The Effects of Phaleria macrocarpa (Scheff.) Boerl Extract on Tumor

Necrosis Factor-Alpha (TNF-α) Level in Preeclampsia-induced

Human Umbilical Vein Endothelial Cell (HUVEC) Culture

Leo Simanjuntak

1,2*

, M. Fidel Ganis Siregar

, Johannes C. Mose

and Sarma N. Lumbanraja

Doctoral Program, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia

Department of Obstetrics and Gynecology, Faculty of Medicine, Nommensen HKBP University,

Medan, Indonesia

Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Sumatera Utara,

Medan, Indonesia

Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Padjadjaran,

Bandung, Indonesia

Keywords : Phaleria Macrocarpa, Preeclampsia, HUVEC, TNF-Α

Abstract : Preeclampsia is a major cause in both maternal and perinatal mortality and morbidity. The

etiopathogenesis of preeclampsia is still not fully elucidated but it is believed to be a multifactor.

Endothelial dysfunction plays a big role in the pathophysiology of preeclampsia. Tumor Necrosis Factor-α

(TNF-α) is considered as one of the potentially specific markers for preeclampsia. In vitro model research is

considered the best and most effective way to understand the disease pathophysiology. HUVEC (Human

Umbilical Vein Endothelial Cell) culture is an in vitro model widely used to study the pathogenesis of

preeclampsia. Phaleria macrocarpa (Scheff.) Boerl also known as Mahkota Dewa is widely used as an anti-

inflammation and antioxidant because of alkaloids, saponins, flavonoids and polyphenols properties. This

study aimed to determine the effects of Phaleria macrocarpa Extract on Tumor Necrosis Factor – Alpha

(TNF- α) Level In preeclampsia-induced Human Umbilical Vein Endothelial Cell (HUVEC). Our results

showed the Phaleria macrocarpa’s extract reduce TNF-α level siginifcantly at concentration of 7.813

μg/mL. Phaleria macrocarpa’s extract at concentration of 62.5 μg/mL reduce TNF-α level to normal level.

Thus, Phaleria macrocarpa’s extract might be used as agent to overcome endothelial dysfunction in

preeclampsia.

1. INTRODUCTION

Preeclampsia is one of the leading causes of

maternal morbidity and mortality worldwide. It is

estimated that maternal deaths worldwide are around

500,000 annually and about 10% - 15% are due to

preeclampsia and eclampsia (Maynard et al. 2008).

In 2006 WHO reported that 16% of maternal deaths

in developed countries due to hypertension in

pregnancy, higher than due to bleeding of 13%,

abortion of 8% and sepsis of 2% (Cunningham et al.

2014).

Preeclampsia and eclampsia also adversely affect

the fetus and the neonates. It is estimated that 15%

of preterm births due to preeclampsia, where labor

has to be performed to prevent the progression of

preeclampsia (Roberts & Gammill 2005).

Although there have been many studies but the

etiopathogenesis of preeclampsia is still not fully

elucidated but it is believed to be a multifactors.

Therefore preeclampsia remains a 'disease of

theories'. The difficulties increase because the

syndrome of preeclampsia usually occurs in the third

trimester when the underlying disorder has occurred

Simanjuntak, L., Siregar, M., Mose, J. and Lumbanraja, S.

The Effects of Phaleria macrocarpa (Scheff.) Boerl Extract on Tumor Necrosis Factor-Alpha (TNF-) Level in Preeclampsia-induced Human Umbilical Vein Endothelial Cell (HUVEC) Culture.

DOI: 10.5220/0008789900100016

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 10-16

ISBN: 978-989-758-438-1

in the early stages of placentation, thus difficult to

understand its progression (Cross 1996).

Endothelial dysfunction plays an important role

in the pathophysiology of preeclampsia. Endothelial

dysfunction is defined as an altered state of

endothelial cell differentiation in response to

sublethal injury or cytokine stimulation (Hubel

1999). Under normal circumstances, endothelial

cells maintain vascular integrity, regulating blood

pressure, preventing intravascular coagulation, and

regulating vascular smooth muscle tone by

producing various substances including nitric oxide

(NO), endothelin, prostacyclin and thromboxane

(Davidge 1998; Scalera , Schlembach & Beinder

2001). Rodgers et al. (1988) suggests that

endothelial dysfunction occurs due to cytotoxic

factors in the circulation. Impaired utero-placental

perfusion in PE causes hypoxia, ischemia, placental

oxidative stress, so the placenta produces free

radicals such as superoxide anions (O

) and H

trophoblast debris, pro-inflammatory cytokines, and

antiangiogenic factors which are thought to cause

vascular endothelial dysfunction and causing

excessive maternal inflammatory responses.

Systemic maternal vascular endothelial dysfunction

is the underlying cause of clinical manifestations in

preeclampsia (Roberts et al. 1989).

