Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Pharmacological basis for the medicinal use of

Holarrhena antidysenterica in gut motility disorders

Anwarul Hassan Gilani, Aslam Khan, Arif-ullah Khan, Samra Bashir, Najeeb-
ur Rehman & Saf-ur-Rehman Mandukhail

To cite this article: Anwarul Hassan Gilani, Aslam Khan, Arif-ullah Khan, Samra Bashir, Najeeb-
ur Rehman & Saf-ur-Rehman Mandukhail (2010) Pharmacological basis for the medicinal
use of Holarrhena antidysenterica in gut motility disorders, Pharmaceutical Biology, 48:11,
1240-1246, DOI: 10.3109/13880201003727960

To link to this article: https://doi.org/10.3109/13880201003727960

Published online: 07 Sep 2010.

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Pharmaceutical Biology, 2010; 48(11): 1240–1246

RESEARCH ARTICLE

Pharmacological basis for the medicinal use of

Holarrhena antidysenterica in gut motility disorders
Anwarul Hassan Gilani1,5, Aslam Khan1,2, Arif-ullah Khan1,3, Samra Bashir1,4, Najeeb-ur-Rehman1,
and Saf-ur-Rehman Mandukhail1
1
Natural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University
Medical College, Karachi, Pakistan, 2Department of Pharmacology, Faculty of Pharmacy, University of Karachi,
Karachi, Pakistan, 3Institute of Pharmaceutical Sciences, Kohat University of Science and Technology, Kohat, Pakistan,
and 4Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan, 5King Saud University, Riyadh,
Saudi Arabia

Abstract
Context: Holarrhena antidysenterica Wall. (Apocynaceae) is widely used in traditional medical system for
treatment of constipation, colic, and diarrhea.
Aim: This study was carried out to provide pharmacological basis for medicinal use of Holarrhena anti-
dysenterica in gastrointestinal disorders.
Materials and methods: Hydro-ethanolic crude extract of Holarrhena antidysenterica (HaCE) and its fractions
were studied in various gastrointestinal isolated tissue preparations.
Results: In guinea pig ileum tissues, HaCE at 0.3-10 mg/mL caused pyrilamine-sensitive spasmogenic effect.
When tested in spontaneously contracting rabbit jejunum preparations, HaCE (0.01-3.0 mg/mL) caused
moderate stimulation, followed by a relaxant effect at next higher concentrations. In presence of pyrilamine,
the contractile effect was blocked and the relaxation was observed at lower concentrations (0.01-0.3 mg/mL).
HaCE inhibited the high K+ (80 mM)-induced contractions at concentration range of 0.01-1.0 mg/mL and
shifted Ca++ concentration response curves to the right, like that caused by verapamil. Activity-directed
fractionation revealed that the spasmogenic component was concentrated in the aqueous fraction, while
the spasmolytic component was concentrated in the organic fraction.
Discussion and conclusion: These results indicate that the gut stimulant and relaxant activities of Holarrhena
antidysenterica are mediated possibly through activation of histamine receptors and Ca++ channel block-
ade, respectively and this study provides sound mechanistic background for its usefulness in gut motility
disorders such as constipation, colic, and possibly diarrhea.
Keywords:  Holarrhena antidysenterica; spasmogenic; histaminergic; spasmolytic; Ca++ antagonist, constipa-
tion; diarrhea

Introduction used traditionally for a variety of health disorders, includ-

Holarrhena antidysenterica Wall. (Apocynaceae), com-
monly known as bitter oleander and locally as “inderjo
tulkh” or “kurchi”, is a small deciduous tree found in
Himalayan and sub-Himalayan tracts, ascending up to
1200 m (Baquar, 1989). Its seeds are 1-2 cm long, linear-
oblong, light brown, marked with linear lines and are bit-
ter in taste (Baquar, 1989; Nadkarni, 1976). The plant is
ing colic, diarrhea, dysentery, constipation, flatulence,
and urethrosis, as well as being considered useful as car-
minative, antispasmodic, astringent, anthelmintic, lithot-
riptic, diuretic, aphrodisiac, tonic, cardiosuppressant,
and antihypertensive (Duke et  al., 2002; Usmanghani
et al., 1997). It is known to contain conessine, ergostenol,
holarrhenine, kurchicine, resin, and tannin (Duke, 1992;
Kapoor, 1990).

