Biology, Medicine, & Natural Product Chemistry ISSN: 2089-6514
Volume 4, Number 1, 2015 | Pages: 5-9 | DOI: 10.14421/biomedich.2015.41.5-9

The Phytoestrogenic Potential of Yam Bean (Pachyrhizus erosus)
on Ovarian and Uterine Tissue Structure of Premenopausal Mice

Cicilia Novi Primiani
Biology Education, Mathematics and Science Faculty, IKIP PGRI Madiun

Jl. Setiabudi 85 Madiun, Tel. +62-351-462986, Fax. +62-351-459400

Author correspondency:
primianibiomipa@yahoo.co.id

Abstract

The use of estrogen hormone by public has significantly been improved either as prevention or treatment of disease. Menopausal issues in
women are often treated using hormone replacement therapy. In regard to this, yam bean is found to contain genistein and daidzein
compounds with a chemical structure that resembles estrogen hormone, therefore yam bean is categorized in the phytoestrogen group. The
purpose of this study was to identify the potential of yam bean on ovarian and uterine histology of mice. This research employed a
Completely Randomized Design of experimental research approach of one factor namely yam bean in three different dosage treatment: 0.3
g/kg, 0.6 g/kg, and 0.9 g/kg of yam bean for 24 days. The surgery and organ harvesting of ovary and uterus were conducted on Day 25
along with the making of histological preparat using paraffin method and Hematoxylin-eosin (HE) staining. The data was then analyzed
descriptively. This research found that there were both secondary and tertiary follicle proliferation as the antrum contains some estrogen
level. Meanwhile, the endometrial tissue of the uterus experienced uterine glandular proliferation. To conclude, yam bean was found to be
a natural estrogen source.

Keywords: yam bean, isoflavone, phytoestrogen, ovarium, uterus, menopause

Introduction

Hormone replacement therapy (HRT) is an action which
is conducted through giving estrogen hormone especially
to women who experience some reduction in the estrogen
hormone. HRT is usually undergone by women
experiencing menopause. In particular, Estrogen
Replacement Therapy (ERT) can also be used for
conducting cardiovascular disease therapy (Stampfer et
al., 1991 and Sourander et al., 1998), reducing the risk of
osteoporosis (Grady et al., 1992 and Nurochmad et al.,
2010), and reducing the symptoms of menopause
(Barrett, 1998). The use of synthetic estrogen compound
in a long term often gives negative impacts so that a
natural alternative is needed to replace estrogene
hormone.

Yam bean plant (Pachyrhizus erosus) as a tuber-
legume crop, is categorised as a legume with the
dissemination area of Sumatera, Java, Bali, Sulawesi and
East Nusa Tenggara (Karuniawan & Wicaksana, 2006).
People usually prefer to consume fresh yam bean either
in the form of salad or rujak (Indonesian traditional fruit
an vegetable salad dish). Yam bean extract is used in the
cosmetics industry in whitening, compact powder, and
moisturizer products. Yam bean contains isoflavone
compounds with estrogen-like chemical structure
(Wanibuchi, 2003; Abid, 2005; and Lukitaningsih, 2009).
The chemical isoflavones structure resembles 17β-
estradiol and has the efficacy like estrogene hormone
(Delmonte and Rader, 2006; Barlow et al., 2007) so that
yam bean is also included in phytoestrogen group
(Urasopon et al., 2008). The largest components of
isoflavone are genistein and daidzein which are often

found in Fabaceae family, including Pachyrhizus erosus
(Kang et al., 2006). Pachyrhizus erosus at least contain
isoflavones such as, daidzein and genistein (Primiani,
2013), the analysis of HPLC daidzein and genistein of
yam bean is 110,454 mg/100 g and 165,530 mg/100 g
respectively.

