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

Keyword: 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.

Suhargo, L. and Winarni, D.

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

DOI: 10.5220/0009842700002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

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

ovariectomized mice (µm)

The

measurement of

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 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).

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.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

2 Experimental

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.

REFERENCES

Burger HG, Hale GE, Robertson DM, Dennerstein L.

2007. A review of hormonal changes during the

menopausal transition: Focus on findings from the

Melbourne Women’s Midlife Health Project. Hum.

Reprod. Update.13(6):559–565

Backer, C.A., Van Den Brink & R.C. Bakhuizen,

1965.Flora of Java. Vol. II. N.V.P. Noordhott.

Groningen.

Fernandez C, Suarez Y, Ferruelo AJ, Gomez-Coronado D,

Lasuncion MA. 2002. Inhibition of cholesterol

biosynthesis by Δ22-unsaturated phytosterols via

competitive inhibition of sterol Δ24-reductase in

mammalian cells. Biochem. J. 366(1):109–119

Guo AJ, Choi RC, Zheng KY, et al. Kaempferol as a

flavonoid induces osteoblastic differentiation via

estrogen receptor signaling. Chin. Med. 2012;7(1):10.

Hamouda AH, Ali.et al. (2018). Study of the Effect of

Aristolochic Acid on Mice Kidney and the Effect of

Withdrawal: Histological and Immunohistochemical

Study.

J Cytol Histol. 9. Vol 9(2).504.

Isnawati, A. 2003.Pemeriksaan Senyawa-senyawa

Turunan Fenol Daun H andeuleum {Graptophyllum

pictum (L) Griff}. National Institute of Health

Research and Development, NIHRD. Percetakan

Negara 29.Jakarta

Kusumawati, I. Dyatmiko, W., Santoso, M.H. dan Maat,

S. 2002. Pengaruh Ekstrak Etanol Daun

Graptophyllum Pictum (L) Griff. Terhadap Fungsi

Fagositosis Serta Pembentukan Imunoglobulin M dan

Tnf-α Pada Mencit. Majalah Farmasi Airlangga.II (2)

: 76.

Kumar Mishra S.2011. Menopausal transition and

postmenopausal health problems: a review on its bio-

cultural perspectives. Health (Irvine. Calif). 3(4):233–

237.

Middleton Jr. E, Kandaswami C, Theoharides TC.

2000.The effects of plant flavonoids on mammalian

cells: implications for inflammation, heart disease, and

cancer. Pharmacol. Rev. 52(4):673–751.

Miller WL. 2005. Disorders of androgen synthesis - From

cholesterol to dehydroepiandrosterone. Med. Princ.

Pract. 14(SUPPL. 1):58–68.

Miep H. Helfrich and Stuart H. Ralston. 2007.

Ovariectomy/Orchidectomy in Rodent. Bone Research

Protocols, Methods in Molecular Biology, vol. 816.

Pinkerton JA V., Aguirre FS, Blake J, et al. 2017.The

2017 hormone therapy position statement of the North

American Menopause Society. Menopause.

24(7):728–753.

Setiawan, R. A. 2012. Keamanan Ekstrak Etanol 96 %

Dau(Pinkerton et al. 2017)n Wungu

(Graptophyllumpictum L.Griff.} Melalui Kajian

Histopat Organ Mencit. Skripsi. Dep.Biokimia.

FMIPA.IPB.Bogor.

Simpson ER. 2002. Aromatization of androgens in

women: current concepts and findings. Fertil. Steril.

77 Suppl 4(4):S6–S10.

Suganya J, Radha M, Naorem DL, Nishandhini M.2014.

In silico docking studies of selected flavonoids -

Natural healing agents against breast cancer. Asian

Pacific J. Cancer Prev. 15(19):8155–8159.

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

Mice