Food and Chemical Toxicology 50 (2012) 816–822
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
Efficacy of Morus nigra L. on reproduction in female Wistar rats
Graziela Tonioni de Queiroz a,⇑, Tatianne Rosa Santos a, Renato Macedo b, Vera Maria Peters a,b,
Magda Narciso Leite c, Rita de Cássia da Silveira e Sá d, Martha de Oliveira Guerra a,b
a
Programa de Pós-Graduação em Ciências Biológicas – Área de concentração em Comportamento e Biologia Animal/Universidade Federal de Juiz de Fora, Campus Universitário,
Bairro São Pedro, Juiz de Fora, Minas Gerais, CEP 36036-330, Brazil
b
Centro de Biologia da Reprodução/Universidade Federal de Juiz de Fora, Campus Universitário, Bairro São Pedro, Juiz de Fora, Minas Gerais, CEP 36036-330, Brazil
c
Departamento de Farmácia e Bioquímica/Universidade Federal de Juiz de Fora, Campus Universitário, Bairro São Pedro, Juiz de Fora, Minas Gerais, CEP 36036-330, Brazil
d
Departamento de Fisiologia e Patologia/Universidade Federal da Paraíba, Campus Universitário, João Pessoa, Paraíba, CEP 58051-900, Brazil
a r t i c l e
i n f o
Article history:
Received 29 August 2011
Accepted 11 December 2011
Available online 17 December 2011
Keywords:
Morus nigra
Mulberry
Toxicology
Reproduction
Wistar rats
a b s t r a c t
Morus nigra L. is a plant employed as a substitute for the conventional hormonal replacement therapy.
This work analyzes the estrogenic effect of M. nigra on the reproductive system and embryonic development of Wistar rats. Female rats were orally treated with M. nigra hydroalcoholic extract (MnHE) at the
dose levels of 25, 50, 75, 350 and 700 mg/kg of body weight over 15 days, and continued through mating
until the 14th day of gestation. Vaginal smears were performed daily and the body weight of the females
was recorded at 5 days intervals. On day 15 of gestation, the females were killed and their kidneys, liver,
spleen and ovaries were removed and weighed. The number of implants, resorptions, and live and dead
fetuses were evaluated. Histological sections of ovaries, measurement of the height of the uterine epithelium and vaginal smears were performed to assess the estrogenic activity. The results showed that the
administration of MnHE did not significantly alter the analyzed variables. Therefore, considering the
experimental model used in this study, the data obtained indicate that M. nigra did not exhibit any estrogenic activity nor did exert a toxic effect on the female reproductive system and on the embryonic development of rats.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Morus nigra L. (Moraceae), known as mulberry (also as ‘‘amoreira’’, ‘‘amora preta’’ or ‘‘negra’’) (Ercisli and Orhan, 2007; Wasano
et al., 2009), is a plant found throughout Brazil and is well known
by the population for its medicinal properties. The fruits, leaves,
barks and roots are used as laxative, sedative, expectorant, refresher, emollient, calmative, diuretic, hypoglycaemic agent, antiseptic, anti-inflammatory, antioxidant, and in the treatment of
eczema, and oral inflammation (Oryan et al., 2003; Naderi et al.,
2004; Ercisli and Orhan, 2007; Padilha et al., 2010). In China, the
leaves of Morus sp. are used as an antioxidant, antimicrobial agent
and anti-inflammatory (Song et al., 2009), however the leaves of
Morus alba L. were reported to have cytotoxic activity in neoplastic
cells (Dat et al., 2010).
The leaves of M. nigra are commonly used by women in menopause as a substitute for the conventional hormonal replacement
therapy, with a similar effect to that obtained after estrogen use
(Silva et al., 2003; Miranda et al., 2010). Moreover, the leaves are
⇑ Corresponding author.
E-mail addresses: grazielaufjf@yahoo.com.br, tgrazy@gmail.com (G.T. de Queiroz), tatiannersantos@yahoo.com.br (T.R. Santos), renatimdalapa@yahoo.com.br (R.
