Study of Embryotoxicity of Mentha piperita L. During Organogenesis in Balb/c Mice

862
Int. J. Morphol.,
29(3):862-867, 2011.
Study of Embryotoxicity of Mentha piperita L. During
Organogenesis in Balb/c Mice
Estudio de la Embriotoxicidad de Mentha piperita L. Durante la Organogénesis en Ratones Balb/c
*Mohammad Jafar Golalipour; **Soraya Ghafari; ***Alireza Maleki; ***Mossa Kiani; **Ebrahim Asadi & ****Mirmerhdad Farsi
GOLALIPOUR, M. J.; GHAFARI, S.; MALEKI, A.; KIANI, M.; ASADI, E. & FARSI, M. Study of embryotoxicity of Mentha
piperita L. during organogenesis in Balb/c mice. Int. J. Morphol., 29(3):862-867, 2011.
SUMMARY: Mentha piperita (Labiatae), commonly known as peppermint is a native Iranian herb which is used in folk medicine
for various purposes. This study was carried out to reveal the teratogenic effect of Mentha piperita on mice fetuses. In this experimental
study, pregnant Balb/c mice divided to four groups. Case group received 600 (treatment I) and 1200 (treatment II) mg/kg/day the hydroalcoholic
extract of Mentha piperita during 6-15 of gestational days and one control group received normal saline during GD6-GD15 by gavages and
other control group did not receive any matter during 6-15 of gestational days. Mice sacrificed at GD18 and embryos were collected.
Macroscopic observation was done by stereomicroscope. 20 fetuses of each group were stained by Alizarin red-S and Alcian blue staining
method. The Mean weight of fetuses decreased in treatment groups rather than control (P<0.05) but CRL there was no significant difference
between treatments and controls groups. In the treatment I (600 mg/kg/day) and treatment II (1200 mg/kg/day), normal saline and control
group, no gross congenital malformations were observed in fetuses. Treated fetuses also had no delayed bone ossification as determined by
Alizarin red-S and Alcian blue staining method. This study showed that the hydroalcoholic extract of Mentha piperita (600 and 1200 mg/
kg/day) has no teratogenic effect in mice fetuses if used continuously during embryonic period.
KEY WORDS: Mentha piperita; Teratogen; Bone ossification; Developmental toxicity; Mice.
INTRODUCTION
More than 80% of the people in the world population
currently rely on traditional medicines and most of these
therapies involve the use of plant extracts (Zhang, 2002).
Recent reports indicate a wide use of medicinal herbs by
pregnant women (Hepner et al., 2002).
Mentha piperita is a herb, which is commonly, used
in folk medicine, in Iran, turkey, India, the Middle East,
Europe and Canada for flatulent colic, appetite, to relieve
abdominal pain, fever, nausea and vomiting and digestion
(Zargari, 1996; Westfall, 2004; Starbuck, 2001).
Also M. piperita is used for preventing vomiting and
morning sickness in pregnant women (Westfall). M. piperita
contains volatile oil, menthol, menthon, methofman, and
limonene (Adkogan et al., 2004a; Baser, 1993).
The chemical components of leave extract and oil of
M. piperita vary with plant maturity, geographical region
and processing conditions (Ruiz Del Castillo et al., 2003;
Xu et al., 2003).
Previous studies have shown antiviral, antibacterial and
anti fungal effects of M. piperita (Minami et al., 2003;
Schuhmacher et al., 2003; Choi et al., 2003; Azuma et al.,
2003; Duarte et al., 2005; Tampieri et al., 2005).
Also, Inoue et al. (2002) and Satsu et al. (2004)
reported anti allergic effects of this herb. Furthermore,
previous studies have been shown anti-inflammatory, analgesic
and antispasmodic effects of M. piperita (Atta & Alkofahi,
1998; Göbel et al., 1995, 1996; Juergens et al., 1998; Asao et
al., 2003; Hiki et al., 2003; Micklefield et al., 2003). Also
Tate et al. (1997) reported anti nausea effect of this herb.
Some studies reported that the extract of M. piperita
reduce symptoms in dyspepsia (Madisch et al., 2001; Rösch
et al., 2002) and IBS patients (Kline et al., 2001).
*Professor, Gorgan Congenital Malformations Center, Department of Anatomical Sciences, Golestan University of Medical Sciences, Gorgan, Iran.
