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The Aga Khan University

eCommons@AKU
Department of Biological & Biomedical Sciences

Medical College, Pakistan

February 2008

Gastrointestinal, selective airways and urinary


bladder relaxant effects of Hyoscyamus niger are
mediated through dual blockade of muscarinic
receptors and Ca(2+) channels
Anwarul Hassan Gilani
Aga Khan University

Arif-Ullah Khan
Aga Khan University

Mustafa Raoof
Aga Khan University

Muhammad Nabeel Ghayur


Aga Khan University

Bina S. Siddiqui
See next page for additional authors

Follow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs


Part of the Physiology Commons
Recommended Citation
Gilani, A., Khan, A., Raoof, M., Ghayur, M., Siddiqui, B., Vohra, W., Begum, S. (2008). Gastrointestinal, selective airways and urinary
bladder relaxant effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca(2+) channels.
Fundamental & Clinical Pharmacology, 22(1), 87-99.
Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/76

Authors

Anwarul Hassan Gilani, Arif-Ullah Khan, Mustafa Raoof, Muhammad Nabeel Ghayur, Bina S. Siddiqui,
Waseem Vohra, and Sabira Begum

This article is available at eCommons@AKU: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/76

doi: 10.1111/j.1472-8206.2007.00561.x
ORIGINAL
ARTICLE

Gastrointestinal, selective airways and


urinary bladder relaxant effects of
Hyoscyamus niger are mediated through
dual blockade of muscarinic receptors
and Ca2+ channels
Anwarul Hassan Gilania*, Arif-ullah Khana,b, Mustafa Raoof a,
Muhammad Nabeel Ghayura,c, Bina S. Siddiquid, Waseem Vohrad,
Sabira Begumd
a

Natural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical
College, Karachi 74800, Pakistan
b
Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan
c
Department of Medicine, McMaster University, St. Josephs Hospital, Hamilton, ON, Canada
d
HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

Keywords
airways and bladder selectivity,
anticholinergic,
Ca2+ antagonist,
Hyoscyamus niger

Received 21 June 2007;


revised 13 September 2007;
accepted 13 November 2007

*Correspondence and reprints:


anwar.gilani@aku.edu

ABSTRACT

This study describes the spasmolytic, antidiarrhoeal, antisecretory, bronchodilatory


and urinary bladder relaxant properties of Hyoscyamus niger to rationalize some of
its medicinal uses. The crude extract of H. niger seeds (Hn.Cr) caused a complete
concentration-dependent relaxation of spontaneous contractions of rabbit jejunum,
similar to that caused by verapamil, whereas atropine produced partial inhibition.
Hn.Cr inhibited contractions induced by carbachol (1 lM) and K+ (80 mM) in a
pattern similar to that of dicyclomine, but different from verapamil and atropine.
Hn.Cr shifted the Ca2+ concentrationresponse curves to the right, similar to that
caused by verapamil and dicyclomine, suggesting a Ca2+ channel-blocking
mechanism in addition to an anticholinergic effect. In the guinea-pig ileum, Hn.Cr
produced a rightward parallel shift of the acetylcholine curves, followed by a nonparallel shift with suppression of the maximum response at a higher concentration,
similar to that caused by dicyclomine, but different from that of verapamil and
atropine. Hn.Cr exhibited antidiarrhoeal and antisecretory effects against castor oilinduced diarrhoea and intestinal fluid accumulation in mice. In guinea-pig trachea
and rabbit urinary bladder tissues, Hn.Cr caused relaxation of carbachol (1 lM) and
K+ (80 mM) induced contractions at around 10 and 25 times lower concentrations
than in gut, respectively, and shifted carbachol curves to the right. Only the
organic fractions of the extract had a Ca2+ antagonist effect, whereas both organic
and aqueous fractions had anticholinergic effect. A constituent, b-sitosterol
exhibited Ca2+ channel-blocking action. These results suggest that the antispasmodic effect of H. niger is mediated through a combination of anticholinergic and
Ca2+ antagonist mechanisms. The relaxant effects of Hn.Cr occur at much lower
concentrations in the trachea and bladder. This study offers explanations for the
medicinal use of H. niger in treating gastrointestinal and respiratory disorders and
bladder hyperactivity.

