Clinical Nutrition Experimental 15 (2017) 9e14

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Clinical Nutrition Experimental

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Punica granatum fruit extract inhibits the production of pro-

inammatory cytokines and angiogenic factors of HUVEC
cells induced by plasma from patients with pre-eclampsia
Dewi Ambarwati a, b, *, Fatmawati Fatmawati b, Mukhamad Nooryanto c,
Sanarto Santoso d, Siti Candra Windu Baktiyani c, Nurdiana Nurdiana e
a
Midwifery Diploma Study Programme, Muhammadiyah University of Purwokerto, Banyumas, Central Java, Indonesia
b
Midwifery Master Study Programme, Faculty of Medicine Brawijaya University, Malang, East Java, Indonesia
c
Obstetric and Ginecology Laboratory, Saiful Anwar General Hospital, Faculty of Medicine Brawijaya University, Malang, East Java,
Indonesia
d
Microbiology Laboratory, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia
e
Pharmacology Laboratory, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia

a r t i c l e i n f o s u m m a r y

Article history: This study aims to nd out whether the ethanol extract of Punica
Received 7 May 2017 granatum fruit can inhibit pro-inammatory cytokines (TNF-a and
Accepted 23 June 2017 IL-6) and anti-angiogenic factors (sFlt-1 and sEng) of endothelial
Available online 1 July 2017
cells given plasma stimulus of pre-eclamptic patients. When the
cells reached conuence, cells would be grouped into ve groups
Keywords:
(ve replicates per group), i.e. endothelial cells exposed to the
Inammation
plasma of normal pregnancy, endothelial cells exposed to the
Anti-angiogenic
In vitro plasma of patients with severe pre-eclampsia, endothelial cells
Endothelial exposed to the plasma of patients of severe pre-eclampsia and
Hypertension of pregnancy given the extract of P. granatum in various doses (14 ppm; 28 ppm,
and 56 ppm). Once induced, measurement of pro-inammatory
cytokine levels and anti-angiogenic factors would be carried out
by using the technique of enzyme-linked immunosorbent assay
(ELISA). Phytochemical analysis showed that the extract contains
phenolic, avonoid and tannin. Plasma from patients with pre-
eclampsia increased the levels of TNF-a, IL-6, sFlt-1 and sEng
signicantly compared with normal pregnancy plasma exposure
group. The increase in the levels of TNF-a, sFlt-1 and sEng was
signicantly inhibited by the administration of extract at a dose of

* Corresponding author. Midwifery Diploma Study Programme, Muhammadiyah University, Jl. Raya Dukuhwaluh, Dukuh-
waluh, Kembaran, Kabupaten Banyumas, Central of Java, 53182, Indonesia.
E-mail addresses: dwambarwt@gmail.com (D. Ambarwati), fatmawatibetty26@gmail.com (F. Fatmawati), siticandrawb@
gmail.com (S.C. Windu Baktiyani).

http://dx.doi.org/10.1016/j.yclnex.2017.06.001
2352-9393/ 2017 The Authors. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
10 D. Ambarwati et al. / Clinical Nutrition Experimental 15 (2017) 9e14

