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Antifertility and Fetotoxic Activities of Acanthus montanus Aqueous Extract

in Wistar Rats

Article  in  Methods and Findings in Experimental and Clinical Pharmacology · October 2008

DOI: 10.1358/mf.2008.30.7.1254614 · Source: PubMed

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Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528
Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.
CCC: 0379-0355/2008
DOI: 10.1358/mf.2008.30.7.1254614

Antifertility and Fetotoxic Activities of Acanthus montanus Aqueous

Extract in Wistar Rats

Emmanuel Acha Asongalem1, Paulin Nana2, Harquin Simplice Foyet2, Théophile Dimo2 and Pièrre
Kamtchouing2
1Pharmacology and Toxicology Unit, Department of Physiological Sciences, Faculty of Medicine & Biomedical
Sciences, and 2Laboratory of Animal Physiology, Department of Animal Biology & Physiology, Faculty of Science,
University of Yaounde 1, Yaounde, Cameroon

SUMMARY
Acanthus montanus T. Anderson (Acanthaceae) possesses several medicinal properties; it is used in Cameroon as a folk medicine to treat pain,
inflammation and threatened abortion. The aim of this study was to determine the effect of A. montanus aqueous extract on the estrous cycle pre- and
postimplantation in rats and its mechanism of action. The estrous cycles of Wistar rats were monitored before, during and after oral administration of
distilled water (control) or aqueous extract (62.5, 125, 250, 500, 1000 mg/kg/day). Furthermore, pregnant rats received the above doses of aqueous
extract on days 1–6 (preimplantation) or 6–15 (postimplantation) of gestation and were sacrificed on day 8 or 20 of pregnancy, respectively. Moreover,
aqueous extract (500 and 1000 mg/kg/day) was given to ovariectomized rats in the presence or absence of exogenously administered estrogen and/or
progesterone and uterine weight and deciduoma count were evaluated. The extract, irrespective of dose, reversibly prolonged the metestrous and occa-
sionally the diestrous stages of the estrous cycle. The extract did not alter the uterine wet weight or deciduoma count, suggesting a lack of estrogenic
and progestational effects. At 1000 mg/kg/day, the extract caused appreciable preimplantation losses of 36.8 ± 6.5% (P < 0.05), while none of the
doses caused postimplantation losses. The extract also caused delayed fetal growth. Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

Key words: Acanthus montanus - Estrous cycle - Antiovulatory - Antifertility - Fetotoxicity - Implantation