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

87

A. H. Gilani et al.

88

INTRODUCTION

MATERIALS AND METHODS

Hyoscyamus niger Linn. (Solanaceae), commonly


known as Henbane and Ajwain-i-khurasani locally
in Pakistan, grows wildly throughout the Himalayan
range at altitudes of 800011 000 feet and in
many other parts of the world [1]. Its seeds are used
in traditional medicine for treating a variety of
ailments including asthma, cough, colic, diarrhoea
and genito-urinary complaints such as irritable bladder [2,3].
Phytochemical studies revealed the presence of
hyoscine (scopolamine), hyoscyamine, anisodine,
anisodamine [4,5], aesculetin, coumarin, kaempferol,
quercetin, rutin, cuscohygrine, chlorogenic acid, linoleic acid, myristic acid, oleic acid, stearic acid, pyridine,
trimethylamine [6], b-sitosterol, grossamide, cannabisin D & G, daucosterol, N-trans-feruloyltyramine,
1-O-octadecanoyl glycerol, 1-O-(9Z,12Z-octadecadienoyl)
glycerol,
1-O-(9Z,12Z-octadecadienoyl)-3-O-(9Z-octadecenoyl) glycerol, vanillic acid [7], calystegines [8]
and withanolides [9]. The plant is known to possess
antispasmodic, analgesic [10], mydriatic [11] and
sedative [12] properties.
Hyoscine and hyoscyamine, previously identified
as the anticholinergic constituents in H. niger, are
considered responsible for its antispasmodic effect [13].
However, it is generally believed that the plant
contains multiple compounds acting on different sites.
The aim of this study was to see if H. niger has an
antispasmodic effect on the gut, airways and urinary
bladder through a combination of mechanisms. Plants
with medicinal use in hyperactive gut and airway
disorders usually possess a Ca2+ channel-blocking
action in combination with other spasmolytic mechanisms [1416]. In this investigation, we report the
presence of a combination of anticholinergic and Ca2+
antagonist effects in H. niger seed extract, studied in
tissues relevant to gastrointestinal, airway and urinary
systems. Activity-directed fractionation revealed that
the anticholinergic component(s) was widely distributed both in the organic and aqueous fractions, while
Ca2+ antagonist was concentrated only in the organic
fraction. Apart from the crude extract and its resultant
fractions, the pure compounds of H. niger namely
b-sitosterol, scopolamine, hyoscyamine, coumarin,
chlorogenic acid and vanillic acid were also studied.
b-sitosterol was found to exhibit spasmolytic effect via
Ca2+ antagonist mechanism.

Plant material, preparation of crude extract and


fractions
The seeds of H. niger (5 kg) were purchased from the
local market, cleaned and coarsely ground. The powdered material was extracted four times with 98%
methanol at room temperature. The soaked material
was filtered through a muslin cloth and then through
Whatman qualitative grade 1 filter paper [17]. This
procedure was repeated thrice and the combined filtrate
was evaporated with a rotary evaporator under reduced
pressure to a thick syrupy mass, the crude extract
(Hn.Cr), at a yield of approximately 19.6%. Activityguided fractionation of the parent extract was carried
out by using solvents of increasing polarity. Hn.Cr was
partitioned between ethyl acetate and water. The ethyl
acetate phase was dried over Na2SO4 (anhydrous), then
concentrated at reduced pressure to yield an ethyl
acetate residue. The water layer was dried to obtain
the aqueous fraction (Hn.Aq). The ethyl acetate residue
was treated with petroleum ether to obtain the petroleum ether fraction (Hn.PE) and insoluble fraction. The
latter was treated with ethyl acetate to give the ethyl
acetate fraction (Hn.EtAc). The fraction left after
extraction with ethyl acetate was dissolved in methanol to obtain the methanolic fraction (Hn.MeOH).
Stock solutions of the plant materials were prepared
in 10% ethanol, and subsequent dilutions in distilled
H2O/saline.
Chemicals
Acetylcholine chloride, atropine sulphate, b-sitosterol,
carbachol, chlorogenic acid, coumarin, dicyclomine,
loperamide hydrochloride, scopolamine, vanillic acid
and verapamil hydrochloride were purchased from
Sigma Chemicals Co (St Louis, MO, USA). Hyoscyamine
sulphate and castor oil were obtained from ChromaDex
(Santa Ana, CA, USA) and KCl Pharma (Karachi,
Pakistan) respectively. Chemicals used for making
physiological salt solutions were potassium chloride
(Sigma Chemicals Co), calcium chloride, glucose,
magnesium chloride, magnesium sulphate, potassium
dihydrogen phosphate, sodium bicarbonate, sodium
dihydrogen phosphate (Merck, Darmstadt, Germany)
and sodium chloride from BDH Laboratory Supplies
(Poole, UK). All chemicals used were of analytical grade
and solubilized in distilled H2O except, b-sitosterol which
was dissolved in 10% ethanol; subsequent dilutions were

