Mechanisms underlying the antispasmodic and bronchodilatory

eCommons@AKU
Department of Biological & Biomedical Sciences
Medical College, Pakistan
March 2008
Mechanisms underlying the antispasmodic and
bronchodilatory properties of Terminalia bellerica
fruit
Anwar Gilani
Aga Khan University
Arif-ullah Khan
Aga Khan University
Tuba Ali
Aga Khan University
Saad Ajmal
Aga Khan University
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Recommended Citation
Gilani, A., Khan, A., Ali, T., Ajmal, S. (2008). Mechanisms underlying the antispasmodic and bronchodilatory properties of Terminalia
bellerica fruit. Journal of Ethnopharmacology, 116(3), 528-538.
Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/99
Available online at www.sciencedirect.com
Journal of Ethnopharmacology 116 (2008) 528–538
Mechanisms underlying the antispasmodic and bronchodilatory
properties of Terminalia bellerica fruit
Anwarul Hassan Gilani a,∗ , Arif-ullah Khan a,b , Tuba Ali a , Saad Ajmal a
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
Received 21 September 2007; received in revised form 17 December 2007; accepted 4 January 2008
Available online 16 January 2008
Abstract
Aim of the study: The present investigation was carried out to provide the pharmacological basis for the medicinal use of Terminalia bellerica in
hyperactive gastrointestinal and respiratory disorders.
Materials and methods: Crude extract of Terminalia bellerica fruit (Tb.Cr) was studied in in vitro and in vivo.
Results: Tb.Cr caused relaxation of spontaneous contractions in isolated rabbit jejunum at 0.1–3.0 mg/mL. Tb.Cr inhibited the carbachol (CCh,
1 ␮M) and K+ (80 mM)-induced contractions in a pattern similar to that of dicyclomine, but different from nifedipine and atropine. Tb.Cr shifted
the Ca++ concentration–response curves to right, like nifedipine and dicyclomine. In guinea-pig ileum, Tb.Cr produced rightward parallel shift of
acetylcholine-curves, followed by non-parallel shift at higher concentration with the suppression of maximum response, similar to dicyclomine, but
different from nifedipine and atropine. Tb.Cr exhibited protective effect against castor oil-induced diarrhea and carbachol-mediated bronchoconstriction in rodents. In guinea-pig trachea, Tb.Cr relaxed the CCh-induced contractions, shifted CCh-curves to right and inhibited the contractions
of K+ . Anticholinergic effect was distributed both in organic and aqueous fractions, while CCB was present in the aqueous fraction.
Conclusions: These results indicate that Terminalia bellerica fruit possess a combination of anticholinergic and Ca++ antagonist effects, which
explain its folkloric use in the colic, diarrhea and asthma.
© 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Terminalia bellerica fruit; Anticholinergic; Ca++ antagonist; Gastrointestinal and respiratory disorders
1. Introduction
Terminalia bellerica Roxb. (family: Combretaceae) commonly known as “belleric myrobalan” and locally as “bahera” is
a large deciduous tree, found throughout Central Asia and some
other parts of the world (Kapoor, 1990). Its fruit is used in folk
medicine to treat anemia, asthma, cancer, colic, cough, diarrhea, dyspepsia, dysuria, headache, hoarseness, hypertension,
inflammations and rheumatism. The half ripe fruit is considered
purgative. It is one of the constituent of “Triphala” which is
prescribed in the diseases of the liver and gastrointestinal tract
(Usmanghani et al., 1997; Duke et al., 2002).
∗
Corresponding author. Tel.: +92 21 4864571;
fax: +92 21 493 4294/494 2095.
E-mail address: [email protected] (A.H. Gilani).
0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2008.01.006
The plant is reported to contain termilignan, thannilignan, 7-hydroxy-3 ,4 -(methylenedioxy) flavone and anolignan
B (Valsaraj et al., 1997), gallic acid, ellagic acid, ␤sitosterol (Anand et al., 1997), arjungenin, belleric acid,
bellericoside (Nandy et al., 1989) and cannogenol 3-O-␤d-galactopyranosyl-(1 → 4)-O-␣-l-rhamnopyranoside (Yadava
and Rathore, 2001).