Address for Correspondence:  Anwarul Hassan Gilani, Ph.D., Professor of Pharmacology, Department of Biological and Biomedical Sciences, The Aga Khan
University, Medical College, Karachi-74800, Pakistan. Tel.: (+92) 21-4864571; Fax: (+92) 21-493 4294, 494 2095; E-mail: anwar.gilani@aku.edu

(Received 08 November 2009; revised 17 January 2010; accepted 24 February 2010)

ISSN 1388-0209 print/ISSN 1744-5116 online © 2010 Informa Healthcare USA, Inc.
DOI: 10.3109/13880201003727960 http://www.informahealthcare.com/phb
 Pharmacological evaluation of H. antidysenterica   1241
Holarrhena antidysenterica has been reported to
possess antimutagenic (Aqil et  al., 2008), antibacterial
(Aqil & Ahmad, 2007) and immunomodulatory (Atal
et al., 1986) properties. In this investigation we provide
evidence that Holarrhena antidysenterica contains gut
stimulatory and inhibitory constituents, mediating their
effects via histaminergic and Ca++ antagonist pathways
respectively, which may explains the folkloric use of the
plant in gastrointestinal motility disorders such as con-
stipation, colic, and diarrhea.
Materials and methods
Plant material, preparation of crude extract and
fractions
Dried seeds of Holarrhena antidysenterica were from
a local herbal store, identified by taxonomist Jhandar
Shah, of the University of Malakand, Chakdara,
NorthWest Frontier Province and a voucher specimen
(HA-SE-01-08-71) was submitted to the herbarium of
the Department of Biological and Biomedical Sciences,
the Aga Khan University, Karachi. The plant material
was cleaned and extracted with 70% aqueous etha-
nol by cold maceration and kept for three days with
occasional shaking. It was filtered through a muslin
cloth and then through a Whatman qualitative grade 1
filter paper. This procedure was repeated twice and the
combined filtrate was evaporated on a rotary evapora-
tor under reduced pressure (-760 mmHg) to a thick,
semi-solid mass of brown color, i.e., the Holarrhena
antidysenterica crude extract (HaCE), yielding approxi-
mately 18% w/w.
Activity-guided fractionation was carried out by using
solvents of increasing polarity (Williamson et al., 1998).
Approximately 40 g of HaCE was dissolved in 500 mL dis-
tilled water. The n-butanol was added to it and shaken
vigorously. The mixture was allowed to separate into
two layers. The n-butanol layer (lower) was removed.
The same process was repeated twice more. All of the
n-butanol layers were combined and evaporated to give
the n-butanol fraction (HaB). The remaining layer was
collected and evaporated to obtain the aqueous fraction
(HaAq).
Chemicals
Acetylcholine perchlorate, atropine sulfate, pyrilamine
sulfate, and verapamil hydrochloride were purchased
from Sigma Chemicals, St. Louis, MO. All chemicals used
were of the analytical grade available. Chemicals used
for making Tyrode’s solution were: potassium chloride
(Sigma), calcium chloride, glucose, magnesium chloride,
sodium bicarbonate, sodium dihydrogen phosphate
(Merck, Darmstadt, Germany) and sodium chloride
(BDH Laboratory Supplies, Poole, UK).
Animals
Animals used in this study, such as guinea pigs (500–
600 g) and rabbits (1–1.2 kg) of local breed and either
sex, were housed at the Animal House of the Aga Khan
University, maintained at 23–25°C and given a standard
diet and tap water. Animals had free access to water but
food was withdrawn 24 h prior to experiment. Guinea
pigs were sacrificed by cervical dislocation and rabbits
by blow on back of the head. Experiments performed
complied with the rulings of the Institute of Laboratory
Animal Resources, Commission on Life Sciences,
National Research Council (1996) and approved by the
Ethical Committee of the Aga Khan University.
Phytochemical analysis
Phytochemical screening of the plant extract was carried
out for the presence of alkaloids, anthraquinones, cou-
marins, flavonoids, saponins, sterols, tannins, and terpe-
nes in accordance to the reported procedures (Akinyemi
et al., 2005; Edeoga et al., 2005). Briefly, alkaloids were
detected by using Dragendorff’s reagent. Presence of
saponins was detected based on the appearance of froth
upon vigorous shaking of diluted samples. The obser-
vation of yellow fluorescence under ultraviolet light on
examination of filter paper previously exposed to the
vapors from boiling plant material indicated the presence
of coumarins. For the detection of sterols and terpenes,
plant material was treated with petroleum ether and sub-
sequently extracted with CHCl3. The gradual appearance
of green to pink (for sterols) and pink to purple (for terpe-
nes) was then noted after treatment of CHCl3 layer with
acetic anhydride and concentrated H2SO4 in succession.
Plant material was detected as positive for flavonoids
when it gave a yellow color with AlCl3 reagent and for
tannins, when green or black was produced with aqueous
FeCl3. Lastly, for detecting anthraquinones, the extract
was dissolved in 1% HCl, then in benzene, and later if the
extract showed pink, violet or red color with NH4OH, that
indicated the presence of anthraquinones.
Experimentations
Guinea-pig ileum
The guinea pig was sacrificed by cervical dislocation; the
ileum was dissected out and kept in Tyrode’s solution
(Gilani et  al., 1997). Segments about 2 cm length were
mounted individually in a 10 mL tissue bath filled with
Tyrode’s solution at 37°C and aerated with 95% O2 in CO2
(carbogen). The composition of the Tyrode’s solution in
mM was: KCl 2.68, NaCl 136.9, MgCl2 1.05, NaHCO3 11.9,
1242   Anwarul Hassan Gilani et al.