INTRODUCTION plant (5, 6). The estrous cycle of rats is controlled by

In many developing countries, the use of medicinal
plant products to treat various ailments is popular and
tends to replace orthodox medicine in some cases. A lack
of information on the adverse effects of these plants has
cast doubt on their safety. A remedy used to treat threat-
ened abortion, dysmenorrhea and uterine spasms in preg-
nant women should be devoid of embryotoxic and ter-
atogenic effects at therapeutic doses, irrespective of the
duration of use. If such a remedy does not possess these
properties, it should not be dispensed to pregnant
women. Acanthus montanus (Nees) T. Anderson
(Acanthaceae) is a medicinal plant used in ethnomedi-
cine for the treatment of an array of illnesses in
Cameroon, including problems associated with pregnan-
cy (1). Scientifically, little attention has been paid to its
phytopharmacology. Chemical investigation of the genus
Acanthus has resulted in the isolation of flavonoids, alka-
loids, triterpenoids and sterols (2-4). We have demon-
strated the antiinflammatory, antipyretic and analgesic
properties and acute toxicity of an aqueous extract of this
many endogenous factors, including estrogen, proges-
terone, histamine and prostaglandins (7), and is charac-
terized by cellular changes in the vagina, divided into
four stages, namely diestrus, proestrus, estrus and
metestrus (8). Any disruption of this cycle may result in
fertility disturbances. Many plant extracts have been
shown to possess antiimplantation effects and a lack of
teratogenic potential (9-11).
The goal of the present study was to assess the effect
of an A. montanus aqueous extract on the estrous cycle of
rats, test its effect on pre- and postimplantation in preg-
nant rats, and determine the mechanism of action.
MATERIALS AND METHODS
Plant collection and identification
A. montanus (Nees) T. Anderson (Acanthaceae)
plants were collected in the Nsimeyong area of Yaounde,
Cameroon, in June 2005 and identified in the National
522 E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528
Herbarium, Yaounde, under the voucher number
1652SRFCAM.
Preparation of plant samples
Using a modified method of the traditional practi-
tioner, an aqueous extract was prepared by maceration of
4.0 kg in 10 l of boiled distilled water for 24 h. This was
followed by filtration and elimination of the solvent
through concentration in a rotor evaporator, and drying in
an oven at 50 oC to obtain 400 g (10.0%) of the extract.
The doses chosen were 6.25%, 12.5%, 25%, 50% and
100% of the adult daily dose usually prescribed by the
traditional practitioner (extract of seven whole leaves of
the plant presented as a concoction taken in two glass-
fuls, three times daily) (12).
Animals
Prior authorization for the use of laboratory animals
in this study was obtained from the Cameroon National
Ethical Committee (Reg. No. FWA-IRB00001954); rats
were handled according to recommendations of the
National and Institutional Ethical Committee. Primi-
parous female albino Wistar rats (60 days old) that were
raised in the Animal House of the Faculty of Science
were used. The average weight was 198 ± 10 g. Males of
similar weight range and age were used for mating. They
were acclimatized in the laboratory for 10 days with
freely available water and standard rat chow.
Effect on estrous cycle or antiovulatory assessment
Stages of estrous cycles of nonpregnant female rats
were monitored daily for 15 days, and those that had
three regular 4- or 5-day cycles were chosen for the
experiment. The selected rats were divided into five
groups of 6 rats each. The vaginal smears were done
using saline (0.9% NaCl) between 7 and 9 a.m. to moni-
tor their estrous cycles for the next 28 consecutive days,
as follows: 10 days prior to, 6 days during and 12 days
after administration of the vehicle or extract. Group 1
received distilled water to serve as control, while groups
2–5 received the aqueous extract at 62.5, 250, 500 and
1000 mg/kg/day. The rats were dissected and ovaries
removed, weighed and fixed in 10% formalin buffer to
perform histological analysis.
Mechanism of action
Test for estrogenic properties
Twenty-four 1.5-month-old rats (110 ± 5 g) were
ovariectomized and assigned to four groups of 6 rats
each. The rats were allowed to adapt for 15 days before
the administration of the vehicle and extract. This adap-
tation period was to enable the uterus to regress to mini-
mum size; vaginal swabs were performed on days 10–15
to ensure that no estrous cycle occurred. 17β-Estradiol
was dissolved in 95% ethanol to obtain a concentration
of 1 mg/ml, which was kept at 4 oC; 0.1 ml of this solu-
tion was diluted in 1.9 ml of corn oil to achieve a final
concentration of 50 µg/ml. One group of rats received 30
µg/kg of 17β-estradiol propionate subcutaneously (s.c.),
while the others received an oral dose (p.o.) of A. mon-
tanus aqueous extract (500 and 1000 mg/kg/day) or dis-
tilled water (p.o.) once daily for 7 consecutive days (13).
After the rats were sacrificed under ether anesthesia, the
uteri were removed and weighed as unblotted (wet) and
blotted weights.
Test for progestational properties
Astwood’s method (14) was adopted to test the prog-
estational effects of the aqueous extract. Four groups
consisting of 6 adult Wistar rats weighing 190–210 g
were overiectomized and, a week later, treated with 0.5
µg s.c. estradiol/animal once daily for 4 consecutive
days. At the end of estradiol administration, group 1
received progesterone in 95% ethanol in corn oil (60
µg/kg s.c.), group 2 received distilled water, and groups
3 and 4 received A. montanus aqueous extract (500 and
1000 mg/kg/day p.o., respectively), each for 9 uninter-
rupted days. On day 5 of the treatment, 0.1 ml/100 g
body weight of histamine (prepared by dissolving 5 mg
in 5 ml of saline to obtain a final concentration of 1
mg/ml) was injected into the lumen of left horns of all
rats to irritate the estrogen/progesterone-primed fallopian
tube, thus inducing deciduoma – a maternal placental
tumor (15). The rats were sacrificed after the last day of
treatment with both uterine horns removed, blotted,
weighed and excised, and the number of deciduoma
counted. These parameters were compared with control
rats.
Preimplantation or antiimplantation assessment
