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Loranthus micranthus ethanol leaf extract (LOME) was screened for anticholinergic activity using both in vivo charcoal meal transit model in rats and in vitro isolated tissue model. Results obtained indicate that LOME at all doses administered elicited a significant (p< 0.05) dose dependent relaxation of the smooth muscles of the rabbit jejunum. In the in vivo work, 100, 200 and 400mg/kg of LOME inhibited the movement of charcoal meal in the rat's gastrointestinal tract by 23.12 ±5.78, 34.82±4.67 and 53.21±2.40% respectively and compared favorably with the effect of atropine (1mg/kg) which yielded a mean % inhibition of 41.28±4.15%. On isolated rabbit jejunum, LOME also elicited a dose dependent relation effect as an EC 50 value of 54.14µg/ml produced 30.92% inhibition, comparing favorably with the effect of noradrenaline(EC50 = 0.057µg/ml) which presented an inhibition of 49.50% and contrasting with that of acetylcholine (EC 50 = 0.012µg/ml) which increased the amplitude of contractions by 152.43%. LOME (EC 50 = 22.40µg/ml), like atropine, significantly (p<0.05) blocked the contractile effect of acetylcholine but showed no effect on the effect of propranolol (a non selective beta receptor blocker). The results therefore suggest that the extract may contain substances with potent anticholinergic properties and may be of value in the management of diseases like diarrhea, Asthma, incontinence, peptic ulcers, muscular spasms etc and may further become a template for yet another synthetic anticholinergic agent of clinical significance.
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Comprehensive Journal of Medical Sciences Vol. 3(1), pp. 20-26, April 2015.
ISSN-2135-6848
Copyright © 2015 Knowledgebase Publishers.
RESEARCH ARTICLE
ANTICHOLINERGIC ACTIVITY OF Loranthus
micranthus (Linn) LEAVES EXTRACT ON THE
GASTROINTESTINAL TRACT SMOOTH
MUSCLES
Madubuike, K.G1, Ebhohon, S.O2, Ijioma, S.N3
Department of Physiology and Pharmacology, College of Veterinary Medicine, Michael Okpara University of
Agriculture, Umudike, Nigeria1,2
Department of Biochemistry, College of Natural Sciences, Michael Okpara University of Agriculture, Umudike,
Nigeria3.
.
Corresponding Author Email: ijiomasolo@yahoo.co.uk
Accepted 23rd February, 2015
Loranthus micranthus ethanol leaf extract (LOME) was screened for anticholinergic activity
using both in vivo charcoal meal transit model in rats and in vitro isolated tissue model.
Results obtained indicate that LOME at all doses administered elicited a significant (p< 0.05)
dose dependent relaxation of the smooth muscles of the rabbit jejunum. In the in vivo work,
100, 200 and 400mg/kg of LOME inhibited the movement of charcoal meal in the rat’s
gastrointestinal tract by 23.12 ±5.78, 34.82±4.67 and 53.21±2.40% respectively and compared
favorably with the effect of atropine (1mg/kg) which yielded a mean % inhibition of
41.28±4.15%. On isolated rabbit jejunum, LOME also elicited a dose dependent relation effect
as an EC50 value of 54.14µg/ml produced 30.92% inhibition, comparing favorably with the
effect of noradrenaline(EC50 = 0.057µg/ml) which presented an inhibition of 49.50% and
contrasting with that of acetylcholine (EC50 = 0.012µg/ml) which increased the amplitude of
contractions by 152.43%. LOME (EC50 = 22.40µg/ml), like atropine, significantly (p<0.05)
blocked the contractile effect of acetylcholine but showed no effect on the effect of
propranolol (a non selective beta receptor blocker). The results therefore suggest that the
extract may contain substances with potent anticholinergic properties and may be of value in
the management of diseases like diarrhea, Asthma, incontinence, peptic ulcers, muscular
spasms etc and may further become a template for yet another synthetic anticholinergic agent
of clinical significance.