NaH2PO4 0.42, CaCl2 1.8 and glucose 5.55, with pH 7.4. dence intervals (CI). CRCs were analyzed by non-linear
A preload of 1 g was applied to each tissue and isotonic regression using GraphPad program (San Diego, CA).
contractions were recorded using a Bioscience transducer
coupled to a Harvard Oscillograph. After an equilibra-
tion period of 30 min, the tissues were repeatedly treated Results
with sub-maximal concentration (0.3 μM) of ACh with
3 min intervals until constant responses were recorded. Phytochemical screening
The contractile effect of the test material was assessed
HaCE was found to contain alkaloids, coumarins, flavo-
as a percentage of the maximum effect, produced by the
noids, saponins and tannins, while testing negative for
control drug, histamine (1 μM).
other classes.
Rabbit jejunum
The jejunum was dissected out after surgical opening Effect of crude extract on ileum
of the rabbit abdomen, kept in Tyrode’s solution and
In guinea pig ileum, HaCE caused a concentration-
cleaned of mesenteries (Gilani et al., 2005a). Segments
dependent (0.3-5.0 mg/mL) contractile effect with
about 2 cm in length were suspended in a 10 mL tissue
decline at next higher concentration (10 mg/mL). The
bath containing Tyrode’s solution, maintained at 37°C
efficacy of the stimulant effect was 6.5 ± 2, 19.2 ± 8, 41 ± 16,
and aerated with carbogen. Intestinal responses were
52.5 ± 12 and 25.8 ± 9.0% (mean ± SEM; n = 6) at 0.3, 1,
recorded isotonically using Bioscience transducers cou-
3, 5, and 10 mg/mL respectively, compared to histamine
pled to a Harvard Oscillograph. Each tissue was allowed
maximum contraction (Figure 1), reflecting partial ago-
to equilibrate for at least 30 min before the addition of
nist effect. Pretreatment of the tissue with pyrilamine
any drug and then stabilized with a sub-maximal concen-
(1 μM) blocked the stimulatory effect of HaCE (Figure 1),
tration of ACh (0.3 μM). Under these conditions, rabbit
similar to that of histamine.
jejunum exhibits spontaneous rhythmic contractions.