**Department of Anatomical sciences, Golestan University of Medical Sciences, Gorgan, Iran.
***Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
****Assistant Professor, Department of Anatomical Sciences, Babol University of Medical Sciences, Babol, Iran.

863
Furthermore, a study reported the histophatological
effect of Mentha piperita on white matter of cerebellum and
proximal convoluted tubules in animal model (Thorup et al.,
1983; Spindler & Madsen, 1992). It was reported that the oil
of Mentha piperita has a genotoxic effect on human
lymphocytes (Lazutka et al., 2001).
Recent researches has shown that spearmint tea has
antiandrogenic properties in both animals and females
(Adkogan et al., 2007; Güney et al., 2006) and anti
spermatogenic activity in rodents (Adkogan et al., 2004;
Sampaio, 2004).
On the other hand some researches showed the
chemoprotective, antimutagenic and anti carcinogenic effects
of M. piperita (Samarth et al., 2006; Samarth & Kumar, 2003;
Samman et al., 1998). In Regard to the use of Mentha piperita
for preventing vomiting and morning sickness in pregnant
women there is a lack of studies about teratogenic effects of
Mentha piperita. This investigation was carried out to reveal
the teratogenic effect of M. piperita on Balb/c mice fetuses.
MATERIAL AND METHOD
Time and setting. The study was performed in 2008 at the
Faculty of Medicine, Gorgan University of Medical Sciences.
Approval for this study was gained from the Animal Care
and Ethics Committee of the Gorgan University of Medical
Sciences.
Materials. Plant material. Mentha piperita leaves were
collected from cultivated plant, from suburb of Gorgan,
Northern Iran.
Methods. Preparation of plant extract. The aerial parts of
Mentha piperita were reduced to small pieces, dried in a
circulating air stove and powdered in a grinder. The powdered
material was then macerated using a hydroalcoholic (60º)
solvent for 48 hours. The ethanol was removed by vacuum
distillation and the resulting residue was filtered and
concentrated at 40Cº to make a jelly-like material. In addition
to thin layer chromatography and purity tests (foreign matter,
total ash, acid insoluble ash and water insoluble ash) for
qualification analysis, monosaccharide-linked another reagent
assay (spectrophotometry) have been carried out to determi-
ne the concentration of polysaccharides in Mentha piperita
leaves for standardization of the extract.
At the time of administration, the prepared powder of
the extract was solved by the saline and the mice were treated
with the solution.
The animals used in this study were experimental,
28-30 gram weight, and 7-8 week old virgin female and
mature male Balb/c mice. The males were part of the ani-
mal house breeding stock with confirmed mating experience.
Dry food pellets and water were provided ad libitum with
animal house conditions maintained at 20-22ºC, 65-68%
relative humidity, and a 12 h: 12 h photoperiod (lights on
0700-1900h). Two females were caged with a male of the
same strain overnight. The presence of vaginal plug the next
(following) morning confirmed that mating had taken place
and was designated as day zero of pregnancy (Gestation Day
0: GD 0). Females that did not mate within 2 estrus cycles
were excluded from the study.
Pregnant mice were randomly divided into the two
experimental groups (600 mg/ kg/day and 1200 mg/ kg/
day of Mentha piperita extract) and the two control groups.
12 mice in the two experimental groups received 600 mg/
kg/day and 1200 mg/ kg/day orally of Mentha piperita
extract respectively. One control group received normal
saline orally, from GD 6 to GD 15, by oral intubation. The
other control group did not receive normal saline. On GD
18 the pregnant mice were sacrificed under chloroform
anesthesia and uterus was opened and umbilical cord cut
close to the fetus; each fetus and placenta were then
weighed. Each fetus was assigned a number according to
its position in the uterine horn, starting with number one at
the ovarian end of the left uterine horn. Fetuses were
assessed as either alive or dead and any resorption was
noted. All live fetuses were measured crown-rump length
(CRL), Bi-parietal diameter (BPD) and were examined
externally for ‘Qlformations or deviations from normal
growth as described. Also, each of the fetuses was weighed
by sensitive electronic measurement serrations PT 210
German and observed for gross malformations by stereo
research microscope, Blue Light US. Fetuses were
eviscerated and the skin removed to facilitate stain
penetration. Skeletal staining of fetuses was performed by
the Alcian Blue- Alizarin Red S method. Differences in
body weight, bi-parietal diameter (BPD) and crown rump
length (CRL) between controls and treatment groups were
analyzed using a one-way ANOVA. A value of P<0.05 was
considered to indicate a significant difference between
groups.
