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59
JOURNAL OF NATURAL REMEDIES
Vol. 2/1 (2002) 59 - 65
Uterine relaxant property of the ethanolic root
extract of Cissampelos mucronata
S. V. Nwafor*, P. A. Akah, C. O. Okoli, O. O.Ndu, E. O. Ichu.
Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria.
Received 29 May 2001 ; Revised and accepted 28 October 2001
Abstract
Objective: To investigate the uterine smooth muscle relaxant property of the root extract of
Cissampelos mucronata and relate it to its traditional use in the prevention of pre-term labour.
Materials and methods: Phytochemical and pharmacological screenings were carried out using
standard procedures. In addition to investigating the effects of the extract on non-gravid and gravid
rat uterus, its effects on contractions induced by known uterine stimulants were assessed. The effects
of the extract on the amplitude and frequency of contractions of gravid rat uterus were also determined.
With the use of glibenclamide, an ATP-sensitive potassium channel blocker, the effect of the extract
on potassium channel opening was studied. Result: Phytochemical constituents present in the root
include carbohydrates, glycosides, sterols/triterpens, flavonoids, tannins and alkaloids. The extract
relaxed the non-gravid rat uterus in a concentration- and time- dependent fashion. It also antagonized
contractions evoked by serotonin, oxytocin, acetylcholine and prostaglandin E2 (known uterine
spasmogens). The uterine relaxant effect of terbutaline (a selective β2-receptor agonist used as a
tocolytic agent) was potentiated by the extract in a concentration-related manner while the
contractions induced by propranolol (a non-selective β-receptor antagonist) were inhibited by the
extract. The frequency and amplitude of contractions of the gravid uterine strips in the absence and
presence of the extract were significantly different (p<0.05). Glibenclamide antagonized the uterine
relaxant effect of the extract, an indication of possible participation of potassium channel in the
actions of the extract. The contractions evoked by calcium chloride in uterine smooth muscles
suspended in Ca2+-free K+-depolarizing solution were inhibited by the extract, suggesting that the
activities of the extract may be non-specific in origin. Conclusion: Ethanolic root extract of C.
mucronata displayed significant (p<0.05) relaxant activity on the isolated gravid and non-gravid
rat uterine smooth muscles. The results justify the use of the plant in traditional medicine as a
tocolytic (uterine relaxant) agent.
Key words: Cissampelos mucronata, Uterine relaxant activity
*Corresponding author
E-mail: epseelon@aol.com
60
1. Introduction
The goal of treatment of preterm labour is to
reduce perinatal mortality and decrease the rate
of prematurity [1]. Despite the liberal use of
tocolytic agents (uterine relaxants) in the recent
decade, the incidence of premature delivery has
not declined and it has continued to be a
therapeutic dilemma for the health-care
professionals [1]. Few of the medications
available for the treatment of premature labour
have proven to be effective for long-term
suppression of uterine contractions.
Risks of these drugs are considerable and may
be life-threatening [2-6]. Pharmacoeconomic
considerations have not been favourable in the
prospects of procuring these drugs by patients
in developing countries. Consequently, a major
priority in obstetric research is the prevention
of prematurity [1].
In many parts of the world, the use of plants
and plant products have been an integral part
of traditional practice in the treatment of
preterm labour. Traditional medicine is an
important component of Nigerian health care
system and plants and herbs belonging to
various families and species are used by
traditional birth attendants and native healers
to prevent premature delivery. Success rates
claimed with some of these plants raise
considerable hope in the prospect of finding a
novel tocolytic agent.
One such plant is Cissampelos mucronata,
traditionally acclaimed as a potent tocolytic agent
in the Eastern parts of Nigeria. The root or leaf
macerated in local gin or water is taken orally
to prevent premature labour. C. mucronata is
a climbing shrub that is widespread in dry parts
of Africa. The leaves are entire, thickly papery,
alternate and about 8 cm long [7] while the root
is fibrous in nature.
In our effort to evaluate Nigerian traditional
medicines [8-11], a screening study was carried
out in vitro to evaluate the ethanolic root extract
of C. mucronata for uterine relaxant property.
2. Materials and methods
2.1. Collection of plant material
Fresh roots of C. mucronata were collected in
May 2000, from Mr. Goddy Mbonu, a herbalist/
traditional birth attendant, in Isuofia. Anambra
State, Nigeria. Mr. A.O.Ozioko of the
Department of Botany, University of Nigeria,
Nsukka (UNN) confirmed botanical
identification, and voucher specimen has been
deposited in the University Herbarium.
2.2. Extraction
The roots were washed, cut into smaller pieces,
air-dried for seven days and reduced to coarse
powder using mortar and pestle. About 90 g of
the coarse powder was macerated for 24 h in
500 ml of 70% ethanol.
This was filtered and freeze-drying of the filtrate
gave a solid yield of 8.13 %. Samples of the
dried filtrate were suspended in 3% Tween 85
to derive the appropriate concentrations used in
the study.
