gastrointestinal selective airways and urinary bladder relaxant

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Department of Biological & Biomedical Sciences Medical College, Pakistan

February 2008

Gastrointestinal, selective airways and urinary bladder relaxant eects of Hyoscyamus niger are

mediated through dual blockade of muscarinicreceptors 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

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Recommended CitationGilani, A., Khan, A., Raoof, M., Ghayur, M., Siddiqui, B., Vohra, W., Begum, S. (2008). Gastrointestinal, selective airways and urinary bladder relaxant eects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca(2+) channels.Fundamental & Clinical Pharmacology, 22(1), 87-99.

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Authors

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

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doi: 10.1111/j.1472-8206.2007.00561.x

O R I G I N A L

A R T I C L E

Gastrointestinal, selective airways and

urinary bladder relaxant effects of

Hyoscyamus niger are mediated through

dual blockade of muscarinic receptorsand Ca2+

channels

Anwarul Hassan Gilania*, Arif-ullah Khana,b, Mustafa Raoof a,

Muhammad Nabeel Ghayura,c, Bina S. Siddiquid, Waseem Vohrad,

Sabira Begumd

aNatural Product Research Division, Department of Biological and Biomedical Sciences, The Aga Khan University Medical

College, Karachi – 74800, Pakistanb

Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi, PakistancDepartment of Medicine, McMaster University, St. Joseph’s Hospital, Hamilton, ON, CanadadHEJ Research Institute of Chemistry, University of Karachi, Karachi – 75270, Pakistan

Keywords

airways and bladder selec-

tivity,

anticholinergic,

Ca2+ antagonist,

Hyoscyamus niger

Received 21 June 2007;

revised 13 September 2007;

accepted 13 November 2007

*Correspondence and reprints:

[email protected]

A B S T R A C T

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) i n a

pattern similar to that of dicyclomine, but different from verapamil and atropine.

Hn.Cr shifted the Ca2+

concentration–response curves to the right, similar to that

caused by verapamil and dicyclomine, suggesting a Ca2+

channel-blockingmechanism 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 non-

parallel 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 oil-

induced 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 antispas-modic 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.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 87–99 87



I N T R O D U C T I O N

Hyoscyamus niger Linn. (Solanaceae), commonly

known as ‘Henbane’ and ‘Ajwain-i-khurasani’ locally

in Pakistan, grows wildly throughout the Himalayan

range at altitudes of 8000–11 000 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 blad-

der [2,3].

Phytochemical studies revealed the presence of

hyoscine (scopolamine), hyoscyamine, anisodine,

anisodamine [4,5], aesculetin, coumarin, kaempferol,

quercetin, rutin, cuscohygrine, chlorogenic acid, lino-

leic acid, myristic acid, oleic acid, stearic acid, pyridine,

trimethylamine [6], b-sitosterol, grossamide, canna-

bisin D & G, daucosterol, N -trans-feruloyltyramine,

1-O-octadecanoyl glycerol, 1-O-(9Z,12Z-octadecadienoyl)

glycerol, 1-O-(9Z,12Z-octadecadienoyl)-3-O-(9Z-octa-

decenoyl) 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 anantispasmodic 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 mecha-

nisms [14–16]. 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 distrib-

uted both in the organic and aqueous fractions, whileCa2+ 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.

M A T E R I A L S A N D M E T H O D S

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 pow-

dered 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%. Activity-

guided 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 Na2

SO4

(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 petro-

leum 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 metha-

nol 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

88 A. H. Gilani et al.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 87–99



prepared in distilled H2O. Vehicles used had no effect on

tissue contractility in control experiments.

Experimental animals

Animals used in this study, adult rabbits (1.0–1.2 kg),

guinea-pigs (500–550 g) and BALB/c mice (20–25 g)

of either sex and local breed, were housed at the

Animal House of the Aga Khan University and main-

tained at 23–25 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, vegeta-

ble 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 Tyrode’s solution and

cleaned of mesentery tissues [19]. Segments about 2 cm

long were suspended individually in a 10-mL tissue bath

containing Tyrode’s solution maintained at 37 C and

aerated with a mixture of 95% O2 and 5% CO2 (carbo-gen). The composition of Tyrode’s 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 acetyl-

choline (0.3 lM) at 3-min intervals until constant

responses were recorded. Under these experimental

conditions, rabbit jejunum exhibits spontaneous rhyth-

mic contractions, allowing the testing of relaxant (spas-molytic) 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].

