ANTIESTROGENIC EFFECT OF TUBER EXTRACT OF CYPERUS ROTUNDUS L. ON THE ENDOMETRIAL THICKNESS OF MICE (MUS MUSCULUS L.)

Abstract

Uterine receptivity for implantation of an embryo is regulated by ovarian hormone estrogen. When function of the hormone is disrupted, for example by anti-estrogenic substances, the implantation process may also be disrupted. Some types of plants, including nut grass, have been known to contain anti-estrogenic compounds. The research aimed to determine the anti-estrogenic effect of tuber extract of Cyperus rotundus on the endometrial thickness of mice (Mus musculus L). By using a completely randomized design, 18 of healthy female mice, aged 3-4 months, weighing 30-40 g were grouped into three each consisted of six mice. Group-1 is mice that are given distilled water as a control. Group-2 is mice treated with anti-estrogenic drug tamoxifen of 0.16 mg/40g body weight. Group-3 is mice given tuber extract of nut garss at a dose of 135mg/40g body weight. The treatment were given once daily for 14 days. On the day 15 all mice were sacrificed, the womb was dissected to be made histological slides of uterus. The result is either tamoxifen 0.16mg/40g body weight as well as tuber extract of nut grass 135 mg/40g body weight are significantly reduce the thickness of endometrium compared with that of

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Kanedi et al. World Journal of Pharmaceutical and Life Sciences
ANTIESTROGENIC EFFECT OF TUBER EXTRACT OF CYPERUS
ROTUNDUS L. ON THE ENDOMETRIAL THICKNESS OF MICE
(MUS MUSCULUS L.)
Hendri Busman1, Yanwirasti2, Jamsari3, Djong Hon Tjong4, Mohammad Kanedi1*
1Department of Biology, Faculty of Mathematics and Natural Sciences, University of
Lampung, Indonesia.
2Department of Pathological Anatomy, Faculty of Medicine, University of Andalas,
Indonesia.
3Department of Biotechnology, Faculty of Agriculture, University of Andalas, Indonesia.
4Department of Biology, Faculty of Mathematics and Natural Sciences, University of
Andalas, Indonesia.
Article Received on 02/11/2016 Article Revised on 23/11/2016 Article Accepted on 13/12/2016
ABSTRACT
Uterine receptivity for implantation of an embryo is regulated by
ovarian hormone estrogen. When function of the hormone is disrupted,
for example by anti-estrogenic substances, the implantation process
may also be disrupted. Some types of plants, including nut grass, have
been known to contain anti-estrogenic compounds. The research aimed
to determine the anti-estrogenic effect of tuber extract of Cyperus
rotundus on the endometrial thickness of mice (Mus musculus L). By using a completely
randomized design, 18 of healthy female mice, aged 3-4 months, weighing 30-40 g were
grouped into three each consisted of six mice. Group-1 is mice that are given distilled water
as a control. Group-2 is mice treated with anti-estrogenic drug tamoxifen of 0.16 mg/40g
body weight. Group-3 is mice given tuber extract of nut garss at a dose of 135mg/40g body
weight. The treatment were given once daily for 14 days. On the day 15 all mice were
sacrificed, the womb was dissected to be made histological slides of uterus. The result is
either tamoxifen 0.16mg/40g body weight as well as tuber extract of nut grass 135 mg/40g
body weight are significantly reduce the thickness of endometrium compared with that of
wjpls, 2016, Vol. 2, Issue 6, 341-347.
Research Article
ISSN 2454-2229
World Journal of Pharmaceutical and Life Sciences
WJPLS
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SJIF Impact Factor: 3.347
*Corresponding Author
Dr. Mohammad Kanedi
Department of Biology,
Faculty of Mathematics
and Natural Sciences,
University of Lampung,
Indonesia.