2.3. Phytochemical analysis
The root was screened for phytochemical
constituents using the methods described by
Evans [12].
2.4. Animals
Adult female white albino rats (110-170 g) in
bred and maintained in the Animal Unit of the
Department of Pharmacology and Toxicology,
UNN, were used in the study.
They were allowed free access to food (guinea
feed PLC, Nigeria) and water prior to the
commencement of the experiment. The animals
used in compliance to the local ethical standard.
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61
2.5. Effect on non-gravid rat uterus
The non-gravid rat uteri were stimulated into
oestrus by pretreating the animals with 0.1 mg/
kg of stilboesterol subcutaneously 24 h before
use and the isolated tissue preparation was set
up using standard procedures [13]. The animals
were killed by a blow on the head and
exsanguinated. The uterus was isolated and each
horn cut open longitudinally into a sheet. Each
sheet was cut into two strips.
Each strip was suspended in a 30 ml organ bath
containing De-Jalon’s solution aerated with 95%
02 and 5% C02 and maintained at 37 ± 1°C.
With a resting tension of 0.5 g, contractions
were recorded using an isotonic transducer, 7006
(Ugo Basile, Italy), connected to a 2-channel
recorder ‘Gemini’ 7070.
After 60 min equilibration period, responses of
the preparation to the extract and standard drugs
(prostaglandin E2, serotonin, acetylcholine,
glibenclamide, calcium chloride and oxytocin)
were established. The effects of the extract on
the responses elicited by the standard drugs were
also evaluated.
The activity of the extract against propranolol-
induced contractions as well as the extract’s
action on the uterine relaxant effect of
terbutaline were evaluated. The inhibitory
effects of the extract on uterine contractions
induced by oxytocin and acetylcholine were
determined in time-dependent manner.
For the study of the effect of calcium ion on
the uterine relaxant activity of the extract, De-
Jalon’s solution was replaced with Ca2+-free, K+-
depolarizing solution of the following
composition (g/L): NaCl 1.58, NaHC03 1.26,
KCl 7.46, MgCl2.7H20 0.25 and glucose 1.98.
The uterine strips were washed for 30 min with
the calcium ion-deficit depolarizing solution to
remove intra- and extra-cellular calcium ions.
Dose-response curves were obtained by non-
cumulative addition of calcium chloride and the
effect of the extract on the contractile effects of
calcium chloride in the uterine strips were
investigated.
2.6. Effect on gravid rat uterus
The experiment was set up as described above
with strips of gravid rat uterus. The influence
of the extract on the spontaneous contractions
of isolated gravid rat uterine
Strips were determined with respect to the
amplitude and frequency of contractions. The
initial values (taken as control) were
determined at the first 10 min after 60 min
equilibration.
Subsequently, the nature of the spontaneous
contractions was evaluated for the following
10 min in the presence of the extract (76 µg/
ml). Three separate determinations were made
in each case and the results compared with the
control.
2.7. Statistical analysis
Results were expressed where appropriate as
mean ± standard error of mean. Means of the
control trials were compared to those of the test
trials using Student’s t - test and results were
regarded as significant at P<0.05.
Table 1
Effect of ethanolic root extract of C.mucronata
on smooth muscle of non-gravid rat uterus.
Extract (µg/ml) Relaxation (cm)
40 0.38 ± 0.11
80 1.62 ± 0.15
120 1.94 ± 0.17
160 2.37 ± 0.10
n=3; Values are Mean±SEM
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62
3. Results
The result of the phytochemical analysis showed
that the root contained carbohydrate, glycosides,
sterols/triterpens, flavonoids, tannins and
alkaloids. Organoleptic examination indicated
that the root tasted bitter with persistent minty
taste. The extract concentration-dependently
relaxed the isolated non-gravid uterine
preparation (Table 1). The extract, 80, 120 and
160 µg/ml, potentiated the relaxant effect of
terbutaline (100 µg/ml) in non-gravid rat uterus
in the order of 19.75 ± 1.75, 72.84 ± 2.56,
and 85.19 ± 1.98 % respectively.
Moreover, the maximal contractions induced
by propanolol (13.3 µg/ml) was inhibited to
the tone of 61.11% by 115 µg/ml of the extract.
The extract inhibited the contractions produced
by known uterine stimulants such as oxytocin,
serotonin, acetylcholine and prostaglandin E2
in a concentration-related manner.
The concentration of the extract producing 50%
inhibition of the maximal contractions produced
by the spasmogens (ID50) is shown in table 2.
The table indicates that the extract was most
potent at inhibiting contractions induced by
oxytocin while that evoked by prostaglandin E2
was least affected. Glibenclamide, an ATP-
sensitive potassium channel blocker,
competitively blocked the relaxant effect of the
extract (Table 3).
In addition, the extract was found to inhibit
in a time-dependent pattern, the contractions
produced by oxytocin and acetylcholine
(Table 4).