KEY WORDS: Acetylcholine, Anticholinergic, Gastrointestinal tract, Loranthus micranthus,
Receptors, Smooth muscles
INTRODUCTION
The role of the muscarinic receptors in the maintenance of the normal physiological contractility of the
gastrointestinal tract is well established (Osim, 2000; Sembulingam and Prema, 2010). While cholinergic
Madubuike et al. 21
activity involving the release of endogenous acetylcholine tends to increase intestinal motility via
muscarinic receptor binding, adrenergic activity does exactly the opposite, producing intestinal relaxation
via the adrenergic receptor binding. The interplay between these cholinergic (parasympathetic) and
adrenergic (sympathetic) arms of the Autonomic nervous system brings about required balance in GIT
peristaltic physiology (Ijioma et al., 2014). Over activity of the cholinergic arm in the gastrointestinal tract
may cause disorders including incontinence, gastrointestinal cramps, gastritis, peptic ulcers, diarrhea,
motion sickness with vomiting etc (Jackquelyn, 2013; Guyton and Hall, 1996). These disorders indeed
result from excessive involuntary muscle movement associated with excess release of acetylcholine, a
neurotransmitter which mediates cholinergic functions (Ijioma et al., 2014).
Anticholinergic agents are substances that block the action of neurotransmitter acetylcholine in the
parasympathetic outflow and thereby inhibit cholinergic nerve impulses by selectively blocking the
muscarinic receptors to which acetylcholine binds. This physiological and biochemical interactions is
therefore the basis for the use of anticholinergic agents to manage disorders caused by over activity of
the cholinergic system. Currently many plants are been studied for the presence of bioactive substances.
Hence, the exploitation of wild plants for medicinal purposes has continued to grow globally (Oluwole and
Peter, 2011). Loranthus micranthus is one of such plants.
Loranthus micranthus is a semi parasitic shrub belonging to family Loranthaceae. The plant in Nigeria
grows by obtaining nutrients and support from a host of trees including Kola acuminata, Kola nitida,
Azadirachta indica, Jatropha curcas and Persia spp. The leaves extracts of Loranthus micranthus has
been used traditionally to treat diabetes mellitus, hypertension and schizophrenia (Bamidele et al., 2011).
The plant has also been reported to have antidiarrheal, antioxidant, hypoglycaemic, antiarthritic/analgesic
and immunological activities (Zorofchian et al., 2013). Previous works done on phytochemical
composition of the plant revealed the presence of terpenoids, steroids, oils, proteins, resins, flavonoids,
tannins, saponins and glycosides (Zorofchian et al., 2013).
In the continued search for anticholinergics and antispasmodics with minimum side effects and maximum
potency, the current study was designed to screen the ethanol leaf extract of Loranthus micranthus for
anticholinergic activity.
MATERIALS AND METHODS
Collection of plant leaves
Loranthus micranthus leaves were collected from a bushy area in Umudike, Ikwuano Local Government
Area of Abia state and were authenticated by Dr. Amosun G. of the Department of Plant Science and
Biotechnology, Michael Okpara University of Agriculture, Umudike, Nigeria. A voucher specimen was
assigned the number MOUAU/CVM/14/13 and was deposited in the Departmental Herbarium.
Preparation of Plant Extract
The leaves were air dried at room temperature for 7 days after which they were ground to coarse powder
using a manual blender. 50g of the powdered material was introduced into the extraction chamber of the
soxhlet extractor and extraction was done using ethanol as solvent. Extraction temperature was
maintained at 700C for 48 hours. At the end of the period, the ethanol was evaporated at low temperature
in an electric oven to obtain a crude extract which weighed 11.20g and represented a yield of 22.40%.