Determination of calcium channel blocking effect Effect of crude extract on jejunum

For the elucidation of Ca++ channel blocking (CCB) activ-
In isolated rabbit jejunum, HaCE (0.01-3.0 mg/mL)
ity, high K+ (80 mM) was used to depolarize the prepara-
caused moderate stimulatory effect followed by weak
tions, as described by Farre et al. (1991). K+ was added to
spasmolytic effect at higher concentrations of 5 and
the tissue bath, which produced a sustained contraction.
Test materials were then added in a cumulative fashion
70
to obtain concentration-dependent inhibitory responses Without pyrilamine
(Van Rossum, 1963). To confirm the Ca++ antagonist action
of the test substance, the tissue was allowed to stabilize in 60 With pyrilamine (1µM)
normal Tyrode’s solution, which was then replaced with
Ca++-free Tyrode’s solution containing EDTA (0.1 mM) 50
for 30 min in order to remove Ca++ from the tissues. This
% Histamine Maximum

solution was further replaced with K+-rich and Ca++-free 40

Tyrode’s solution, having the following composition
(mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90,
30
NaH2PO4 0.42, glucose 5.55 and EDTA 0.1. Following
an incubation period of 30 min, control concentration-
response curves (CRCs) of Ca++ were obtained. When the 20
control Ca++ CRCs were found super-imposable (usually
after two cycles), the tissue was pretreated with the plant 10
extract for 60 min to test the possible CCB effect. The
CRCs of Ca++ were reconstructed in the presence of dif- 0
ferent concentrations of the test material.
0.3 1.0 3.0 5.0 10.0
[HaCE] mg/mL
Statistical analysis
Figure 1.  Bar diagram showing the spasmogenic effect of the crude
Data were expressed as mean ± standard error of mean extract of Holarrhena antidysenterica (HaCE) on isolated guinea pig
(SEM, n = number of experiments) and the median ileum preparations in the absence and presence of pyrilamine. Values
effective concentrations (EC50 values) with 95% confi- shown are mean ± SEM, from three to six determinations.
 Pharmacological evaluation of H. antidysenterica   1243

10 mg/mL (Figure 2), because of the accompanied of 0.07 mg/mL (0.04-0.1, n = 5) as shown in Figure 2.
relaxant component. The spasmogenic effect was HaCE concentration-dependently (0.03-0.1 mg/mL)
blocked in the presence of pyrilamine (0.1 μM) and the shifted the Ca++ concentration-response curves to
spasmolytic effect was observed at lower concentra- the right (Figure 3A), like that caused by verapamil
tions with EC50 value of 0.03 mg/mL (0.02-0.04, 95% CI, (Figure 3B).
n = 4). When tested against high (80 mM) K+-induced
contractions, HaCE caused relaxation with EC50 value
Effect of fractions on Ileum
In ileum, HaB was found devoid of any spasmogenic
175
effect, while HaAq exhibited pyrilamine-sensitive
Spontaneous
(without pyrilamine) marked stimulatory effect. The efficacy of the contrac-
150
tile effect was 12.5 ± 6.4, 21 ± 7.5, 47 ± 9.8 and 60 ± 9%
at 0.3, 1, 3, and 5 mg/mL respectively, while at next
125
higher concentration (10 mg/mL) did not produce
further increase in response, rather a trend of decline
100 in response was observed, as the response at this con-
% Control