Terminalia bellerica is scientifically proven to possess
antioxidant activity (Saleem et al., 2001). The plant is known
to lower the levels of lipid in hypercholesterolemic animals
(Thakur et al., 1988; Shaila et al., 1995). It was found effective
against several pathogens including Bacillus subtilis, Proteus
vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa,
Escherichia coli and Staphylococcus aureus (Ahmad et al.,
1998). It exhibited inhibitory effect on human immunodeficiency virus-1 reverse transcriptase (El-Mekkawy et al., 1995).
Terminalia bellerica reduced the serum glucose level both in normal and alloxan-induced diabetic rats (Sabu and Kuttan, 2002)
A.H. Gilani et al. / Journal of Ethnopharmacology 116 (2008) 528–538
and also showed preventive effect against the myocardial necrosis in rats (Tariq et al., 1977). Dwivedi et al. (1994), Srivastava
et al. (1992) and Khan and Gilani (in press) reported that Terminalia bellerica lowers the arterial blood pressure. A water
soluble fraction obtained from the defatted fruits of Terminalia
bellerica caused protection against CCl4 -induced liver injury in
rodents (Anand et al., 1994). In a clinical study, Terminalia bellerica was found to possess antispasmodic, antiasthmatic and
antitussive effects (Trivedi et al., 1979). A polyherbal formulation containing Terminalia bellerica exhibited antimutagenic
activity (Kaur et al., 2002).
We have experienced that plants with medicinal use in
hyperactive gut and airways disorders usually exhibit spasmolytic effect through combination of mechanisms (Gilani et
al., 2005a,b, 2008). In this investigation, we report the presence
of a combination of anticholinergic and Ca++ antagonist effects
in the Terminalia bellerica, which justify its medicinal use in
such conditions.
2. Materials and methods
2.1. Plant material, preparation of crude extract and
fractions
The fruits of Terminalia bellerica were bought from a
local market in Dhaka (Bangladesh) and was authenticated
by Assistant Professor, Mehboob-ur-Rehman, Department of
Botany, Govt. Postgraduate Jehanzeb College, Saidu Sharif,
Swat, N.W.F.P., Pakistan. A sample voucher (TB-FR-10-95-30)
was submitted to the herbarium of Department of Biological
and Biomedical Sciences, Aga Khan University, Karachi. After
cleaning of adulterant material, 432 g of fruits were crushed
and soaked in 3 L of 70% aqueous-methanol for three days
with occasional shaking. It was filtered through a muslin cloth
and then through a Whatman qualitative grade 1 filter paper
(Williamson et al., 1998). This procedure was repeated thrice
and the combined filtrate was evaporated on rotary evaporator to obtain the crude extract (Tb.Cr), yielding approximately
9.25%.
Activity-guided fractionation was carried out by using solvents of increasing polarity to obtain organic and aqueous
fractions. Tb.Cr was dissolved in about 150 mL of distilled water.
The chloroform was added to it with vigorous shaking. The chloroform layers (lower) were collected thrice and evaporated on
rotary evaporator to give the chloroform fraction (Tb.CHCl3 ).
The other layer (upper) was again taken into a separating funnel, ethyl acetate was added into it, separated and was also
evaporated in rotary evaporator to give the ethyl acetate fraction (Tb.EtAc). The remaining lower layer was collected and
evaporated to obtain the aqueous fraction (Tb.Aq).
2.2. Drugs and animals
Acetylcholine (ACh), atropine, carbachol (CCh), dicyclomine, loperamide and nifedipine were purchased from
Sigma Chemicals Co., St. Louis, MO, USA. Salbutamol,
pentothal sodium (thiopental sodium) and castor oil were respec-
529
tively obtained from Glaxo Wellcome, Abbot Laboratories and
KCL Pharma, Karachi, Pakistan. Chemicals used for making
physiological salt solutions were potassium chloride (Sigma
Chemicals Co.), calcium chloride, glucose, magnesium chloride,
magnesium sulfate, potassium dihydrogen phosphate, sodium
bicarbonate, sodium dihydrogen phosphate (Merck, Darmstadt,
Germany) and sodium chloride from BDH Laboratory Supplies,
Poole, England. All chemicals used were of the analytical grade
available and solubilized in distilled water.