56 ppm (P < 0.05). For IL-6, the increase was signicantly inhibited
by the extract at doses of 28 and 56 ppm. It is concluded that P.
granatum fruit extract provides protection to the endothelial cells
through suppressing the production of pro-inammatory cyto-
kines (TNF-a and IL-6) and anti-angiogenic factors (sFlt-1 and
sEng) due to plasma stimulus of patients with severe pre-
eclampsia.
2017 The Authors. Published by Elsevier Ltd on behalf of
European Society for Clinical Nutrition and Metabolism. This is an
open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Pre-eclampsia (PE) and HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count)
are two specic diseases for pregnant women which are multifactorial and involve genetic components
and environmental inuences [1]. Symptoms of pre-eclampsia include hypertension and proteinuria
after 20 weeks of pregnancy. Pre-eclampsia occurs in 3e5% of pregnancies, as a cause of maternal and
fetal morbidity and mortality [2,3]. The pathogenesis of this syndrome is underlain by the mismatch of
trophoblast invasion and endothelial cell dysfunction that triggers placental dysregulation [4].
The imbalance between pro- and anti-inammatory cytokines is also involved in the development
of preeclampsia [5]. Pre-eclampsia is characterized by a shift in Th-1-type maternal immune response
in the form of the production of pro-inammatory cytokines [5e7]. Plasma levels of TNF-a were higher
in pre-eclamptic pregnancy with early and late onset than normal pregnancy [8]. In addition, the levels
of IL-6 increased in severe preeclampsia, but not increased in mild pre-eclampsia [5]. In addition to
inammatory factors, anti-angiogenic factors of soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble
endoglin are also released in the placenta of pre-eclampsia and is involved in endothelial dysfunction
[9,10].
In tropical countries, many plants which can be used as a medicine. The utilization of herbs for pre-
eclampsia is still very limited [11e14]. P. granatum is a typical Indonesian plant which has been used as
a medicine in many areas and is believed to be safe and non-toxic [15]. Anti-inammatory effects of this
plant have been proved in previous studies [16,17], but its anti-angiogenic effects and application for
pre-eclampsia have never been performed. Under these conditions, this study is directed to investigate
the effects of P. granatum extracts in the modulation of pro-inammatory cytokines (TNF-a and IL-6)
and the anti-angiogenic factors (sFlt-1 and sEng) in the cells of human umbilical vascular endothe-
lial cells induced by plasma from patients with pre-eclampsia.

2. Material and method

2.1. Taking off newborn's umbilical cord

The umbilical cord of newborn baby was taken off by caesarean section. Endothelial cell culture was
carried out in a range of 12 h after delivery. Before taking off the umbilical cord, bottle containing cord
solution had to be prepared stored in
4  C. Immediately after birth, the umbilical cord was cut along
10 cm, and was directly put into the cord solution. The umbilical cord was then transferred to the
laboratory in cold and sterile conditions.

2.2. Isolation of plasma of normal pregnancy and severe pre-eclampsia

Normal pregnant women or patients with severe pre-eclampsia whose blood would be collected
had given a statement of willingness on the consent form. The blood was collected by aseptic technique
 D. Ambarwati et al. / Clinical Nutrition Experimental 15 (2017) 9e14 11

as much as 2 ml using dispossible 5 cc syringe and then the blood was inserted into sterile vacutainer.
The blood was then kept so that plasma and blood cells were separated. Plasma was taken by using a
2 ml sterile pipette then inserted into the tube to be centrifuged at 3500 rpm for 10 min. After being
disentruged, the plasma was isolated and stored at
40 C or
80 C (long term storage).

2.3. Extraction

P. granatum was obtained from Bandungan village, Semarang, Central Java, Indonesia. One kilogram
of pomegranate arils was dried for approximately 24 h in an oven at 40  C. This process produced dry
red pomegranates as many as 100 g. Furthermore, the dried fruits were blended until smooth, then
dried pomegranate powder was soaked by using 900 ml of ethanol solvent (3  24 h) and evaporation
process was carried out. The nal result of evaporation process was the total extracts. In this study, total
of 44 g of pomegranate extracts were obtained from 100 g of dried pomegranates in the form of
essential oils.

2.4. Culture of HUVECs

The production of HUVEC culture was based on the method developed in Biomedical Laboratory,
Faculty of Medicine, University of Brawijaya [18]. Materials for the isolation of endothelial cells derived
from umbilical veins. The umbilical cord was cleaned with alcohol from tissues and blood clots, and
then cannula was inserted into one end of the vein as deep as 1.5 cm, then tied. The vein was washed
in a solution of Phosphate-Buffered Saline A (PBS) by using a pipette. After being clean, the end having
no cannula was clamped rmly. A total of 10 ml, 0.5 mg/ml of collagenase was inserted into the vein
with the syringe was kept attached and clamped, then grasped by hand for 8 min. Centrifuged at
700 g (1000 rpm) for 8 min. After being centrifuged, the supernatant was discarded and the pellet was
resuspended to be homogeneous by 4 ml of culture media.
Culture media were composed of plasma free (Medium 199, penicillin, streptomicyn, bicarbonate
phenol red, glutamine) plus Phosphate-Buffered Saline (FBS). The pellet was inserted into the well as
many as 24 pieces having been given a 0.2% gelatin previously, the media were grown in an incubator at
a temperature of 370 C, 5% CO2 concentration and 95% humidity (to maintain the pH) until monolayer,
the cells were observed every 2 days, washed with plasma-free media if needed and replaced by new
media. The umbilical cord whose endothelial cells having been taken was cleaned, wrapped and buried
in the ground.