Female rats were assessed for pregnancy by monitor-
ing the presence of vaginal sperm plugs at 6 a.m. follow-
ing an overnight cohabitation of 2 females and a male rat.
Confirmation corresponded to day 0 of pregnancy, and
the rats were then divided into six groups of 6 animals
each. The control group was administered distilled water
and the treated groups received 62.5, 125, 250, 500 or
1000 mg/kg/day extract. The extract was given orally by
gavage once daily from day 1 to day 6 of gestation. Daily
observations were made to check for maternal behavioral
changes and mortality. The animals were sacrificed on
day 8 to allow for clear macroscopic observation of
implanted and unimplanted embryos. From the exposed
uteri, the number of follicles (atretic and nonatretic) (16),
corpora lutea, ovulated oocytes and implantations on
both uterine horns was evaluated using a binocular
microscope (Olympus CH2, Model CHS). An atretic fol-
licle was identified by a “glassy membrane” appearance
or a collapsed zona pellucida. The uteri were weighed
and their thickness and diameters assessed.
 E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528
Postimplantation evaluation
Evaluation of maternal effects
This procedure was similar to that described above.
The extract was administered once daily for 10 consecu-
tive days, i.e., days 6–15, which corresponds to the
organogenesis stage of the rat gestational period. At the
end of the administration, the organs of the exposed
fetuses were allowed to develop from day 15 to day 20.
Each rat was monitored daily (days 0–20) for behavioral
changes (rearing, gnawing, movements), mortality, body
weight and quantity of water and food consumed. On day
13, their vaginas were checked for any blood stains,
which signified the process of abortion and possible fetal
expulsion, which was then confirmed by sacrificing the
animal.
Evaluation of fetal effects
On day 20, each of the pregnant rats was weighed and
sacrificed. Parameters evaluated were: number of corpo-
ra lutea and implants, uterine weight, live fetuses, fetal
resorption, fetal death, fetal body weight, crown–rump
length, tail length, placental weight and sex ratio; uteri of
nonpregnant rats were immersed in a 10% ammonium
sulfide solution for 10 min to evaluate those with early
fetal loss (17). A dark coloration corresponded to an early
resorption. Other parameters measured were absolute
and relative weights of the lungs, heart, liver, spleen and
kidneys. Live fetuses were examined for any external
morphological anomaly. They were divided into two
halves and stored in Bouin’s (for evaluation of visceral
and soft tissue abnormalities) and formol (for skeletal
anomaly assessment) solutions (18). The fetal skeleton
was prepared using Alizarin red S (1 mg/dl) dissolved in
2% KOH to assess poor bone ossification and supernu-
merary ribs. Both binocular and dissecting microscopes
were used for the observations.
Statistics
Maternal body weight, body weight gain, uterine
weight, corrected body weight and organ weights
(absolute and relative), fetal body and placental weights,
crown–rump and tail lengths, and food and water con-
sumption were tested by Bartlett’s test for equality of
variance. Depending on the outcome of the test, para-
metric and nonparametric one-way ANOVA was per-
formed. If the F value was significant (P < 0.05), the data
were further subjected to Dunnett’s multiple-comparison
test (to compare the treatment groups with the control) or
the Mann–Whitney U test (to compare two independent
random samples).
To analyze the influence of body weight on organ
weights, analysis of covariance (ANCOVA) was per-
formed on variables of treatment and body weight.
Estrogen and progesterone groups were compared with
the control using the unpaired t test following ANOVA,
523
whereas the extract-treated groups were tested for signif-
icance using ANOVA only. Preimplantation loss, resorp-
tions and fetal alterations were assessed with the
Wilcoxon rank sum test. Corpora lutea, deciduoma
count, implantations, litter size and fetal deaths were ana-
lyzed with the Kruskal–Wallis test. Fetal sex ratio was
analyzed using a binomial distribution test. The results
were expressed as mean ± SEM and P < 0.05 was con-
sidered significant.
RESULTS
Effect on estrous cycle or antiovulatory assessment
All doses of the extract resulted in irregular estrous
cycles with prolonged metestrous and occasionally pro-
longed diestrous stages (Table 1, data for 500 mg/kg/day
not included). Reversibility of the extract-altered estrous
cycle occurred in at least 90% of the animals used, begin-
ning at least 9 days posttreatment. No weight differences
or histological abnormalities were observed on the
ovaries (data not shown).
Mechanism of action
There was a significant increase (P < 0.01) in wet
uterine weight in the estradiol-treated group compared to
the aqueous extract group (Table 2). All blotted weights
remained unchanged. Histamine-irritated horn induced
deciduomas in the progesterone-treated group but not in
the control or extract-treated rats (Table 3).
Preimplantation loss or antiimplantation assessment
When the extract was given on days 1–6 of gestation,
36.8% (P < 0.05) preimplantation loss was recorded at
1000 mg/kg/day (Table 4). Ovarian qualitative histologi-
cal analysis did not reveal any changes in developing and
numbers of atretic (regressing) follicles in the treated
rats. The difference in corpora lutea counts was insignif-
icant. Uterine thickness, diameter and weight remained
unchanged compared to control and irrespective of the
dose used. No behavioral alterations or maternal mortal-
ity were observed (data not shown).
Postimplantation loss evaluation
Maternal effects
Dams with nonviable litters were excluded from the
analysis of the results. No mortality or treatment-related
clinical signs of maternal toxicity were observed. When
compared with control, no significant differences were
observed in the intake of food and fluid, uterine weight,
corrected body weight, and absolute organ and maternal
weights. However, there were significant reductions in
maternal weight gains for the 62.5 (P < 0.05), 125 (P <
0.05) and 1000 mg/kg/day (P < 0.05) groups in compar-
TABLE 1. Effect of Acanthus montanus aqueous extract on normal female rat estrous cycle. 524