Macedo), peters.vera@ujfj.edu.br (V.M. Peters), magda.narciso@hotmail.com (M.N.
Leite), Brasil.ritacassia.sa@bol.com.br (Rita de Cássia da Silveira e Sá), martha.guerra@ufjf.edu.br (Martha de Oliveira Guerra).
0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2011.12.014
also used by younger women as a reliever of the symptoms of
the premenstrual tension. The use of M. nigra usually involves
the preparation of a ‘‘tea’’ (i.e. through decoction or infusion of
the leaves) and is reported to ameliorate the symptoms of menopause (Miranda et al., 2010), particularly hot flashes (Castro,
2010), which are related to the sudden vasodilatation that causes
the feeling of intense heat and redness of the skin, namely the face.
However, the experimental studies are controversial in regard
to the tests employed to verify the estrogenic effect on oophorectomized and pubescent animal models (Vanoni, 2006; Bolzan,
2008; Castro, 2010). For instance, Silva et al. (2003) assessed the
estrogenic activity of an infusion of Morus sp. leaves and did not
find any estrogenic effect on oophorectomized rats.
Previous studies have shown the presence of various substances
in different species of the genus Morus sp., such as fatty acids, vitamin C (originated from the plants primary metabolism), phenylflavonoid (morusine) (Petlevski et al., 2001); flavonoids (Naderi
et al., 2004; Lin and Tang, 2007; Ercisli and Orhan et al., 2007; Pawlowska et al., 2008; Song et al., 2009), phenolic compounds (Ercisli
and Orhan, 2007; Song et al., 2009; Kim et al., 2010), organic acids
(Koyuncu, 2004), anthocyanins, karotenoids (Dugo et al., 2001; Hassimotto et al., 2007), alkaloids (Song et al., 2009), originating from
the secondary metabolism of the plants. M. nigra fruit in particular
has high concentrations of phenols and flavonoids (Ercisli and Orhan, 2007) in addition to four types of anthocyanins (Pawlowska
G.T. de Queiroz et al. / Food and Chemical Toxicology 50 (2012) 816–822
et al., 2008), whereas terpenoids and steroids, such as b-sitosterol,
have been found in the leaves of this species (Padilha et al., 2010).
Several pharmacological activities have already been described
in relation to some of the components mentioned above, such as
the ability to change gene expression and anticarcinogenic activity
due to the presence of phenolic substances and b-sitosterol (Tapiero et al., 2002; Nakamura et al., 2003; Awad et al., 2005). Some
flavonoids have been reported to have estrogenic activity, such as
quercetin, with broad pharmacological application, such as antiinflammatory, anticarcinogenic. The anthocyanins inhibit Cox-1
and Cox-2, enzymes that are involved in the synthesis of prostaglandins (Seeram et al., 2001).
Moreover, some studies have reported the possible estrogenic
potential of M. nigra leaves in pubescent (Vanoni, 2006) and
oophorectomized Wistar rats (Silva et al., 2003; Vanoni, 2006),
and in oophorectomized rats submitted to chronic treatment (Castro, 2010). The pharmacologic activities previously mentioned and
the phytoestrogens may have an adverse effect on the reproductive
process by: interfering with the ovarian hormone synthesis and the
vaginal cyclicity; the embryo cell proliferation (anticarcinogenic
activity); the mechanisms involved in the gene expression during
the complete development and differentiation of the embryo or
the prostaglandin F2-a or E production, which are related to many
reproductive mechanisms, such as ovulation (PGF2-a) and blastocyst implantation (PGE).
Most of the information available in the literature about the
estrogenic effect of M. nigra was concerned with young females.
Therefore, considering the use of this plant by women and the popular belief of its effects on the female organism, the present study
was aimed at investigating the estrogenic activity of M. nigra leaves
in adult females, its reproductive toxicity and the embryonic
development.