centration was 50.7 ± 6.7%, of histamine maximum

75 response (Figure 4).
Spontaneous
(with pyrilamine)
50
K+(80 mM) Effect of fractions on jejunum
25 Like the parent crude extract, HaAq caused a concentra-
tion-dependent (0.01-3.0 mg/mL) spasmogenic effect in
0 rabbit jejunum followed by relaxation at higher concen-
trations (5 and 10 mg/mL). In the presence of pyrilamine
0.003 0.03 0.3 3 10 (0.1 μM), the spasmogenic effect was blocked and the
[HaCE] mg/mL spasmolytic effect was observed at lower concentra-
tions (Figure 5) with EC50 value of 0.01 mg/mL (0.005-
Figure 2.  Concentration-response curves of the crude extract of
0.02, n = 3). In jejunum, relatively low concentration of
Holarrhena antidysenterica (HaCE) on spontaneous contractions of
isolated rabbit jejunum in the absence and presence of pyrilamine (0.1 pyrilamine was used, in comparison to that in ileum, so
μM) as well as on K+-induced contractions in jejunum preparations. that it could not affect rhythmicity of jejunum spontane-
Values shown are mean ± SEM, from four to seven determinations. ous contraction. When tested against high K+-induced

A 100 Control
B 100 Control
HaCE 0.03 mg/mL
Verapamil 0.03 µM
HaCE 0.1 mg/mL Verapamil 0.1 µM
75 75
% Control Maximum

% Control Maximum

50 50

25 25

0 0

−4.5 −3.5 −2.5 −1.5 −4.5 −3.5 −2.5 −1.5

Log (Ca++) M Log (Ca++) M

Figure 3.  Concentration-response curves of Ca++ in the absence and presence of increasing concentrations of (A) crude extract of Holarrhena anti-
dysenterica (HaCE) and (B) verapamil in isolated rabbit jejunum preparations. Values shown are mean ± SEM, from five to six determinations.
1244   Anwarul Hassan Gilani et al.

80 Without pyrilamine 100

70 With pyrilamine (1µM)

60 75
% Histamine Maximum

50

% Control
40 50

30

25 Spontaneous
20

10 K+(80 mM)

0 0

0.3 1 3 5 10 0.003 0.03 0.3

[HaB] mg/mL
[HaAq] mg/mL
Figure 6.  Concentration-response curves of n-butanol fraction of
Figure 4.  Bar diagram showing the concentration-dependent spas-
Holarrhena antidysenterica (HaB) on spontaneous contractions of
mogenic effect of the aqueous fraction (HaAq) of Holarrhena anti-
isolated rabbit jejunum and on K+-induced contractions in rabbit
dysenterica on isolated guinea pig ileum preparations. Values shown
jejunum preparations. Values shown are mean ± SEM, from four to
are mean ± SEM, from four determinations.
six determinations.

175 Spontaneous
Discussion
(without pyrilamine)
150 In view of the well-known medicinal use of Holarrhena
antidysenterica in constipation, its crude extract was
125 tested for possible stimulatory effect on guinea pig ileum,
a quiescent gut preparation considered useful for this
100
purpose (Ghayur & Gilani, 2005b), where it produced
% Control

excitatory effect, like that caused by histamine and ACh.