The chemical structure of geninstein and daidzein can
bind to estrogen receptor and compete with endogenous
estrogen such that they may provide both estrogenic and
anti-estrogenic effects (Adlercreutz, 1990; Griffiths et al.,
1996; Adlercreutz and Mazur, 1997). Utilization of yam
bean as phytoestrogen is not conducted much, a study by
Nurrochmad et al., (2010) showed that providing yam
bean extract of 400 mg/kg and 800 mg/kg dosage for 4
weeks to mice through ovariectomy could prevent bone
fragility; phytoestrogen was then proven to be able to
improve uterine mass (Ford et al., 2006). Meanwhile,
Genistein affects on the increasing of weight of uterus by
stimulating uterine endometrial thickening (Santell,
1997). Genistein dose 26,6 mg/day eqiuvalen with human
dose 0,625 mg/day within 6 months on monkey cause
vaginal maturation (Marquez et al., 2012).

Research Methods

Research Design

The research was conducted using experimental research
approach with a completely randomized design, the
treatment was completed through providing grated yam
bean in three different dosage: 0.3 g/kg, 0.6 g/kg, 0.9
g/kg. The observation is focused on the changes of
ovarian and uterine tissue structure.



6 Biology, Medicine, & Natural Product Chemistry 4 (1), 2015: 5-9

Tools and Materials

The research tools used in this study were: a gavage tube,
plastic mice cages (50 x 30 x 20cm size), drinking bottles
for mice, a digital scale (HM-200 brand), 1ml syringes
with 3ml disposable needles (G23), an incubator, optical
miscroscope, digital optilab camera microscope, a triple
beam balance ohaus 700 series scale, analytical balance
type HM-200 with the capacity of 210 grams and
accuracy level of 0.1 mg, PR-50 microtome, surgical
equipment, a surgical board, object glasses and lenses,
glass beakers, spiritus lamp, a cube made of calendar
paper, blender, flour sifter, a nife, a pipette, and a couple.

The research materials were yam bean obtained from
Takeran village Madiun Indonesia, ovarian and uterine
tissue, milk A granule for the mice food produced by PT.
Charoen Pokphand Indonesia, husk, cotton, tissue paper,
aqua destillata, water tap, paraffin, 0.9% physiological
saline, bouin fixative solution, 50%, 70%, 90% and
absolute alcohol, pure xylol, xylol-alcohol mixture, with
xylol and acohol comparison consecutively 1:3, 2:2, and
3:1, Li2CO3 solution, 1% HCl, 3% formalin, and haupt
adhesive.

Animal Experiments

The experimental animals used were mice (Mus
musculus) strain Balb/c female, in a good health
condition, aged 12 months, 24 mice in total. Every mouse
had an initial body weight ranging from 20-25 grams
before treatment, the mice were kept in a cage located in
the Biology Education IKIP PGRI MADIUN, Indonesia

Maintenance of mice and manufacture of test materials

The mice were placed in the mice cages, given food and
drink ad libitum and acclimatized for 14 days prior to

induction treatment. The mice were maintained at the
room temperature of (± 270C), relative humidity between
50-60% and 12 hour lighting cycle. Every day the mice
were weighed as the basis for determining the provision
of grated yam bean. The manufacture of grated yam bean
as a research material was conducted through grating the
yam bean using a grater. The amount of the yam bean was
then weighed and complied with the amount given to the
mice.

The provision of yam bean and manufacture of preparat
of ovarian and uterine histology

The provision of yam bean was coducted through direct
induction to the stomach using a gavage tube once a day
for 24 days. The mice were then dislocated on the 25th
day and dissected. The organ harvesting of their ovaries
and uterus were then performed. The manufacture of the
preparat of ovarian and uterine histology was completed
using paraffin method in attempt to determine any
ovarian and uterine tissue structure changes.

Data analysis

The ovarian and uterine tissue structure changes were
analyzed descriptively based on the changes that
happened in the ovarian follicle, myometrium layer,
endometrium and uterine mucosa.

Results and Discussions

The results of observation on the ovarian tissue showed
that there were changes in the ovarian follicles (Figure 1).

Figure 1. The ovaries of mice (Mus
musculus); HE stained sections; 400x
Description: A. Control (P1); B. The dose of
0.3 g/kg (P2); C. The dose of 0.6 g/kg; D. The
dose of 0.9 g/kg (P4).
A. Ovary with the control treatment, there are

some corpus albikans.
B. Ovary with mature follicles and granulosa

layer.
C. Secondary follicular proliferation, tertiary

follicles begin to develop.
D. Tertiary follicles with follicular fluid.

