Article
Revista Brasileira de Farmacognosia
Brazilian Journal of Pharmacognosy
20(6): 950-955, Dez. 2010
Received 6 Aug 2009; Accepted 12 Apr 2010; Available online 27 Aug 2010.
Preliminary assessement of the estrogenic potential of Apuleia
leiocarpa (Vogel) J.F. Macbr., Fabaceae, Platypodium elegans Vogel,
Fabaceae, and Brosimum guianense (Aubl.) Huber, Moraceae,
on the wistar rat reproductive system
Rita de Cássia da Silveira e Sá,*,1 Vera Maria Peters,2 Martha de Oliveira Guerra2
1
Departamento de Fisiologia e Patologia, Universidade Federal da Paraíba, Campus Universitário, 58059-900
João Pessoa, PB, Brazil,
2
Centro de Biologia da Reprodução, Universidade Federal de Juiz de Fora, Juiz de Fora, Campus Universitário,
36036-330 Juiz de Fora-MG, Brazil
RESUMO: Avaliação preliminar do potencial estrogênico de Apuleia leiocarpa (Vogel) J.F.
Macbr., Fabaceae, Platypodium elegans Vogel, Fabaceae, and Brosimum guianense (Aubl.)
Huber, Moraceae, no sistema reprodutor de ratos Wistar ”. Um grupo de primatas da espécie
Alouatta guariba foi estudado em seu habitat natural na Mata Atlântica, onde foi observada uma
drástica redução populacional dessa espécie. Suspeita-se que essa redução se deve à inibição da
fertilidade das fêmeas devido ao consumo de Apuleia leiocarpa (Vogel) J.F. Macbr., Fabaceae,
Platypodium elegans Vogel, Fabaceae e Brosimum guianense (Aubl.) Huber, Moraceae. Estudos
fitoquímicos indicaram a presença de cumarinas, especialmente em B. guianense e P. elegans,
cujo efeito adverso no desenvolvimento de folículos ovarianos foi previamente relatado em ratas.
Este trabalho investiga a atividade estrogênica dessas plantas no útero e vagina utilizando ratas
castradas como modelo experimental. Ratas Wistar pubescentes castradas foram tratadas por
cinco dias com os extratos hidroalcoólicos de A. leiocarpa, P. elegans and B. guianense (50 mg/
rata). Foram analisados os seguintes parâmetros: peso de útero e hipófise, cornificação e abertura
vaginal. Os resultados preliminares obtidos mostraram que a administração dos extratos não
alterou significativamente as variáveis analisadas, indicando, indiretamente, a ausência de efeito
estrogênico no sistema reprodutor das ratas tratadas com as plantas citadas. Esses dados sugerem
que o consumo dessas plantas não está relacionado com a redução populacional observada no grupo
de primatas da espécie A. guariba.
Unitermos: Alouatta guariba, Apuleia leiocarpa, Brosimum guianense, atividade estrogênica,
Platypodium elegan, rato Wistar.
ABSTRACT: A group of primates (Alouatta guariba) was studied in its natural habitat, where a
drastic population reduction was detected. It is suspected that this reduction is due to the inhibition of
fertility caused by the consumption of Apuleia leiocarpa (Vogel) J.F. Macbr., Fabaceae, Platypodium
elegans Vogel, Fabaceae, and Brosimum guianense (Aubl.) Huber, Moraceae. These plants are
reported to have cumarins, which have been shown to affect ovarian follicular development in
rats. This work investigates the estrogenic activity of these plants on the uterus and vagina using
castrated rats as the biological model. Pubescent castrated rats were treated for five days with A.
leiocarpa, P. elegans and B. guianense hydroalcoholic extracts (50 mg/rat). Uterus and pituitary
gland weight, vaginal cornification and opening were evaluated. The results showed that the
administration of the extracts did not significantly alter the parameters analyzed. This preliminary
investigation indirectly indicates the absence of estrogenic effect on the rat reproductive system and
no relation to the population reduction of this particular group of primates.
Keywords: Alouatta guariba, Apuleia leiocarpa, Brosimum guianense, estrogenic activity;
Platypodium elegan, Wistar rat.
