The use and awareness of medicinal plants has increased over the years. In
some African countries, up to 90% of the population still relies exclusively
on plants as a source of medicine (Hostettmann et al.,
One plant used by traditional healers in Nigeria is Ficus exasperata
Vahl Enum. Pl. vahl. The plant belongs to the family Moraceae (Umerie
et al., 2004) and is locally known as sandpaper tree in Nigeria (Ijeh
and Ukweni, 2007) due to the well known rough or scabrous surface of its
leaves. Its ethnic names include: Kawusa (Nupe); Ameme (Edo); Erepin (Yoruba);
Anwerenwa (Igbo) and Umwemwe (Etsako).
Ijeh and Ukweni (2007) reported the use of the aqueous extract of the bark
in hastening the expulsion of placenta in cows, after calf delivery. In Congo,
extracts of the bark are used by traditional birth attendants to ease childbirth
(Ijeh and Ukweni, 2007). In Ivory Coast, the leaves are
used to counteract dysmenorrhoea (Ake, 1990). Baerts
and Lehmann (1991) reported the use of the leaves as an oxytocic to hasten
childbirth and as an abortifacient.
From the foregoing, it would appear that the leaves of F. exasperata
are used traditionally for relaxing the uterus by some herbal medical practitioners
(Ake, 1990) and in enhancing contractions of the uterus
by others as Baerts and Lehmann (1991). Our unpublished
data indicate that the aqueous leaf extract of the plant at 1.0x10-2
mg mL-1 significantly inhibited oxytocin-induced uterine contractions.
This study is therefore aimed at investigating the effects of higher concentrations
of the extract on the isolated uterus, in order to determine if the reported
dual uterine actions of the extract are concentration-dependent.
MATERIALS AND METHODS
Preparation of the Plant Material
The leaves of F. exasperata were collected in September, 2006 from
the premises of the University of Benin, Benin City, Nigeria. The plant was
identified by Dr. B. Ayinde of the Department of Pharmacognosy and authenticated
by Mr. Felix Usang of the Forest Research Institute of Nigeria, Ibadan, where
a herbarium sample with voucher number F.H.I.107312 was prepared and deposited.
A specimen voucher was also deposited in the Department of Pharmacognosy, University
The fresh leaves were manually rendered free of adulterants, cleaned by
rinsing in clean water and ground. The aqueous leaf extract was obtained by
macerating the ground leaves (2 kg) in distilled water (2 L) for 24 h. The resulting
decoction was decanted, filtered and concentrated under pressure in a rotary
evaporator (R110 Buchi, Switzerland) at 60°C and dried to a constant weight
in an oven set at 40°C. The dried extract gave a yield of 20.14% w/w and
was stored in an air-tight container at about 4°C until required.
Adult female Sprague-Dawley rats (160-200 g) bred in the animal house Department
of Pharmacology and Toxicology, University of Benin, Nigeria were used. The
animals were maintained under standard conditions and had free access to standard
diet (Ladokun Feeds Ltd, Ibadan, Nigeria) and water. They were handled according
to standard guidelines for use of laboratory animals (National Institute of
Health USA: Public Health Service Policy on Humane Care and Use of Laboratory
Preparation of Uterine Tissues
The animals were treated with diethylstilboesterol (0.2 mg kg-1
i.p.,) 24 h prior to the commencement of the experiment. Oestrus was confirmed
by microscopic observation of vaginal smears and macroscopic observation of
the vulva. The rats were sacrificed under chloroform anaesthesia and uterine
segments, 2 cm in length, were rapidly dissected out and freed of adhering connective
tissues and fat. The segments were mounted in 40 mL organ baths containing physiological
salt solution of the following composition in 5 g L-1: NaCl 45.0,
NaHCO3 2.5, D-Glucose 2.5, KCl 2.1 and CaCl2.2H2O
1.32. The lower end of a segment of the uterine tissue was attached to a tissue
holder by means of silk suture and the upper end to a Ugo Basile isometric force-displacement
transducer (model 82145) connected to a Ugo Basile unirecorder (Model 7050).
The solution was maintained at 37°C and continuously aerated (Eferekeya
and Nworgu, 1985). The preparations were equilibrated for 45 min at resting
tension of 0.75 g before the start of the experiment.
High Concentrations of the Aqueous (aq) Leaf Extract of F. exasperata
or Acetylcholine on Spontaneous Uterine Contractions
After equilibration, the baseline (100%) amplitude and frequency were recorded
in the first 10 min (Perusquia and Navarette, 2005).
This was followed by subsequent 10 min exposure of the tissue to 2.5x10-2
mg mL-1 of the extract followed by increasing cumulative concentrations,
Kurowicka et al. (2005) of 2.5x10-2
to 250x10-2 mg mL-1. These were compared with the effect
of acetylcholine (0.0005x10-2 to 0.5x10-2 mg mL-1).
Salbutamol on the aq. Leaf Extract of F. exasperata or Acetylcholine
Induced Uterine Contraction
Concentration - response relationships were done over a higher range of
concentration of extract 2.5x10-2 to 500x10-2 mg mL-1
in the absence and presence of 10-6 mg mL-1 of salbutamol.
The experiments were repeated with acetylcholine (0.0005x10-2 to
0.5x10-2 mg mL-1) as the contractile agent.
Phytochemical Screening of the Extract
The aqueous leaf extract of F. exasperata was screened for glycosides,
tannins, saponins, alkaloids, triterpenes and anthracenes by using the methods
of Evans (2002).