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

Anticholinergic and Ca2+ antagonist activities of Hyoscyamus niger

prepared in distilled H2O. Vehicles used had no effect on


tissue contractility in control experiments.
Experimental animals
Animals used in this study, adult rabbits (1.01.2 kg),
guinea-pigs (500550 g) and BALB/c mice (2025 g)
of either sex and local breed, were housed at the
Animal House of the Aga Khan University and maintained at 2325 C. Animals were given tap water ad
libitum and a standard diet consisting of (g/kg): flour
380, chokar 380, molasses 12, sodium chloride 5.8,
nutrivet L 2.5, potassium meta-bisulphate 1.2, vegetable oil 38, fish meal 170 and powdered milk 150.
Rabbits and guinea-pigs had free access to water, but
food was withdrawn 24 h prior to experiments. They
were killed by a blow on the back of the head and
cervical dislocation respectively. Experiments performed
complied with the recommendations of the Institute of
Laboratory Animal Resources, Commission on Life
Sciences, National Research Council [18] and approved
by the Ethics Review Committee of the Aga Khan
University.
Isolated tissue preparations
Rabbit jejunum
The jejunum was dissected, kept in Tyrodes solution and
cleaned of mesentery tissues [19]. Segments about 2 cm
long were suspended individually in a 10-mL tissue bath
containing Tyrodes solution maintained at 37 C and
aerated with a mixture of 95% O2 and 5% CO2 (carbogen). The composition of Tyrodes solution in mM was:
KCl 2.68, NaCl 136.9, MgCl2 1.05, NaHCO3 11.90,
NaH2PO4 0.42, CaCl2 1.8 and glucose 5.55. Intestinal
contractions were recorded isotonically using Bioscience
transducers and oscillograph (Harvard Apparatus,
Holliston, MA, USA). Tissues were allowed to equilibrate
for at least 30 min before the addition of any drug, then
stabilized with a sub-maximal concentration of acetylcholine (0.3 lM) at 3-min intervals until constant
responses were recorded. Under these experimental
conditions, rabbit jejunum exhibits spontaneous rhythmic contractions, allowing the testing of relaxant (spasmolytic) activity directly without the use of an agonist.
For the determination of Ca2+ channel-blocking
activity, high K+ concentration (80 mM) was used to
depolarize the preparations as described by Farre et al.
[20]. K+ was added to the tissue bath, which produced a
sustained contraction. Test materials were then added in
cumulative fashion to obtain concentration-dependent
inhibitory responses [21].

89

To confirm the Ca2+ antagonist action of the test


substance, the tissue was allowed to stabilize in normal
Tyrodes solution, which was then replaced with Ca2+free Tyrodes solution containing ethylenediaminetetraacetic acid (EDTA; 0.1 mM) for 30 min in order to remove
Ca2+ from the tissues. This solution was replaced with K+rich and Ca2+-free Tyrodes solution, having the composition (mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3
11.90, NaH2PO4 0.42, glucose 5.55 and EDTA 0.1.
Following an incubation period of 30 min, control
concentrationresponse curves of Ca2+ were constructed.
When the control Ca2+ concentrationresponse curves
were super-imposable (usually after two cycles), the
tissue was pretreated with the plant extract for 60 min
to test for a possible Ca2+ antagonist effect. The concentrationresponse curves of Ca2+ were reconstructed in
the presence of different concentrations of the test
material.
Guinea-pig ileum
The ileum was dissected and kept in Tyrodes solution.
The segments, about 2 cm long, were mounted individually in a 10-mL tissue bath, filled with Tyrodes
solution, at 37 C and aerated with carbogen. An initial
load of 0.7 g was applied to the tissue and isotonic
contractions were recorded with a Bioscience transducer
coupled to a Harvard oscillograph (Harvard Apparatus,
Holliston, MA, USA). An equilibrium period of 30 min
was required before administration of drugs. After
equilibration, each tissue preparation was repeatedly
treated with sub-maximal concentrations (0.3 lM) of
acetylcholine at 3-min intervals until constant responses
were recorded. Control acetylcholine concentration
response curves were then constructed by addition of
increasing concentration of agonist and washing between doses. Acetylcholine curves were then re-determined in the presence of increasing concentrations of
test and control drugs as described previously [22].
Guinea-pig trachea
Trachea was dissected and kept in Krebs solution. The
tracheal tube was cut into rings, 23 mm wide, each
containing about two cartilages. Each ring was opened
by a longitudinal cut on the ventral side opposite the
smooth muscle, forming a tracheal chain with smooth
muscle in the centre and cartilaginous portions on the
edges. The preparation was mounted in a 20-mL tissue
bath containing Krebs solution at 37 C, aerated with
carbogen. The composition of Krebs solution was (mM):
NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

A. H. Gilani et al.

90

1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). A tension of


1 g was applied to the tracheal strips continuously
throughout the experiment. The tissue was equilibrated
for 1 h before the addition of any drug. In some
preparations, carbachol (1 lM) or K+ (80 mM) were used
to stabilize the respective preparations until constant
responses of each agonist were achieved (usually three to
four concentrations). When sustained contractions were
obtained, the relaxant effect of the test substance was
assessed by adding it in a cumulative fashion. The
carbachol cumulative concentrationresponse curves
were plotted using increasing concentrations of agonist.
When a threefold increase in concentration produced no
further increment in response, the tissue was washed to
re-establish the baseline tension (usually 3035 min).
The carbachol curves were then replotted in the presence
of increasing concentrations of an extract [23]. Isometric
responses were recorded on a Grass model 7 Polygraph
(Grass Instrument Company, Quincy, MA, USA).
Rabbit urinary bladder
The whole urinary bladder was divided into four vertical
strips [24]. Each preparation was mounted in a 20-mL
tissue bath containing KrebsHenseleit solution (mM):
NaCl 119, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4
1.18, MgSO4 1.17, EDTA 0.027, glucose 5.5 and HEPES
10 aerated with carbogen to yield a pH of 7.4 at 37 C. A
tension of 1 g was applied continuously throughout the
experiment. Tissues were equilibrated for 1 h before the
addition of any drug. As was the case with trachea, in
some preparations carbachol (1 lM) and K+ (80 mM)
were used to stabilize the respective preparations until
constant responses of agonists were achieved. Once
sustained contractions were obtained, inhibitory effects
of the plant material were assessed by adding it in
a cumulative fashion. The carbachol concentration
response curves were plotted in the absence and presence
of increasing concentrations of the extract. Isometric
responses were recorded on a Grass model 7 Polygraph.
In vivo experiments
Castor oil-induced diarrhoea
Mice were fasted for 24 h before the experiment.
Animals were housed in individual cages and divided
in four groups, each containing five mice. The first group
received saline (10 mL/kg p.o.) and served as a negative
control. Extract doses were selected on trial basis and
then two increasing doses of the extract were given
orally. A group of mice was treated with loperamide
(10 mg/kg p.o.), as a positive control. One hour after