Tumor Necrosis Factor-α (TNF-α) is considered

as one of the potentially specific markers for

preeclampsia and contributes to the formation of free

radicals such as peroxides (H

), and superoxide

) (Amash et al. 2010). In endothelial cells TNF-α

causes endothelial dysfunction by increasing

oxidation of low-density lipoprotein (LDL)

(Maziere, Auclair & Maizere 1994), inhibiting

eNOS enzymes causing NO levels decrease (Zhang

et al. 2009) and increasing free radical production by

xanthine oxidase enzyme, then binds to endothelial

cells and produces an O

anion in endothelial cells

(Page et al., 1997).

Wang & Walsh (1996) found that TNF-α levels

in placenta preeclampsia were significantly higher

than normal pregnancies (17.32 ± 1.97 pg / mg

protein vs 11.62 ± 0.93 pg / mg protein) with ELISA

method and allegedly TNF-α increases oxidative

stress by stimulating the formation of ROS. Hayashi

et al. (2005) found that TNF-α level in preeclampsia

serum is significantly higher than in the serum of

normal pregnancies (4.68 pg/mL vs 3.31 pg/mL).

Zuspan (1978) suggested that PE treatment will

only be successful and rational if based on

understanding the disease pathophysiology. In an

attempt to determine the pathophysiology of a

disease, in vitro model research is considered the

best and most effective way (Orendi et al. 2011).

HUVEC (Human Umbilical Vein Endothelial Cell)

cell line culture and trophoblast cell line is an in

vitro model widely used to study the pathogenesis of

preeclampsia.

The preventions of preeclampsia consist of

primary, secondary, and tertiary prevention. Primary

prevention aims to prevent the onset of disease by

avoiding pregnancy with contraception because the

pathogenesis of preeclampsia remains unclear.

Secondary prevention aims to inhibit the disease

progression before the onset of clinical

manifestations. Tertiary prevention aims to prevent

the complications of a disease, in preeclampsia, the

complications such as seizures, HELLP syndrome,

and IUGR, which tertiary prevention can be

interpreted as treatment Tertiary prevention includes

regular antenatal examination, appropriate referral,

anti-hypertensive administration, anti-convulsant

administration, and appropriate timing of delivery

(Dekker & Sibai 2001)..

The use of traditional medicines in Indonesia is

part of a culture that has been going on since long

time ago. Act No. 381 of 2007 on national

traditional drug policy regulates the development of

traditional medicines in order to obtain good quality,

safe, and scientifically tested traditional medicine

(Departemen Kesehatan RI 2007).

Herbs or medicinal plants have been used

traditionally as alternative medicine since ancient

times. Phaleria macrocarpa (Scheff.) Boerl also

known as Mahkota dewa belongs to the

Thymelaceae family, that originated from Papua

province, is very popular in Indonesia used in the

treatment of various diseases such as cancer,

hemorrhoids, diabetes mellitus, allergies, liver

disease, heart disease, kidney disease, hypertension,

migraine, skin diseases and others (Hendra et al.

2011; Anggraini & Lewandowsky 2015; Alara,

Alara & Olalere 2016). Phaleria macrocarpa

(Scheff.) Boerl is widely used as an antioxidant

because of alkaloids, saponins, flavonoids and

polyphenols properties. The phenol and flavonoid

compounds in the extract of Phaleria macrocarpa

have antioxidant and anti-inflammatory activity

(Tiwari 2001; Hendra et al. 2011).

To date there has been no research on the effect

of the Phaleria macrocarpa’s extract on the levels of

TNF-α in preeclampsia-induced Human Umbilical

Vein Endothelial Cell (HUVEC). The aim of this

study is to determine the tffects of Phaleria

macrocarpa (Scheff.) Boerl Extract on Tumor

Necrosis Factor – Alpha (TNF- α) Level In

The Effects of Phaleria macrocarpa (Scheff.) Boerl Extract on Tumor Necrosis Factor-Alpha (TNF-) Level in Preeclampsia-induced Human

Umbilical Vein Endothelial Cell (HUVEC) Culture

preeclampsia-induced Human Umbilical Vein

Endothelial Cell (HUVEC).

2. MATERIALS AND METHOD

Serum samples used were obtained from women

at >20 - 42 weeks of gestational age, which were

diagnosed preeclampsia and normal pregnancy at

Dr. Hasan Sadikin General Hospital. Research

subjects have fulfilled inclusion and exclusion

criteria.