2. Materials and methods
The experimental protocol was approved by the Ethical Committee of the Centro de Biologia da Reprodução at the Federal University of Juiz de Fora (UFJF – Juiz
de Fora, Minas Gerais State, Brazil) (Protocol No. 048/2009-CEEA), which follows the
international principles in ethics for animal experimentation.
2.1. Plant material
M. nigra leaves were collected in the medicinal garden of the Pharmacy and Biochemistry Faculty (UFJF) in November 2009. The plant was identified by Prof. Luiz
Menine Neto in the Herbarium Professor Leopoldo Krieger, Department of Botany
(UFJF), where a voucher specimen registered under the number CESJ 48362 is
deposited.
M. nigra hydroalcoholic extract (MnHE) was produced in the Pharmacognosy
Laboratory of the Pharmacy and Biochemistry Faculty (UFJF). It was prepared using
50 g of air-dried leaves that were powdered and extracted with 70% ethanol (Soxhlet apparatus). The solvent was evaporated in a rotavapor (Büchi Rotavapor R-114,
Büchi Waterbath B-480, Alemanha) for the complete elimination of the ethanol and
then filtrated for the complete elimination of the chlorophyll. The final solution was
dissolved in distilled water at the appropriate concentrations (25, 50, 75, 350 and
700 mg/kg) for the experiment.
For the extracts at the dose levels of 25, 50 and 75 mg/kg, a final solution concentration of 16 mg/mL was used. For the dose level of 350 mg/kg, the final solution
concentration was 50 mg/mL, and for the 700 mg/kg dose level, the concentration
was 106 mg/mL.
817
mals were housed individually inside climatized cabinets (Alesco, Brasil), under
standard laboratory conditions (temperature of 22 °C ± 2 °C and relative air humidity of 40–60%), with a 12 h light/12 h dark photoperiod. They were fed on rat chow
pellets (25 g daily – Nuvilab) and received water ad libitum.
2.4. Bioassay
The development of this work was based on the protocol from the International
Committee for Harmonization – stage I, (ICH, 2005) with minor modifications, as
shown below.
Female Wistar rats were randomly divided into three groups of 15 animals each
(T1, T2, T3) and three groups of 20 animals each (T4, T5 and control) that were treated as follows: (1) T1 (MnHE 25 mg/kg); (2) T2 (MnHE 50 mg/kg); (3) T3 (MnHE
75 mg/kg); (4) T4 (MnHE 350 mg/kg); (5) T5 (MnHE 700 mg/kg); (6) Control (distilled water). The dose of 700 mg/kg was based on that used by Vanoni (2006)
and the other doses represented decreasing intervals of the highest dose. The animals were treated orally with MnHE for 15 days, during which the cyclicity was
determined through the vaginal cytology, performed daily at 8 a.m.
In order to observe the estrogenic effect, on the 16th day, five animals from
groups T4, T5 and control (Okuda et al., 2010) were killed by exsanguination
through cardiac puncture under Ketamine 2% (10 mg/kg, i.p.) and Xylazine 5%
(90 mg/kg, i.p.) anesthesia. The ovaries and uterus were removed and fixed in formol calcium (Baker), embedded in paraffin, and sectioned at 5 lm thickness for routine haematoxylin-eosin staining. A light microscope connected to a video camera
(AxioCam IcC3, Carl Zeiss, Jena, Germany) was used for morphological evaluation
and image recording of the slides. Transversal sections of the uterus were used
for measurement of the epithelium thickness, using image analysis software (AxioVision 4.7 REL Carl Zeiss, Jena, Germany) (Fig. 1). For each tubular section, 16 measurements per group were obtained.
The remaining rats from all groups continued to be treated through mating until
the 14th day of gestation. The presence of spermatozoa in the vaginal smear indicated successful mating and was considered as day one of gestation (Keshri et al.,
2003).
For 60 min after treatment, and then once daily, the females were observed for
clinical signs of toxicity, such as piloerection, stereotypy, hyper or hypoactivity,
diarrhea, sialorrhea, polyuria or death (Christian, 2001). Body weight was recorded
before the beginning of treatment and at 5 days intervals. Food consumption was
monitored daily, considering a fixed amount of pellets (25 g) placed on the cage
lid and what was left of it on the following day.