Animals used in this study such as Chinchilla rabbits (1–1.2 kg), Himalayan guinea-pigs (500–550 g),
Sprague–Dawley rats (200–250 g) and Balb-C mice (20–25 g)
of either sex were housed at the Animal House of the Aga Khan
University, maintained at 23–25 ◦ C. Animals were given tap
water ad libitum and a standard diet. Rabbits and guinea-pigs
had free access to water, but food was withdrawn 24 h prior
to experiment and sacrificed 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.
2.3. Isolated tissue preparations
2.3.1. Rabbit jejunum
The jejunum was dissected out, kept in Tyrode’s solution and
cleaned of mesenteries (Ghayur and Gilani, 2005). Each segment
of about 2 cm length was suspended in a 10 mL tissue bath containing Tyrode’s solution, maintained at 37 ◦ C and aerated with a
mixture of 95% oxygen and 5% carbon dioxide (carbogen). The
composition of the Tyrode’s solution in mM was KCl 2.68, NaCl
136.9, MgCl2 1.05, NaHCO3 11.90, NaH2 PO4 0.42, CaCl2 1.8
and glucose 5.55. Intestinal responses were recorded isotonically using Bioscience transducers and oscillograph. Each tissue
was allowed to equilibrate for at least 30 min before the addition
of any drug and then stabilized with a sub-maximal concentration of ACh (0.3 ␮M) with a 3 min interval in between 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 Ca++ channel blocking (CCB) activity, high K+ (80 mM) was used to depolarize the preparations
as described by Farre et al. (1991). 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 a substance causing inhibition of high K+ -induced contraction is considered as a blocker of Ca++ influx (Godfraind et al.,
1986). To elucidate the presence of any additional spasmolytic
effect, the plant extract was tested on CCh-induced contractions.
CCh is a cholinergic agonist which causes smooth muscle contraction through activation of muscarnic receptors (Jenkinson,
2002). Once plateau of the induced contractions was achieved
(usually within 5–10 min), the test materials were added in a
cumulative fashion to obtain concentration-dependent inhibitory
responses (Van-Rossum, 1963).
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To confirm the Ca++ antagonist property of the test substance,
the tissue was allowed to stabilize in normal Tyrode’s solution,
which was then replaced with Ca++ -free Tyrode’s solution containing EDTA (0.1 mM) for 30 min in order to remove Ca++
from the tissues. This solution was further replaced with K+ -rich
and Ca++ -free Tyrode’s solution, having the following composition (mM): KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90,
NaH2 PO4 0.42, glucose 5.55 and EDTA 0.1. Following an incubation period of 30 min, control concentration–response curves
(CRCs) of Ca++ (CaCl2 ) were obtained. When the control Ca++
CRCs were found super-imposable (usually after two cycles),
the tissue was pretreated with the crude extract for 60 min to
test the possible CCB effect. The CRCs of Ca++ were reconstructed in the presence of different concentrations of the test
material.
2.3.2. Guinea-pig ileum
The ileum was dissected out and kept in Tyrode’s solution.
The segments, each of about 2 cm length, were mounted individually 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.
After equilibration period of 30 min, each tissue preparation was
repeatedly treated with sub-maximal concentration (0.3 ␮M)
of ACh until constant responses were recorded. Control bolus
curves to ACh were then constructed by the addition of increasing concentrations of agonist. The ACh curves were then
re-determined in the presence of increasing concentrations of
the crude extract and control drugs (dicyclomine, nifedipine and
atropine) as described earlier (Choo and Mitchelson, 1978).
2.3.3. Guinea-pig trachea
The trachea was dissected out and kept in Kreb’s solution.
