The Utilization of Graptophyllum pictum (L) Griff. Extract as the

Endometrial and Myometrial Hyperplasia Inhibitor of

Ovariectomized Mice

Listijani Suhargo* and Dwi Winarni

Department of Biology, FST, Airlangga University, Surabaya

Keywords: Graptophyllum pictum L.Griff. extracts, endometrium, myometrium, ovariectomized

Abstract: Endometrial and myometrial hyperplasia was caused as a side effect of estradiol hormone using for

menopausal condition and it could change to be cancer. Graptophyllum pictum (L) Griff. (Daun wungu)

ethanol extract with its flavonoid competes with estradiol to bind the estrogen reseptor and decreases the

side effect of estradiol. The aim of this research was conducted to evaluate the utilization of daun wungu

ethanol extracts as the endometrial and myometrial hyperplasia inhibitor of ovariectomy mice. This

researches used 30 ovariectomized mice. The ovariectomized mice were grouped in 5 groups, one

group control group with 3 µg/kg ethynil estradiol/EE), 4 groups for the treatment with 3 µg/kg EE with

25 mg/kg, 37,5 mg/kg, 50 mg/kg and 62,5 mg/kg daun wungu extracts in coconut oil. All treatments

were done by oral for 30 days. At the end of the treatments, all mice was sacrificed and were made

histology preparation of uterus. The result showed that daun wungu ethanol extracts could inhibit

endometrium and myometrium hyperplasia that were showed in the thickness of those tissues of

ovariectomized mice. And 37,5 mg/kg daun wungu extract was optimal dose for hyperplasia inhibitor of

those tissues.

1 INTRODUCTION

In general, the female reproductive cycle stops at the

age of 45 to 50 years due to the absence of a

growing follicle. That condition is called

menopausal condition. At this time the estrogen

hormone is not produced by follicles in the ovary.

Decreased levels of estrogen cause several

symptoms such as heat fluctuation, heart

palpitations, sleep disorders, irritability, headache,

tingling, libido disorders, obstipation and weight

gain (Burger, 2007). Not all postmenopausal women

experience menopausal symptoms because estrogen

is also produced in fatty and muscle tissues through

aromatization of adrostenedione. Androstenedione is

produced by the adrenal glands (95%) and ovaries

(5%) (Simpson, 2002).

In menopausal women the presence of

estrogen produced by muscle and fat cells can also

affect the growth of endometrial and myometrial

cells. The growth of these cells might increase and

was called hyperplasia in overweight women or who

use estrogen therapy during menopause. Estrogen

therapy using synthetic estrogens such as diethyl

stilbestrol, hexestrol, dienestrol and ethinyl estradiol

could increase cell proliferation through synthetic

estrogen binding to estrogen receptors. Increased

cell proliferation or cell hyperplasia might lead to

change in cell genetic properties and may further

lead to the occurrence of cancer cells (Pinkerton,

2017).

The use of natural ingredients to suppress

the effects of estrogen needs to be investigated with

the aim of inhibiting the hyperplasia of uterine cells.

One of the natural ingredients is daun wungu

(Graptophyllum pictum L. Briff.) which is known to

contain flavonoids and phytosterols (Isnawati,

2003). Flavonoids contained in daun wungu are

myricetin and kaempferol (Kusumawati et al.,

2002).

The study was aimed to find out the role of

daun wungu (Graptophyllum pictum L. Griff.) as

inhibitors of endometrial and myometrial cell

proliferation in ovariectomized mice.

Ovariectomized mice are model animals of

menopausal conditions. Table 1 shew the mean data

of endometrial thickness and myometrium of

ovariectomy mice with daun wungu and EE (Ethynil

estradiol) treatment.

102

Suhargo, L. and Winarni, D.

The Utilization of Graptophyllum pictum (L) Griff. Extract as the Endometrial and Myometrial Hyperplasia Inhibitor of Ovariectomized Mice.

DOI: 10.5220/0008358101020105

In Proceedings of BROMO Conference (BROMO 2018), pages 102-105

ISBN: 978-989-758-347-6

2 METHODS

2.1 Daun Wungu Extraction Procedure

Daun Wungu were weighed as much as 1 kg, it

were dried in oven to remove water content with

temperature 40

C, then were mashed with a blender

to form a leaf powder. Daun wungu powder was

macerated with 90% ethanol, the filtration was

evaporated with rotary evaporator for 48 hours. The

results obtained are ethanol extract of daun wungu

(Setiawan, 2012).

2.2 Ovariectomy Via Ventral Route

Anesthetize mice with 0.5 cc ketalar 10 mg / bw via

intramuscular injection on femur, the mice were

stretched and feathered was wetted by ethanol 70%

around the midline of the lower abdomen. and made

incision in the middle as long as 1 cm for skin,

muscle and peritoneal layer. Ovarium was removed

and binding of fallopian tubes and ovarian cutting.