To confirm the Ca2+ antagonist action of the test

substance, the tissue was allowed to stabilize in normal

Tyrode’s solution, which was then replaced with Ca2+-

free Tyrode’s solution containing ethylenediaminetetra-

acetic 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 Tyrode’s solution, having the compo-

sition (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

concentration–response curves of Ca2+ were constructed.

When the control Ca2+ concentration–response 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 concen-

tration–response 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 Tyrode’s solution.

The segments, about 2 cm long, were mounted individ-

ually in a 10-mL tissue bath, filled with Tyrode’s

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 repeatedlytreated 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 be-

tween doses. Acetylcholine curves were then re-deter-

mined in the presence of increasing concentrations of

test and control drugs as described previously [22].

Guinea-pig trachea

Trachea was dissected and kept in Kreb’s solution. The

tracheal tube was cut into rings, 2–3 mm wide, eachcontaining 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 Kreb’s solution at 37 C, aerated with

carbogen. The composition of Kreb’s solution was (mM):

NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4

Anticholinergic and Ca2+

antagonist activities of Hyoscyamus niger 89




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 concentration–response 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 30–35 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 Krebs–Henseleit 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, insome 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 diarrhoeaMice 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 admin-

istration 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 concen-

trations (EC50 values) with 95% confidence intervals. The

statistical parameters applied were Student’s 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).

R E S U L T S

Effect on jejunum

Figure 1a compares inhibitory effects of Hn.Cr with those

of dicyclomine, verapamil and atropine on the sponta-

neous contractions of isolated rabbit jejunum. The

respective EC50 values were 5.6 lg/mL (4.1–7.6, 95%

confidence interval, n = 5), 0.53 lM (95% CI 0.40–0.70,

n = 4), 0.23 lM (95% CI 0.17–0.32, n = 5) and

0.001 lM (95% CI 0.0008–0.01, n = 4). The spasmo-

lytic effect was concentration-dependent with the excep-

tion of atropine, where further increments in theconcentration 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 4.4–12, n = 4) as compared with 300 lg/mL (95% CI

209.8–428.4, n = 4) for K+. Dicyclomine also showed a

similar pattern of inhibition (Figure 1c) with respective





Jejunum

0

25

50

75

100

0.0003 0.003 0.03

[Atropine] µM

% o

f c o n t r o l

0

25

50

75

100

0.3 3.0 30030

(b)

(a)

CCh (1 mM)

K+ (80 mM)

[Hn.Cr] µg/mL

% o

f c o n t r o l

% o

f c o n t r o l

0

25

50

75

100

0.01 0.1 101

(c)

[Dicyclomine] µM

0

25

50

75

100

0.001

Atropine

Verapamil

Dicyclomine

Hn.Cr

Control 1 min

0.03

0.3 1 3 10 30 100 µg/mL

0.1 0.3 1 3

0.03 0.1 0.3 1

0.003 0.01 0.03 0.1 0.3 1 3 µ M

µ M

µ M

0.003 0.03 0.3

(d) (e)

[Verapamil] µM

% o

f c o n t r o l

CCh (1 µM)

K+ (80 mM)

CCh (1 µM)

K+ (80 mM)

CCh (1 µM)

K+ (80 mM)

Figure 1 (a) Tracing showing spasmolytic effect of the crude extract of Hyoscyamus niger (Hn.Cr), dicyclomine, verapamil and atropine on

spontaneous contractions and concentration–response 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 = 4–8.






EC50 values of 0.13 (95% CI 0.11–0.19, n = 6) and

3.2 lM (95% CI 1.9–5.3, n = 5). Verapamil on the other

hand, was more potent against K+-induced contractions

with EC50 value of 0.04 lM (95% CI 0.03–0.05, n = 5),

compared with carbachol [0.16 lM (95% CI 0.13–0.19,

n = 5)] as shown in Figure 1d . Atropine relaxed the

carbachol-induced contractions potently with EC50 value

of 0.003 lM (95% CI 0.002–0.004, 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).

Effect on ileum

Hn.Cr at 1.0 lg/mL caused a rightward parallel shift in

the acetylcholine concentration–response curves with-

out 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 (0.01–0.03 lM) produced a non-parallel

rightward shift with suppression of the maximum

response (Figure 3c) and atropine (0.01–0.03 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 (300–1000 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 intestinal fluid accumulation

When tested against castor oil-induced intestinal fluid

accumulation in mice, Hn.Cr had a dose-dependent

(300–1000 mg/kg) antisecretory effect (Figure 4). Intes-

tinal fluid accumulation in the saline-treated group was

114.4 ± 0.77 g, while in castor oil-treated group it was

Jejunum

0

25

50

75

100(a) (b)

(c) (d)

Control

Hn.Cr 100 µg/mL

Hn.Cr 300 µg/mL

% o

f c o n t r o l m a x .