The tracheal tube was cut into rings, 2–3 mm wide. Each ring
was opened by a longitudinal cut on the ventral side opposite to
the smooth muscle layer, forming a tracheal strip with a central
part of smooth muscle in between the cartilaginous portions on
the edges. The preparation was then mounted in a 20 mL tissue bath containing Kreb’s solution, at 37 ◦ C and aerated with
carbogen. The composition of Kreb’s solution was (mM): NaCl
118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2 PO4 1.3, MgSO4
1.2 and glucose 11.7 (pH 7.4). A tension of 1 g was applied to
each of the tracheal strips and was kept constant throughout
the experiment. The tissue was equilibrated for 1 hr before the
addition of any drug. In some preparations, CCh (1 ␮M) and
K+ (80 mM) were used to stabilize the respective preparations
until constant responses of each agonist were achieved. Then
their sustained contractions were obtained and relaxant effect
of the crude extract and control drugs (dicyclomine, nifedipine
and atropine) was assessed by adding in a cumulative fashion.
Cumulative curves to CCh were constructed using increasing
concentration of agonist. When a 3-fold increase in concentration produced no further increment in response, the tissue
was washed to re-establish the base-line tension. The CCh
CRCs were repeated in the presence of increasing concentrations
of crude extract, dicyclomine, nifedipine and atropine (Gilani
et al., 1997). Isometric responses were recorded on a Grass
model 7 Polygraph (Grass Instrument Company, Quincy, MA,
USA).
2.4. In vivo experiments
2.4.1. Castor oil-induced diarrhea
Mice were fasted for 24 h before the experiment (Borelli et
al., 2006). The animals were housed in individual cages and
divided in four equal groups. The first group received saline as
the vehicle control (10 mL/kg, p.o.) and so acted as the negative
control. The dose of the crude extract was selected on a trial basis
and then two increasing doses of the crude extract were given
to the two different group animals. A fourth group of mice was
treated with loperamide (10 mg/kg, p.o.), as the positive control.
One hour after the treatment, each animal received 10 mL/kg
of castor oil orally through a feeding needle. Afterwards, the
cages were inspected for the presence of the typical diarrheal
droppings; their absence was noted as a positive result, indicating
protection from diarrhea at that time.
2.4.2. Bronchodilatory activity
Rats were anaesthetized with sodium thiopental
(80–100 mg/kg, i.p.), than incubated with a tracheal tube
and ventilated with a volume ventilator (Miniature ideal
pump, Bioscience, UK) adjusted at a rate of 70–80 strokes/min
(to deliver 7–10 mL/kg of room air) in the supine position
(Channa et al., 2005). A polyethylene catheter was inserted
into the jugular vein for drugs administration. Changes in
airways resistance was measured by connecting to a side
arm of tracheal cannula a pressure transducer (MLT1199)
and recorded by a PowerLab 4/25 via bridge amplifier (Quad
Bridge Amp, ML112) running Chart software, ver. 5.5 (ADInstruments, Bella Vista, NSW, Australia). Bronchoconstriction
was induced with carbachol (CCh 1 ␮M/kg), which was
reversed within 7–10 min. The test drug was given to the
animals 5–8 min prior to administration of CCh. The responses
were expressed as the per cent reduction of the CCh-induced
bronchospasm.
2.4.3. Acute toxicity test
Animals were divided in groups of five mice each. The
test was performed using increasing doses of the plant extract,
given orally, in 10 mL/kg volume to different groups serving as
test groups (Sanmugapriya and Venkataraman, 2006). Another
group of mice was administered saline (10 mL/kg, p.o.) as negative control. The mice were allowed food ad libitum and kept
under regular observation for 6 h while the lethality was recorded
after 24 h.
2.5. Statistical analysis
All the data expressed are mean ± standard error of mean
(S.E.M., n = number of experiments). Inhibitory effects are
expressed as the median inhibitory concentrations (IC50 ), while
A.H. Gilani et al. / Journal of Ethnopharmacology 116 (2008) 528–538
531
effects of the contractile agents as maximum effective concentrations (Emax) and median effective concentrations (EC50 )
with 95% confidence intervals (CI). CRCs were analyzed by
non-linear regression using GraphPad3 program (GraphPAD,
San Diego, CA, USA). The statistical parameter applied is Student’s t-test except in the antidiarrheal study where Chi-square
test was used. P < 0.05 was considered significantly different.