treatment, each animal received 10 mL/kg of castor oil


orally through a feeding needle [25]. Afterwards, the
cages were inspected for the presence of diarrhoea
droppings; their absence was noted as a positive result,
indicating protection from diarrhoea at that time.
Intestinal fluid accumulation
Different groups of overnight fasted mice were treated
with increasing doses of extract intraperitoneally
through detachable U-100 insulin syringe with 25G
1 (0.50 25 mm) needle, 60 min before oral administration of castor oil (10 mL/kg). The mice were killed
30 min later by cervical dislocation and the entire
intestine was removed and weighed with care, not
allowing any intestinal fluid to leak out [25]. The results
were expressed as (Pi/Pm) 1000 where Pi is the weight
(g) of the intestine and Pm is the weight of the animal.
Statistical analysis
Data are mean standard errors of mean (SEM, n =
number of experiments) for the median effective concentrations (EC50 values) with 95% confidence intervals. The
statistical parameters applied were Students t-test for
intestinal fluid accumulation, and chi-squared test for
antidiarrhoeal activity. Difference of P < 0.05 was
considered statistically significant. Concentration
response curves were analysed by nonlinear regression
using GraphPad program (GraphPAD, San Diego, CA,
USA).
RESULTS
Effect on jejunum
Figure 1a compares inhibitory effects of Hn.Cr with those
of dicyclomine, verapamil and atropine on the spontaneous contractions of isolated rabbit jejunum. The
respective EC50 values were 5.6 lg/mL (4.17.6, 95%
confidence interval, n = 5), 0.53 lM (95% CI 0.400.70,
n = 4), 0.23 lM (95% CI 0.170.32, n = 5) and
0.001 lM (95% CI 0.00080.01, n = 4). The spasmolytic effect was concentration-dependent with the exception of atropine, where further increments in the
concentration beyond 0.3 lM did not show additional
inhibition. When tested against carbachol (1 lM) and K+
(80 mM)-induced
contractions,
Hn.Cr
inhibited
(Figure 1b) carbachol-induced contractions at a much
lower concentration with EC50 value of 7.2 lg/mL (95%
CI 4.412, n = 4) as compared with 300 lg/mL (95% CI
209.8428.4, n = 4) for K+. Dicyclomine also showed a
similar pattern of inhibition (Figure 1c) with respective

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

Anticholinergic and Ca2+ antagonist activities of Hyoscyamus niger

91

Jejunum
(a)

1 min

(b)

% of control

Control

Hn.Cr

100

75

50

25

0.3

10

30

100

CCh (1 mM)

g/mL

K+ (80 mM)

Dicyclomine

3.0

0.3

0.1

0.03

0.3

(c)

30

300

10

[Hn.Cr] g/mL

100

0.03

0.1

0.3

% of control

Verapamil

Atropine

75

50

CCh (1 M)

25

K+ (80 mM)

0.001

0.003

0.01

0.03

0.1

0.01

0.1

[Dicyclomine] M

(e)

100

100

75

75

% of control

% of control

(d)

0.3

50

25

CCh (1 M)

50

25

K+ (80 mM)

K+ (80 mM)

0
0.003

CCh (1 M)

0.03

0.3

[Verapamil] M

0.0003

0.003

0.03

[Atropine] M

Figure 1 (a) Tracing showing spasmolytic effect of the crude extract of Hyoscyamus niger (Hn.Cr), dicyclomine, verapamil and atropine on
spontaneous contractions and concentrationresponse curves representing comparison of (b) Hn.Cr; (c) dicyclomine; (d) verapamil and
(e) atropine for the inhibitory effect against carbachol (CCh) and K+-induced contractions in isolated rabbit jejunum preparations. Symbols
represent mean SEM, n = 48.