On the 15th day of gestation, the animals were killed by exsanguination
through cardiac puncture under Ketamine 2% (10 mg/kg, i.p.) and Xylazine 5%
(90 mg/kg, i.p.) (Köning, S.A., Buenos Aires, Argentina) anesthesia. Immediately
after death, the animals underwent laparotomy for the removal and weighing of
the liver, kidneys and spleen. The female reproductive tract was also removed
and the ovaries were weighed and the corpora lutea counted under a stereoscopic
microscope (SZ40 Olympus Tokio, Japão). The uterine horns were longitudinally
sectioned for counting the number of live (presence of heart beat) and dead fetuses.
The placenta and fetuses were weighed to obtain the average weight of the litter
and the placenta. Subsequently, the fetuses were fixed in Bouin for 60 min and observed under a stereoscopic microscope for the identification of alterations on the
front and hind legs, face morphology, and verification of the neural tube closure.
2.2. Phytochemical analysis
The phytochemical analysis of MnHE was performed according to Matos (1997),
in order to verify the presence of the following classes of secondary metabolism
products: flavonoids, tannins, cumarins, heterosides, triterpenes/steroids, saponins,
alkaloids and anthraquinones.
2.3. Animals
Ninety adult nulliparae Wistar rats (Rattus norvegicus Berkenhout, 1769)
(60 days old and weighing about 250 g) were obtained from the vivarium of the
Federal University of Juiz de Fora (UFJF), where they were born and bred. The ani-
Fig. 1. Histological section of the rat uterus, showing the thickness of the
epithelium.
G.T. de Queiroz et al. / Food and Chemical Toxicology 50 (2012) 816–822
818
29
C o n t ro le
T1 (2 5 m g / k g )
T2 (5 0 m g / k g )
Food consumption (g)
26
T3 (7 5 m g / k g )
T4 (3 5 0 m g / k g )
T5 (7 0 0 m g / k g )
23
20
17
14
11
0
2
4
6
8
10
12
14
16
Preinsemination day
18
20
22
24
26
28
30
32
Postinsemination day
Fig. 2. Daily food consumption of control and M. nigra extract-treated Wistar rats at the dose levels of 25, 50, 75, 350 and 700 mg/kg, administered during the pre and
postinsemination period (v.o., N = 15).
The total number of live fetuses, corpora lutea, resorptions and implants were
determined for the calculation of the following indexes: implants per group (total
number of implants/total number of corpora lutea) 100; preimplantation loss
per group (100 implantation index); and postimplantation loss per group [(total
number of resorptions + total number of dead fetuses)/total number of implants] 100 (Parker, 2006).
2.5. Statistical analysis
The data were analyzed using the one-way analysis of variance (ANOVA) followed by Dunnett’s test. For the non homoscedastic and without normal distribution data the Kruskal–Wallis test followed by the Mann–Whitney test was used
(a = 0.05). The litter, not the mother, was the experimental unit when comparing
the litter weight and placenta weight.
3. Results
3.1. Phytochemistry
The phytochemical evaluation of MnHE M. nigra showed the
presence of flavonoids, tannins, cumarins, and triterpenes/steroids.
Flavonoids and triterpenes, are known to possess estrogen-like effects (Ibarreta et al., 2001; Jefferson, 2003).
3.2. Reproductive toxicology and embryonic development
During the experimental procedure, no deaths or any clinical
signs of toxicity were observed. Food consumption (Fig. 2) and
Controle
58
T1 (25 mg/kg)
54
T2 (50 mg/kg)
Weight gain (g)
50
T3 (75 mg/kg)
46
T4 (350 mg/kg)
42
T5 (700 mg/kg)
38
34
30
26
22
18
14
10
6
2
0
1
2
3
Preinsemination day
4
5
6
7
Postinsemination day
Fig. 3. Body weight gain of control and M. nigra extract-treated Wistar rats at the dose levels of 25, 50, 75, 350 and 700 mg/kg, administered during the pre and
postinsemination period (v.o., N = 15).