2.1. Cell Culture

HUVEC cell line was obtained from American

Type Collection Culture with ATCC CRL-1730

code number. HUVEC cell line was growth into

tissue culture flask (25 cm

) containing RPMI 1640

media, 20% (v/v) FBS qualified (fetal bovine serum)

supplementation, 10% endothelial supplement, 1%

Penicillin G - Streptomycin solution stabilized, and

1% antimycotic Fungizone Amphotericin B and 1%

gentamicin. The cells were then incubated at 37

and 5% CO

(v/v). Culture medium is replaced every

2 - 3 days. Then cells are passaged every seven days

until reach 80-90% confluence.

2.2. Phaleria macrocarpa’s Extract

Phaleria macrocarpa (Scheff.) Boerl was

obtained from the Research Institute for Industrial

Plants at Manoko, Lembang, West Java, Indonesia.

The plant species was identified by the laboratory of

Plant Taxonomy staff at Herbarium Bogoriense,

Bogor, Indonesia.

2.3. Measurement of TNF- α Level

As many as 6x10

cells/mL induced with normal

and preeclampsia serum, were placed into 60-well

plate, then incubated at 37

C and 5% CO

(v/v).

Each well then was washed with 37

C PBS 3-4

times. Furthermore, various concentrations of

Phaleria macrocarpa’s extract ((0,977; 1,953; 3,906;

7,813; 15,625; 31,25; 62,5; 125; and 250 μg/mL)

were added into each well, then incubated for 24 and

72 hours 37

C and 5% CO

(v/v). Each well then

was washed with 37

C once for five minutes.

Transfer the cells into centrifugation tube using 1.5

mL pipette. Centrifuged at 1.500 rpm for 10 minutes

at 4°C. Use the supernatant as a sample for the

ELISA method measurement, then the rest of the

sample can be stored at -80 °C.

2.4. Data Analysis

Data distribution was analyzed with Shapiro-

Wilk normality test. Data was analyzed with

repeated ANOVA (analysis of variance) test and

followed by Bonferroni test as post hoc comparison

test.

3. RESULTS

Figure 1. TNF-α levels in normal and preeclampsia-induced HUVEC based on incubation time.

NORMAL 24H NORMAL 72H PE 24H PE 72H

SAMPLE

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

As shown in figure 1 TNF-α levels in

preeclampsia HUVEC culture model is higher than

normal pregnancy HUVEC culture model. The

TNF-α levels at 72 hours incubation time was lower

than the 24 hours incubation time in both normal and

preeclampsia models.

Variables tested in this study were normally

distributed both in normal and preeclampsia model

treated with Phaleria macrocarpa’s extract in various

concentrations incubated for 24 and 72 hours.

Table 1. TNF-α levels (pg/mL) in preeclampsia and normal serum-induced HUVEC culture model treated with Phaleria

macrocarpa’s extract in various concentrations incubated for 24 and 72 hours.

Phaleria macrocarpa’s

extract concentration

(μg/mL)

24 H INCUBATION TIME 72 H INCUBATION TIME

NP* (Mean ± SD) PE (Mean ± SD) NP* (Mean ± SD) PE (Mean ± SD)

Control

8.718 ± 0.043 18.709 ± 0.007 7.858 ± 0.029 17.848 ± 0.035

0.977

8.218 ± 0.003 18.273 ± 0.051 7.445 ± 0.015 17.395 ± 0.007

1.953

7.886 ± 0.005 17.888 ± 0.001 6.987 ± 0.006 16.778 ± 0.015

3.906

7.382 ± 0.071 15.295 ± 0.087 6.335 ± 0.02 14.666 ± 0.312

7.813

6.814 ± 0.007 14.533 ± 0.000 5.950 ± 0.057 13.763 ± 0.000

15.625

6.009 ± 0.003 12.778 ± 0.000 5.103 ± 0.007 11.492 ± 0.708

31.25

6.000 ± 0.000 10.089 ± 0.000 5.077 ± 0.014 9.325 ± 0.000

62.5

5.994 ± 0.006 8.578 ± 0.000 5.009 ± 0.000 7.668 ± 0.001

125

5.455 ± 0.001 7.654 ± 0.000 4.662 ± 0.000 6.834 ± 0.001

250

5.003 ± 0.000 6.089 ± 0.000 4.101 ± 0.001 5.404 ± 0.000

NP : Normal Pregnancy

Table 1. shows TNF-α levels mean in difference

preeclampsia and normal serum-induced HUVEC

culture model treated with Phaleria macrocarpa’s

extract in various concentrations incubated for 24

and 72 hours.

Table 2. TNF-α levels (pg/mL) mean comparison before and after various concentrations of Phaleria macrocarpa’s extract

treatment at 24 hours and 72 hours incubation time in preeclampsia HUVEC culture model.