950
*E-mail: ritasa@ltf.ufpb.br, Tel. +55 32 3229 3251, Fax +55 32 3229 3255
ISSN 0102-695X
DOI: 10.1590/S0102-695X2010005000011
Preliminary assessement of the estrogenic potential of Apuleia leiocarpa (Vogel) J.F. Macbr., Fabaceae, Platypodium elegans
INTRODUCTION
A group of primates from the species Alouatta
guariba (Alouatta fusca Geoffrey, 1812, previously),
popularly known as brown-howler monkey and classified
as near threatened on the International Union for the
Conservation of Nature and Natural Resources (IUCN) red
list 2007, was studied in its natural habitat in the Atlantic
Forest located at Mata da Boa Vista in the district of
Comendador Levy Gasparian, Rio de Janeiro State (Brazil),
where a drastic populational reduction of this species was
detected (Limeira, 1996). A populational density survey
showed that the number of animals found at Mata da Boa
Vista was much lower than that of other groups of the
same primate species from other areas in Brazil, namely
Reserva da Cantereira and Lageadinho (São Paulo State),
and Estação Biológica de Caratinga (Minas Gerais State)
(Limeira, 1996).
The differences observed in the population
density of primates can be due to several factors, including
habitat destruction, food availability, hunting, diseases and
aggressive social behavior. One additional factor that is
seldom questioned and might explain this reduction is the
inhibition of fertility caused by the consumption of certain
types of plants found in the Atlantic Forest which could
be interfering with the reproductive cycle of primates. For
instance, in many plants it is common the occurrence of
substances, such as flavonoids and phytosterols (sitosterol
and stigmasterol), which have similar activity to that of the
natural hormones (Salah et al., 2002). A quantitative study
of the diet of A. guariba at Mata da Boa Vista indicated that
the most frequently consumed plant species were Apuleia
leiocarpa (Vogel) J.F. Macbr., Fabaceae (Correa, 1984b),
Platypodium elegans Vogel, Fabaceae (Silva Jr, 1981;
Correa, 1984a), and Brosimum guianense (Aubl.) Huber,
Moraceae (Correa, 1984b; Limeira, 1996). Phytochemical
studies have shown the presence of sitosterol and
stigmasterol in P. elegans and cumarins in B. guianense and
P. elegans (Amaral et al., 2001; Amaral, 1997) which have
been reported to affect ovarian follicular development in
rats (Ulubelen et al., 1994). The relationship between the
chemical composition of plants and feeding strategies of
primates has been investigated by many authors (Glander,
1979; Milton, 1979; Rodriguez et al., 1985; Barton et al.,
1993). In 1995, Stryer observed that female primates of
the species Brachyteles arachnoides (muriqui) showed a
specific feeding habit by increasing the consumption of A.
leiocarpa and P. elegans only during mating season.
The investigation of how plant constituents may
act or affect the organism has already been addressed in
various animal species. In rodents, many studies have
reported the estrogenic and anti-estrogenic effects of
plant extracts on the female reproductive system, giving
emphasis to their action on the uterus and vagina through
the occurrence of histological alterations and cornification
of the vaginal epithelium (Shukla et al., 1989; Tewari
et al., 1989; Johri et al., 1991), their interference in the
implantation (Montanari & Bevilacqua, 2002; Salah et al.,
2002), and their abortifacient activity (Lutterodt, 1988;
Badami et al., 2003).
Considering the issue of species preservation
and the ecological implications that may result from its
disappearance, it is important to identify the food sources
that have compounds among their constituents capable of
interfering with fertility, which could be responsible for
the reduction of primates’ populations. Such compounds
may have an effect similar to that of hormones, such as
estrogen, that are known to affect fertility. Therefore, the
purpose of this work was to investigate the estrogenic
activity of A. leiocarpa, P. elegans and B. guianense using
rats as the biological model.
MATERIALS AND METHODS
Plant material
Apuleia leiocarpa (Vogel) J.F. Macbr., Fabaceae,
Platypodium elegans Vogel, Fabaceae, and Brosimum
guianense (Aubl.) Huber, Moraceae were collected in Mata
da Boa Vista, district of Comendador Levy Gasparian, Rio
de Janeiro State, Brazil. The species were authenticated by
H. B. Lima from Instituto Jardim Botânico (Rio de Janeiro)
and voucher specimens were deposited at the Herbarium
of Museu Nacional-UFRJ, Rio de Janeiro (Amaral et al.,
1997; Amaral, 2001). The hydroalcoholic extracts were
prepared by Dr. Gilda Guimarães Leitão and Dr. Suzana
Guimarães Leitão from the Biology Laboratory, Natural
Products Research Center (Federal University of Rio de
Janeiro-UFRJ) using aerial parts of these plants.