G.T. de Queiroz et al. / Food and Chemical Toxicology 50 (2012) 816–822
819
Fig. 4. Wistar rats’ ovaries during the progestational (A, B, C) and estrogenic phases (D, E, F), showing follicles on different phases of development. Control animals (A and D)
and M. nigra extract-treated group at the dose level of 350 mg/kg (T4) (B and E), and 700 mg/kg (T5) (C and F).
body weight (Fig. 3) gain were similar between control and treated
animals. Death and clinical signs of toxicity as well as increase or
reduction of food intake are suggestive of maternal toxicity, which
could indicate that the alteration in the embryonic development is
not due to MnHE but could be related to the maternal toxicity.
The analysis of the vaginal smears showed a similar vaginal
cytology among the experimental groups (data not shown). The
presence of ovarian structures (corpora lutea and follicles at various stages of development) suggests that the ovaries are physiologically normal. Here, the histological sections of the ovaries
showed the presence of such structures. In addition, there were
no signs of edema, cystic follicles or retained oocytes (Fig. 4). The
thickness of the uterine epithelium of the treated animals did not
change significantly when compared to the control ones: estrogenic
phase
(C = 23.33 ± 1.15 lm;
T4 = 23.56 ± 1.64 lm;
T5 = 22.88 ± 1.45 lm); progestational phase (C = 14.49 ± 1.28 lm;
T4 = 14.13 ± 1.29 lm; T5 = 14.76 ± 1.29 lm). If the extract had exerted estrogenic effect, it would have been expected to find thinner
uterine walls, proliferative endometrium and a vaginal cytology
showing predominant cornified superficial cells (Li and Davis,
2007).
The absolute and relative weight of the liver, kidneys, spleen
and ovaries is shown in Table 1. Reduction or increase in the
weight of these organs suggests maternal toxicity. The number of
corpora lutea, live fetuses and resorptions, implants, pre and
postimplantation losses, live and dead fetuses per group, average
weight of the fetuses and placenta per litter is shown in Table 2.
Reduction in the number of live fetuses, increase in the number
of resorptions and pre- and post-implantation losses indicate toxicity to the embryonic development. No significant alterations of
these variables were observed (p > 0.05), and no external malformations were detected during the macroscopic analysis of the
fetuses.
4. Discussion
Owing to the popular use of M. nigra leaves as a reliever of hot
flashes and the symptoms of premenstrual tension (Miranda et al.,
2010), it would be possible to suppose that substances capable of
exerting some estrogenic or progesteronic effect would be present
among the constituents of this plant’s leaves. The phytochemical
analysis showed the presence of flavonoids, tannins, cumarins,
polyphenols, triterpenes/steroids, of which two of these compounds, flavonoids and triterpenes, are known to possess estrogen-like effects (Ibarreta et al., 2001; Jefferson, 2003), which may
justify its popular use.
The female reproductive function was assessed through the
vaginal cytology (for evaluation of cyclicity), the histological analysis of the ovaries and the histomorphometry of the uterus. Cycles
that do not follow the sequence proestrus, estrus, metestrus and
diestrus are considered irregular and abnormal (Marcondes et al.,
G.T. de Queiroz et al. / Food and Chemical Toxicology 50 (2012) 816–822
820
Table 1
Organ weights gain of control and M. nigra extract-treated Wistar rats at the dose levels of 25, 50, 75, 350 and 700 mg/kg, administered during the pre and postinsemination
period (v.o.).