Phaleria macrocarpa’s

extract concentration

(μg/mL)

24 H INCUBATION TIME 72 H INCUBATION TIME

PE (Mean ± SD) P value* PE (Mean ± SD) P value*

Control 18.709 ± 0.007 17.848 ± 0.035

0.977 18.273 ± 0.051 1.000 17.395 ± 0.007 1.000

1.953 17.888 ± 0.001 0.227 16.778 ± 0.015 0.362

3.906 15.295 ± 0.087 0.470 14.666 ± 0.312 1.000

7.813 14.533 ± 0.000 0.034 13.763 ± 0.000 0.175

15.625 12.778 ± 0.000 0.024 11.492 ± 0.708 1.000

31.25 10.089 ± 0.000 0.017 9.325 ± 0.000 0.082

62.5 8.578 ± 0.000 0.014 7.668 ± 0.001 0.070

125 7.654 ± 0.000 0.013 6.834 ± 0.001 0.065

250 6.089 ± 0.000 0.012 5.404 ± 0.000 0.058

* : statistically significant if p < 0.05

The Effects of Phaleria macrocarpa (Scheff.) Boerl Extract on Tumor Necrosis Factor-Alpha (TNF-) Level in Preeclampsia-induced Human

Umbilical Vein Endothelial Cell (HUVEC) Culture

Table 2 shows TNF-α level decreased in

preeclampsia serum-induced HUVEC ATCC CRL

1730 following increased Phaleria macrocarpa’s

extract concentration. TNF-α level significantly

decreased after exposure of Phaleria macrocarpa’s

extract on concentration 7.813 μg/mL. (p<0,05).

Figure 2. TNF-α levels in relation with Phaleria macrocarpa’s extract concentration

Figure 2 shows that Phaleria macrocarpa’s

extract at concentration of 62.5 μg/mL reduce

TNF-α level in preeclampsia model to normal

pregnancy level.

4. DISCUSSION

This were the first study to evaluate the effects of

Phaleria macrocarpa (Scheff.) Boerl extract on

Tumor Necrosis Factor – Alpha (TNF- α) level in

Preeclampsia-Induced Human Umbilical Vein

Endothelial Cell (HUVEC). Preeclampsia and

eclampsia have been known since ancient times but

their pathophysiology is still not clearly understood.

Abnormal trophoblast invasion and placental

perfusion disorders are thought to be the underlying

cause of preeclampsia.

There is compelling evidence that endothelial

dysfunction plays a role in the pathophysiology of

preeclampsia. A consistent finding is the presence of

glomerular endotheliosis in more than 70% of

primiparous preeclampsia patients and this

glomerular endotheliosis will disappear after

delivery (Roberts et al., 1989).

To date, invitro research using HUVEC has been

done a lot recently. Previous invitro research on

HUVEC cultures by treating with anti-inflammatory

and antioxidant compounds such as curcumin and

Papua ant nest (Myrmecodia pendens) decrease

oxidative stress and inflammation characterized by

decreased levels of MDA, and TNF-α. These studies

conclude that the Papuan ant nests and curcumin

have a therapeutic effect on preeclampsia (Yeni et

al., 2017, Gunardi et al., 2016).

Tumor Necrosis Factor-α (TNF-α) is considered

as one of the potentially specific markers for

preeclampsia and contributes to the formation of free

radicals such as peroxides (H

), and superoxide

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

) (Amash et al. 2010). The study by Yoshizumi

et al. (1993) showed TNF-α decrease NOS mRNA

levels in HUVEC, indicating a decrease in NO levels

as vasodilators.

In this study results showed TNF- α levels in

preeclampsia HUVEC culture model was higher

than normal pregnancy HUVEC culture model.

TNF-α level decreased in preeclampsia and normal

serum-induced HUVEC ATCC CRL 1730 culture

following increased Phaleria macrocarpa’s extract

concentration. TNF-α level significantly decreased

after exposure of Phaleria macrocarpa’s extract on

concentration 7.813 μg/mL. Phaleria macrocarpa’s

extract at concentration of 62.5 μg/mL reduce TNF-

α level to normal level.

The result of present study suggests that Phaleria

macrocarpa’s extract contains anti-inflammatory

activity proven by decreased level of TNF-α. It was

also described that TNF-α level decreased in

preeclampsia and normal serum-induced HUVEC

ATCC CRL 1730 following increased Phaleria

macrocarpa’s extract concentration. Thus, Phaleria

macrocarpa’s extract might be used as agent to

restore endothelial dysfunction in preeclamsia.

5. CONCLUSION

The Phaleria macrocarpa’s extract reduce

TNF-α level siginificantly at concentration of 7.813

μg/mL in preeclampsia-induced HUVEC ATCC

CRL 1730 culture. Phaleria macrocarpa’s extract at

concentration of 62.5 μg/mL reduce TNF-α level to

normal level.

ACKNOWLEDGEMENT

The authors whose names are listed above certify

that they have no affiliations with or involvement in

any organizations or entity with any financial

interests in the subject matter or materials discussed

in this manuscript.

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