2.5. Qualitative screening

Qualitative screening of alkaloids, avonoids, terpenoids, tannins, saponins and steroids in P.

granatum extracts was performed in accordance with the detailed procedures carried out in the pre-
vious study [19].

2.6. Analysis of TNF-a and IL-6 levels

The levels of TNF-a and IL-6 in the HUVEC medium were analyzed by an enzyme-linked immu-
nosorbent assay technique. The study procedures were carried out in accordance with the detailed
instructions on the kit of Human IL-6 Immunosorbent Assays (Biolegend, USA, Catalog series 430507)
and Human TNF-a Immunoassay (Biolegend, USA, Catalog series 430207).

2.7. Analysis of sFlt-1 and sEng levels

The levels of sFlt-1 and sEng in the HUVEC medium were analyzed by an enzyme-linked immu-
nosorbent assay technique. The study procedures were carried out in accordance with the detailed
instructions on the kit of (eBIOSCIENCE, catalog # BMS268/3/BMS268/3 TEN) and Human TNF-a
Immunoassay (Biolegend, USA, Catalog series 430207) and RayBio Human Endoglin ELISA kit (Ray-
BioTech, Inc; catalog ELH-Endoglin).
12 D. Ambarwati et al. / Clinical Nutrition Experimental 15 (2017) 9e14

2.8. Ethics

This study has passed an ethical review from the Research Ethics Committee, Faculty of Medicine,
University of Brawijaya, Malang, Indonesia.

2.9. Statistical analysis

All data will be shown in mean standard deviation. Differences between treatment groups will be
analyzed using one-way ANOVA test with the assistance of SPSS 17.0 statistical package software. If
signicance is found in the ANOVA test, post Hoc test will be conducted. Value of P < 0.05 is set as a
value of statistically signicant difference.

3. Results

Analysis of qualitative phytochemical screening is intended to nd out the content of active in-
gredients in P. granatum fruit extracts, which are presented in Table 1. This study proved that P.
granatum fruit extract contains phenolic, avonoid, tannin. P. granatum fruit extract does not contain
steroids, terpenoids, saponins and alkaloids.
Table 2 shows the levels of TNF-a and IL-6 in the HUVEC culture media from the various treatment
groups. The levels of TNF-a and IL-6 are signicantly higher in the HUVECs exposed to plasma of pre-
eclamptic patients than HUVECs exposed to plasma of normal pregnancy. Of the three doses of P.
granatum extract, only doses of 28 and 56 ppm can inhibit TNF-a level signicantly (P < 0.05), even
reaching level comparable to HUVECs exposed to plasma of normal pregnancy (P > 0.05). This inhi-
bition also occurs in the IL-6 level, but only found at the highest dose (56 ppm), capable of reaching
level comparable to HUVECs exposed to plasma of normal pregnancy (P > 0.05).
For anti-angiogenic factor, induction of plasma of patients with pre-eclampsia against HUVECs
increases the levels of sFlt-1 and sEng signicantly compared with HUVECs exposed to plasma of
normal pregnancy (P < 0.05). The administration of P. granatum extract at a dose of 56 ppm signi-
cantly lowers the levels of sFlt-1 and sEng compared with the group of HUVECs exposed to plasma of
pre-eclamptic patients (P < 0.05), and reaches the level comparable to HUVECs exposed to plasma of
normal pregnancy (P > 0.05) (Table 3).

4. Discussion

Inammation was involved in the pathomechanism of pre-eclampsia. In this study, we found that
the levels of IL-6 and TNF-a increased signicantly in the group of HUVECs exposed to pre-eclampsia
plasma compared with HUVECs exposed to plasma of normal pregnancy (P < 0.05). These ndings
indicate that the plasma of patients with pre-eclampsia contains several soluble molecules or factors
which can trigger pro-inammatory cytokine upregulation of HUVECs. Previous ndings stated that
the various materials are detected in the plasma of pre-eclampsia, including fetal cells, pro-

Table 1
Qualitative screening of phytoconstituents of the Punica granatum
extract.