Group Estrous cycle (days)

Before administration During administration After administration

Dose (mg/kg/day)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Control 0
D P E M D P E M D E M D P E M D E M D P E M D E M D P E
E M D P E M D E M D P E M D P E M D E M D P E M D E M D
P E M D P E M D E M D P E M D P E M D E M D P E M D E M
M D D P E M D D P E M D D M D D P E M D D P E M D D P E
E M D P E M D E M D P E M D P E M D E M D P E M D E M D

Extract 62.5
D P E M D P E M D P M D M D M M M M M M M M M D P E M D
P E M D P E M D E M M M M M M M M M M M M M M M D P E M
E M D P E M D P E M D M M M M M M M M M M M M M M M D P
M D P E M D P E M D P M M M M M M M M M M M M M M D P E
P E M D P E M D E M M M M M M M M M M M M M M M D P E M

Extract 250
E E M D P E M D E M M M M M M M M M M M M M M D M D P E
P E M D P E M D E M M M M M M M M M M M M M M M M M D P
P E M D P E M D E M M M M M M M M M M M M M M M M D P E
E M D D P E M D D P M M M M M M M M M M M M M M D D P E
M D D P E M D D E M D M D D M D M M D M D M M D D P E M
Extract 1000
P E M D P E M D E M M M M M M M M M M M M M M M M M D M
E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528

P E M D D P E M D E M M M M M M M M M M M M M M M D D P
P E M D D P E M D E M M M M M M M M M M M M M M M D D P
E M D D P E M D D P E D M M D D M D M M D D M D D P E M
M D D P E M D D P E M M M M M M M M M M M M M M D D E M

D, diestrus; P, proestrus; E, estrus; M, metestrus. The extract maintained the cycle mainly in metestrous and intermittently in diestrous stages; n = 6 rats per group.
 E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528 525

TABLE 2. Estrogenic effect of Acanthus montanus on rat uterus.

Parameter Control Estradiol Aqueous extract
(30 mg/kg) 500 mg/kg/day 1000 mg/kg/day
Wet uterine weight (10–2 g) 4.86 ± 0.33 7.35 ± 0.63* 5.79 ± 0.43 5.72 ± 0.46
Blotted uterine weight (10–2 g) 4.33 ± 0.34 5.34 ± 0.65* 4.21 ± 0.44 3.98 ± 0.42
*P < 0.01 between control and estradiol-treated groups (ANOVA). Each value is expressed as mean ± SEM; n = 6 rats per group.

TABLE 3. Progestational effect of Acanthus montanus on rat uterus.

Parameter Control Progesterone Aqueous extract
(60 mg/kg) 500 mg/kg/day 1000 mg/kg/day
Blotted uterine weight (10–2 g)
Right 0.11 ± 0.01 0.12 ± 0.03* 0.04 ± 0.01 0.04 ± 0.01
Left 0.11 ± 0.01 0.12 ± 0.02* 0.04 ± 0.01 0.04 ± 0.01
No. deciduomas 0 5.4 ± 0.24* 0 0
Values are expressed as mean ± SEM; n = 6 rats per group; *P < 0.05, statistically analyzed by Kruskal–Wallis.

TABLE 4. Preimplantation loss induced by Acanthus montanus administered on days 1–6 of gestation in pregnant rats.
Parameter Aqueous extract (mg/kg/day)
Control 62.5 125 250 500 1000
No. of rats 6 6 6 6 6 6
No. of atresic follicles/rat 0 0 1.1 ± 0.01 1.0 ± 0.01 2.2 ± 0.01 3.1 ± 0.3
No. of corpora lutea 9.4 ± 0.6 10.2 ± 0.8 9.7 ± 0.5 10.5 ± 0.8 9.3 ± 0.4 11.3 ± 0.2
No. of implantations 8.2 ± 0.5 8.3 ± 0.4 7.8 ± 0.5 8.2 ± 0.3 8.3 ± 0.5 7.2 ± 0.6
Preimplantation loss (%) 15.4 ± 6.5 16.8 ± 4.3 17.2 ± 4.6 19.9 ± 6.5 10.2 ± 5.5 36.8 ± 6.5*
Statistics: Mann–Whitney U test and Kruskal–Wallis test. Values expressed as mean ± SEM; n = 6 rats per group; *P < 0.05.

ison to the control group (Table 5). A similar trend was
not observed for the 250 and 500 mg/kg/day groups.
Fetal effects
Postimplantation loss was assessed as the difference
between the number of corpora lutea and the number of
implants. The difference was attributed to resorption and
fetal death (Table 6). None of the treatment groups
showed significant postimplantation losses. There was a
tendency, although not significant (P < 0.07), for an
increase in fetal resorption in the 1000 mg/kg/day group.
The resorptions were predominantly early and complete
(94.6%; mid: 5.4%, late: 0%). Fetotoxic properties of the
plant were assessed based on fetal resorption, death
(fetuses with no signs of life), litter size, body and pla-
cental weights, sex ratio and crown–rump and tail
lengths. Fetal death and litter size were unaffected by the
treatment, although at 500 mg/kg/day there was a ten-
dency for increasing litter size. The reductions in fetal
body weight, placental weight and crown–rump and tail
lengths were more pronounced as the dose increased. On
sex ratio, both sexes were equally unaffected.
Macroscopic examination of the fetuses did not
reveal any significant morphological, visceral or soft tis-
sue changes. Skeletal abnormalities were absent; howev-
er, cranial, sternal, coccygeal and phalanx bones showed
various degrees of delayed ossification from 500
mg/kg/day (data not shown).
DISCUSSION
Effect on estrous cycle or antiovulatory effect
of A. montanus
A. montanus was found to significantly prolong the
metestrous stage, and sometimes the diestrous stage,
both of which constitute the luteal phase of the rat estrous
cycle. The disruption was annulled at least 9 days after
stopping the administration of the extract, thus showing
the reversibility of the effect. Normally, during the devel-
opment and maturation of follicles, changes occur which
depend on ovarian and extraovarian hormonal balance.
For example, ovarian estrogen stimulates the release of
histamine, which acts on hormonally prepared
endometrium to induce decidualization and preparation
of decidual tissues into which the blastocyte can embed.
If this balance is distorted, irregularity occurs in the func-
tioning of the ovaries and estrous cyclization, which may
526 E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528

