Pharmaceutical Biology, 2009; 47(8): 660–664

RESEARCH ARTICLE

Insight into the possible mechanism of antidiarrheal

and antispasmodic activities of piperine
Syed Intasar Husain Taqvi2, Abdul Jabbar Shah1,3, and Anwarul Hassan Gilani1
1
Drug Discovery and Natural Products Research Division; Department of Biological and Biomedical Sciences,
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Aga Khan University Medical College, Karachi, Pakistan, 2Department of Pharmaceutical Sciences, Federal Urdu
University of Arts, Science And Technology, Gulshan Campus, Karachi, Pakistan, and 3Department of Pharmaceutical
Sciences. COMSATS, Institute if Information Technology, University Road, Abbottabad, Pakistan

Abstract
Piperine is a piperidine-ring containing alkaloid and a major constituent of Piper nigrum Linn. and Piper
longum Linn. species, belonging to the Piperaceae family. The present study explored their mode of action
in gastrointestinal disorders, such as diarrhea and colic. Piperine at the dose of  10 mg/kg provided complete
protection from castor oil-induced diarrhea in mice, similar to that of loperamide. In isolated rabbit jejunum
preparations, piperine exhibited concentration-dependent inhibition of spontaneous contractions with an
EC50 value of 149.1 µM (89.26–249.20, 95% CI). When used to treat high K+ (80 mM)-induced sustained con-
For personal use only.

tractions, piperine inhibited such contractions with an EC50 value of 80.86 µM (56.10–116.50, 95% CI), which
suggested a calcium channel blocking (CCB) effect. The CCB effect was further confirmed when pretreat-
ment of the tissues with piperine (10–100 µM) caused a rightward shift in the Ca++ ­concentration–response
curves (CRCs) in Ca++-free medium, similar to that caused by verapamil. Loperamide also caused the inhibi-
tion of spontaneous and high K+-induced contractions as well as shifted the Ca++ CRCs to the right at con-
centrations of 1–10 μM. These data indicate that piperine exhibits antidiarrheal and antispasmodic activi-
ties, mediated possibly through calcium channel blockade.
Keywords:  Piperine; rabbit jejunum; antidiarrheal; antispasmodic; calcium antagonist

Introduction provided protection against castor oil-, MgSO4-, and ara-

Piperine is an alkaloid containing the piperidine ring
found in Piper nigrum Linn. and Piper longum Linn.
(Johri & Zutshi, 1992), belonging to the Piperaceae fam-
ily (Nadkarni, 1976). Peppers are common spices used
worldwide, in addition to their medicinal use in anti-
diarrheal formulations (Bajad et al., 2001). Piperine was
found to alter the intestinal motility of rabbit and guinea-
pig ileum (Neogi et al., 1971; Annamalai & Manavalan,
1990; Takaki et al., 1990). It was also reported that pip-
erine exhibited an inhibitory effect on gastrointestinal
motility both in vivo (Takaki et  al., 1990) and in vitro
(Izzo et al., 2001). Efforts have been made to explore the
possible mode of action in diarrhea and to rationalize the
use of peppers in antidiarrheal formulations, whereas
piperine was found to be a major component which
chidonic acid-induced diarrhea (Bajad et al., 2001; Stohr
et al., 2001). Th ere are some studies which suggest that
piperine reduces castor oil-induced diarrhea possibly
through capsaicin-sensitive neurons (Izzo et  al., 2001;
Capasso et al., 2002). McNamara et al. (2005) suggested
the involvement of the vanilloid receptor (TRPV1) in
mediating the effects of piperine on gastrointestinal
function. All these studies reflect that ambiguity prevails
over the mode of action in inducing the antidiarrheal
effect of piperine.
We have previously reported that piperidine-
containing compounds suppress the spontaneous con-
traction of smooth muscle through calcium channel
blockade (Taqvi et al., 2006a). The present study reports
the calcium channel blocking effect of piperine, being
another possible mode of action for its antidiarrheal

Address for Correspondence:  Anwarul Hassan Gilani, PhD, Professor of Pharmacology, Department of Biological and Biomedical Sciences, Aga Khan
University Medical College, Karachi-74800, Pakistan. Tel: (+92) 21-486 4571. Fax: (+92) 21-493 4294, 493 2095. E-mail: anwar.gilani@aku.edu
(Received 1 March 2008; revised 30 April 2008; accepted 30 April 2008)

ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK Ltd

DOI: 10.1080/13880200902918352 http://www.informapharmascience.com/phb

and antispasmodic activities. The antispasmodic and
calcium channel blocking effects of piperine were found
to be comparable to those of verapamil, a standard
calcium antagonist (Triggle, 1992) and loperamide, a
known antidiarrheal agent, which also contains the pip-
eridine ring (Reynolds et al., 1984).
Materials and methods
Drugs and chemicals
The following reference chemicals were obtained from
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the sources specified: verapamil hydrochloride, lop-
eramide hydrochloride, potassium chloride, piperine
(Sigma-Aldrich Chemie GmbH, Steinheim, Germany),
dimethylsulfoxide (Sigma Chemical Company, St. Louis,
MO, USA), and Tween 80 (Chemie S.S., Barcelona, Spain).
Castor oil was purchased from Karachi Chemicals Labs,
Karachi, Pakistan. All chemicals used were of the high-
est purity grade available. Stock solutions of physiologi-
cal salt solution and verapamil were prepared in dis-
tilled water, whereas loperamide was dissolved in 10%
dimethylsulfoxide (DMSO) and piperine was dissolved
For personal use only.
in Tween 80. Dilutions were made fresh on the day of
the experiment.
Animals
Experiments complied with the rules of the Institute
of Laboratory Animal Resources, Commission on Life
Sciences, National Research Council (1996), and were
approved by the Ethics Committee of the Aga Khan
University, Karachi. Rabbits of local breed (Oryctolagus
cuniculus) weighing 1.5–2.0 kg, belonging to the
Leporidae family (Wilson & Reeder, 1993), and Balbc mice
(20–25 g) of either sex used in this study were housed at
the animal house of Aga Khan University under control-
led conditions (23–25°C). Animals were fed a standard
diet consisting of (g/kg): flour 380, choker 380, molasses
12, salt 5.8, nutrivetL 2.5, potassium metabisulfate 1.2,
vegetable oil 38, fish meal 170, and powdered milk 150.
Castor oil-induced diarrhea
Mice (20–25g) of either sex were used as described ear-
lier (Izzo et  al., 1994; Gilani et  al., 2005). The animals
were housed in individual cages and divided into five
groups, five animals in each. Animals were fasted for
24 h prior to experiments. The first group received saline
(10 mL/kg; p.o.) as vehicle and so acted as the negative
control. The dose of test compound was selected on a
trial basis, and then three increasing doses were given
orally to the animals. A group of mice were treated with
loperamide ( 10 mg/kg) as the positive control. One hour
Piperine and calcium channel blockade   661
after treatment, each animal received 10 mL/kg of castor
oil orally through a feeding needle. After 4 h cages were
inspected for the presence of typical diarrhea droppings;
their absence was noted as a positive result, indicating
protection from diarrhea at that time.
Isolated tissue preparations
Isolated tissue experiments were carried out as described
previously (Gilani et al., 2005). The animals were starved
for 24 h prior to the experiment but had free access to
water. The animals were sacrificed by cervical disloca-
tion; the abdomen was cut open and the jejunum was
isolated. Segments of  2 cm each were cut and mounted
in a 10 mL tissue bath containing normal Tyrode’s solu-
tion, aerated with carbogen (95% O2 and 5% CO2). The
composition of the Tyrode’s solution in mM was: KCl
2.7, NaCl 136.9, MgCl2 1.1, NaHCO3 11.9, NaH2PO4 0.4,
glucose 5.6, and CaCl2 1.8 (pH 7.4). A preload of 1 g was
applied and the tissues were allowed to equilibrate for
30 min. Then, control responses to a submaximal con-
centration of acetylcholine (0.3 µM) were recorded.
Reproducible responses determined the stability of the
tissues.
Under these experimental conditions, rabbit jejunum
exhibits spontaneous rhythmic contractions, allowing
testing of the relaxant (spasmolytic) activity directly
without the use of an agonist (Gilani et al., 1994).
Determination of spasmolytic and calcium antagonist
activity
To assess the spasmolytic activity of piperine, the tis-
sues were treated with different concentrations of pip-
erine in ascending order and in a cumulative fashion.
To explore whether the spasmolytic effect was due to
blockade of the calcium channel, high K+ (80 mM) was
added to depolarize the rabbit jejunum (Farre et  al.,
1991). K+ (80 mM) was added to the tissue bath, which
produced sustained contraction. The test compound
was then added in a cumulative fashion to obtain
concentration-dependent inhibitory responses (van
Rossum, 1963). Relaxation was expressed as a percent
of the control response mediated by high K+.
To confirm the calcium antagonist activity of piper-
ine, first the tissues were allowed to stabilize in normal
Tyrode’s solution, and then the solution was replaced
with Ca++-free Tyrode’s solution containing ethylen-
ediaminetetraacetic acid (EDTA; 0.1 mM) for 30 min in
order to remove calcium from the tissues. This solution
was further replaced with K+-rich and Ca++-free Tyrode’s
solution having the following composition in mM: KCl
50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90, NaH2PO4
0.42, glucose 5.55, and EDTA 0.1. Following an incuba-
tion period of 30 min, control concentration–response
 662   Syed Intasar Husain Taqvi et al.