Diethylstilboesterol and acetylcholine were obtained from Sigma (UK). Salbutamol
was obtained from Glaxo Smithkline (England). The drugs were prepared fresh
on the day of the experiment by dissolving in physiological salt solution (composition
stated above). Diethylstilboesterol for the induction of oestrus was constituted
in 95% ethanol (Sigma, UK).
All values were expressed as Mean±SEM and n
represents the number of rats from which uterine segments were obtained. The
EC50 (concentration which produced 50 % of maximum response) and
Emax (maximum achievable response) were computed for each concentration-response
experiment. Comparisons were made using one-way ANOVA with Dunnett multiple
comparison test or Student’s t-test.
p<0.05 indicated statistical significance in all cases.
Effects of the aq. Leaf Extract of F. exasperata (AET) and Acetylcholine
on Amplitude and Frequency of Spontaneous Contractions
The higher concentrations of AET and those of acetylcholine each significantly
(p<0.05) increased the frequency but not the amplitude of spontaneous contractions
(Table 1, 2).
||Effect of the aqueous leaf extract of F. exasperata (AET) on the
frequency and amplitude of spontaneous uterine contractions
|*p<0.05 and **p<0.01 compared to baseline; n = 7 rats
||Effect of acetylcholine (ACh) on the frequency and amplitude of spontaneous
*p<0.05 and **p<0.01 compared to baseline; n = 7
Concentrations of AET and ACh producing effects at 50% of maximal response
(EC50) alone and in the presence of salbutamol (Sbl)
*p<0.001 compared to Aet alone; **p<0.001 compared
to ACh alone; n = 8 rats
Concentration-response curves showing the effects of acetylcholine
and the aq. extract of F. exasperata on the isolated uterus. There
was no significant difference in the Emax of both uterine stimulants
n = 8 rats
Effect of Salbutamol on the Aq. Leaf Extract of F. exasperata (AET)
and Acetylcholine Induced Uterine Contraction
AET and acetylcholine each caused a concentration-dependent contractile
response in the isolated uterus. There was no significant difference in their
Emax (Fig. 1). Salbutamol significantly (p<0.05) increased the
EC50 of both extract and acetylcholine (Table 3) and significantly
(p<0.01) depressed the Emax (Fig. 2).
||Concentration-response curves showing the effects of salbutamol on acetylcholine
induced contraction. Salbutamol significantly (p<0.001) depressed the
Emax of ACh. n = 8 rats
||Phytochemical constituents of the aqueous leaf extract of F. exasperata
Phytochemical Constituents of the Extract
Results of the preliminary phytochemical analysis of the aqueous leaf extract
of F. exasperata showed the presence of reducing sugars, cardiac glycosides,
saponins and tannins (Table 4).
The observed increase in the frequency of spontaneous contractions of the rat
uterine smooth muscle by the higher concentrations of the extract, may be due
to an increase in the open state probability of the voltage-dependent calcium
channels, allowing an influx of extracellular calcium thereby enhancing contractions
(Aaronson et al., 2006) or the extract may regulate
the opening of voltage-gated potassium channels which Aaronson
et al. (2006) proposed as a major contributing factor to basal myometrial
contractility. The extract may also promote directly or indirectly the production
of prostaglandins in situ thereby increasing the inherent uterine contractility
(Vane and Williams, 1973). However, this remains to be
It was also observed that the higher concentrations of the extract which increased
the frequency of spontaneous contractions did not have any effect on the amplitude.
A probable explanation for this might be that the extract had no direct effect
on the endogenous pacemaker cells, which was reported by Mackler
et al. (1999), to reside in uterine tissues and promote increase
in amplitude of uterine contraction. Thus, the extract would have no effect
on gap junction assembly and would not enhance or inhibit cellular communication,
culminating relatively in unaffected amplitude of uterine contraction. Otherwise,
due to the increased sensitivity induced by the administration of diethylstilboesterol,
the tissues were contracting at the maximum attainable amplitude of contraction
and addition of a stimulant or an agonist would not produce any observable change.
The contractile effect of the extract was compared to that of acetylcholine
(ACh), a stimulator of uterine smooth muscle contraction via activation of M2
and M3 receptors located within the myometrium (Pennefather,
1994). The extract and ACh appeared to contract the uterus similarly, though
ACh was the more potent of the two producing a greater increase in the frequency
of spontaneous contractions; a lesser concentration of ACh was required to elicit
contractions and a shift in the concentration response curve of the extract,
this was also confirmed from the potency ratio computation. Despite being crude,
the higher concentrations of the extract exhibited a good potential as an oxytocic
agent. It is hoped that further studies on the extract will produce a clinically
useful oxytocic agent with minimal side effects for the facilitation of labour
and as an abortifacient. The extract was also observed to be made up of tannins,
flavonoids, saponins and cardiac glycosides which have been reported to have
varied effects on the uterus. Sugimoto (1913) and Norris
(1961) reported the stimulation of the uterus by cardiac glycosides. Calixto
et al. (1986) reported that tannic acid affects calcium availability
necessary for smooth muscle contraction and it dose-dependently and non-competitively
antagonized contractions to several agonists in the rat uterus. Edward et
al. (1996) reported that saponins inhibited the metabolism of prostaglandin
E2 and prostaglandinF2α. The culminative effect of
these interacting phytoconstituents may contribute to the dual effect of the
extract on the uterus.
This study has shown that higher concentrations of the aqueous leaf extract of F. exasperata stimulate contractions of the isolated rat uterus in a manner similar to acetylcholine and also increase the frequency of rhythmic spontaneous uterine contractions.
The researchers wish to express their profound gratitude to Dr. A. Bafor, O. Uwumarongie and Dr. S. Okpo for their assistance.