TABLE 5. Maternal outcomes following exposure of pregnant rats to Acanthus montanus on days 6–15 of gestation.
Parameter Aqueous extract (mg/kg/day)

Control 62.5 125 250 500 1000

No. of dams 6 6 6 6 6 6
No. of dead dams 0 0 0 0 0 0
No. of dams alive 6 6 6 6 6 6
Food consumption (g/kg/day) 83.0 ± 3.8 84.2 ± 1.3 84.1 ± 4.8 85.2 ± 2.7 78.8 ± 1.8 74.7 ± 5.1
Water consumption (ml/kg/day) 75.9 ± 0.9 73.7 ± 2.8 74.0 ± 4.9 70.7 ± 2.0 68.5 ± 2.1 66.4 ± 2.2
Maternal weight, day 0 (g) 189.6 ± 4.6 192.8 ± 4.8 191.0 ± 4.0 186.5 ± 3.7 192.0 ± 3.5 181.2 ± 3.3
Maternal weight, sacrificing day (g) 239.0 ± 6.9 227.0 ± 2.7 222.4 ± 5.8 225.1 ± 7.6 232.5 ± 6.9 212.1 ± 10.5
Uterine weight (g) 33.7 ± 3.2 28.0 ± 3.3 25.5 ± 3.4 29.4 ± 2.3 32.3 ± 1.5 23.0 ± 3.0
(P < 0.36) (P < 0.13)
Corrected body weight (g) 205.3 ± 9.0 199.7 ± 4.2 196.3 ± 4.3 193.1 ± 6.9 200.2 ± 6.9 188.7 ± 11.0
Maternal weight gain (g), days 0–19 49.6 ± 6.2 32.4 ± 3.7* 31.0 ± 3.8* 38.5 ± 5.3 40.1 ± 3.5 27.7 ± 6.0*

Absolute organ weight (g)

Lungs 1.46 ± 0.08 1.54 ± 0.07 1.68 ± 0.04 1.33 ± 0.07 1.42 ± 0.12 1.55 ± 0.20
Heart 0.64 ± 0.02 0.64 ± 0.0 0.68 ± 0.0 0.65 ± 0.0 0.70 ± 0.0 0.64 ± 0.0
Liver 8.30 ± 0.2 8.02 ± 0.3 7.49 ± 0.3 7.34 ± 0.3 8.12 ± 0.5 7.47 ± 0.3
Spleen 0.75 ± 0.06 0.78 ± 0.02 0.75 ± 0.03 0.70 ± 0.07 0.74 ± 0.06 0.90 ± 0.01
Kidneys 1.22 ± 0.07 1.09 ± 0.04 1.13 ± 0.02 1.07 ± 0.05 1.16 ± 0.05 1.15 ± 0.0

Values expressed as mean ± SEM; *P < 0.05 (significance based on Dunnett’s multiple-comparison test); % relative weight = absolute weight/
corrected body weight x 100.

TABLE 6. Fetotoxicity of rat offspring following maternal exposure to Acanthus montanus extract on days 6–15 of gestation.

Parameters Aqueous extract (mg/kg/day)

