents mean SEM of 3 observations. LogEC50 = -8.818.

Effect of Atropine on Ach Induced Contraction on

Rat Ileum

a parallel rightward shift of the Ach dose response curves

without affecting the maximum response (fig.2).

The use of atropine in conjunction with Ach produced

- 90 -

Jordan Journal of Pharmaceutical Sciences, Volume 7, No. 1, 2014

Figure 2. Figure illustrate the inhibitory effect of atropine on Ach dose response curve.
Each point represents mean SEM of 3 observations. Atropine caused a concentration-dependent rightward shift of the Ach dose response curve where
LogEC50 for Ach alone was -7.555 and in the presence of 1.3 nM atropine was -6.619, and with 2.6 nM of atropine was -5.686. LogEC50 was significantly
different for each data set (P < 0.0001). HillSlope was not significantly different for any of the data sets (P < 0.0001).

Analysis of this data utilizing a Schild plot showed a

different from unity, and a pA2 (pKB) value of - 9.612

slope of -0.9824 0.02419, which was not significantly

(fig.3).

Figure 3. Figure illustrates the schild plot for atropine.

Where dr = EC50 for Ach in the presence of atropine divided by EC50 for Ach. Slope = -0.9824 0.02419 and pA2 value = 9.612.

- 91 -

Peppermint Aqueous

Amjad Abuirmeileh et al

Effect of Peppermint Aqueous Extract on Illial

Contraction Induced by Ach

used, 2.5 % and 3.75 %, where there was a concentrationdependent rightward shift of the Ach dose response

The dose response curve for Ach was evaluated in the

curve. Also peppermint aqueous extract brought about a

presence and absence of peppermint aqueous extract.

significant concentration-dependent reduction in the

Here, two different concentrations of the extract were

maximal response (fig. 4).

Figure 4. Effect of peppermint extract on rat ileum contraction induced by Ach.

Each point represents mean SEM of 3 observations. Peppermint aqueous extract caused a concentration dependent rightward shift in the Ach dose
response curve where LogEC50 for Ach was -7.734 for Ach alone, -7.130 in the presence of 2.5% peppermint and -6.820 in the presence of 3.75%
peppermint. This extract also brought about a concentration dependant reduction in the maximal response where Hill Slope for Ach was 7.593 for Ach
alone, 2.571 in the presence of 2.5% peppermint and 2.321 in the presence of 3.75% peppermint. LogEC50 was significantly different for each data set (P
< 0.0001). HillSlope was also significantly different for each data set (P < 0.0001).

Effect of Peppermint Aqueous Extract on Illial

presence of two different concentrations of mint aqueous

extract. Here, a concentration-dependant inhibition of

Contraction Induced by KCl:

Peppermint extract caused a dose-dependent relaxant

illial muscle contractility was observed and the dose

effect on the contraction of rats ileum. Figure 5

response curve of KCl suffered a rightward shift without

illustrates the dose response curve obtained from using

affecting the maximal response.

KCl as an inducer of illial muscle contractility in the

- 92 -

Jordan Journal of Pharmaceutical Sciences, Volume 7, No. 1, 2014

Figure 5. Effect of peppermint extract on rat ileum contraction induced by KCl.

The dose response curve of KCl suffered a rightward shift without affecting the maximal response. Here, LogEC50 for KCl alone was -5.253 and in the presence of
2.5% of peppermint aqueous extract it was -4.938, and in the presence of 3.75% of the extract it was -4.568. Each point represents mean SEM of 3 observations.
LogEC50 was significantly different for each data set (P < 0.0001). HillSlope was not significantly different for any of the data sets (P < 0.0001).