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Kanedi et al. World Journal of Pharmaceutical and Life Sciences
control. Thus it can be concluded that the tuber extract of C. rotundus has anti-estrogenic
effects on the endometrial thickness of mice.
KEYWORDS: Cyperus rotundus, nut grass, tamoxifen, antiestrogenic effect, endometrial
thickness
INRODUCTION
Implantation involves interaction between the blastocyst and the uterine endometrium that
goes through a series of unique stages[1] on a right time and place.[2] The end stages of the
implantation is the invasion of blastocyst into the maternal tissues, from which the embryo
obtain nutrients essential to life and development.[3] Uterine receptivity for implantation of an
embryo is regulated by ovarian hormones estrogen and progesterone. Several cytokines and
growth factors also play an important role in embryo implantation under the influence of
ovarian hormones. As some of the molecules involved in the interaction between the embryo
and maternal tissue during the implantation process takes place. It has also been known that,
if the function of these molecules during implantation failure, the implantation is also
impaired, and even lead to infertility.[4] Biochemical and molecular aspects of the
endometrium is strongly associated with the ability of embryo implantation. In an
endometrial biopsy there was molecules known to be associated with the endometrial
response. A number of factors could cause interference with the implantation, low receptivity
of endometrium will lead implantation failure.[5,6] One of the causes of the implantation
failure is biochemical and molecular disruption in endometrium.[7] In addition,
malfunctioning of the endometrium can be caused by antiestrogenic compounds that known
to be contained in the nut grass Cyperus rotundus.[8] Antiestrogenic drugs often used in the
treatment of breast cancer, because physiologically estrogen stimulate normal breast growth.
Antiestrogen acted by modifying or antagonize the action of estrogen and among the
antiestrogenic drugs is tamoxifen.[9] A study to compare the effect of estrogen with
antiestrogen reported by Papaconstantinou et al.[10] showed that the estrogen compounds
causing increases the weight of uterus, on the contrary anti-estrogen made the uterine weight
decreased. Antiestrogen is substances that fight or lessen the effects of estrogen and able to
eliminate a part or the whole action of estrogen.
To ascertain the antiestrogenic effects of crude extract of nut grass plant (Cyperus rotundus)
on the uterine wall thickness, methanolic tuber extract of the plant has been applied to female
mice for 14 days.

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Kanedi et al. World Journal of Pharmaceutical and Life Sciences
MATERIALS AND METHODS
Plant Samples and Extraction
The samples of nut grass plant (Cyperus rotundus L.) were collected from suburbs Bandar
Lampung. To prepare simplicia, fresh tubers of the grass which has been previously washed
were sun dried. Once dry, the tubers were ground to be a powder form. Then, by using
Soxhlet apparatus the simplicia was extracted using methanol solvent at the temperature of
35oC and the rotation of 60 rpm for 60 minutes.
Experimental Mice and Treatment
Female Swiss albino mice (Mus musculus L.) aged 3-4 moths, weighing 30-40 grams were
used for the study. The animals and the food pellets were obtained from Lampung Veterinary
Center, Indonesia. Mice were housed in a room at the temperature of 25°C and 12:12-hour
light-dark cycle with free access to water and pellets ad libitum. All animal care and
treatment procedures were approved by the Ethics Committee, Faculty of Medicine,
University of Lampung, Indonesia.
By using a completely randomized design, the animals grouped into three consisted of six
rats each. Group-1 is mice that are given distilled water as a control. Group-2 is mice treated
with anti-estrogenic drug tamoxifen of 0.16 mg/40g body weight. Group-3 is mice given
tuber extract of nut grass at a dose of 135mg/40g body weight. The treatment were given
once daily for 14 days. By the day 15, after being deeply anesthetized using chloroform, all
mice were sacrificed, the womb was dissected to be made histological slides of uterus.
Study Parameters and Data Analysis
Study parameters assessed in this study is the thickness of endometrial layers of the uterine
wall. The data are presented as Mean ± SD and analyzed statistically using a one-way
ANOVA. Least Significance Difference (LSD) test was used as the post hoc test. All of the
statistics that were applied are programmed in SPSS version 21.
RESULTS NAD DISCUSSION
The descriptive data of the effects of tamoxifen and tuber extract of C. rotundus on thickness
of endometrial layers of mice uterine wall are presenterd in Table 1. The results of analysis of
variance (ANOVA) of the data in the Table 1 are tabulated in Table 2.