Animals
Thirty five mice (20-25g), 25 rats (90-140g) and 5 rabbits (1.8-2.5kg) obtained from the Animal production
unit of the College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, were used
for the study. They were housed under specific pathogen free (SPF) conditions with 13 H/11 H light/dark
schedule and were provided standard feed and water ad libitum, but starved for 12hours prior to
commencement of experiment. All animal experiments were conducted in compliance with NIH guidelines
for Care and Use of Laboratory Animals (Pub. No. 85-23, Revised 1985), as reported by Akah et al,
22. Madubuike et al
(2009). The study was carried out in the Physiology Laboratory of the Department of Physiology,
Pharmacology and Biochemistry, Michael Okpara University of Agriculture, Umudike, Nigeria.
Acute toxicity test of Loranthus micranthus leaf extract (LOME)
Thirty mice of both sexes weighing 20-25g were divided into 6 groups of 5 mice each and were assigned
graded oral doses of LOME in the order 500, 1000, 2000, 3000, 4000 and 5000mg/kg body weight. The
mice were kept in aluminum cages after administration and allowed free access to feed and water.
Observation was made for toxicity signs and number of deaths in each group within 24 hours for LD50
determination using the method of Karber, as expressed by Enegide et al., (2013).
In vivo effect of LOME on charcoal meal transit in rats
Twenty five rats of both sexes were divided into 5 groups of 5 rats each. Group 1 was administered 0.2ml
normal saline orally. Group 2 received 1mg/kg Atropine (i.p). Groups 3, 4 and 5 received oral
administrations of LOME at doses 100, 200 and 400mg/kg respectively. Thirty minutes later, 0.2ml of
activated charcoal meal was given orally to all the rats. The animals were all sacrificed in a further 30
minutes by suffocation in a chloroform chamber. Each animal was opened and the full length of the small
intestine was measured. The distance travelled by the charcoal meal was also measured and expressed
as a percentage of the length of the intestine using the formula:
Percentage distance moved by charcoal meal
= Distance moved by charcoal x 100
Full length of intestine
Percentage Inhibition for the in vivo study was evaluated using the expression
Percentage Inhibition = A-B x 100
B
Where A = % distance moved by charcoal in control
B = % distance moved by charcoal in test. Ijioma et al, (2014).
Preparation of intestinal smooth tissue for in vitro isometric contraction effect of LOME
The method of Uchendu, (1999) was adopted. In this method the rabbits were killed by stunning and
decapitation. The abdomen was cut open and the jejunum was carefully isolated and transferred into
tyrode solution that was continuously bubbled with air and maintained at 370C (pH 7.4) The tyrode
solution had the following composition: NaCl ( 8g), KCl (0.2g), CaCl2 (0.2g), NaHCO3(1g), NaH2PO4(1g),
MgCl2(0.1g) and Glucose(2g).
The jejunum, about 2-3cm in length was cut out and suspended vertically in a 35ml organ bath by
means of ligatures attached at one end to a tissue holder and at the other end to an isometric force
displacement transducer connected to a digital physiological recorder (Medicaid Physiopac) and
computer screen for displaying isometric contractions. Resting tension in the muscle strip was readjusted,
just sufficient to remove the slack. The preparation was allowed to equilibrate within 30 minutes of
mounting.
After regular rhythmic contractions were recorded, dose-response relationships were established for
acetylcholine, noradrenaline and LOME. EC50 values of the drugs were also administered in the presence
their respective antagonists, atropine for acetylcholine and propranolol for noradrenaline. Following
results obtained, EC50 of acetylcholine was repeated but this time in the presence of LOME. For all
administrations, a minimum time of 1 minute was allowed for individual tissue responses before been
washed 3 times with Tyrode solution. Concentration of test substances given in the text are all Final Bath
concentrations (FBC), except otherwise indicated.
Madubuike et al. 23
Statistical Analysis
Results were expressed as Means+ standard error of mean (SEM) and analysed using one way analysis
of variance. P-values less than 0.05 at 95% level of significance were considered as being significant.
RESULTS
Acute Toxicity
No death was recorded at the end of the 24 hours of acute toxicity study, even at the highest dose
administered (5000 mg/kg) body weight. The mice instead had normal disposition both physically and
mechanically in addition to surviving the 24 hours period of the study.