3. Results
3.1. Effect of Tb.Cr on rabbit jejunum
Tb.Cr, dicyclomine, nifedipine and atropine caused relaxation of rabbit jejunum spontaneous contractions with the
respective IC50 values of 1.2 mg/mL (0.9–1.7, 95% CI, n = 5),
0.6 ␮M (0.42–0.8, n = 4), 0.15 ␮M (0.10–0.22, n = 4) and
0.006 ␮M (0.002–0.01, n = 4) as shown in Fig. 1. When tested
against CCh (1 ␮M) and K+ (80 mM)-induced contractions,
Tb.Cr was found (Fig. 2A) to inhibit the CCh-induced contractions at lower concentration with IC50 value of 1.05 mg/mL
(0.6–1.8, n = 4) as compared to its effect against K+ with IC50
value of 8.1 mg/mL (4.7–14.1, n = 4). Dicyclomine, also showed
a similar pattern of inhibitory effect (Fig. 2B) with respective
IC50 values of 0.21 (0.14–0.31, n = 6) and 2.9 ␮M (1.9–4.7,
n = 6), whereas nifedipine was more potent against K+ -induced
contractions with IC50 value of 0.02 ␮M (0.015–0.03, n = 5),
when compared with CCh-induced contractions [0.20 ␮M
(0.12–0.29, n = 5)] as shown in Fig. 2C. Atropine relaxed the
CCh (1 ␮M)-induced contraction potently with IC50 value of
Fig. 1. Concentration–response curves representing spasmolytic effect of the
crude extract of Terminalia bellerica fruit (Tb.Cr), dicylomine, nifedipine and
atropine on spontaneous (Spont.) contraction of isolated rabbit jejunum preparations. Test material was added into the tissue bath in a cumulative fashion and
the concentrations shown are the final bath concentrations. Results are expressed
as mean ± S.E.M., n = 4–5.
0.0025 ␮M (0.002–0.004, n = 8), without any effect on K+
(80 mM)-induced contractions, n = 8 (Fig. 2D), as expected.
When tested for the possible interaction with Ca++ channels,
Tb.Cr produced rightward shift in the Ca++ CRCs (Fig. 3A),
similar to that caused by nifedipine (Fig. 3B) and dicyclomine
(Fig. 3C). Respective Emax values of Ca++ CRCS in the absence
and presence of Tb.Cr, nifedipine and dicyclomine are given in
Table 1.
Fig. 2. Concentration–response curves showing comparison of (A) crude extract of Terminalia bellerica fruit (Tb.Cr), (B) dicylomine, (C) nifedipine and (D) atropine
for the inhibitory effect against carbachol and K+ -induced contractions in isolated rabbit jejunum preparations. Results are expressed as mean ± S.E.M., n = 4–8.
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Fig. 3. Concentration–response curves of Ca++ in the absence and presence of the increasing concentrations of (A) crude extract of Terminalia bellerica fruit (Tb.Cr),
(B) nifedipine and (C) dicyclomine in isolated rabbit jejunum preparations. Results are expressed as mean ± S.E.M., n = 4.
3.2. Effect of Tb.Cr on guinea-pig ileum
Tb.Cr at 1.0 mg/mL caused a rightward parallel shift in
the ACh-curves without suppression of maximum contractile
response, followed by non-parallel shift with the suppression
of maximum response at 3.0 mg/mL (Fig. 4A). Dicyclomine
(0.03–0.1 ␮M) also showed a similar pattern of shift (Fig. 4B),
while nifedipine (0.03–0.1 ␮M) produced a non-parallel rightward shift with the suppression of the maximum response
(Fig. 4C). Atropine (0.01–0.03 ␮M) caused rightward parallel
Fig. 4. Concentration–response curves of acetylcholine in the absence and presence of (A) crude extract of Terminalia bellerica fruit (Tb.Cr), (B) dicyclomine, (C)
nifedipine and (D) atropine in isolated guinea-pig ileum preparations. Results are expressed as mean ± S.E.M., n = 4.