curves (CRCs) of Ca++ were constructed. When the
control CRCs of Ca++ were found to be superimposable
(usually after 2–3 cycles), the tissues were pretreated
with test compound for 1 h to test the possible calcium
channel blockade. The CRCs of Ca++ were reconstructed
in the presence of an ascending concentration of test
compound.
Statistical analysis
All data are expressed as mean ± standard error of the
mean (SEM), and median effective concentrations
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The effect of piperine on gut motility was evaluated
in in vitro studies. For this purpose, isolated spontane-
ously contracting rabbit jejunum preparations were
exposed to cumulative additions of piperine, which
caused concentration-dependent inhibition (Figure
1), with an EC50 value of 149.1 µM (89.26–249.20,
95% CI) (Figure 2A). Verapamil and loperamide also
inhibited spontaneous contractions (Figure 1). The
contraction of smooth muscle is dependent on an
increase in the cytoplasmic free Ca++, which activates
the contractile elements (Karaki & Wiess, 1983). The
increase in intracellular Ca++ occurs via either influx

(EC50 values) are given with 95% confidence intervals
(CIs). The statistical parameter applied was Student’s
t-test, with p < 0.05 noted as significantly different.
Concentration–response curves were analyzed by non-
linear regression. Results for antidiarrheal activity were
analyzed by 2 test, where p < 0.05 was noted as sig-
nificantly different. GraphPad program version 4 was
used for all statistical analysis (GraphPad, San Diego,
CA, USA).
Results and discussion
For personal use only.
through voltage-dependent Ca++ channels (VDCs) or
its release from intracellular stores in the sarcoplasmic
reticulum. Periodic depolarization and repolarization
regulates the spontaneous movements of the intestinal
smooth muscles. At the height of depolarization, the
action potential induces a rapid influx of Ca++ via VDCs
(Brading, 1981).
Thus the inhibitory effect of piperine observed in this
study may be due to interference of either Ca++ influx
through VDCs or Ca++ release. Our previous studies
(Taqvi et al., 2006a, 2006b) reported a calcium channel
blocking (CCB) effect of other piperidine derivatives,

When tested against castor oil-induced diarrhea, pip-

Control
erine (1– 10 mg/kg) caused dose-dependent protection,
similar to loperamide, which also provided 100% pro-
tection at  10 mg/kg (Table 1); this dose is in accordance
with the dose range as previously reported (Bajad et al.,
2001). Castor oil is known to induce diarrhea due to the
action of ricinoleic acid formed as a result of hydroly-
sis of the oil (Iwao & Terada, 1962), which produces Piperine 1 min
changes in the transport of water and electrolytes. This
results in a hypersecretory response and the generation
of huge contractions in the transverse and distal colon,
observed as diarrhea in the animal. Thus, a poten-
tial antidiarrheal agent may exhibit its antidiarrheal 30 100 300 µM
effect by inhibiting the contractions and/or secretion,
observed as the absence of diarrheal droppings (Croci Loperamide
et  al., 1997). These data suggest that piperine has an
inhibitory effect on either gut motility and/or electro-
lyte outflux.
30 100 300 µM