In vivo effect of LOME on charcoal meal transit in rats
All doses of LOME significantly (P<0.05) reduced the distance moved by charcoal meal in the rat’s
gastrointestinal tract when compared to control as100, 200 and 400mg/kg of LOME inhibited charcoal
transit by 23.12±5.78, 28.87±4.67 and 33.89±2.40% respectively and compared favorably with the effect
of atropine (1mg/kg) which yielded a mean 41.28±4.15% inhibition (Table 1).
Table 1: In vivo effect of LOME on charcoal meal transit in rats
Group
Treatment (mg/kg)
Mean % distance moved
by charcoal
Mean% inhibition of
charcoal movement
1
0.2 normal saline
92.77 ± 6.68
-
2
Atropine (1)
60.98 ± 7.32*
41.28 ± 4.15
3
LOME (100)
71.32 ± 5.29*
23.12 ± 5.78
4
LOME (200)
65.98 ± 4.45*
28.87 ± 4.67
5
LOME (400)
61.33 ± 7.35*
33.89 ± 2.40
*= p< 0.05 for test versus control
Table 2: In vitro effect of Acetylcholine on an isolated rabbit jejunum
FBC (µg/ml)
Mean basal amplitude
(mm)
Amplitude in response to
Ach(mm)
% rise in amplitude
0.014
7.00 ± 0.00
10.33 ± 0.52*
47.57
0.029
7.00 ± 0.00
13.24 ± 0.48*
89.14
0.057
7.00 ± 0.00
17.67 ± 0.17*
152.43
0.114
7.00 ± 0.00
22.00 ± 0.52*
214.29
0.229
7.00 ± 0.00
24.15 ± 0.34*
245.00
*= p< 0.05 for test versus basal values
In vitro effect of LOME on an isolated rabbit jejunum
Responses of the rabbit jejunum to acetylcholine were characterized by a dose dependent increase in the
amplitude of the rhythmic contractions (Table 2), while noradrenaline on the other hand exhibited a
relaxation effect which also appeared to be dose dependent (Table 3). LOME also relaxed the smooth
24. Madubuike et al
muscles of the isolated jejunum in a manner comparable to the adrenergic effect of noradrenaline (Table
4). The effect of acetylcholine (EC50 = 0.035µg/ml) was effectively blocked by atropine (0.029µg/ml)
(fig.4), while that of noradrenaline (EC50 = 0.012µg/ml) was also blocked by propranolol. While LOME
(EC50 = 22.40µg/ml) had no effect on the activity of propranolol but effectively blocked that of
acetylcholine in a manner which compared favorably to that of atropine, a muscarinic receptor blocker
(fig. 4)
Table 3: In vitro effect of Noradrenaline on an isolated rabbit jejunum
FBC (µg/ml)
Basal amplitude (mm)
Amplitude in response to
NA (mm)
% inhibition
0.014
11.15 ± 0.13
6.20 ± 0.49*
44.39
0.029
13.20 ± 0.42
7.00 ± 0.81*
46.07
0.057
14.10 ± 0.23
7.12 ± 0.25*
49.50
0.114
11.00 ± 0.19
6.18 ± 0.11*
43.82
0.229
12.25 ± 0.09
4.25 ± 0.20*
65.31
*= p< 0.05 for test versus basal values
Table 4: In vitro effect of LOME on an isolated rabbit jejunum
FBC (µg/ml)
Basal amplitude (mm)
Amplitude in response to
LOME (mm)
% inhibition
14.28
09.00 ± 0.25
7.63 ± 0.09*
15.22
28.57
10.00 ± 0.09
6.15 ± 0.13*
38.50
57.14
14.00 ± 0.22
9.67 ± 0.12*
30.92
114.30
12.00 ±0.14
8.20 ± 0.15*
31.67
228.60
09.00 ± 0.12
4.33 ± 0.14*
51.89
*= p< 0.05 for test versus basal values
Fig. 1: Effects of graded doses of Acetylcholine on an isolated rabbit jejunum
0.114 µg/ml
Madubuike et al. 25
Fig. 2: Effects of graded doses of Noradrenaline on an isolated rabbit jejunum
Fig. 3: Effects of graded doses of LOME on an isolated rabbit jejunum
Fig. 4: Effects of Atropine and LOME on Ach. Induced contractions on the rabbit jejunum
DISCUSSION
At an oral dose of 5000 mg/kg body weight, LOME produced no death/ toxicity symptoms. This attest to
the high safety margin of Loranthus micranthus leaf extract (LOME) and may account for why the plant
has been used over the years for the management of diseases. This toxicity result agrees with Osadebe
et al., (2012), who reported that Loranthus micranthus when administered to laboratory animals produced
no death even at very high doses.