The slices on the peritoneum were stitched, then the

muscle and skin sections were stitched. (Miep et al.,

2007).

2.3 Animal Treatment

In this research, 50 mice were used as control group,

one group was treated with 3 µg/kg ethinyl estradiol

(EE), and 4 other groups were given 3 µg/kg EE and

daun wungu extract treatment with dose 25 mg/kg,

37.5 mg/kg, 50 mg/kg and 62.5 mg/kg with coconut

oil as solvent. All the treatment were done by oral,

each mouse was given 0,1 ml EE and daun wungu

extract for 30 days.

2.4 Histology Procedure

Uterus were quickly removed at sacrifice and fixed

in Bouin Solution. After fixation, tissues were

dehydrated in 70%, 80%, 96% ethanol and , then

embedded in paraffin and cut on a rotation

microtome in 5-6 µm sections, and then stained with

hematoxylin & eosin (Hamouda et al., 2018). The

endometrial and myometrial thickness were

measured with micrometer in microscope.

3. RESULT

The results of the research indicated that the ethanol

extract of daun wungu (Graptophyllum pictum L.

Griff.) had a maximum role as endometrial and

myometrial cell proliferation inhibitor at 37,5

mg/kg dose. Ovariectomized mice as experimental

animals were given ethinyl estradiol for increased

proliferation of epithelial cells, fibroblast and

endothelial cells. Flavonoid of daun wungu with

kaempferol and myricetin content had chemical

structures that resemble estradiol so they could bind

to estrogen receptors. Flavonoids competed with

estradiol to bind estrogen receptors. The estrogenic

effect of flavonoid was lower than EE to induce cell

proliferation on endometrium and myometrium, so

daun wungu extract could reduce the high estrogenic

effects of EE. Flavonoid of daun wungu also can

inhibit aromatase activity that plays a role to alter

androgens to estrogen and further decrease the effect

of cells proliferation on endometrial and myometrial

cells (Suggest et al., 2014 and Guo et al., 2012).

Tabel 1. The effect of daun wungu (Graptophyllum pictum L.Griff.) extract on endometrial and myometrial

thickness of ovariectomized mice (µm)

The measurement

The treatment for ovariectomized mice with 3 µg/kg EE plus daun wungu extract with doses

Control (EE)

25 mg/kg

37,5mg/kg

50 mg/kg

62,5mg/kg

Endometrial

thickness

441,9+59,8

397,9+30,1

297,7+85,7

422,1+54,2

411,9+72,3

Myometrial

thickness

63,6+8,6

69,3+9,6

50,2+8,3

58,7+8,8

59,9+10,1

The Utilization of Graptophyllum pictum (L) Griff. Extract as the Endometrial and Myometrial Hyperplasia Inhibitor of Ovariectomized

Mice

103

Figure 1. Uterus section of ovariectomy mice with 5 treatment.A. Control (EE). B.C.D.E. treatment with EE and daun

wungu extract (25 mg/kg, 37,5mg/kg, 50mg/kg and 62,5mg/kg)

Daun Wungu contains phytosterols that plays a role

to inhibit cholesterol absorption and increase its

excretion so as to reduce total cholesterol

(Fernandez et al., 2002). Cholesterol is the main

ingredient for estrogen formation. Cholesterol is

transferred to the mitochondria of the adrenal gland

cells and converted to pregnenolone. Pregnenolone

was transfered to the cytoplasm and formed

progesterone and then became androstenedione

(Miller, 2005). Androstenedione leads to the

endometrial cells through the blood vessels and was

converted by aromatase enzyme to estrone and then

became estradiol. Estradiol increased proliferation of

endometrial and myometrial cells. The phytosterol

content of daun wungu extract would decrease the

synthesis of estradiol (Simpson, 2002).

At the lowest dose 25 mg/kg of daun

wungu extract could not inhibit cell proliferation

due to dominan induction of ethynil estradiol and

less flavonoid of daun wungu extract couldn’t

competed with ethynil estradiol in β-estrogen

receptor. In higher doses 50 mg/kg and 62.5 mg /

kg of daun wungu extract also couldn’t inhibit cell

proliferation, it was caused that higher doses of

flavonoid and fitosterol bound to β-estrogen receptor

could increase the proliferation of endometrial and

myometrial cells in addition to the effects of Ethinyl

estradiol, consequently the inhibitory effect of

proliferation cells are insignificant and cell

proliferation still increased.

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The Utilization of Graptophyllum pictum (L) Griff. Extract as the Endometrial and Myometrial Hyperplasia Inhibitor of Ovariectomized

Mice

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