DISCUSSION

right. And since the scenario did not follow the

The results of this study confirm the ability of

competitive muscarinic receptor behavior as seen with

peppermint extract to directly relax gastrointestinal

atropine (fig.2 and fig.3) it is safe to assume that ability

smooth muscles. These results are in accordance with

of peppermint to antagonize Ach induced contraction was

relevant findings on the effect of peppermint oil on

due to its interference with intracellular calcium

smooth muscle contractility in vitro and in vivo. In the

availability in illial smooth muscle cells.

latter, peppermint oil was examined for its clinical

Rat illial smooth muscle contractions induced by KCl

viability in patients suffering GIT disorders and proved

were antagonized by the peppermint aqueous extract, and

clinically effective in managing irritable bowel syndrome

the dose response curve suffered rightward without

and colonic spasms during endoscopy

8,9

affecting the maximum response. Agents which inhibit

Using Ach in vitro on illial tissue to induce

contractions induced by KCl are considered calcium

contraction serves as a model where excessive enteric

channel blockers, where exposure of smooth muscle cells

nervous system activation would be responsible for GIT

to K+ stimulates contractions through the opening of

spasms. Agents which counteract these spasms are

voltage-dependent L-type Ca+2 channels and consequent

considered antispasmodic drugs which exert their effects

influx of extra cellular Ca+2 16. The previous suggests that

by either blocking muscarinic receptors, relaxing smooth

the peppermint extract used in this study may have at

muscles, or blocking calcium-channels

13,14,15

. The results

least partially conveyed its effects in this manner.

obtained in this study indicate that peppermint aqueous

The obtained data also illustrate that peppermint aqueous

extract reduced the Ach induced contractions in rat ileum

extract antagonized KCl induced contractions without

and shifted the dose response curves downward and to the

affecting maximum response. This is supported by Taylor et

- 93 -

Peppermint Aqueous

Amjad Abuirmeileh et al

al. where similar antagonism was reported after using

17

psychologically induced relaxing effect. In this study it

. The

was clearly established that peppermint aqueous extract

authors of the previous study suggested that the produced

and hence peppermint tea is of value in treating GIT

+2

antagonism was a result of interference with cellular Ca

symptoms, such as spasms and cramps, secondary to

influx. The alterations in the dose response curve produced

excessive illial smooth muscle stimulation.

menthol, a major constituent of peppermint oil

by KCl in this study suggest a similar mode of action. The

+2

CONCLUSION

interference of peppermint aqueous extract with Ca influx

From the results obtained in this study we conclude that

could be attributed to direct action on voltage gated calcium

peppermint aqueous extract is a useful herbal remedy for

channels preventing their natural response to alterations in

treating intestinal spasms. While many herbal remedies used

18

potential . On the other hand, peppermint extract could be

in Jordan have questionable benefits 4, mint aqueous extract

indirectly hyperpolarizing smooth muscle cells through

was actually one that has proven to have significant effects

interfering with either cyclic adenosine monophosphate

on GIT motility and thus, valuable in treating intestinal

levels or the intracellular calcium pool. This would then

spasms and related GIT symptoms.

+2

hinder Ca

influx through voltage gated channels which

18,19

The mechanism of action of the peppermint aqueous

. In any

extract is presumed to be achieved through interfering

case, the consequent outcome would be reduced smooth

with calcium channel natural responses and not via

muscle contractility.

directly blocking muscarinic receptors. The effects of

normally respond to muscle cell depolarizing

When other research had proven the usefulness of

peppermint aqueous extract on the calcium current

peppermint oil in counteracting intestinal spasms and

resembles those caused by menthol, suggesting that

hyper motility, it remained not completely clear if

menthol, which is the major constituent of peppermint,

peppermint aqueous extract would be of value in such

maybe implicated in mediating these actions.

situations. In particular, plant oil extracts usually do not

harbor similar compounds as water extracts would. This
would

have

previously

questioned

the

value

of

peppermint tea in such situations and that it merely had a

REFERENCES
(1) Kim H.R., Appel S., Vetterkind S., Gangopadhyay S.
S., Morgan K. G. Smooth muscle signalling pathways
in health and disease, J. Cell. Mol. Med. (2008).,12(6A)
pp. 2165-2180
(2) Samuels L. A. Review: Pharmacotherapy Update:
Hyoscine Butylbromide in the treatment of abdominal
spasms. Clinical Medicine: Therapeutics (2009)., 1:
647655
(3) Fabricant D. S. and Farnsworth N. R. The value of
plants used in traditional medicine for drug discovery.

- 94 -

ACKNOWLEDGMENT
Many thanks to Mr.mohammad Abu Shkhedem for all
of his help with the animals used in this study.