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

A. H. Gilani et al.

92

EC50 values of 0.13 (95% CI 0.110.19, n = 6) and


3.2 lM (95% CI 1.95.3, n = 5). Verapamil on the other
hand, was more potent against K+-induced contractions
with EC50 value of 0.04 lM (95% CI 0.030.05, n = 5),
compared with carbachol [0.16 lM (95% CI 0.130.19,
n = 5)] as shown in Figure 1d. Atropine relaxed the
carbachol-induced contractions potently with EC50 value
of 0.003 lM (95% CI 0.0020.004, n = 8), without any
effect on K+ (Figure 1e), as was expected. When tested for
the possible interaction with Ca2+ channels, Hn.Cr
caused a rightward shift of the Ca2+ concentration
response curves (Figure 2a), similar to that caused by
verapamil (Figure 2b) and dicyclomine (Figure 2c).

rightward shift with suppression of the maximum


response (Figure 3c) and atropine (0.010.03 lM)
caused a rightward parallel shift without suppression of
the maximum contractile effect (Figure 3d).
Effect on castor oil-induced diarrhoea
Hn.Cr exhibited a dose-dependent (3001000 mg/kg)
inhibitory effect against castor oil-induced diarrhoea in
mice. The negative control treatment (saline) did not
protect the animals from diarrhoea. Pretreatment with
the plant extract led to 40% protection from diarrhoea
at 300 mg/kg and 80% protection at 1000 mg/kg
(P < 0.05 vs. saline group). Loperamide (10 mg/kg) led
to complete protection from diarrhoea in the positive
control group (Table I).

Effect on ileum
Hn.Cr at 1.0 lg/mL caused a rightward parallel shift in
the acetylcholine concentrationresponse curves without suppressing maximum contractile response, followed
by a non-parallel shift with suppression of the maximum
response at 3.0 lg/mL (Figure 3a). Dicyclomine (0.03
0.1 lM) also showed a similar pattern of shift (Figure 3b),
while verapamil (0.010.03 lM) produced a non-parallel

Effect on intestinal fluid accumulation


When tested against castor oil-induced intestinal fluid
accumulation in mice, Hn.Cr had a dose-dependent
(3001000 mg/kg) antisecretory effect (Figure 4). Intestinal fluid accumulation in the saline-treated group was
114.4 0.77 g, while in castor oil-treated group it was

Jejunum
(a)

(b)

100

Control

100

Hn.Cr 100 g/mL

Verapmil 0.1 M

75

% of control max.

% of control max.

Hn.Cr 300 g/mL

50

25

4.5

3.5

Log

2.5

[Ca++]

50

25

4.5

1.5

3.5

2.5

1.5

Log [Ca++] M

(c)

(d)

100

Control
Dicyclomine 3 M
Dicyclomine 10 M

75

% of control max.

% of control max.

75

100

Control
Verapamil 0.03 M

50

25

Control
-sitosterol 100 g/mL
-sitosterol 300 g/mL

75

50

25

0
4.5

3.5

Log

2.5

[Ca++]

1.5

4.5

3.5

2.5

Log [Ca++] M

1.5

Figure 2 Concentrationresponse
curves of Ca2+ in the absence and
presence of the increasing concentrations of (a) crude extract of Hyoscyamus
niger (Hn.Cr); (b) verapamil; (c) dicyclomine and (d) b-sitosterol in isolated
rabbit jejunum preparations. Symbols
represent mean SEM, n = 46.

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

Anticholinergic and Ca2+ antagonist activities of Hyoscyamus niger

93

Ileum
(b) 100

100

Control

Control
Hn.Cr
(1 g/mL)

75

Dicyclomine
(0.03 M)

% of ACh max.

% of ACh max.

(a)

Hn.Cr
(3 g/mL)

50
25

7
6
5
Log [ACh] M

25

(d)

100

Control
Verapmil
(0.01 M)
Verapmil
(0.03 M)

75
50
25

100

7
6
5
Log [ACh] M

Control
Atropine
(0.01 M)

75

Atropine
(0.03 M)

50
25
0

0
8

7
6
5
Log [ACh] M

Table I Effect of the crude extract of Hyoscyamus niger (Hn.Cr) on


castor oil (c.oil, 10 mL/kg)-induced diarrhoea.
Treatment (p.o.)

No. mice/five with diarrhoea

Saline (10 mL/kg) + (c.oil)

Hn.Cr (300 mg/kg) + (c.oil)

40

Hn.Cr (1000 mg/kg) + (c.oil)

1*

Loperamide (10 mg/kg) + (c.oil) 0**

50

% of ACh max.

% of ACh max.

Figure 3 Concentrationresponse
curves of acetylcholine (ACh) in the
absence and presence of (a) crude extract
of Hyoscyamus niger (Hn.Cr); (b) dicyclomine; (c) verapamil and (d) atropine in
isolated guinea-pig ileum preparations.
Symbols represent mean SEM,
n = 36.

Dicyclomine
(0.1 M)

(c)

75

% Protection

80
100

*P < 0.05, **P < 0.01 compared to saline group, chi-squared test.