Animals and housing
Pubescent female Wistar rats (Rattus norvegicus
Berkenhout, 1769), weighing around 40 g, were obtained
from the vivarium of UFJF, where they were born and bred.
They were housed individually under standard laboratory
conditions with a 12 h light/12 h dark photoperiod. They
were fed on rat chow pellets and received water ad libitum.
Animal care and the experimental protocol followed the
principles and guidelines suggested by the Brazilian College
of Animal Experimentation (COBEA) and were approved
by the Ethical Committee of the Federal University of Juiz
de Fora (protocol number 019/2004 – CEA - UFJF).
Bioassay
The biological assay used to study the estrogenic
effect follows the guidelines proposed by the Organization
for Economic Cooperation and Development (OECD
Reprotox Protocol) with modifications. In brief, this
protocol comprises the analysis of vaginal cornification,
vaginal opening, pituitary gland and uterus weight gain.
Rev. Bras. Farmacogn.
Braz. J. Pharmacogn.
20(6): Dez. 2010
951
Rita de Cássia da Silveira e Sá, Vera Maria Peters, Martha de Oliveira Guerra
The presence of cornified cells in the vagina and the
presence of mitosis in the uterin glands and endometrium
were used as indicatives of occurrence of estrogenic effect
(OECD, 2007).
The rats were randomly divided into eight groups
of ten animals each and underwent bilateral ovariectomy
at 28 days of age. Briefly, the animals were anesthetized
with 10 mg/kg xylazine and 90 mg/kg ketamine i.p. and
following hair removal at the site of the operation, an
antiseptic wash with an organic iodine preparation was
applied on the surface of the skin to prevent infection. The
anaesthetized animal was laid on its ventral surface and a
small midline dorsal skin incision was made approximately
half way between the middle of the back and the base
of the tail. Entrance to the peritoneal cavity was gained
through muscle incisions made half to two-thirds of the
way down the side of the body, where the ovary was
removed after being separated from its surrounding fat
deposits (Waynforth & Flecknell, 1994)). After surgery,
the animals were given 15 days to recover prior to drug
administration. They were then treated for five days (Gray
et al., 2004) as follows:
Group 1: hydroalcoholic extract of A. leiocarpa (50 mg/
kg) - v.o.
Group 2: hydroalcoholic extract of P. elegans (50 mg/kg)
- v.o.
Group 3: hydroalcoholic extract of B. guianense (50 mg/
kg) - v.o.
Group 4: estrogen dissolved in corn oil (50 µg/kg) - i.p.
Group 5: corn oil (0.1 mL/kg) - i.p.
Group 6: hydroalcoholic solution 50% (0.5 mL/kg) - v.o.
Group 7: Ovariectomy – no treatment
Group 8: colony control - no treatment.
The hydroalcoholic extracts were administered
at a dose level which corresponded to 1% of the highest
dose used on reproductive toxicological assays (5 g/
kg) (Chan & Hayes, 1994). During the experiment, the
animals were inspected daily for detection of clinical
signs of toxicity, such as piloerection, and alterations in
locomotor activity (Christian, 2001). Body weight was
recorded before the beginning of treatment, and at the
end of treatment. The animals were killed by an overdose
inhalation of anesthetic (halothane) 24 h after the last
treatment. Before death, the genital region was inspected
for observation of the vaginal opening and, immediately
after death, the animals underwent laparotomy for removal
of the uterus, vagina and pituitary gland. The uterus and
pituitary gland were weighed. After that, the uterus and
vagina were fixed in Bouin for histological examination.
The tissues were dehydrated in a graded series of ethanol,
embedded in paraffin and sectioned at 7 µm thickness for
routine haematoxylin and cromotrope staining, and light
microscope examination (Michalany, 1998).