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Table 1: The descriptive data of the effects of tamoxifen and tuber extract of C.
rotundus on thickness of endometrial layers of mice uterine wall.
N
Control
Tamoxifen
Extract
1
203.5
154
145.2
2
196.5
147
138.1
3
189
140.5
131.5
4
182.9
133.5
124.5
5
175
126
117
6
168.5
119.5
110.5
Mean
185.90
136.75
127.85
SD
13.13
12.96
13.03
Table 2: The results of Analysis of Variance.
Source
DF
Adj SS
Adj MS
F-Value
P-Value
Treatment
2
11743
5871.4
34.5
0.000
Error
15
2552
170.1
Total
17
14295
As the F-value of the ANOVA (=34.5) and the P-value <0.01 it can be suggested that the
difference between groups of treatment is highly significant. By using Least Significant
Difference (LSD) against the above descriptive data resulted in the lower bound of
LSD=16.046 and the upper bound=22.191. The notation of the difference of mean values
between the treatments are presented in Table 3.
Table 3: The difference of mean thickenss of endometrium between treatment group of
mice.
Treatments
Mean
Difference
Control
Tamoxifen
Control
185,90
Tamoxifen
136,75
49,15**
Extract
127,80
58,10**
8,95ns
Notes: ** mean highly significant different at P<0.01
‘ns’ mean not significant different at P>0.05
Based on the results of statistical test above, it can be affirmed that both tamoxifen and tuber
extract of nut grass effectively reduce the thickness of the endometrial layers of uterine wall
of mice.
The endometrial surface consists of mucosal epithelial cells, which its main function to
facilitate implantation of an embryo.[11] The endometrial receptivity determined by the
molecular and genetic markers in the form of cytokines, growth factors, transcription factors

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and ovarium hormones, such progesterone, the most essential for implantation and pregnancy
of all mammals, as well as the specific estrogen hormones.[12, 4] In this study, tuber extract of
C. rotundus showed antiestrogenic effect on reducing thickness of the endometrial lining and
caused no proliferation on endometrial epithelial cells. It was allegedly due to the absence of
estrogen influence on the endometrium which imply that tuber extract of C.rotundus possess
an anti estrogenic activity. Estrogen is required for the proliferation of uterine epithelial cells
and enhance the action of progesterone through the induction of progesterone receptor genes.
The progesterone plays a role in controlling proliferation, differentiation, maintenance of
endometrial stroma, glandular and myometrial cells.[12]
The syntesis of progesterone by corpus luteum will stimulate proliferation and diffentiation of
stroma cells. Furthermore, implantation of an embryo the will promote secretion of estradiol-
17β that in turn stimulate proliferation and differentiation of the uterine epithelial cells.[13]
It was also suggested that the endometrium may undergo histological changes, such as
vascularization of the endometrium, development of endocrine glands and pinopod to be
luminal surface of the epithelium.[14] These changes are known as morphological changes
due to the influence of the endometrial estrogen and progesterone.[15] A series action of
estrogen and progesterone of endometrium will result in secretion of epithelial gland and a
series of decidual transformation in the stroma cells.[16] Estrogen and progesterone are
important for regulating the uterine receptivity to embryo development and the success of
pregnancy. The differentiation of stromal cells into decidual cells is responded by the
progesterone hormone during the decidualization process, which is characterized by
morphological changes and prolactin secretion.[17] Finally endometrium undergo
morphological and functional changes, including growth, differentiation and desquamation.
Finally endometrium undergo morphological and functional changes, including growth,
differentiation and desquamation. Simultaneously physiological changes initiated in
preparation for endometrial receptivity and control the invasion of cells tropoblas in the mid-
secretory phase of implantation process.[18]
CONCLUSION
Either tamoxifen as well as tuber extract of nut grass equally give effect in reducing
endometrial thickness of mice. Thus it can be concluded that tuber methanolic extract of C
rotundus allegedly possess antiestrogenic activities and potentially as an antiimplatation drug.

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