Control 62.5 125 250 500 1000

No. of pregnant dams 6 6 6 6 6 6

No. of aborted dams 0 0 0 0 0 0
No. of corpora lutea/litter
On the right ovary 6.2 ± 0.6 4.7 ± 0.3 4.0 ± 0.6 4.8 ± 0.7 5.8 ± 0.6 4.8 ± 0.8
On the left ovary 3.2 ± 0.5 4.3 ± 0.4 5.2 ± 0.6 4.7 ± 0.4 4.8 ± 0.7 4.2 ± 0.8
Total 9.4 ± 0.6 9.0 ± 0.2 9.1 ± 0.6 9.5 ± 0.7 10.7 ± 0.7 8.5 ± 0.3
No. of implantations/litter 8.2 ± 0.5 7.2 ± 0.4 7.5 ± 0.3 7.8 ± 0.8 9.2 ± 0.5 6.2 ± 0.8
Postimplantation loss (%) 12.3 ± 3.7 19.9 ± 3.1 17.5 ± 2.5 24.3 ± 9.8 17.6 ± 4.4 32.6 ± 11.0
(P < 0.14)
No. of resorptions/litter
On the right horn 1.0 ± 0.6 0.5 ± 0.2 0.8 ± 0.3 1.0 ± 0.4 0.7 ± 0.3 1.2 ± 0.5
On the left horn 0.2 ± 0.2 1.3 ± 0.4 0.8 ± 0.4 0.7 ± 0.4 0.8 ± 0.2 2.0 ± 0.7
Total 1.2 ± 0.7 1.8 ± 0.3 1.7 ± 0.3 1.7 ± 0.5 1.5 ± 0.7 3.2 ± 0.9
(P < 0.07)
No. of fetal deaths/litter 0 0 0 0 0 0.16 ± 0.16
Litter size
On the right horn 5.2 ± 0.8 4.2 ± 0.4 3.2 ± 0.6 3.8 ± 0.6 5.2 ± 0.5 3.7 ± 0.9
(P < 0.052)
On the left horn 3.0 ± 0.5 3.0 ± 0.6 4.3 ± 0.5 4.0 ± 0.6 4.0 ± 0.8 2.2 ± 0.7
Total 8.2 ± 0.5 7.2 ± 0.4 7.5 ± 0.3 7.8 ± 0.8 9.2 ± 0.5 5.8 ± 1.1
(P < 0.10)
Fetal body weight (g) 2.54 ± 0.05 2.49 ± 0.06 2.55 ± 0.05 2.11 ± 0.01**** 2.05 ± 0.02**** 2.05 ± 0.05****
Placental weight (g) 0.43 ± 0.01 0.43 ± 0.01 0.44 ± 0.01 0.37 ± 0.01**** 0.37 ± 0.01**** 0.39 ± 0.02***
Crown–rump length (cm) 3.39 ± 0.04 3.35 ± 0.05 3.20 ± 0.04** 3.12 ± 0.03**** 3.07 ± 0.03**** 2.92 ± 0.06****
Tail length (cm) 1.34 ± 0.01 1.30 ± 0.02 1.34 ± 0.03 1.21 ± 0.02*** 1.16 ± 0.02**** 1.09 ± 0.02****
Fetal sex ratio (M:F) 0.85:1 0.86:1 1:0.80 0.88:1 0.86:1 0.75:1
Values expressed as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
 E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528
result in fertility disturbances (19–21). The metestrous
phase is characterized by low estrogen levels, and the
diestrous stage by high progesterone levels. Thus, two
possibilities exist to explain the prolongation of the mete-
strous phase in these rats.
First, there is a possibility that the plant blocked
estrogen release and the luteinizing hormone surge, con-
sequently leading to a lack of ovulation and a longer
luteal phase. During the estrous cycle, the weight of
ovarian tissue increases under the influence of
gonadotropins and estrogen. This work confirmed the
lack of estrogenic activity of the plant. The unchanged
ovarian weight and histology and the inability of the
extract to augment uterine wet weight compared to estro-
gen corroborated this point. This study could neither con-
firm nor refute the antiestrogenic properties of the plant
either at the receptor level or any position on the hypo-
thalamic–hypophysial–gonadal axis (22). The possibility
of an antiprogestational effect was not ruled out either;
however, the plant had no progestational potential since
it failed to show any deciduoma formation, as observed
in the progesterone-treated group.
The second explanation for the prolongation of the
metestrous phase could be attributed to the antihistaminic
properties of the plant (23). Histamine concentration has
been found to be increased and to play a role in the decid-
ualization process (24). This raised the possibility of a
role for antihistamines, such as the extract, in inhibiting
histamine-induced deciduoma formation, as observed
during the experiment.
Antiimplantation effect of A. montanus
In this study, the extract prevented implantation by
36.8% at the highest dose used. Its antiimplantation
effect was somewhat weak compared to Lepidium capi-
talum, where the hexane-soluble fraction of 90% ethano-
lic extract showed 100% pregnancy inhibition at 250
mg/kg (25). It is a well-known fact that for implantation
to occur an exact equilibrium of estrogen and proges-
terone secretion is necessary. Any imbalance can culmi-
nate in antiimplantation or abortifacient effects. The non-
estrogenic and progestational properties of the plant are
discussed above. The lack of uterine changes (weight,
thickness and diameter) in pregnant rats treated with var-
ious extract concentrations may mean that the abortifa-
cient activity or preimplantation loss at the highest dose
may not be due to estrogenic activity of the extract. On
the other hand, prostaglandins play critical roles in decid-
ualization and vascular permeability associated with
implantation, and administration of nonsteroidal antiin-
flammatory drugs blocks blastocyst implantation (26,
27). We previously demonstrated the antiinflammatory
activity of A. montanus and its in vitro antagonist effect
against PGF2α, thereby confirming its observed antiim-
plantation activity, probably through a reduction in vas-