0

25

50

75

100 Control

Verapamil 0.03 µM

Verapmil 0.1 µM

% o

f c o n t r o l m a x .

0

25

50

75

100 Control

β-sitosterol 100 µg/mL

β-sitosterol 300 µg/mL

% o

f c o n t r o l m

a x .

–4.5 –3.5 –2.5 –1.5

0

25

50

75

100ControlDicyclomine 3 µM

Dicyclomine 10 µM

Log [Ca++] M

–4.5 –3.5 –2.5 –1.5

Log [Ca++] M

–4.5 –3.5 –2.5 –1.5

Log [Ca++] M

–4.5 –3.5 –2.5 –1.5

Log [Ca++] M

% o

f c o n t r o l m

a x .

Figure 2 Concentration–response

curves of Ca2+ in the absence and

presence of the increasing concentra-

tions of (a) crude extract of Hyoscyamus

niger (Hn.Cr); (b) verapamil; (c) dicyclo-

mine and (d) b-sitosterol in isolated

rabbit jejunum preparations. Symbols






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 0.5–1.3, n = 4), compared

with that against K+ (80 mM) contractions with EC50

value of 12.3 lg/mL (95% CI 7.9–18.9, n = 5) as shownin Figure 5a. Similar to that in ileum, Hn.Cr produced a

rightward parallel displacement of the carbachol curves

without suppression of the maximum contractile re-

sponse 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 0.3–1.0, n = 5) and 6.2 lg/mL (95% CI 4.2–9.1,

n = 5) as shown in Figure 5c. Hn.Cr caused a rightwardparallel 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

Ileum

0

25

50

75

100Control

Atropine(0.01 µM)

Atropine(0.03 µM)

% o

f A C h m a x .

0

25

50

75

100Control

Dicyclomine

(0.03 µM)

Dicyclomine

(0.1 µM)

% o

f A C h

m a x .

0

25

50

75

100(a) (b)

(c) (d)

Control

Hn.Cr(1 µg/mL)

Hn.Cr

(3 µg/mL)

% o

f A C h m a x .

–8 –7 –6 –5 –4

0

25

50

75

100Control

Verapmil(0.01 µM)

Verapmil(0.03 µM)

Log [ACh] M

–8 –7 –6 –5 –4

Log [ACh] M

–8 –7 –6 –5 –4

Log [ACh] M

–8 –7 –6 –5 –4

Log [ACh] M

% o

f A C h m a x

.

Figure 3 Concentration–response

curves of acetylcholine (ACh) in the

absence and presence of (a) crude extract

of Hyoscyamus niger (Hn.Cr); (b) dicyclo-

mine; (c) verapamil and (d) atropine in

isolated guinea-pig ileum preparations.

Symbols represent mean ± SEM,

n = 3–6.

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 % Protection

Saline (10 mL/kg) + (c.oil) 5 0

Hn.Cr (300 mg/kg) + (c.oil) 3 40

Hn.Cr (1000 mg/kg) + (c.oil) 1* 80

Loperamide (10 mg/kg) + (c.oil) 0** 100

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






(Figure 6a–c) with respective EC50 values of 9.0 (95% CI

6.0–13.6, n = 3) and 59.6 (95% CI 30.2–117.8, n = 3),

236.8 (95% CI 171.3–327.2, n = 3) and 1117 (95% CI

738.7–1690, n = 3), 11.7 (95% CI 7.1–19.1, n = 3)

and 130.3 lg/mL (95% CI 93.3–226.4, n = 5), while

Hn.Aq relaxed carbachol (1 lM) contractions with EC50

value of 8.6 lg/mL (95% CI 6.2–12.0, n = 4) without

any effect on K+ (80 mM), n = 5 (Figure 6d ).

Effect of pure compounds

Some of the known commercially available pure com-

pounds 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 7.1–13.1,

n = 5), when compared with carbachol [218.6 lg/mL

(95% CI 129.5–369.0), n = 4] and shifted Ca2+ concen-

tration–response curves to the right (Figure 2d ). Scopol-

amine and hyoscyamine relaxed only carbachol (1 lM)

contractions with respective EC50 values of 0.004 lg/mL

(95% CI 0.003–0.005, n = 6) and 0.001 lg/mL (95% CI

0.0009–0.002, n = 4) without any effect on K+ (Fig-

ure 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.

