Antiproliferative and Apoptotic Induction of n-Hexane Fraction of

Picria Fel-Terrae Lour. Herbs on T47D Cell Line

Denny Satria

1,4*

, Jansen Silalahi

, Ginda Haro

, and Syafruddin Ilyas

Department of Pharmaceutical Biology,

Department of Pharmaceutical Chemistry,

Department of Biology

1,2,

Faculty of Pharmacy,

FMIPA, University of Sumatera Utara

Faculty of Health Sciences and Pharmacy, Universitas Sari Mutiara Indonesia

Keywords: Antiproliferative, Apoptotic, Picria fel-terrae Lour., Herbs, N-hexane.

Abstract: A recent research reported that breast cancer is leading to the estimated new cancer cases, and the second

most incidence death cause of women affictioning from cancer. This research aim is to evaluate cytotoxic,

antiproliferative and apoptotic induction activities of n-hexane fraction (nHF) of Picria fel-terrae Lour.

herbs. Cytotoxic activity of nHF was determined with MTT method, cell cycle and apoptotic analysis were

determined with flow cytometry method towards T47D cell line. Cytotoxic activity from nHF with MTT

assay measured as IC50 was 75.87 ± 0.75 µg/mL, nHF at 15 µg/mL caused accumulation in G2-M

(37.47%) and S phase accumulation (19.41%) and increased early (24.25%) and late apoptosis (4.26%). The

results reveal that nHF of Picria fel-terrae Lour. herbs have antiproliferative and apoptotic induction

activities. Our further study is to isolation anticancer compounds from Picria fel-terrae Lour. herbs.

1. INTRODUCTION

Breast cancer take place when breast cells start to

grow with uncontrollably. Cells could invade

nearby tissues and spread pass through the body.

Each kind of tissue in the breast can form a cancer,

but the cancer generally arises in the milk ducts or

glands. Factors which influence the risk of breast

cancer are reproductive factors (e.g. no children

and first pregnancy at an advanced age), the length

of exposure to hormones, dietary factors and lack

of physical activity, radiation during breast

development, hormone replacement therapy, as

well as congenital genetic factors (Barnett, et. al.,

2008). WHO reported that breast cancer is one of

the main cause of death and the most common

incidence of cancer type amongst women

worldwide in 2012 (WHO, 2015).

Poguntano (Picria fel-terrae Lour.) have been

used for treat of colic, diuretic, fever, malaria, and

skin disease (Perry, 1980). The modern

pharmacological assessment indicated that the

Picria fel-terrae Lour. exerts antidiabetic,

antioxidant, anti-inflammatory, anthelmintic,

diuretic, antipyretic, hepatoprotective,

cardioprotective, and analgesic activities (Sitorus,

et al., 2014; Dalimunthe, et al., 2015; Sihotang, et

al., 2016;Huang, et al., 1994; Thuan, et al., 2007;

Zhong, et al., 1979; Zou, et al., 2005; Harfina, et

al., 2012; Patilaya and Husori, 2015). Moreover,

Picria fel-terrae inhibits hepatitis B (HB) e-antigen

excreted by HepG2 2215 cell lines, suggesting to

have anti-HB virus activity (Zheng, et al., 2010). It

can be developed as co-chemotherapeutic regimen

and inhibit metastasis for breast cancer by inducing

apoptosis, cell cycle arrest, suppressing cyclin D1

and Bcl-2 expression, suppressing expression of

COX-2 and VEGFR2 based on the recent studies

(Satria, et al., 2015; Lestari, et al., 2013, Harahap,

et al., 2018). The aim of this study was to assess

the antiproliferative and apoptosis induction

activities of n-hexane fraction of Picria fel-terrae

Lour. Herbs.

2. MATERIALS AND METHODS

2.1 Plant and Chemicals Material

Fresh herbs of Picria fel-terrae Lour. was collected

from Tiga Lingga village, Dairi regency, Sumatera

Utara province, Indonesia. Picria fel-terrae Lour.

Satria, D., Silalahi, J., Haro, G. and Ilyas, S.

Antiproliferative and Apoptotic Induction of n-Hexane Fraction of Picria fel-terrae lour. Herbs on T47D Cell Line.