RESULTS
During the whole experiment, no maternal deaths and
behavioral changes were recorded in any group. The treated
females consumed as much food and water as the controls
and gained comparable weight.
GOLALIPOUR, M. J.; GHAFARI, S.; MALEKI, A.; KIANI, M.; ASADI, E. & FARSI, M. Study of embryotoxicity of Mentha piperita L. during organogenesis in Balb/c mice.
Int. J. Morphol., 29(3):862-867, 2011.

864
There were no signs of maternal toxicity due to
Mentha piperita treatment. No signs of toxicity were noted
in any of the animals. All pregnant animals appeared healthy
at sacrifice. The weight of fetuses in treatment I (1.29 ± 0.02)
and treatment II (1.37 ± 0.02) were lower than controls
(p<0.05)(Table 1). Also BPD in the treatment I group
(7.15±0.12) and treatment II group (7.05±0.05) was not
significantly lower than the control (7.25±0.02) groups
(Table 1). Crown rump length in treatment and controls was
similar.
Implantation and number of fetuses in the left horn
(55%) of uterine of the treatment group was higher than the
right horn (45%). While in the control group this percent
was similar, 48% in the right and 52% in the left uterine
horn. Major congenital malformations in fetuses were not
found in treatment and control groups. Alizarin red S and
Alcian blue staining did not show skeletal malformations
and delayed bone ossification in the treated groups as
comparing with control groups.
DISCUSSION
The findings of this developmental toxicity study
showed that the Mentha piperita extract did not cause any
major birth defects and delayed bone ossification in fetuses
if used continuously during the embryonic period. There are
very rare studies on the effect of Mentha piperita. Inoue
study in Turkey showed that the M. piperita has a cytotoxic
effect on rats (Inoue et al., 2001). Romero-Jiménez & Cam-
pos-Sánchez (2005) reported that M. piperita oil has a
cytotoxic effect and it induced the changes in chromatids
chromosomes.
Furthermore, Lazutka et al. reported that M. piperita
oil according to Smart method has a cytotoxic effect.
Gordon et al. (1987) reported that cytotoxic effect of
Mentha piperita can be related to high level of pulegone
which is a toxic substance or menthol.
Furthermore in a study, Akdogan et al. (2004b) showed
that M. piperita decreased testosterone level and increased
LH, FSH concentration in male rats. Also, he reported that
this herb has effect on hypophyseal-testis hormonal axis
causing alterations in germinal layer of seminiferous tubules
in adult male rats thus Akdogan et al. concluded that M.
piperita has antispermatogenic effect in rats.
In the literature we did not find any article about
teratogenic effect of this plant. In our study we did not ob-
serve any birth defect or bony deletion in fetuses treated
with Mentha piperita extract. Morphologic cytotoxic and
hormonal alterations of this herb may be due in intracellular
or DAN of cells. Also the different effects of M. piperita in
different studies can be due to various chemical components
of leave or oil extract of M. piperita which depend on plant
maturity, species, geographical region and processing
conditions (Ruiz del Castillo et al.; Xu et al.)
In this study, the fetuses of the treatment group had
decreasing weight.
The decreasing weight of fetus in treated experimen-
tal groups may be due to the blocking of cell growth which
may be due to genotoxic effect of this herb. Also the
decreasing weight of fetuses in treated experimental groups
can be due to reabsorbing of extra cellular liquid in the fetus.
because extra cellular liquid in the fetus makes up the main
part of weight and volume of fetuses. This mechanism was
explained in previous studies which reported that saffron or
crocin with reabsorbing of extra cellular liquid in the fetus
causes decreasing weight in fetuses (Golalipour et al., 2008;
Garcia-Olmo et al., 1999).
The growth of the fetus during intrauterine life is
reflected in the weight at birth. Fetal growth is largely
determined by the availability of nutrients from the mother,
as well as placental capacity to supply these nutrients in
sufficient quantities to the fetus. Of course there is evidence
that both placental volume and the rate of placental growth
may influence fetal size. Also the decreasing of weight of
fetuses can be due to blocker effect on cell growth.