Maximal contractions produced in the presence
of the spasmogens alone were re-established in
the presence of 192 βg/ml of the extract by non-
cumulative addition of increasing concentration
of the spasmogens, pointing to surmountable
antagonism. CaCl2 elicited contractions in non-
gravid uterine smooth muscles suspended in
calcium ion-deficit K+-depolarized solution.
These contractions were antagonized by the
extract (Table 5).
Additionally, the extract completely blocked
the spontaneous rhythmic movement of the
isolated gravid rat uterus. The amplitude of
contractions of gravid rat uterine strip
monitored for 10 min each were 9.0 ± 0.2 cm
and 5.7 ± 0.9 cm while the frequency were 14
± 3 per 10 min and 7 ± 2 per 10 min in the
absence and presence of the extract (76.67 µg/
ml) respectively.
4. Discussion
The results of this study show that the ethanolic
root extract of C. mucronata contain
pharmacologically active substances capable of
relaxing smooth muscles of the isolated gravid
and non-gravid rat uterus.
Table 2
Concentrations of the C.mucronata extract that
inhibited 50% (ID50) of the maximal contractions
elicited by different uterine spasmogens.
Uterine spasmogens ID50 value (µµ
µµ
µg/ml)
Oxytocin 56.67
Serotonin 71.33
Acetylcholine 87.66
Prostaglandin E2 103.00
Table 3
Percentage inhibition of the relaxant activity of
the extract (180 µg/ml) by glibenclamide in non-
gravid rat uterus
Glibenclamide (µg/ml) Percentage inhibition
of relaxation
1.67 20.00 ± 1.21
3.33 46.76 ± 2.43
6.67 66.64 ± 1.76
13.33 85.89 ± 2.23
n=3; Values are Means±SEM
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63
The ability of the extract to inhibit contractions
induced by acetylcholine in the rat uterine
smooth muscle probably indicated inhibitory
action through the parasympathetic
(cholinergic) nervous pathway since the uterus
has been shown to be partly innervated through
the parasympathetic axis [14]. Serotonin can
directly stimulate the smooth muscles of the
uterus to contract [15].
Hence, the blockade of serotonin-induced
contraction is a likely indication of anti-
serotonergic activity. Prostaglandin E2 causes
contraction of the myometrium especially from
the late second trimester [16].
On the other hand, oxytocin stimulates both
the frequency and the force of contractile
activity in uterine smooth muscle [14].
Deriving from these, the inhibitory activity of
the extract on the uterine contraction elicited
by oxytocin and prostaglandin E2 may point
to inhibitory activity at the respective receptors
or blockade in one of the pathways through
which they exert their contractile effects.
Furthermore, the extract significantly
decreased the amplitude and frequency of rat
uterine smooth muscle. Such activities are
the desired properties of a good uterine
relaxant.
Table 4
Time-dependent inhibitory effect of the extract (76.67 µg/ml) on contractions
produced by oxytocin and acetylcholine (2.67 µg/ml each) in non-gravid rat uterus.
Time (min) Percentage inhibition of maximal contractions
Oxytocin Acetylcholine
0.0 30.00 ± 2.15 2.03 ± 0.31
2.5 74.12 ± 3.98 4.40 ± 0.86
5.0 83.63 ± 6.72 60.32 ± 3.54
10.0 94.42 ± 5.68 100.00 ± 0.00
15.0 100.00 ± 0.00 -
n=3; Values are Means±SEM
Myometrial relaxation is partly mediated by
stimulation of the β2- receptors. This stimulation
ultimately decreases myometrial contractility by
decreasing intracellular calcium [17,18]. This
may explain the potentiation of uterine relaxant
effect of terbutaline (a typical β2-receptor
agonist) by the extract and antagonism of
propranolol (non-selective β-receptor antagonist)
-induced contractions by the extract.
It is not known whether the observed uterine
relaxant effect of the extract was partially as a
result of direct effect on µ2-receptors or due to
influence on intracellular calcium concentration.
Potassium channel opening would hyperpolarize
the plasma membrane, which in turn would
prevent activation of voltage-dependent calcium
Table 5
Effect of increasing concentration of the extract
on maximal contractions produced by CaCl2 (0.04
µg/ml) in non-gravid rat uterus.
Increasing concentration Percentage inhibition
of the extract (µµ
µµ
µg/ml)
76.67 60.2 ± 3.6*
115.00 77.4 ± 2.4*
153.34 100.0 ± 0.0*
n=3; * (P<0.05) (maximal contraction induced by CaCl2
vs contraction produced by the presence of ext.)
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64
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of tension development [19]. Glibenclamide, a
blocker of ATP-sensitive potassium channel [19],
was found to antagonize the uterine relaxant effect
of the extract.
Consequently, it is possible that the uterine
relaxant effect of the extract may be partly
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The ethanolic root extract of C. mucronata
exhibited potent uterine relaxant property. The
activity may be attributed to one or more of the
bioactive constituents present in the root. The
mechanism(s) responsible for the observed
effects has not been elucidated but may most
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This suggestion is supported by the efficacy
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