122.6 0.70 g (P < 0.001 vs. saline group). Hn.Cr


at the doses of 300 and 1000 mg/kg reduced the castor
oil-induced fluid accumulation to 118.6 1.10 g
(P < 0.05 vs. castor oil group) and 114.4 0.70 g
(P < 0.001 vs. castor oil group) respectively.
Effect on trachea
In tracheal preparations, Hn.Cr inhibited carbachol
(1 lM) contractions at low concentrations with EC50
value of 0.8 lg/mL (95% CI 0.51.3, n = 4), compared
with that against K+ (80 mM) contractions with EC50

7
6
5
Log [ACh] M

value of 12.3 lg/mL (95% CI 7.918.9, n = 5) as shown


in Figure 5a. Similar to that in ileum, Hn.Cr produced a
rightward parallel displacement of the carbachol curves
without suppression of the maximum contractile response at 1.0 lg/mL, followed by a non-parallel shift
with suppression of the maximum effect at 3.0 lg/mL
(Figure 5b).
Effect on urinary bladder
Hn.Cr inhibited carbachol (1 lM) and K+ (80 mM)
contractions with respective EC50 values of 0.6 (95%
CI 0.31.0, n = 5) and 6.2 lg/mL (95% CI 4.29.1,
n = 5) as shown in Figure 5c. Hn.Cr caused a rightward
parallel shift of the carbachol curves without suppression
of the maximum contractile response at 1.0 lg/mL,
followed by a non-parallel shift with the suppression of
maximum effect at 3.0 lg/mL (Figure 5d).
Effect of fractions
Hn.PE, Hn.EtAc and Hn.MeOH inhibited carbachol
(1 lM) and K+ (80 mM) contractions of rabbit jejunum

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

A. H. Gilani et al.

94

Intestinal fluid accumulation


(Pi/Pm) x 1000

125
120

Saline
Castor oil
Castor oil + Hn.Cr

#
*

***

115
110
105
100
300
[Hn.Cr] mg/kg

1000

Figure 4 Effect of increasing doses of the crude extract of


Hyoscyamus niger (Hn.Cr) on castor oil-stimulated fluid accumulation in small intestine of mice. Results shown are mean SEM of
five animals for each experimental group. Intestinal fluid accumulation is expressed as Pi/Pm 1000 (g) where Pi is the weight of the
small intestine and Pm is the weight of the mouse. #P < 0.001 vs.
saline group, *P < 0.05 and ***P < 0.001 vs. castor oil group,
Students t-test.

(Figure 6ac) with respective EC50 values of 9.0 (95% CI


6.013.6, n = 3) and 59.6 (95% CI 30.2117.8, n = 3),
236.8 (95% CI 171.3327.2, n = 3) and 1117 (95% CI
738.71690, n = 3), 11.7 (95% CI 7.119.1, n = 3)
and 130.3 lg/mL (95% CI 93.3226.4, n = 5), while
Hn.Aq relaxed carbachol (1 lM) contractions with EC50
value of 8.6 lg/mL (95% CI 6.212.0, n = 4) without
any effect on K+ (80 mM), n = 5 (Figure 6d).
Effect of pure compounds
Some of the known commercially available pure compounds of H. niger were also tested in the jejunum.
b-sitosterol, like verapamil was found more potent
against K+ (80 mM)-induced contractions (Figure 7a),
with an EC50 value of 9.6 lg/mL (95% CI 7.113.1,
n = 5), when compared with carbachol [218.6 lg/mL
(95% CI 129.5369.0), n = 4] and shifted Ca2+ concentrationresponse curves to the right (Figure 2d). Scopolamine and hyoscyamine relaxed only carbachol (1 lM)
contractions with respective EC50 values of 0.004 lg/mL
(95% CI 0.0030.005, n = 6) and 0.001 lg/mL (95% CI
0.00090.002, n = 4) without any effect on K+ (Figure 7b,c). Coumarin, chlorogenic acid and vanillic acid
failed to relax either carbachol (1 lM) or K+ (80 mM)
contractions up to 1000 lg/mL.

DISCUSSION
Hyoscyamus niger is known to contain tropane alkaloids,
i.e. hyoscine and hyoscyamine, which are well known
for their anticholinergic effect [13]. In view of the wide
distribution of Ca2+ antagonists in medicinal plants,
which usually exist in combination with other spasmolytics [1416], the present study was undertaken to see
whether the antispasmodic action of H. niger is the result
of a combination of anticholinergic and Ca2+ antagonist
mechanisms. When tested in isolated rabbit jejunum
preparations, the plant extract completely inhibited
spontaneous contractions, like that caused by verapamil,
a standard Ca2+ channel blocker [26]. Atropine, a
muscarinic receptor antagonist [27], caused partial
relaxation of spontaneous contractions. Gastrointestinal
motility is regulated by multiple physiological mediators,
mainly acetylcholine, histamine, 5-hydroxytryptamine,
bradykinins, prostaglandins, substance P and cholecysto
kinins which achieve their contractile effects through an
ultimate increase in cytosolic Ca2+ [28]. Hence, substances that evoke non-specific inhibition, like Ca2+
antagonists, are considered to more effectively suppress
gut motility, whereas muscarinic receptor antagonists
produce partial inhibition, as cholinergic innervation is
one of the several mechanisms responsible for regulating
intestinal motility [29,30]. This suggests the presence of
a non-specific spasmolytic component(s), most likely
Ca2+ antagonist in H. niger in addition to the anticholinergic constituents (hyoscine and hyoscyamine). This
speculation was strengthened when the plant extract
reversed both carbachol and K+-induced contractions
(being more potent against carbachol), indicating
the presence of at least two different spasmolytic
mechanisms, similar to dicyclomine, a dual blocker of
muscarinic receptors and Ca2+ influx [31]. Verapamil,
however, was found to be more potent against the
contractions of K+ than carbachol, and atropine relaxed
carbachol-induced contractions only.
High K+ (>30 mM) is known to cause smooth muscle
contractions through opening of voltage-dependent
L-type Ca2+ channels, thus allowing influx of extracellular Ca2+ [32] and a substance that inhibits high K+
contractions is considered as a blocker of Ca2+ influx
[33]. The Ca2+ antagonist effect was further confirmed
when H. niger extract caused a rightward shift in the
Ca2+ concentrationresponse curves similar to that
caused by verapamil and dicyclomine.
The concept of a dual mode of inhibition involving
anticholinergic and Ca2+ channel blockade received