Groups Live Fetuses (n) Absorbed
Fetuses (n)
BPD (mm) CRL (mm) Weight (gr)
Control 54 2 7.25 – 0.02 22.96 – 0.38 1.43 – 0.61
Control (Normal Saline) 38 3 7.20 – 0.04 22.85 – 0.22 1.41 – 0.02
Treatment I (6oo mg/kg Mentha) 45 6 7.15– 0.12 22.38 – 0.22 1.29 – 0.02*
Treatment II (1200 mg/kg Mentha) 52 1 7.05– 0.05 22.03 – 0.16 1.37 – 0.02*
Table I. Number of live, absorbed fetuses, BPD, CRL, weight of fetuses in controls and treatments. *p<0.05 compared to controls.
GOLALIPOUR, M. J.; GHAFARI, S.; MALEKI, A.; KIANI, M.; ASADI, E. & FARSI, M. Study of embryotoxicity of Mentha piperita L. during organogenesis in Balb/c mice.
Int. J. Morphol., 29(3):862-867, 2011.

865
This study showed that Mentha piperita extract has
no teratogenic effect in mice fetus if it is used continuously
during embryonic period, although this herb caused
decreasing weight of fetuses. Therefore, regarding our results
we suggest that pregnant women avoid high consumption
of M. piperita during the organogenesis period.
Further study is needed to determine the exact
mechanisms of decreasing fetus weight due to Mentha
piperita consumption in organogenesis period.
ACKNOWLEDGMENTS
The authors appreciate the Department of Research
Gorgan University of Medical Sciences for their financial
support.
GOLALIPOUR, M. J.; GHAFARI, S.; MALEKI, A.; KIANI, M.; ASADI, E. & FARSI, M. Estudio de la embriotoxicidad de
Mentha piperita L. durante la organogénesis en ratones Balb/c.Int. J. Morphol., 29(3):862-867, 2011.
RESUMEN: Mentha piperita (Labiatae), comúnmente conocida como menta, es una hierba nativa de Irán, que se utiliza en
la medicina tradicional para diversos fines. Este estudio fue realizado para descubrir el efecto teratogénico de la Mentha piperita en
fetos de ratones. Los ratones Balb/c preñadas fueron divididas en cuatro grupos. Los grupos recibieron 600 (tratamiento I) y 1200
(tratamiento II) mg/kg/día del extracto hidroalcohólico de Mentha piperita durante los días 6-15 de gestación (DG), mientras que un
grupo control recibió solución salina normal durante los DG 6-15 vía oral y otro grupo control sano no recibió substancia durante los
DG 6-15. Los ratones fueron sacrificados el DG 18, recolectando los fetos. Se realizó la observación macroscópica mediante un
estereomicroscopio. 20 fetos de cada grupo se tiñeron por el método de rojo de alizarina-S y azul de Alcián. La media de peso de los
fetos disminuyó más en los grupos de tratamientos que los controles (p <0,05), pero CRL no presentó diferencias significativas entre
los tratamientos y los grupos control. En los fetos del grupos tratamiento I (600 mg/kg/día), tratamiento II (1200 mg/kg/día), solu-
ción salina normal y control no se observó ninguna malformación congénita grave. Los fetos tratados tampoco tuvieron osificación
ósea retrasada según lo determinado por el método de rojo de alizarina-S y azul de Alcián. Este estudio mostró que el extracto
hidroalcohólico de Mentha piperita (600 y 1200 mg/kg/día) no tiene efectos teratogénicos en fetos de ratones al ser utilizado
continuamente durante el período embrionario.
PALABRAS CLAVE: Mentha piperita; Teratógenos; Osificación ósea; Toxicidad para el desarrollo; Ratones.
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Correspondence to:
Dr. Mohammad Jafar Golalipour
Gorgan Congenital Malformations Research Center
Department of Anatomical Sciences
Golestan University of Medical Sciences
Gorgan, P.O.
Box: 49175-1141
IRAN
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Tel/Fax: +98 (171) 44 25 165, 44 25 660
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Email: mjgolalipour@yahoo.com
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Received: 15-01-2011
Accepted: 03-06-2011
GOLALIPOUR, M. J.; GHAFARI, S.; MALEKI, A.; KIANI, M.; ASADI, E. & FARSI, M. Study of embryotoxicity of Mentha piperita L. during organogenesis in Balb/c mice.
Int. J. Morphol., 29(3):862-867, 2011.