Environ Health Perspect. (2001)., 109(1): 6975.

(4) Afifi F.U. and Abu-Irmaileh B. Herbal medicine in
Jordan with special emphasis on less commonly used
medicinal herbs. Journal of Ethnopharmacol. (2000).,
72(1-2):101-10.
(5) Aburjai T., Hudaib M, Tayyem R, Yousef M. and
Qishawi M.
Ethnopharmacological survey of
medicinal herbs in Jordan, the Ajloun Heights region.
(2007)., 110: 2, 21,: 294304.
(6) Keifer D., Ulbricht C., Abrams T., Basch E., Giese N.,
Giles M., DeFranco K.C., Miranda M. and Woods J.

Jordan Journal of Pharmaceutical Sciences, Volume 7, No. 1, 2014

Peppermint (Mentha xpiperita): An evidence-based

systematic review by the Natural Standard Research
Collaboration. Journal of Herbal Pharmacotherapy.
(2007)., 7 (2) (pp 91-143).
(7) Yuliya D., Cinzia M.B., Andrea O., Simone B., and
Massimo E.M. UV-B modulates the interplay between
terpenoids and flavonoids in peppermint (Mentha
piperita L.). Journal of Photochemistry and
Photobiology. (2010)., 100: 2, 2, pp 6775.
(8) Kingham, J. G. C. Peppermint oil and colon
spasm. Lancet. (1995)., 346(8981): 986.
(9) Merat, S, Khalili S, Mostajabi P, Ghorbani A, Ansari R.
and Malekzadeh R. The effect of enteric-coated,
delayed-release peppermint oil on irritable bowel
syndrome. Digestive
diseases
and
sciences(2010)., 55 (5): 138590.
(10) Taylor BA., Luscombe DK., Duthie HL., Inhibitory
effect of peppermint oil on gastrointestinal smooth
muscle. Gut. (1983) 24:A992.
(11) Hills J.M. and Aaronson P.I. The mechanism of action
of peppermint oil on gastrointestinal smooth muscle. An
analysis using patch clamp electrophysiology and
isolated tissue pharmacology in rabbit and guinea pig.
Gastroenterology. (1991)., 101:55-65.
(12) Hawthorne M., Ferrante J., Luchowski E., Rutledge A.,
Wei X., Triggle DJ. Menthol and peppermint oil actions

on calcium channel dependent rocesses in cardiac,

intestinal and neuronal preparations. Aliment
Pharmacol Therap. (1988)., 2:101- 118.
(13) Hosey, M. M. Diversity of structure, signaling and
regulation within the family of muscarinic cholinergic
receptors. FASEB J. (1992)., 6(3): 845-852.
(14) Singh, R.K., Pandey, H.P., and Singh, R.H. Irritable
Bowel Syndrome: Challenge ahead. Current Science.
(2003)., 84(2): 1525-1533.
(15) Christen M. O. Action of pinaverium bromide, a
calcium-antagonist, on gastrointestinal motility
disorders. Gen. Pharmacol. (1990)., 21: 821825.
(16) Godfraind, T., Miller, R., Wibo, M. Calcium
antagonism
and
calcium
entry
blockade.
Pharmacological Reviews (1986)., 38: 321416.
(17) Taylor BA., Luscombe DK., Duthie HL. Inhibitory
effect of peppermint oil and menthol on human isolated
coll. Gut. ( 1984)., 25:A1168-A1169.
(18) Bean BP., Sturek M., Puga A., Hermsmeyer K. Calcium
channels in muscle cells isolated from rat mesenteric
arteries: modulation by dihydropyridine drugs. Circ
Res. (1986)., 59:220-235.
(19) Bean BP. Nitrendipine block of cardiac calcium
channels: high affinity binding to the inactivated state.
Proc Natl Acad Sci USA. (1984)., 81:6388-6392.

- 95 -

Peppermint Aqueous

Amjad Abuirmeileh et al

1 1 1 1 1
1

.
.
.
.
. 37
.
.
.
.
.
.
.
.
:
.

____________________________________________

2013/7/28 .2013/12/31
- 96 -