D I S C U S S I O N

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 spasmo-lytics [14–16], 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 cholecystokinins which achieve their contractile effects through an

ultimate increase in cytosolic Ca2+ [28]. Hence, sub-

stances 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 anticho-

linergic 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 extracel-

lular 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+ concentration–response curves similar to that

caused by verapamil and dicyclomine.

The concept of a dual mode of inhibition involving

anticholinergic and Ca2+ channel blockade received

300 1000

100

105

110

115

120

125

Saline

Castor oil

Castor oil + Hn.Cr

*

#

[Hn.Cr] mg/kg

I n t e s t i n a l f l u i d a c c u m u l a t i o n

( P i / P m ) x 1 0 0 0

***

Figure 4 Effect of increasing doses of the crude extract of

Hyoscyamus niger (Hn.Cr) on castor oil-stimulated fluid accumula-tion in small intestine of mice. Results shown are mean ± SEM of

five animals for each experimental group. Intestinal fluid accumu-

lation 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,

Student’s t-test.





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 similarto that of extract, while verapamil resulted in a right-

ward but non-parallel shift with suppression of the

maximum contractile effect at both concentrations used.

Atropine caused a rightward parallel shift of the acetyl-

choline curves without suppression of the maximum

response. This confirms the presence of a combination of

muscarinic receptors and Ca2+ channel-blocking activ-

ities 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 [39–41].As H. niger has uses in hyperactive respiratory and

urogenital ailments, the plant extract was further studied

for possible bronchodilatory and urinary bladder relax-

ant 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

Trachea Urinary bladder

0

25

50

75

100(a) (c)

(b) (d)

K+ (80 mM)

CCh (1 µM)

% o

f c o

n t r o l

0

25

50

75

100

K+ (80 mM)

CCh (1 µM)

0.1 101 100

[Hn.Cr] µg/mL0.1 101 100

[Hn.Cr] µg/mL

% o

f c o

n t r o l

0

25

50

75

100Control

Hn.Cr 1 µg/mL

Hn.Cr 3 µg/mL

% o

f C C h m a x .

–8.5 –7.5 –6.5 –5.5 –4.5 –3.5

0

25

50

75

100Control

Hn.Cr 1 µg/mL

Hn.Cr 3 µg/mL

Log [CCh] M

–8.5 –7.5 –6.5 –5.5 –4.5 –3.5

Log [CCh] M

% o

f C C h m a

x .

Figure 5 (a, c) represents concentra-tion-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 concentration–response

curves in the absence and presence of

Hn.Cr in isolated guinea-pig tracheal

and rabbit urinary bladder preparations.

Symbols represent mean ± SEM,

n = 4–6.






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 hetero-

geneous and different Ca2+ antagonists have selectivity

for different organ systems [20,42]. A parallel shift of the

cholinergic agonists’ concentration–response 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 anticholin-

ergic constituents of H. niger , i.e. hyoscine and hyoscy-

amine [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.

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+ channel-

blocking 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

Jejunum

0

25

50

75

100

0.1 1 10

[Hn.Aq] µg/mL

% o

f c o n t r o l

0

25

50

75

100

CCh (1 µM)

K+ (80 mM)

0.3 3 30 300

[Hn.MeOH] µg/mL

% o

f c o n t r o l

0

25

50

75

100

1 10 100 1000

[Hn.EtAc] µg/mL

% o

f c o n t

r o l

0

25

50

75

100(a) (b)

(c) (d)

K+ (80 mM)

CCh (1 µM)

K+ (80 mM)

CCh (1 µM)

K+ (80 mM)

CCh (1 µM)

0.3 3 30

[Hn.PE] µg/mL

% o

f c o n t r o

l

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






against carbachol and K+-induced contractions in the

organic fractions was less than that in the parent

extract, probably because the Ca2+ antagonist compo-

nent is entirely concentrated in these fractions, while

anticholinergic component is distributed in both organicand aqueous fractions. Among the tested plant constit-

uents, 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 combina-

tion 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 selec-

tivity for the latter two preparations presents an inter-

esting scenario, which warrants further studies to find

out whether this tissue-selective behaviour of the plant isdue to the selectivity of the Ca2+ antagonist component

or the better synergistic interaction of the plant-active

constituents in trachea and urinary bladder.

A C K N O W L E D G E M E N T S

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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0

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CCh (1 µM)

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