Maternal variables
Groups
Control
N = 15
T1
(25 mg/kg)
N = 15
T2
(50 mg/kg)
N = 15
T3
(75 mg/kg)
N = 15
T4
(350 mg/kg)
N = 15
T5
(700 mg/kg)
N = 15
Liver
Absolute weight (g)
Relative weight
7.72 ± 0.53
4.01 ± 0.20
7.44 ± 0.88
3.89 ± 0.44
7.52 ± 0.56
3.94 ± 0.26
7.79 ± 0.93
3.98 ± 0.32
7.55 ± 0.50
4.01 ± 0.21
7.17 ± 0.60
3.92 ± 0.22
Kidney*
Absolute weight (g)
Relative weight
0.75 ± 0.05
0.39 ± 0.02
0.71 ± 0.04
0.37 ± 0.02
0.73 ± 0.06
0.38 ± 0.03
0.75 ± 0.06
0.38 ± 0.02
0.71 ± 0.05
0.37 ± 0.02
0.68 ± 0.04
0.37 ± 0.01
Spleen
Absolute weight (g)
Relative weight
0.53 ± 0.07
0.27 ± 0.03
0.54 ± 0.09
0.28 ± 0.04
0.52 ± 0.06
0.27 ± 0.03
0.53 ± 0.04
0.27 ± 0.02
0.47 ± 0.06
0.25 ± 0.02
0.48 ± 0.06
0.26 ± 0.02
Results expressed in mean ± standard deviation. P > 0.05.
*
Left and right kidney average weight.
Table 2
Number of corpora lutea, live fetuses, implants and resorptions, implantation, preimplantation and postimplantation loss indexes, average of live fetuses per group, average
fetuses body and placenta weights of control and M. nigra extract-treated Wistar rats at the dose levels of 25 mg/kg, 50 mg/kg, 75 mg/kg, 350 mg/kg and 700 mg/kg, administered
during the pre and postinsemination period (v.o.).
Variables
Corpora lutea
Implants
Live fetuses
Resorption
Implantation/group (%)
Preimplantation loss/group (%)
Postimplantation loss/group (%)
Live fetuses/group
Fetuses‘weight/ litter (mg)
Placenta weights /litter (mg)
Groups
Control
N = 15
T1
(25 mg/kg)
N = 15
T2
(50 mg/kg)
N = 15
T3
(75 mg/kg)
N = 15
T4
(350 mg/kg)
N = 15
T5
(700 mg/kg)
N = 15
176
167
161
6
94.88 (167/176)
5.11 (9/176)
3.59 (6/167)
10.73 ± 1.22
164.56 ± 12.95
140.29 ± 15.42
166
156
152
4
93.97(156/166)
6.02(10/166)
2.56 (4/156)
10.13 ± 1.06
164.20 ± 9.42
144.82 ± 20.45
177
167
163
4
94.35 (167/177)
5.64 (10/177)
2.39 (4/167)
10.86 ± 0.99
166.91 ± 11.93
140.71 ± 15.68
166
153
148
5
92.16 (153/166)
7.83 (13/166)
3.26 (5/153)
9.86 ± 2.13
162.61 ± 7.10
140.41 ± 24.27
176
166
158
8
94.31 (166/176)
5.68 (10/176)
4.81 (8/166)
10.53 ± 1.92
167.33 ± 8.91
150.72 ± 15.10
166
152
144
8
91.56 (152/166)
8.43 (14/166)
5.26 (8/152)
9.40 ± 1.40
168.55 ± 9.78
145.81 ± 22.24
Results expressed in mean ± standard deviation. P > 0.05.
2002). Moreover, persistent vaginal cornification with increased
estrous cycle duration, and prolonged diestrus or estrus could all
be indicative of ovulation arrest (EPA, 1996; Goldman et al.,
2007). According to Li and Davis (2007) studies involving toxicity
in the reproductive system can be used as a useful tool in the female reproductive physiology by evaluating the ovary, vagina
and uterus ‘‘as a synchronous system where all the parts should
tell the same story’’.
In this study, the highest doses (350 and 700 mg/kg) of the extract did not produce morphological changes nor did interfere with
the vaginal cyclicity of the rats. In fact, the occurrence of ovulation
was detected in the treated group 4, as indicated by the presence of
two oocytes in the uterine horn (Fig. 5). In addition to the normal
cyclicity, no significant differences in the general morphology of
the ovaries and the uterus morphology and epithelium thickness
were observed. Therefore, considering the experimental protocol
here employed, the MnHE does not seem to exhibit an effect similar to that displayed by the ovarian hormones.