It is established that the smooth muscles of the gastrointestinal tract is host to numerous muscarinic
receptors of both M2 and M3 subtypes which play major role in intestinal contractility and peristaltic activity
0.029µg/ml
Atp. (0.029µg/ml) +
Ach.(0.035µg/ml)
LOME (22.40µg/ml) +
Ach.(0.035µg/ml)
26. Madubuike et al
resulting from parasympathetic innervations. While the M3 receptors does so by triggering
phosphoinositide hydrolysis, Ca2+mobilization and direct contractile response, M2 subtype does same by
inhibiting adenylcyclase and Ca2+ activated K+ channels and potentiating Ca2+ dependent, non selective
conductance (Eglen, 2001; Ehlert et al., 1999; Ehlert, 2003).Thus, the administered acetylcholine the in
vivo and in vitro experiments generated inositol, 1,4,5-triphosphate (IP3) which evoked Ca2+ release from
intracellular storage sites in the rabbit GIT smooth muscle cells and thus eliciting contractions in the
isolated tissue (Uchendu, 1999).This was the underlying physiological principle for the movement of the
charcoal meal along the intestines of the rats in the in vivo experiment since the contractions are
responsible for moving intestinal contents forward (Guyton and Hall, 1996; Sembulingam and Prema,
2010).
Atropine (1mg/kg), a standard anticholinergic drug inhibited the contractions induced by acetylcholine in
the experiments conducted by competitively binding to the muscarinic receptors to which acetylcholine
molecules should bind to and so reduced the effect of the acetylcholine (Rang et al., 2007).
Noradrenaline in contrast caused relaxation of the isolated jejunum by binding to adrenergic receptors
also found in the smooth muscle with propranolol, a non selective beta blocker providing sufficient block
to its effect.
Loranthus micranthus ethanol leaf extract (LOME) like adrenaline exhibited a dose dependent inhibition
of intestinal contractions in the in vivo and in vitro study models and also significantly (p< 0.05) blocked
acetylcholine induced contractions with no significant effect on the activity of propranolol. The results
suggest that LOME may contain active principles with anticholinergic activities. Zorofchian et al., (2013)
had reported the presence of various phytochemicals including terpenoids, steroids, oils, proteins, resins,
flavonoids, tannins, saponins and glycosides in leaf extracts of Loranthus micranthus some of which have
been implicated in the relaxation of gastrointestinal smooth muscles. These results therefore suggest that
LOME may have achieved its effects by binding to the muscarinic receptors, thus, antagonizing the
activity of acetylcholine and inhibiting intestinal peristaltic contractions. This work agrees with existing
literature which had reported that the extract exhibited antimotility effect on the gastrointestinal tract
(Osadebe et al., 2012) and further explains its use in traditional medicine for the management of diarrhea
(Zorofchian et al., (2013).
Conclusively, the inhibitory effect of Loranthus micranthus ethanol leaf extract on the rhythmic
contraction of the rabbit jejunum coupled with its significant blockade of acetylcholine induced
contractions in the in vivo and in vitro experiments suggests that the extract may contain substances with
potent anticholinergic properties and may be of value in the management of diseases like diarrhea,
Asthma, incontinence, peptic ulcers, muscular spasms etc and may further become a template for yet
another synthetic anticholinergic agent of clinical significance.
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