Phytochemicals Punica granatum extract

Phenolic
Terpenoid e
Saponin e
Flavonoid
Tannin
Alkaloid e
Steroids e

indicates the presence of constistuents; indicates the absence of

constistuents
 D. Ambarwati et al. / Clinical Nutrition Experimental 15 (2017) 9e14 13

Table 2
Level of pro-inammatory cytokines in endothelial cells induced by plasma preeclamptic patients.

Level NP PP PP Punica granatum

14 ppm 28 ppm 56 ppm

a ab ab
IL-6 (pg/ml) 51.10 5.27 435.4 41.21 164.30 70.96 150.87 31.63 59.57 19.05b
TNF-a (pg/ml) 2808 550.20 15368 1277.40a 12492 3123.50ab 4088 1981b 3236 643.30b

Note: values are presented as mean SD; ap < 0.05; in comparison with NP group; bp < 0.05; in comparison with PP group; TNF-
a: tumor necrosis factor-a; IL-6: interleukin-6; NP: plasma from normal pregnancy; PP: plasma from preeclamptic patients;
ppm: part per million; pg/ml: picogram/mililiter.

Table 3
Level of anti-angiogenic factors in endothelial cells induced by plasma preeclamptic patients.

Level NP PP PP Punica granatum

14 ppm 28 ppm 56 ppm

sFlt-1 (ng/dL) 4.40 0.42 61.70 5.48a 18.39 1.72ab 17.47 0.68ab 5.64 1.33b
sEng (pg/ml) 1801.30 199.90 7411 806.80a 6352.70 290.50ab 4796 726.10ab 2467 809.70b

Note: values are presented as mean SD; ap < 0.05; in comparison with NP group; bp < 0.05; in comparison with PP group; sFlt-
1: soluble FMS like tyrosine kinase-1; sEng: soluble Endoglin; NP: plasma from normal pregnancy; PP: plasma from pre-
eclamptic patients; ppm: part per million; ng/dL: nanogram/desiliter; pg/ml: picogram/militer.

inammatory cytokines (IL-6 and TNF-a), chemokines (IL-8, IP-10 and MCP-1), and adhesion molecules
(ICAM-1 and VCAM-1) [20e22]. Thus, an increase in pro-inammatory cytokines in the HUVEC me-
dium can be caused by the content of given plasma or upregulation process involving various signals in
HUVECs. The increase in the levels of IL-6 and TNF-a can be inhibited by the administration of P.
granatum extract at doses of 56 ppm (IL-6 and TNF-a) and 28 ppm (TNF-a). This indicates that the
active ingredients from P. granatum extract, i.e. phenolic, avonoid, tannin can suppress the modula-
tion signal of pro-inammatory cytokines in HUVECs exposed to pre-eclampsia plasma. These ndings
are supported by previous studies that pomegranate extract can suppress transcription factors (NF-kB)
for the production of pro-inammatory cytokines in various cells [23e26].
In this study, the induction by pre-eclampsia plasma increased the levels of sFlt-1 dan sEng
signicantly compared with the induction by plasma of normal pregnancy. We hypothesized that
plasma from patients with pre-eclampsia contained sFlt-1 and sEng and was capable of modulating
transcriptional activity of p38 and NF-kB for the production of sFlt-1. It is expanding consistent with
previous ndings [6]. The administration of P. granatum extract at a dose of 56 ppm was capable of
reducing the levels of sFlt-1 and sEng signicantly compared with the group of HUVECs exposed to pre-
eclampsia plasma (P < 0.05), and reached the levels comparable to controls (P > 0.05). These ndings
conrm that the active ingredients of P. granatum are anti-angiogenic presumably through the action of
phenolic, avonoid, tannin in inhibiting NF-kB signal. Your ndings are consistent with previous study
[25].
In conclusion, P. granatum fruit extract provides protection to endothelial cells through suppressing
the production of pro-inammatory cytokines (TNF-a and IL-6) and anti-angiogenic factors (sFlt-1 and
sEng) due to plasma stimulus of pre-eclamptic patients. Thus, this plant can be a candidate to suppress
endothelial dysfunction in patients with pre-eclampsia.

Conict of interest

None.

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