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Statistical analysis
The data were analyzed using the One-way
analysis of variance (ANOVA) followed by Tukey’s
multiple comparison test (α=0.05). The presence or absence
of mitosis and vaginal opening were analyzed using the X2
test (α=0.05) (Sokal & Rohlf, 1996).
RESULTS
During the experimental procedure no deaths
and no clinical signs of toxicity such as locomotor
activity alterations and piloerection were detected in
any of the groups. Table 1 shows that at the end of the
five-day treatment period body weight gain was not
significantly different when comparing the extract,
vehicle, ovariectomized and estrogen-treated groups with
the colony control group. The weight of the uterus was
significantly increased in the estrogen-treated group and,
as expected, castration reduced the weight of the uterus of
all other groups; however, the extract did not induce any
uterotrophic effects (Table 2). The weight of the pituitary
gland of the vehicles, extract and estrogen-treated groups
was not significantly altered when compared to the colony
control. In particular, P. elegans-treated rats showed a
reduction in the pituitary gland weight, but the mean
value obtained did not differ statistically from that of the
ovariectomized group (Table 2).
Table 1. Body weight of Wistar rats treated with Apuleia leiocarpa,
Brosimum guianense and Platypodium elegans extract, corn oil,
estrogen, alcohol 50%, ovariectomized and colony control.
Groups
Weight (g)
Body 1
Body 2
A. leiocarpa
44.62±1.25
75.74±2.58
B. guianense
41.61±6.73
77.14±7.28
P. elegans
39.61±3.05
67.40±7.56
Alcohol 50%
42.27±1.65
74.71±3.65
Corn oil
37.16±1.60
69.73±4.03
Ovariectomy
48.79±4.26
79.11±6.11
Estrogen
36.43±2.05
63.59±4.07
Colony control
38.09±3.56
67.88±9.03
The results were expressed by mean ± standard deviation (SD). p>0.05.
Body 1 and 2 – body weight before the beginning and at the end of
treatment, respectively. n= 0.
Premature opening of the rat's vaginal membrane
follows the administration of estrogen. In this experiment,
at the time of death (48 days old), the vaginal opening was
observed in all estrogen-treated animals whereas in the
extract-treated and vehicle groups only 50% and 30%, of
the animals, respectively, exhibited this trait.
The uterus and vagina sections showed that the
administration of the hydroalcoholic extract of A. leiocarpa,
Preliminary assessement of the estrogenic potential of Apuleia leiocarpa (Vogel) J.F. Macbr., Fabaceae, Platypodium elegans
P. elegans and B. guianense, during five days, did not
cause any visible morphologic alteration of the uterine and
vaginal walls of castrated rats. The administration of these
plant extracts did not induce cellular proliferation, which
is represented by the occurrence of mitotic figures in the
epithelium of the uterus and the endometrial glands. The
uterus wall showed a simple columnar epithelium lining
its mucosa, the adjacent muscle layers (the myometrium)
and the perimetrium with no morphological alteration due
to treatment (Figures 1a, b and c). The vagina presented a
normal stratified squamous epithelium without any signs
of cornification typical of the estrogen effect.
In the estrogen-treated control rats, the uterine
wall showed increased activity of cell division and mitotic
figures. Epithelium proliferation was observed in the
endometrial glands (Figure 1f) as well as in the uterine
lining which showed a pseudostratified epithelium. The
exposure to estrogen enhanced mitosis in the vaginal
epithelium showing areas of pseudostratified epithelium
and also areas of keratinized stratified squamous epithelium.
The other control groups did not show any morphologic
alterations in the uterus (Figures 1d and e) or vagina.
Table 2. Uterus and pituitary gland weights of Wistar rats treated
with Apuleia leiocarpa, Brosimum guianense and Platypodium
elegans extract, corn oil, estrogen, alcohol 50%, ovariectomized
and colony control.
Groups
Weight (mg)
Pituitary gland
Uterus
A. leiocarpa
3.50±0.38
18.06±4.25
B. guianense
3.64±0.94
14.32±2.32
P. elegans
2.98±0.40
16.52±5.03
Alcohol 50%
3.48±0.28
15.27±2.53
Corn oil
3.98±0.40
29.48±9.84
Ovariectomy
3.87±0.73
15.53±2.28
Estrogen
3.77±0.76
101.30±12.81*
Colony control
3.19±0.90
45.24±18.00
The results were expressed by mean±standard deviation (SD). p<0.001.
n=10.