Verapamil
Table 1.  Effect of piperine on castor oil-induceda diarrhea in mice
(n = 5).
Treatment (mg/kg, p.o.) No. of mice with diarrhea % Protection
Saline (10 mL/kg) 5 0
Piperine ( 1 mg/kg) 2 60
Piperine ( 3 mg/kg) 1 80 µM
Piperine ( 10 mg/kg) 0** 100 0.1 0.3 1
Loperamide ( 10 mg/kg) 0** 100
Figure 1.  A typical tracing of showing inhibitory effect of piperine,
**p < 0.001 compared to saline group, 2 test. loperamide and verapamil on spontaneously contracting isolated
a
1h after administration of castor oil (10 mL/kg, p.o.). rabbit jejunum preparations.
 Piperine and calcium channel blockade   663

A 100 A Control
100 Piperine 10 µM
Piperine 30 µM
% of Control 75 Piperine 100 µM

75

% of Control Max.
50

Spontaneous 50
25 +
K (80 mM)

0 25
0 10 100 1000
[Piperine] µM 0
B 100 −4.5 −3.5 −2.5 −1.5
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Log [Ca++] M
75
% of Control

B Control
100 Verapamil 0.1 µM
50 Verapamil 0.3 µM
Verapamil 1 µM
Spontaneous

% of Control Max.
25 + 75
K (80 mM)

0
50
0.03 0.1 0.3 1 10
[Verapamil] µM
25
C 100
For personal use only.

75 0
% of Control

−4.5 −3.5 −2.5 −1.5

50 Log [Ca++] M

C Control
25 Spontaneous Loperamide 1 µM
+ 100 Loperamide 3 µM
K (80 mM)
Loperamide 10 µM
0
% of Control Max.

75
0.03 0.3 3 30 300 3000
[Loperamide] µM
50
Figure 2.  Inhibitory effect of piperine (A), verapamil (B) and lopera-
mide (C) on spontaneous and high K+-induced contractions in iso-
lated rabbit jejunum preparations. Values shown are mean ± SEM, 25
n = 4.
0
also selectively in smooth muscle. Therefore, further −4.5 −3.5 −2.5 −1.5
experiments were carried out to see whether piperine Log [Ca++]M
exhibits a CCB effect.
To determine the calcium channel blocking activ- Figure 3.  Effect on the Ca++ concentration-response curves of piper-
ity of piperine, high K+ was added in the tissue bath, ine (A), loperamide (B) and verapamil (C) in isolated rabbit jejunum
which produced sustained contraction, followed by preparations in Ca++ free medium. Values shown are mean ± SEM,
n=3.
the cumulative addition of piperine. Piperine relaxed
the high K+-induced sustained contractions in a
­concentration-dependent manner with an EC50 value of considered to be a CCB (Godfraind et  al., 1986). The
80.86 µM (56.10–116.50, 95% CI), similar to verapamil or CCB activity of piperine was further confirmed when
loperamide. pretreatment of tissues with piperine (10–100 µM)
The sustained contractions induced by high K+ caused a rightward shift in the Ca++ CRCs (Figure 3A),
(>30 mM) are dependent on the influx of Ca++ into cells constructed in Ca++-free medium, similar to vera-
through VDCs (Bolton, 1979). A substance that can pamil, a standard CCB (Fleckenstein, 1977), or lop-
inhibit the high K+-induced contraction is therefore eramide, which is also known to possess a CCB effect
 664   Syed Intasar Husain Taqvi et al.
at antidiarrheal doses (Wang et  al., 2005). These data
indicate that piperine also possesses CCB activity,
which may be responsible for its antidiarrheal activity,
similar to loperamide. This activity may be attributed
to the piperidine ring, which is shared by both lopera-
mide and piperine. Moreover, this is in accordance with
the known fact that CCBs such as verapamil possess
antidiarrheal and antispasmodic activities (Pasricha
2006). The present data suggest that piperine mediates
its antidiarrheal effect through CCB, though additional
mechanism(s) cannot be ruled out.
In summary, these data indicate that piperine pos-
sesses a CCB effect, which may be another possible
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mechanism involved in its antidiarrheal and antispas-
modic activities in addition to those already reported.
Further studies are recommended to evaluate the poten-
tial of piperine for development of a new antidiarrheal
agent.
Declaration of interest: This study was partially sup-
ported by a research grant from the Pakistan Science
Foundation.
For personal use only.
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