A.H. Gilani et al. / Journal of Ethnopharmacology 116 (2008) 528–538
533
Table 1
The maximum effective concentrations (Emax ) and median effective concentrations (EC50 ) values of the Ca++ , acetylcholine (ACh) and carbachol
concentration–response curves in the absence (Control) and presence of different concentrations (Conc.) of the crude extract oiTerminalia bellerica fruit (Tb.Cr),
dieyclomine, nifedipine and atropine
The values given with percentage (%) represent Emax and those with ␮M are EC50 . Values represent geometric means along with 95% confidence intervals in
parenthesis. ** P < 0.01, *** P < 0.001 vs. respective control, Student’s t-test.
shift without the suppression of the maximum contractile effect
(Fig. 4D). Table 1 shows respective Emax and EC50 values of ACh
in the absence and presence of Tb.Cr, dicyclomine, nifedipine
and atropine.
pressure in anaesthetized rats (Fig. 5A). Salbutamol was used
as a positive control which caused suppression of CChinduced bronchoconstriction at 0.01, 0.03 and 0.1 mg/kg by
22.75 ± 1.31, 42.5 ± 3.2 and 69.35 ± 1.03% (n = 4), respectively
(Fig. 5B).
3.3. Effect of Tb.Cr on castor oil-induced diarrhea in mice
3.5. Effect of Tb.Cr on guinea-pig trachea
Tb.Cr exhibited a dose-dependent (300–1000 mg/kg) protective effect against castor oil-induced diarrhea in mice. The
negative control group (saline treated) did not show any protection against castor oil-induced diarrhea. Pretreatment of animals
with the plant extract, showed 20% protection from diarrhea at
300 mg/kg dose and 60% protection at 1000 mg/kg (P < 0.05
versus saline group). Loperamide (10 mg/kg) showed 80% protection from diarrhea in the positive control group (Table 2).
3.4. Effect of Tb.Cr on carbachol-induced
bronchoconstriction in rats
Tb.Cr at the doses of 30, 100 and 300 mg/kg produced
18 ± 2.8, 39.4 ± 4.05 and 97.6 ± 0.81% (n = 5) respective inhibition of CCh (1 ␮M/kg)-induced increase in inspiratory
In tracheal preparations, Tb.Cr caused inhibition of CCh
(1 ␮M)-induced contractions at low concentrations with IC50
value of 3.3 mg/mL (1.7–6.7, n = 4) compared to that against
K+ (80 mM)-induced contractions with IC50 value of 8.5 mg/mL
Table 2
Effect of the crude extract of Terrrinalia bellerica fruit (Tb.Cr) on castor oil (C.
oil, 10 mL/kg)-induced diarrhea
Treatment (p.o.)
No. of mice/5 with diarrhea
% Protection
Saline (10 mL/kg) + C. oil
Tb.Cr (300 mg/kg) + C. oil
Tb.Cr (1000 mg/kg) + C. oil
Loperamide (10 mg/kg) + C. oil
5
4
2a
1a
0
20
60
80
a
P < 0.05 vs. saline group, Chi-square test.
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3.6. Acute toxicity study of Tb.Cr in mice
Tb.Cr did not cause any mortality up to the dose of 10 g/kg.
3.7. Effect of fractions on rabbit jejunum
When tested on spontaneously contracting rabbit jejunum,
all resultant fractions, Tb.CHCl3 , Tb.EtAc and Tb.Aq inhibited
the spontaneous contractions with respective IC50 values of 2.6
(1.7–4.1, n = 4), 1.1 (0.7–1.9, n = 4) and 2.9 mg/mL (1.8–5.0,
n =n = 4). Tb.CHCl3 and Tb.EtAc relaxed the CCh (1 ␮M)induced contractions with IC50 values of 0.47 (0.3–0.8, n = 4)
and 0.13 mg/mL (0.09–0.18, n = 3) respectively without any
effect on K+ (80 mM), while Tb.Aq relaxed both CCh and
K+ (80 mM)-induced contractions (being more potent against
CCh) with respective IC50 values of 0.21 (0.15–0.3, n = 4) and
7.7 mg/mL (3.9–15.3, n = 5) as shown in Fig. 8.