DISCUSSION
The mammal feminine genital tract and the ovary
are under the control of hormones capable of causing
histological and physiological alterations that directly
influence sexual maturation and the reproductive behavior
of the species (Eckert et al., 1988). When a hormonal
imbalance occurs, these hormones can interfere in the
natural process of animal reproduction, inhibiting the
proliferation of the species, which may lead to disastrous
consequences to the environment (Tewari et al., 1989).
The most important hormones involved in the regulation
of the alterations detected in the feminine genital tract
are estrogen and progesterone, which are regulated by
pituitary-secreted gonadotropin hormones (Brann et al.,
1995).
The hypertrophic response of the rodent uterus is
one of the widely used tests in monitoring the estrogenic/
antiestrogenic potency of various drugs and chemicals
(Khan-Dawood & Satyaswaroop, 1995). Previous works
have reported the endogenous and exogenous estrogenic
and antiestrogenic activity of different compounds in this
group of animals (Brann et al., 1995); many of them have
shown that ovariectomy results in depletion of endogenous
estrogens, and the exposure to exogenous estrogens or
phytoestrogens can enhance mitosis and therefore provoke
epithelium proliferation of the genital tract (Ravichandran
et al., 2007). Another group of phytochemicals found in
the plants employed in this study, namely sitosterol and
stigmasterol are phytosterols that are chemically similar
to animal cholesterol. Sitosterol is mainly known and
used for its cholesterol lowering property and stigmasterol
is used as a precursor in the manufacture of synthetic
progesterone, that plays an important physiological role
in the regulatory and tissue rebuilding mechanisms related
to estrogen effects, as well as acting as an intermediate
in the biosynthesis of androgens, estrogens, and corticoids
(Peterson, 1995). Moreover, cumarin, a natural product also
found in the plants of this study, has also been reported to
have antifertility activity in mature female rats (Ulubelen
etal., 1994).
In the present work, ovariectomized Wistar rats
treated with A. leiocarpa, P. elegans and B. guianense
hydroalcoholic extracts did not show any histological
alterations on the uterine and vaginal wall. The most
common estrogenic effects, such as cell proliferation in the
endometrium, immature vaginal opening and cornification,
which were observed in the estrogen-treated rats of this
experiment, were absent from the groups treated with the
plant extracts. The weight of the pituitary gland and uterus
was not significantly altered in the groups treated with the
extracts and neither was any early vaginal opening detected
in those groups. These facts indirectly indicate the absence
of functional alterations caused by estrogenic influence.
In conclusion, considering the bioassay used
in this work, A. leiocarpa, P. elegans and B. guianense
extracts did not seem to have estrogenic effect on the Wistar
rat reproductive system, given that the histoarchitecture
of the uterus and vagina of these animals was kept intact.
These data suggest that the ingestion of A. leiocarpa, P.
elegans and B. guianense could not be held accountable
for the populational reduction of the howler monkeys in
the Brazilian Atlantic forest.
ACKNOWLEDGEMENTS
The authors are grateful to Paulo Sérgio do Carmo
for the technical assistance and Dr. Dr. Gilda Guimarães
Leitão and Dr. Suzana Guimarães Leitão for supplying the
plant extracts and Ms Angela Harman for reviewing the
English version of the manuscript.
Rev. Bras. Farmacogn.
Braz. J. Pharmacogn.
20(6): Dez. 2010
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Rita de Cássia da Silveira e Sá, Vera Maria Peters, Martha de Oliveira Guerra
Figure 1. Transverse section of the uterus of ovariectomized rats treated with (a) A. leiocarpa, (b) P. elegans and (c) B. guianense
hydroalcoholic extract, (d) hydroalcoholic solution 50 % and (e) untreated showing no morphologic alteration of the uterine wall. (f)
estrogen-treated ovariectomized rats showing the uterine wall with increased epithelium of the endometrial gland (crossed arrows).
Arrow - simple columnar epithelium; black star – stroma of the endometrium. (100x).
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Preliminary assessement of the estrogenic potential of Apuleia leiocarpa (Vogel) J.F. Macbr., Fabaceae, Platypodium elegans
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