A B

C D



Cicilia Novi Primiani – The Phytoestrogenic Potential of Yam Bean (Pachyrhizus erosus) … 7

The results of observation on the uterine tissue showed that there were changes in the uterus endometrium tissue
(Figure 2).

Figure 2. The uterus of mice (Mus musculus); HE
stained; 400x Description: A. Control (P1); B. The
dose of 0.3 g/kg (P2); C. The dose of 0.6 g/kgD. The
dose of 0.9 g/kg (P4).
A. Uterine wall consists of tunica serosa, tunica

muscularis and tunica mucosa.
B. Proliferation of tunica mucosa (uterus

endometrium layer).
C. Proliferation of uterine glands in uterine

endometrium layer.
D. Proliferation of endometrium and myometrium

layers, proliferation of uterine glands.

The observation results of ovarian histology using
hematoxylin-eosin (HE) staining are depicted in Figure 1.
The control treatment (P1) shows follicles that
experienced atresia (corpus albikans) and there is no
indication of any primary follicle proliferation. The
atresia folikuli phase shows histological features that
represent disintegration in the form of fat drops and
coarse granules in the ova (Figure 1A). In the ovary that
is given grated yam bean of 0.3 g/kg dose, some follicles
with a thick layer under the tunica albuginea occur and
they have a unique characteristic that the ova contained
do not have any vitelina membrane (Figure 1B). The ova
are surrounded by layers of follicular cells which form
granula layers on the more mature follicles afterward. In
the provision of yam bean of 0.6 g/kg dose, secondary
follicular growth takes place, the follicles are more egg-
shaped (oval) and has been moving away from the cortex
approaching the medulla ovary and forming a room that
is filled with fluid (antrum) around the ova and the
granulosa cells layer are coating it (1C). Liquid called
liquor folliculi makes the follicles developed into tertiary
follicles (apparent from the picture of ovary with 0.9 g/kg
dose yam bean treament) (1D).

Changes in the uterine tisue structure of the mice after
being given grated yam bean are depicted in Figure 2. The
results of observation on uterine histology with
hematoxylin-eosin staining (HE) show that the uterine
wall of the control treatment (P1) is relatively thick and
formed in 3 layers: (1) tunica serosa or outer perimetrium
in the form of connecting tissue that consists of a layer of
mesothelium supported by a thin connective tissue, (2)
tunica muscularis or myometrium is the thickest layer
which is composed of smooth muscle tissue.
Myometrium is the thickest tunica that consists of
bundles of smooth muscle fibers which are separated by

connective tissue. The endometrium consists of epithelial
siliata and non siliata as well as lamina propria or stroma
endometrialis containing simple tubular glands (uterine
glands) that are branched on the inside (near the
endometrium) (Figure 2A).

The provision of grated yam bean of 0.3 g/kg (P2)
causes endometrium and myometrum proliferation of the
uterus, uterine luminal has been narrowing due to the
epithelialization process. The proliferation of uterine
glands of the uterus begin to exist despite the decrease of
premeneopausal period proliferation (Figure 2B). The
grated yam bean provided to the mice at a dose of  0.6
g/kg (P3) dan 0,9 g/kg (P4) apparently causes
proliferation of the uterine endometrium and
myometrium layers and increases the number of uterine
glands (Figure 2C and 2D).

Genistein and daidzein are categorised in isoflavone
group with the chemical structure that is similar to
estrogen hormone and able to work as estrogen. The
similarity of the structure of genistein and daidzein
demonstrate the ability to bind to estrogen receptors
(Setchell & Cassidy, 1999). The decrease of ovarian
follicles proliferation during the premenopause causes
delays of tertiary follicle growth such that the secretion of
estrogen hormone in the tertiary follicle antrum does not
occur. The provision of grated yam bean can optimize the
level of estrogen so that it is able to stimulate the
development of primary follicles (primordial), secondary
follicles, and tertiary follicles.