cular permeability, which is necessary for normal
implantation (28).
527
The antihistaminic activity of the extract probably
played a role in blocking implantation. Brandon and
Wallis (29) reported a decrease in the number of sites of
blastocyte attachment following treatment of pregnant
rats with a combination of two antihistamines, mepyra-
mine and burimamide, demonstrating the antifertility
properties of these drugs. In another experiment, the
administration of cimetidine and ranitidine to rats on
days 1–7 of gestation caused antiimplantation effects
which were not linked to their estrogenic or antiestro-
genic properties, but probably to histamine inhibition
(30).
It is possible that biochemical alterations, such as
changes in superoxide anion radical levels and superox-
ide dismutase activity, contribute to the antiimplantation
activity of the plant, as observed in the endometrium of
mice treated with Hibiscus rosa-sinensis (31).
Fetotoxic effects
The absence of clinical signs of fetotoxic effects is
probably indicative of the lack of maternal toxicity of the
extract at the doses used. However, the reduction in
maternal weight gain observed in some of the treated
groups may be linked to a decrease in the number of
implants and litter size rather than absolute body weight
loss. This decrease may be due mainly to early resorption
of the fetuses. This is corroborated by the insignificant
differences in the corrected body weights.
Unlike preimplantation (36.8%, P < 0.05), rats that
received 1000 mg/kg/day of extract had increased but
insignificant postimplantation losses (32.6%), suggesting
a greater inhibitory impact on implantation than on
implanted embryos. Similar findings were obtained for
Coleus barbatus B. At 880 mg/kg, significant preimplan-
tation and fetal developmental retardation were recorded,
but no postimplantation losses were seen in the experi-
mental groups (32).
At doses above 62.5 mg/kg/day, the extract retarded
perinatal fetal growth, manifested by reductions in fetal
weight, crown–rump and tail lengths, bone ossification
and placental weight. The delayed skeletal ossification
may not be attributed to maternal toxicity, as some stud-
ies have indicated (33). The extract had a direct fetotox-
ic effect without causing maternal toxicity, as observed
earlier with Solanum lycocarpum (34). Despite these
fetal reductions, the extract failed to cause any external,
visceral or skeletal morphological abnormalities. The
absence of these fetal morphological abnormalities may
not signify the nonexistence of tissue or organ dysfunc-
tion, since some biochemical and physiological changes
induced by the extract would be perceptible only during
weaning and maturation of the fetus.
In conclusion, A. montanus aqueous extract main-
tained rat estrous cycles at the metestrous stage, and
occasionally at the diestrous stage, culminating in fertili-
ty disturbances due to its antiovulatory and antiimplanta-
 528 E.A. Asongalem et al./Methods Find Exp Clin Pharmacol 2008, 30(7): 521-528
tion effects. It also caused fetotoxicity. These effects
were linked to its antiinflammatory activity and not to
estrogenic or progestational properties.
ACKNOWLEDGEMENTS
This research was supported by the International
Foundation for Science, Stockholm, Sweden, and by
United Nations University, Tokyo, Japan, through a grant
to E.A. Asongalem.
REFERENCES
1. Noumi, E., Fozi, F.L. Ethnomedical botany of epilepsy treatment in
Fongo–Tongo village, Western province, Cameroon. Pharmaceut
Biol 2003, 41(5): 330-9.
2. Kokpol, U., Chittawong, V., Miles, D.H. Chemical constituents of
roots of Acanthus illicifolius Linn and biological activity. Nat Prod
1986, 49(2): 355-6.
3. Anam, E.M. Novel pentacyclic triterpenoids from Acanthus mon-
tanus. Ind J Chem 1997, 30(1): 110-3.
4. Amer, M.A., Abou-Shoer, M.I., Abdel-Kader, M.S., El-Shaibany,
A.M.S., Abdel-Salam, N.A. Alkaloids and flavone acyl glycosides
from Acanthus aboreus. J Braz Chem Soc 2004, 15(2): 262-6.
5. Asongalem, E.A., Foyet, H.S., Ekobo, S., Dimo, T., Kamtchouing,
P. Anti-inflammatory, lack of central analgesia and antipyretic
properties of Acanthus montanus. J Ethnopharmacol 2004, 95(1):
63-8.
6. Nana, P., Asongalem, E.A., Foyet, H.S., Dimo, T., Kamtchouing, P.
Acute toxicological studies of Acanthus montanus (Nees T.
Anderson) Acanthaceae in Wistar rats. Pharmacologyonline 2007,
1: 339-48.
7. Van Orden, D.E., Goodale, D.B., Baker, H.A., Farley, D.B.,
Bhatnagar, R.K. Uterine catecholamines and prostaglandins dur-
ing the estrous cycle of the rat. Endocrinology 1980, 106(5): 1650-
4.
8. Marcondes, F.K., Bianchi, F.J., Tanno, A.P. Determination of the