The normal maternal physiology is paramount for the normal
development of the fetus and the offspring (Chahoud et al.,
1999). Hence the evaluation of maternal body and organs weight
and food consumption provides important data for the assessment
of the maternal conditions. The analysis of the physical signs is also
important because they indicate maternal stress, such as piloerection, diarrhea, sialorrhea and reduced ambulation, that could interfere with cortisone production and affect the embryonic
development (Christian, 2001; Hood and Miller, 2006; Chernoff
et al., 2008). None of these factors, however, was observed in the
treated animals, which indicates a probable nontoxicity of MnHE.
Ercisli and Orhan (2007) have reported that a high concentration of phenols and flavonoids, particularly quercetin, is found in
the M. nigra fruits. Quercetin has been shown to have affinity for
beta estrogenic receptors (Havsteen, 2002), which are present in
all endometrial cells and also in the cells involved with the decidualization process, namely the subepithelial stroma cells (Li and
Davis, 2007). The presence of flavonoids in the MnHE suggested a
possible role of this plant in the decidualization process and consequently in the embryonic development. In addition, the change in
the estrogen/progesterone hormonal balance can interfere with the
zygote/embryo uterine horn transit and also the implantation in
the endometrium, which could reduce the number of embryos/fetuses (Croxatto et al., 1991).
In the present study, the implantation index, considered an
indicator of success of the blastocyst implantation in the endometrium (Tyl and Marr, 2006), was similar between the control and
treated groups, suggesting that the necessary hormonal concentrations for transit, development and implantation were not altered.
This fact was corroborated by the similar number of resorptions,
which are indicative of abnormal postimplantation development
(Kalter, 1980).
According to Chahoud et al. (1999), the normal growth of the fetuses depends on the combination of immunological, nutritional,
vascular, genetic, endocrine and environmental factors, to the extent that any change in any of these factors can interrupt the
G.T. de Queiroz et al. / Food and Chemical Toxicology 50 (2012) 816–822
Fig. 5. Uterine horn of M. nigra extract-treated group at the dose level of 350 mg/kg
(T4), showing the presence of oocytes ().
growth and normal development of the embryo/fetus. The hypoglycemic effect attributed to the M. nigra leaves (Oryan et al.,
2003) and the antineoplastic activity attributed to the phenolic
compounds in addition to the ability to change the gene expression
(Tapiero et al., 2002; Nakamura et al., 2003), could disturb the
embryonic development. Nevertheless, the data from this experiment showed that no alterations were detected on the number of
fetuses, their body weight, the placenta weight or the external
morphology, suggesting absence of embryotoxic effects of MnHE
during the development. These data need to be further investigated by more detailed studies on the organogenesis, fetogenesis
and postnatal development of the offspring.
Another aspect that should be considered is related to the possibility that the flavonoids present in the MnHE could be in their
heteroside form and it is known that the absorption of glycosylated
flavonoids is low in the organism. In order to be absorbed, they
would need to undergo hydrolysis and acquire the aglycone form
(Day et al., 1998; Gee et al., 1998; Graefe et al., 2001). Thus, it is
possible that deficient flavonoid absorption may have resulted in
low or lack of interaction with the estrogenic receptors.
In conclusion, the study of the possible female reproductive toxicity of MnHE indicates that there is neither antifertility activity
nor any interference with the embryonic development. The results
of the estrogenic effect experiment also showed that the extract
did not exhibit any estrogen-like activity after a period of treatment which covered at least three estrus cycles.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors are grateful to Prof. Luis Cláudio Ribeiro (Statistics
Department/UFJF) for doing the statistical analysis and ‘‘Rede
Mineira de Bioterismo (172/08), ‘‘Rede Mineira de Toxicologia e
Farmacologia de Produtos terapéuticos’’ (173/08), FAPEMIG, Brazil,
for financial support and Roy Robinson for reviewing the english
version of the manuscript.
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