The provision of phytoestrogen during the
premenopause period improves the secretion of estrogen
hormone which later causes an increase in the
endometrium proliferation and epithelial cells
replacement to cover the surface of mucosa.
Phytoestrogens have a similar structure to that of estradiol

A B

C D



8 Biology, Medicine, & Natural Product Chemistry 4 (1), 2015: 5-9

which may also occupy estrogen receptors and lead to
estrogen-like effects such as endogenous itself (Harrison
et al., 1999). Myometrium may develop due to the
phytoestrogens yam bean effect.

Genistein and daidzein in yam bean have an effect on
uterine epithelium resulting in proliferation and
cornification of epithelial cells as well as in the
optimization of the secretion of estrogen. Decreased
estrogen level in premenopausal and postmenopausal
period, resulting in an excess amount of estrogen
receptors that are not bound. Genistein and daidzein
ability to bind to estrogen receptors called sex hormone
binding globulin (HGB) serves to increase the production
of steroid hormones and is responsible for binding
estrogen and circulating it through the blood vessels
(Setchell et al., 1998). Genistein as one of the isoflavone
compounds has the effect of increasing the weight of the
uterus by stimulating uterine endometrial thickening
(Santell et al., 1997).

Decreased levels of endogenous estrogen in
premenopausal period can cause the estrus phase to not
occur, thus giving yam bean to premenopausal mice can
stimulate the estrus phase. Genistein and daidzein as a
group of phytoestrogens bind to estrogen receptors in the
ovaries and uterus. The resulting response when estrogen
levels are low is that the receptor and phytoestrogens
binding will help in balancing estrogen levels. The
function of estrogen in relation to reproduction is causing
proliferation to occur and tissue in the reproductive
organs to grow.

The use of natural materials as hormone replacement
therapy in postmenopausal women is one of the
alternatives that can be done to overcome the side effects.
Component compounds contained in natural materials are
very complex and often interact each other to provide a
physiological effect (Ioannides, 2002 and Zhou et al.,
2003). Multi component contained in herbal medicine is
a compounds complexity that provides optimal effect
(Lan and Jia, 2010).

Conclusions

Yam bean can be used as natural estrogen hormone
source which can increase the proliferation and
maturation of ovarian follicles and proliferation of uterine
endometrial glands of the uterus in premenopausal
period. Following up the need for estrogen therapy for
premenopausal and postmenopausal women, it is
necessary to study the effectiveness and safety ranging
from experimentin on animals to humans, so its use as
hormone therapy can be trustworthy.

References

Abid, M. 2005. Pharmacological Evaluation of Pachyrhizus erosus
(L) Seeds for CNS Activity. Disertasi, Bangalore: Rajiv Gandhi
University of Health Science.

Adlercreutz, H. 1990. Western Diet and Western Diseases: Some
Hormonal Biochemical Mechanism and Associations.
Scandinavian J. Clin. Lab. Invest. 50s 201:3-23.

Adlercreutz, H., Mazur, W. 1997. Phyto-oestrogens and Western
Diseases. J. Anayls of Med. 29:95-120.

Barlow, J., Johnson, J.A., Scofield, L. 2007. Fact sheet on the
phytoestrogen genistein. NIEHS/NCI Environment Research
Centers.

Barrett, C.E. 1998. Hormone Replacement Therapy. BMJ.
317:457-461.

Delmonte, P., Rader, J. 2006. Analysis of isoflavones in foods and
dietary supplements. Journal of AOAC International,
89(4)1138-1146,

Ford, J.A., Clark, S.G., Walters, E.M.,  Wheeler,  M.B.,  Hurley,
W.L.  2006. Estrogenic Effects of Genistein on Reproductive
Tissues of Ovariectomized gilts. Anim Sci. 84:834-842.

Grady, D., Rubin, S.M., Petitti, D.B. 1992. Hormone Therapy to
Prevent Disease and Prolong Life in Postmenopausal Women.
Ann Intern Med. 117:1016-1037.

Griffiths, K., Adlercreutz, H., Boyle, P. 1996. Nutrition and
Cancer. Oxford: Isis Medical Media.

Harrison, R.M., Phillippi, P.P., Swan, K.F., Henson, M.C., 1999.
Effect of genistein on steroid hormon production in the
pregnant rhesus monkey. Society for Experimental Biology
and Medicine, vol. 222.