4. Discussion
Fig. 5. Effect of increasing doses of (A) crude extract of Terminalia bellerica
fruit (Tb.Cr) and (B) salbutamol on carbachol-mediated bronchoconstriction in
anaesthetized rats. Results are expressed as mean ± S.E.M., n = 4–5.
(2.4–30.5, n = 4) as shown in Fig. 6A. Dicyclomine, also showed
a similar pattern of inhibitory effect (Fig. 6B) with respective
IC50 values of 0.3 (0.20–0.41, n = 4) and 4.24 ␮M (2.5–7.1,
n = 4), whereas nifedipine was more potent against K+ -induced
contractions with IC50 value of 0.03 ␮M (0.02–0.05, n = 4),
when compared with CCh-induced contractions [0.33 ␮M
(0.24–0.50, n = 4)] as shown in Fig. 6C. Atropine relaxed
the CCh (1 ␮M)-induced contraction potently with IC50 value
of 0.003 ␮M (0.002–0.005, n = 4), without any effect on K+
(80 mM)-induced contractions, n = 4 (Fig. 6D). Like in ileum,
Tb.Cr produced rightward parallel displacement of the CChcurves without suppression of the maximum contractile response
at 1.0 mg/mL, followed by non-parallel shift with suppression
of the maximum effect at 3.0 mg/mL (Fig. 7A). Dicyclomine
(0.03–0.1 ␮M) also showed a similar pattern of shift (Fig. 7B),
while nifedipine (0.03–0.1 ␮M) produced a non-parallel rightward shift with the suppression of the maximum response
(Fig. 7C). Atropine (0.01–0.03 ␮M) caused rightward parallel
shift without the suppression of the maximum contractile effect
(Fig. 7D). Table 1 shows respective Emax and EC50 values of CCh
in the absence and presence of Tb.Cr, dicyclomine, nifedipine
and atropine.
Due to the folkloric reputation of Terminalia bellerica as
a gastrointestinal relaxant, its fruit extract was tested for its
possible spasmolytic effect in spontaneously contracting rabbit
jejunum preparations, where it inhibited spontaneous contractions, thus showing an antispasmodic action.
To assess the possible mechanisms of spasmolytic effect, the
extract was tested on the induced contractions. Tb.Cr reversed
the CCh and K+ -induced contractions, being more potent against
CCh. Dicylomine, a dual blocker of muscarinic receptors and
Ca++ influx (McGrath et al., 1964; Downie et al., 1977) showed
similar pattern of inhibition, while nifedipine, a standard Ca++
channel blocker (Fleckenstein, 1977) was more potent against
the induced contractions of K+ than CCh and atropine, a muscarinic receptor antagonist (Arunlakhshana and Schild, 1959)
relaxed the CCh-induced contractions only. It is thereby suggested that the inhibitory effect of the plant extract is mediated
possibly through a combined blockade of Ca++ influx and muscarinic receptors.
The presence of dual mode of inhibition, involving Ca++
antagonist and anticholinergic was confirmed through constructing the Ca++ and ACh CRCs in the presence of different
concentrations of the extract. In jejunum, Tb.Cr shifted the Ca++
curves to the right accompanied by the suppression of maximum
response, similar to that caused by nifedipine and dicyclomine.
The anticholinergic activity was evaluated by acetylcholineconcentration–response curves constructed in guinea-pig ileum.
A parallel displacement of ACh-curves without suppression of
the maximum effect was observed at the lower concentration,
a characteristic of a competitive or specific antagonist, like
atropine (Gilani and Cobbin, 1986; Eglen and Harris, 1993),
followed by non-parallel shift with suppression of the maximum effect at next higher concentration, pointing towards the
presence of non-competitive inhibition (VanDen Brink, 1973),
known with Ca++ antagonists (Irie et al., 2000). Dicyclomine
also shifted the ACh-curves to the right, similar to that of the
crude extract, while nifedipine resulted in rightward but nonparallel shift with suppression of the maximum effect at both the
A.H. Gilani et al. / Journal of Ethnopharmacology 116 (2008) 528–538
535
Fig. 6. Concentration–response curves showing comparison of (A) crude extract of Terminalia bellerica fruit (Tb.Cr), (B) dicylomine, (C) nifedipine and (D) atropine
for the inhibitory effect against carbachol and K+ -induced contractions in isolated guinea-pig tracheal preparations. Results are expressed as mean ± S.E.M., n = 4.