75
Pretreatment of the tissues with pyrilamine, a histamine
Spontaneous receptor (H1) antagonist (Haley, 1983) blocked the con-
(with pyrilamine) tractile effect, while atropine, a muscarinic receptor
50
antagonist (Arunlakshana & Schild, 1959), did not alter
K+(80 mM)
it, indicating that crude extract of Holarrhena anti-
25
dysenterica causes gut stimulation possibly via activation
of histaminergic receptors. There is enough evidence for
0
histamine being an important cellular messenger of the
0.003 0.03 0.3 3 10 gastrointestinal tract (Schworer et al., 1994) and stimu-
[HaAq] mg/mL lates various smooth muscles including the gut through
activation of H1 receptors (Hill, 1990). The observed
Figure 5.  Concentration-response curves of aqueous fraction of stimulant effect of Holarrhena antidysenterica explains
Holarrhena antidysenterica (HaAq) on spontaneous contractions of its medicinal use in hypomotility disorders of the gut.
isolated rabbit jejunum in the absence and presence of pyrilamine (0.1 The maximum spasmogenic effect of the extract was
μM) as well as on K+-induced contractions in jejunum preparations.
Values shown are mean ± SEM, from three to four determinations.
around 52% of histamine maximum response and was
followed by relaxation at next higher concentration, indi-
contraction, HaAq caused concentration-dependent cating the co-existence of spasmolytic constituents. The
relaxation with EC50 value of 0.05 mg/mL (0.02-0.06, combination of gut inhibitory with stimulatory effect in
n = 4) as shown in Figure 5. HaB showed only inhibi- Holarrhena antidysenterica is probably meant by nature
tory effect on spontaneous and (80 mM) K+-induced not to allow the spasmogenic effect to go beyond the
contraction with EC50 values of 0.02 mg/mL (0.02-0.04, therapeutic limits beyond which it could have caused
n = 6) and 0.06 mg/mL (0.04-0.08, n = 4), respectively abdominal cramps, as is the case with chemical drugs
(Figure 6). used in constipation (Gilani et al., 2005a).
 Pharmacological evaluation of H. antidysenterica   1245
The presence of spasmolytic constituents may also
explain the use of Holarrhena antidysenterica in hyper-
active diseases of the gut such as abdominal spasm and
diarrhea. Therefore, the spasmolytic effect was further
investigated on rabbit jejunum, a spontaneously contract-
ing gut preparation (Gilani et al., 2005c), thus allowing
relaxant effect to be studied without induced contraction.
When tested on rabbit jejunum, a pyrilamine-sensitive
spasmogenic effect followed by spasmolytic effect was
observed.
In earlier studies we observed that the relaxant
effect of medicinal plants is usually mediated through
blockade of calcium channels (Gilani et  al., 2000,
2005b). To investigate whether the spasmolytic effect
of Holarrhena antidysenterica is also mediated via a
similar mechanism the extract was tested on high K+-
induced contractions. High K+ (> 30 mM) is known to
cause smooth muscle contractions through opening of
voltage-dependent L-type Ca++ channels, thus allowing
influx of extracellular Ca++ causing a contractile effect
(Bolton, 1979) and the substance causing inhibition of
high K+-induced contraction is considered an inhibi-
tor of Ca++ influx (Godfraind et al., 1986). HaCE relaxed
the high K+-induced contractions, like that caused by
verapamil, a standard Ca++ antagonist (Fleckenstein,
1977), indicating its CCB action. The Ca++ antagonist
effect of Holarrhena antidysenterica was further con-
firmed when it shifted the Ca++ CRCs to the right, like
that caused by verapamil. Ca++ antagonists have been
shown to be beneficial in gut disorders resulting from
hyperactivity such as abdominal cramps and diarrhea
(Pasricha, 2006), hence the observed CCB effect justi-
fies the medicinal use of Holarrhena antidysenterica in
such conditions. Interestingly, such combinations of
gut stimulatory and relaxant activities have been found
commonly in plants used for two contrasting states of
gut, i.e., constipation and diarrhea (Ghayur & Gilani,
2005b; Gilani et al., 2005a).
Activity-guided fractionation revealed that the spas-
molytic component(s) is distributed among both the
organic and aqueous fractions, but the spasmogenic
component is separated in the aqueous fraction,
which showed stimulant effect slightly stronger than
the parent crude extract. The observed histaminergic
and Ca++ antagonist effects of the plant might be due
to the presence of tannins and flavonoids evident in
the phytochemical screening, as the compounds of
these classes have been reported to possess histamine-
(Read et al., 1970) and CCB-like (Di Carlo et al., 1993;
Revuelta et al., 1997) actions, respectively. Saponins are
also known as spasmogenic (Awe et al., 1999; Ghayur
& Gilani, 2005a); however, the contribution of other
constituents accounting for reported effects cannot be
ignored.
Conclusions
In conclusion, these data indicate that Holarrhena
antidysenterica exhibits spasmogenic and spasmolytic
effects, mediated through stimulation of histaminergic
receptors and Ca++ antagonist mechanisms respectively,
though additional mechanisms cannot be ruled out.
Thus, this study provides sound pharmacological basis
for the medicinal use of Holarrhena antidysenterica in gut
disorders such as constipation, colic and diarrhea.
Declaration of interest
This study was supported by the Higher Education
Commission of Pakistan, as indigenous M.Phil/PhD
scholarship awarded to Aslam Khan.
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