Ioannides, C. 2002. Pharmacokinetic Interactions Between Herbal
Remidies and Medicinal Drugs. Xenobiotica. 32:451-478.

Kang, S., Chung, J.H., Lee, J.H., Fisher, G.J., Wan, Y.S., Duell,
E.A. 2003. Topical N-acetyl cysteine and genistein prevent
ultraviolet-light-induced signaling that leads to photoaging in
human hkin in vivo. J Invest Dermatol. 120:835-841.

Karuniawan, A., Wicaksana, N. 2006. Kekerabatan genetik
populasi bengkuang Pachyrhizuz erosus berdasarkan karakter
morfologi dan daun. Bul Agrom. (34)(2):98-105.

Lan, K., Jia, W. 2010. An integrated metabolomics and
pharmacokinetics strategy for multi-component drugs
evaluation. Current Drug Met. 11:105-114.

Lukitaningsih, E. 2009. The Exploration of Whitening and Sun
Screening Compounds in Benguang Roots (Pachyrhizus
erosus. Disertasi, Wurzburg: Bayerischen Julius Maximillians
University.

Marquez, S.R., Hernandez, H., Flores, J.A., Gutierrez, M.M.,
Duarte, G., Vielma, J., Rodriguez, G.F., Fernandez, I.G.,
Keller, M., and Delgadillo, J.A. 2012. Effects of
Phytoestrogens on Mammalian Reproductive Physiology.
Tropical and Subtropical Agroecosystems. 15 SUP 1:S129-
S145.

Nurrochmad, A., Leviana, F., Wulancarsari, C.G.., &
Lukitaningsih, E. 2010. Phytoestrogens of Pachyrhizuz erosus
Prevent Bone Loss in an Ovariectomized Rat Model of
Osteoporosis. Int. J. Phytomed. 2:363-372.

Primiani, C.N. 2013. Dinamika Senyawa Daidzein Umbi
Bengkuang (Pachyrhizus erosus) dalam Darah Serta
Potensinya pada Tikus Betina. The Proceeding of 10th National
Seminar of Biology, Environment and their Educational
Implementation. Biology Education Department, Faculty of
Pedagogy, Sebelas Maret University, Surakarta. 6 July 2013.
ISBN 978-602-8580-94-6. 3:502-510.

Santell, R.C., Chang, Y.C., Muralee, G.N., William, G.H.  1997.
Dietary genistein exerts  estrogenic  effects  upon  the  uterus,
mammary  gland  and  the hypothalamic/pituitary axis in rats.
Nutr. 127: 263–269.

Setchell, K., Nechemias, L.Z., Cai, J., Heubi, J. 1998. Isoflavone
content of instant formulas and the metabolic fate of these
phytoestrogens in early life. Am J Soc Clin Nutr.68:1453-1461.

Setchell, K.D.R., Cassidy, A. 1999. Dietary Isoflavones: biological
effects and relevance to human health. J Nutr. 129:758s-767s.

Sourander, L., Rajala, T., Raiha, I., Makinen, J., Erkkola, R., dan
Helenius, H. 1998. Cardiovascular and cancer death in



Cicilia Novi Primiani – The Phytoestrogenic Potential of Yam Bean (Pachyrhizus erosus) … 9

postmenopausal women on estrogen replacement therapy
(ERT). Lancet. 352:1965-1969.

Stampfer, M.J., Colditz, G.A., dan Willett, W.C.1991.
Postmenopausal Estrogen Therapy and Cardiovascular
Disease. N Engl J Med. 325:756-762.

Urasopon, N., Hamada, Y., Asaoka, K., Poungmali, U., dan
Malavijitnond, S. 2008. Isoflavone content of rodent diets and
its estrogenic effect on vaginal cornification in Pueraria
mirifica treated rats, Science Asia, 34:371-376.

Wanibuchi, H., Kang, J.S., Fukushima, S. 2003. Toxicity vs
Benefecial Effects of Phytoestrogens. Pure Appl Chem. 75(11-
12):2047-2053.

Zhou, S., Gao, Y., Jiang, W., Huang, M., Xu, A., Paxton, J.W.
2003. Interactions of herbs with cytochrome P450. Drug Metab
Rev. 35:35-98.




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