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

Anticholinergic and Ca2+ antagonist activities of Hyoscyamus niger

95

Trachea

Urinary bladder
(c)

100

75

50

25

100

% of control

% of control

(a)

75

50

25

CCh (1 M)
K (80 mM)

0
0.1

10

100

0.1

[Hn.Cr] g/mL
100
75

(d)
Control
Hn.Cr 1 g/mL

100

Hn.Cr 3 g/mL

50
25
0

10

100

[Hn.Cr] g/mL

% of CCh max.

Figure 5 (a, c) represents concentration-dependent inhibitory effect of the


crude extract of Hyoscyamus niger
(Hn.Cr) on carbachol (CCh) and K+induced contractions, while (b) and
(d) show CCh concentrationresponse
curves in the absence and presence of
Hn.Cr in isolated guinea-pig tracheal
and rabbit urinary bladder preparations.
Symbols represent mean SEM,
n = 46.

% of CCh max.

(b)

CCh (1 M)
K+ (80 mM)

Control
Hn.Cr 1 g/mL
Hn.Cr 3 g/mL

75
50
25
0

8.5 7.5 6.5 5.5 4.5 3.5

8.5 7.5 6.5 5.5 4.5 3.5

Log [CCh] M

Log [CCh] M

additional support from acetylcholine concentration


response data in guinea-pig ileum, as this preparation is
considered more useful to quantify contractile responses
of an agonist in the presence of an inhibitor, thus
allowing exploration of the nature of antispasmodic
effect [22]. The plant extract at a low concentration
caused a rightward parallel shift in the acetylcholine
curves without suppressing the maximum response, a
characteristic of a competitive or specific antagonist, like
atropine [27], then at higher concentration caused a
non-parallel shift with suppression of the maximum
effect, pointing towards an additional non-competitive
inhibition [34], as expected for Ca2+ antagonists [35,36].
Dicyclomine also shifted the acetylcholine curves similar
to that of extract, while verapamil resulted in a rightward but non-parallel shift with suppression of the
maximum contractile effect at both concentrations used.
Atropine caused a rightward parallel shift of the acetylcholine curves without suppression of the maximum
response. This confirms the presence of a combination of
muscarinic receptors and Ca2+ channel-blocking activities in H. niger, similar to dicyclomine.

In view of the medicinal use of H. niger in diarrhoea,


its extract was tested for possible protective effect against
castor oil-induced diarrhoea in mice. Diarrhoea induced
by castor oil is a result of the action of ricinoleic acid
formed during hydrolysis [37], which affects transport of
electrolytes and water and generates giant contractions
of the transverse and distal colon [38]. The observed
antidiarrhoeal effect of H. niger extract, following oral
administration, could have resulted from a local or a
combination of local and systemic actions. H. niger also
protected the mice against castor oil-induced intestinal
fluid secretions. This is expected, as both anticholinergic
drugs and Ca2+ antagonists possess an antidiarrhoeal
and antisecretory actions [3941].
As H. niger has uses in hyperactive respiratory and
urogenital ailments, the plant extract was further studied
for possible bronchodilatory and urinary bladder relaxant activity. In trachea and bladder preparations, as with
the gut, the extract inhibited both carbachol and K+induced contractions and displaced carbachol curves to
the right in a parallel fashion without suppression of the
maximum response at low and higher concentrations it

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

A. H. Gilani et al.

96

Jejunum
100

(b) 100

75

75

% of control

% of control

(a)

50
CCh (1 M)

25

50
CCh (1 M)

25

K+ (80 mM)

K+ (80 mM)

0
0.3

(d)

100
75

10
100
1000
[Hn.EtAc] g/mL

100
75

50
CCh (1 M)

25

% of control

% of control

(c)

3
30
[Hn.PE] g/mL

50
CCh (1 M)

25

K+ (80 mM)

K+ (80 mM)

0.3

300
3
30
[Hn.MeOH] g/mL

0.1

caused a non-parallel shift with suppression of the


maximum effect. Interestingly, the plant extract was
found to be a more potent spasmolytic in trachea and
bladder than in the gut (Table II). Tissue-selective
behaviour of H. niger could be due to a Ca2+ antagonist
component, as Ca2+ channels are known to be heterogeneous and different Ca2+ antagonists have selectivity
for different organ systems [20,42]. A parallel shift of the
cholinergic agonists concentrationresponse curves
constructed in different tissues by similar concentrations
of the plant extract may rule out any possibility of the
tissue-selective nature of the anticholinergic component,
as expected from the non-selective profile of anticholinergic constituents of H. niger, i.e. hyoscine and hyoscyamine [43]. Alternatively, a possibility exists that a
difference in physiological modulators among various
tissues and/or the extent of their regulatory influences
[44] may cause plant constituents to exert a better
synergistic interaction in trachea and bladder compared
with the gut.