estrous cycle phases of rats: Some helpful considerations. Braz J
Biol 2002, 62(4a): 1-8.
9. Chamorro, G., Salazar, M., Fournier, G., Pages, N. The anti-implan-
tation effects of various savine extracts on the pregnant rat. J
Toxicol Clin Exp 1990, 10(3): 157-60.
10. Singh, M.M., Chowdhury, S.R., Kulshreshtha, D.K., Kamboj, V.P.
Antigestagenic activity of Ixora finlaysoniana in rats.
Contraception 1993, 48(2): 178-91.
11. Weidner, M.S., Sigwart, K. Investigation of the teratogenic poten-
tial of a Zingiber officinale extract in the rat. Reprod Toxicol 2001,
15(1): 75-80.
12. Adjanohoun, J. E., Aboubakar, N., Dramane, K. et al. Acanthus
montanus. In: OAU/STRC Traditional Medicine and
Pharmacopoeia: Contribution to Ethnobotanical and Floristic
Studies in Cameroon. CNPMS, Porto Novo, Benin, 1996, 442.
13. Kanno, J., Onyon, L., Peddada, S., Ashby, J., Jacob, E., Owen, W.
The OECD program to validate the rat uterotrophic bioassay.
Phase 2: Dose-response studies. Environ Health Persp 2003,
111(12): 1-39.
14. Astwood, E.B. An assay method for progesterone based on the
decidual reaction in rats. Endocrinology 1939, 1: 49-55.
15. Hatanaka, K., Kitamura, Y., Maeyama, K., Watanabe, T.,
Matsumoto, K. Deciduoma formation in uterus of genetically mast
cell-deficient W/Wv mice. Biol Reprod 1982, 27(1): 25-8.
View publication stats
16. Vogel, H.G., Hock, F.J, Maas, J., Mayer, D. Drug discovery and
evaluation: Safety and pharmacokinetics assay. SpringerLink,
Berlin Heidelberg, 2006, 841-8.
17. Devine, P.J., Payne, C.M., McCuskey, M.K., Hoyera, P.B.
Ultrastructural evaluation of oocytes during atresia in rat ovarian
follicles. Biol Reprod 2000, 63(5): 1245-52.
18. True, R.M. Staining of embryonic and small mammalian skeletal
systems. Stain Techno 1947, 22: 107-8.
19. Valsala, S., Karpagaganapathy, P.R. Effect of Mimosa pudica root
powder on estrous cycle and ovulation in cycling female albino rat,
Rattus norvegicus. Phytother Res 2002, 16(2): 190-2.
20. Mathur, R., Vinita, S., Prakash, A.O. Effect of Pueraria tuberosa
DC on the estrous cycle of adult rats. Acta Eur Fertil 1984, 15(5):
393-4.
21. Shivalingappa, H., Satyanarayan, N.D., Purohit, M.G.,
Sharanabasappa, A., Patil, S.B. Effect of ethanol extract of Rivea
hypocrateriformis on the estrous cycle of the rat. J Ethnopharmacol
2002, 82(1): 11-7.
22. Benie, T., Duval J., Thieulant, M.-L. Effects of some traditional
plant extracts on rat compared with Clomid. Phytother Res 2003,
17(7): 748-55.
23. Foyet, H.S., Asongalem, E.A., Dimo, T., Kamtchouing, P. Tocolytic
effect of Acanthus montanus on rat uterus. Pharmacologyonline
2006, (3): 9-17.
24. Shelesnyak, M.C. Histamine and nidation of the ovum.
Endocrinology 1959, 6: 84-8.
25. Singh, M.M., Wadhwa, V., Gupta, D.N., Pal, R., Khanna, N.M.,
Kamboj, V.P. Postcoital contraceptive efficacy and hormonal pro-
file of Lepidium capitalum. Planta Med 1984, 50(2): 154-7.
26. Kennedy, T.G., Squires, P.M., Yee, G.M. Mediator involved in
decidualization. In: Blastocyst Implantation. Yoshinaga, K. (Ed.).
Adarns Publishing Group, Boston, 1989, 135-43.
27. Akil, M., Amos, R.S., Stewart, P. Infertility may sometimes be asso-
ciated with NSAID consumption. Br J Rheumatol 1996, 35(1): 76-
8.
28. Finn, C.A. Implantation, menstruation and inflammation. Biol Rev
Camb Philos Soc 1986, 61(4): 313-28.
29. Brandon, J.M., Wallis, R.M. Effect of mepyramine, a histamine H1-
and burimamide, a histamine H2-receptor antagonists, on ovum
implantation in the rat. J Reprod Fertil 1977, 50(2): 251-4.
30. Agrawal, S.S., Aravinda, S. Anti-implantation activities of some H2
blockers. Ind J Pharmacol 1995, 27: 40-2.
31. Nivsarkar, M., Patel, M., Padh, H., Bapu, C., Shrivastava, N.
Blastocyst implantation failure in mice due to “nonreceptive
endometrium”: Endometrial alterations by Hibiscus rosa-sinesis
leaf extract. Contraception 2005, 71(3): 227-30.
32. Almeida, F.C., Lemonica, I.P. The toxic effects of Coleus barbatus
B. on the different periods of pregnancy in rats. J Ethnopharmacol
2000, 73(1-2): 53-60.
33. Ariyuki, F., Khihara, H., Higaki, K., Yasuda, M. A study of fetal
growth retardation in teratological tests: Relationship between
body weight and ossification of the skeleton in rat fetuses.
Teratology 1982, 26(3): 263-7.
34. Veiga, C.C., Carvalho, F.A., De Paula Reis, J.E., De Oliveira,
G.M., Peters, V. M. Fetal toxicity of Solanum lycocarpum
(Solanaceae) in rats. J Ethnopharmacol 2002, 81(2): 265-9.
Address for correspondence: Dr. Emmanuel Acha Asongalem,
Pharmacology and Toxicology Unit, Department of Physiological
Sciences, Faculty of Medicine & Biomedical Sciences, University of
Yaounde 1, P.O. Box 8283, Yaounde, Cameroon. E-mail: cpehw@
yahoo.com