Fig. 7. Concentration–response curves of carbachol in the absence and presence of (A) crude extract of Terminalia bellerica fruit (Tb.Cr), (B) dicyclomine, (C)
nifedipine and (D) atropine in isolated guinea-pig tracheal preparations. Results are expressed as mean ± S.E.M., n = 3–5.
536
A.H. Gilani et al. / Journal of Ethnopharmacology 116 (2008) 528–538
the oil (Iwao and Terada, 1962) which produces changes in the
transport of electrolytes and water resulting in the generation of
giant contractions of the transverse and distal colon (Izzo et al.,
1994; Croci et al., 1997). Thus, a potential antidiarrheal agent
may exhibit its effect by inhibiting bowel contractions (Di-Carlo
et al., 1993). The effect of Tb.Cr was in accordance with the
expectation, as both anticholinergic drugs and Ca++ antagonists
were reported to possess an antidiarrheal action (Reynolds et al.,
1984; Rang et al., 1999).
Based on the use of Terminalia bellerica in hyperactive
respiratory ailments, the plant extract was evaluated for bronchodilatory effect in rats under anesthesia. The extract protected
the CCh-evoked bronchospasm in a dose-dependent fashion.
Tb.Cr was then studied in isolated trachea to investigate the
possible mode of brochodilatory action. Similar to the gut, the
crude extract caused inhibition of CCh-induced contractions and
displaced the CCh-curves to the right in a parallel fashion without suppression of the maximum response at low concentration,
followed by non-parallel shift with suppression of the maximum effect at the next higher concentration. Tb.Cr also relaxed
the K+ -induced contractions at higher concentration, suggesting
the co-existence of anticholinergic and Ca++ antagonist properties, like dicyclomine. Nifedipine was more potent against
K+ than CCh-induced contractions and caused a rightward nonparallel shift of CCh-curves with suppression of the maximum
effect. Atropine inhibited the CCh-induced contractions, without any effect on K+ and caused a rightward parallel shift of
CCh-curves without suppression of maximum response. Both
the crude extract and dicyclomine were slightly more effective in the gut preparations than in the trachea. This could
possibly be due to difference in the physiological modulators
among various tissues and/or the extent of their regulatory influences (Gayton and Hall, 1996) may cause a better synergistic
interaction of the different spasmolytic mechanisms in the gut
compare to airways, though species difference cannot be ruled
out.
The study on the activity-directed fractionation revealed that
the Ca++ antagonist component was concentrated in the aqueous fraction, while anticholinergic agent was distributed both
in organic and aqueous fractions, with the ethyl acetate fraction
exhibiting the most potent in its antispasmodic effect.
5. Conclusions
Fig. 8. Concentration–response curves showing effect of the Terminalia bellerica fractions (A) chloroform, (B) ethyl acetate and (C) aqueous (Tb.Aq) on
spontaneous, carbachol and K+ -induced contractions in isolated rabbit jejunum
preparations. Results are expressed as mean ± S.E.M., n = 3–5.
concentrations used. Atropine caused a rightward parallel shift
of the ACh-curves without suppression of maximum response.
The in vivo antidiarheal property of Terminalia bellerica fruit
was determined by its protective effect against castor oil-induced
diarrhea in mice. The induction of diarrhea with castor oil results
from the action of ricinoleic acid formed in the hydrolysis of
These results show that Terminalia bellerica offers a combination of anticholinergic and Ca++ antagonist properties, which
provide pharmacological basis for its usefulness in the hyperactive gastrointestinal and respiratory disorders. Moreover, this
study by reporting the in vivo antidiarrheal and bronchodilatory effects of Terminalia bellerica fruit contributes towards
evidence-based phytomedicine.
Acknowledgement
This study was supported by funds made available by the
Higher Education Commission of Pakistan under the scheme of
Distinguished National Professor Research Allowance.
A.H. Gilani et al. / Journal of Ethnopharmacology 116 (2008) 528–538
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