1
10
[Hn.Aq] g/mL

Figure 6 Concentration-response
curves showing effect of the Hyoscyamus
niger fractions: (a) petroleum ether
(Hn.PE); (b) ethylacetate (Hn.EtAc);
(c) methanolic (Hn.MeOH) and (d)
aqueous (Hn.Aq) on carbachol (CCh)
and K+-induced contractions in isolated
rabbit jejunum preparations. Symbols
represent mean SEM, n = 35.

The presence of a Ca2+ channel-blocking component


in H. niger reported here for the first time may enhance
the medicinal benefits of the plant, as Ca2+ antagonists
have the potential to depress cardiac arrhythmias, which
usually occur when anticholinergics are used alone [45].
In smooth muscles, a combination of anticholinergic and
Ca2+ antagonist components could synergize, making
the plant more effective for therapy against spasmodic
conditions [16]. For instance dicyclomine, which
possesses dual muscarinic receptors and Ca2+ channelblocking action, is considered to be a more efficacious
spasmolytic, compared with pure anticholinergics [46].
The activity-directed fractionation study revealed that
the Ca2+ antagonist component, as opposed to the
anticholinergic was concentrated in the petroleum ether
fraction (around 10 times more potent than the parent
extract), while the aqueous fraction was devoid of it. This
observation supports our previous findings that the Ca2+
channel-blocking component is concentrated in the
organic fractions [47,48]. The relative potency difference

2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 8799

Anticholinergic and Ca2+ antagonist activities of Hyoscyamus niger

Table II Comparative inhibitory effects of the crude extract of


Hyoscyamus niger (Hn.Cr) on different isolated tissue preparations.

Jejunum
(a)

97

100
Preparation
Jejunum

% of control

75
Trachea

50

Bladder

25

CCh (1 lM)

7.2 (4.412), n = 4

K+ (80 mM)

300 (209.8428.4), n = 4

CCh (1 lM)

0.8 (0.51.3), n = 4

K+ (80 mM)

12.3 (7.918.9), n = 5

CCh (1 lM)

0.6 (0.31.0), n = 5

K+ (80 mM)

6.2 (4.29.1), n = 5

Values represent geometric means along with 95% confidence intervals in

K+ (80 mM)

parenthesis. n represents number of observations.

0.03

30
300
3
0.3
[-sitosterol] g/mL

(b) 100

75
% of control

EC50 values (95% CI; lg/mL)

CCh (1 M)

50

25

Parameter

CCh (1 M)
K+ (80 mM)

0
0.0003

0.003
0.03
[Scopolamine] g/mL

0.3

(c) 100

against carbachol and K+-induced contractions in the


organic fractions was less than that in the parent
extract, probably because the Ca2+ antagonist component is entirely concentrated in these fractions, while
anticholinergic component is distributed in both organic
and aqueous fractions. Among the tested plant constituents, b-sitosterol was found to possess Ca2+ antagonist
effect, though the presence of additional compounds with
Ca2+ channel-blocking mechanisms cannot be ruled out.
In conclusion, H. niger offers an interesting combination of spasmolytic mechanisms (anticholinergic and
Ca2+ antagonist) which might be responsible for its
medicinal use in treating disorders of the gut and
airways, and urinary bladder hyperactivity. The selectivity for the latter two preparations presents an interesting scenario, which warrants further studies to find
out whether this tissue-selective behaviour of the plant is
due to the selectivity of the Ca2+ antagonist component
or the better synergistic interaction of the plant-active
constituents in trachea and urinary bladder.

% of control

75

ACKNOWLEDGEMENTS
50

25
CCh (1 M)
K+ (80 mM)

0
0.0003

0.003
0.03
[Hyoscyamine] g/mL

0.3

Figure 7 Concentrationresponse curves showing the effect of


Hyoscyamus niger active ingredients: (a) b-sitosterol; (b) scopolamine; and (c) hyoscyamine on carbachol (CCh) and K+ (80 mM)induced contractions in isolated rabbit jejunum preparations.
Symbols represent mean SEM, n = 45.

This study was supported by funds made available by the


Higher Education Commission of Pakistan under the
scheme of Distinguished National Professor Research
Allowance. We would like to thank Prof. John Connor,
Department of Biological and Biomedical Sciences, Aga
Khan University Medical College, Karachi for editorial
suggestions.
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