DOI: 10.5220/0009844300002406

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

ISBN: 978-989-758-347-6

was identified in Research Centre for Biology,

Indonesian Institute of Science, Bogor, and the

voucher specimen was deposited in

herbarium. Chemicals used were annexin-V

(BioLegend), distilled water, DMSO (Sigma), [3-

(4,5-dimethylthiazole-2-yl)-2,5-diphenyl

tetrazolium bromide] (MTT) (Sigma), propidium

iodide kit (BioLegend).

2.2 Preparation of n-Hexane Fraction

(nHF)

The air-dried and powdered herbs of Picria fel-

terrae Lour. (1 kg) were repeatedly fractionated by

cold maceration with n-hexane (3x3 d, 7.5 L) at

room temperature with occasional stirring. The

filtrate was collected, and then evaporated under

reduced pressure to give a viscous fraction and

then freeze dried to dry (Satria, et al., 2015;

Anggraeni, et al., 2015; Hasibuan, et al., 2015).

2.3 Cytotoxicity Assay

The cells were treated with nHF. In this test, T47D

cell line was grown in RPMI 1640 medium,

medium containing 10% Fetal Bovine Serum

(Gibco), 1% penicillin-streptomycine (Gibco), and

fungizone 0.5% (Gibco) in a flask in a humidified

atmosphere (5% CO

) at 37

C. The inoculums

seeded at 1x10

cells/mL at an optimal volume of

0.1 mL per well. After 24 h incubation, the

medium was discharged and treated by nHF. After

incubation 24 h, the cells were incubated with 0.5

mg/mL MTT for 4 h in 37

C. Viable cells reacted

with MTT to produce purple formazan crystals.

After 4 h, SDS 10% as stopper (Sigma) in 0.01N

HCl (Merck) was added to dissolve the formazan

crystals. The cells were incubated for 24 h in room

temperature and protected from light. After

incubation, the cells were shaken, and absorbance

was measured using microplate reader at λ 595 nm.

The data which were absorbed from each well

were converted to percentage of viable

cells

(Hasibuan, et al., 2015; Satria, et al., 2014).

2.4 Preparation of Cells for

Flowcytometry Analysis

T47D cells (5x10

cells/well) were seeded into 6-

well plate and incubated for 24 h. After that, the

cells were treated with nHF and then incubated for

24 h. Both floating and adherent cells were

collected in conical tube using tripsin 0.025%. The

cells were washed thrice with cold PBS and

centrifuged 2500 rpm for 5 min. The supernatant

was separated, while the sediment was collected

(Satria, et al., 2015; Anggraeni, et al., 2015).

2.5 Cell Cycle Analysis

Cells were fixed in cold 70% ethanol in PBS at -

C for 2 h. The cells were washed thrice with

cold PBS and resuspended then centrifuged at

3000 rpm for 3 min and PI kit (containing PI 40

µg/mL and RNAse 100 µg/mL) added to sediment

and resuspended and incubated at 37

C for 30 min.

The samples were analyzed using FACScan flow

cytometer. Based on DNA content, the percentage

of cells in each of stage in cell cycle (G1, S and

G2/M) were calculated using ModFit Lt. 3.0.s.

2.6 Apoptosis Analysis

Annexin V kit was added to sediment and

suspended and incubated at 37

C for 30 min. The

samples were analyzed using FACScan flow

cytometer (Harahap, et al., 2018).

2.7 Statistical Analysis

Data were expressed as mean ± SD with

descriptive analysis. All statistics were analyzed

using the SPSS 21 software.

3. RESULTS AND DISCUSSION

3.1 Inhibitory Concentration 50%

(IC

)

MTT method was used to determine cell viability

after incubation for 24 h. In every treatment nHF

was shown to inhibit cells growth. The IC

value

of nHF was 75.87 ± 0.75 µg/mL.The natural

product is suspected to have cytotoxic properties

based on their active compound in Picria fel-terrae

Lour. Triterpenoids/steroids are suspected to be the

main active compound (Yadav, et al., 2012).

Triterpenoids are also considered as one of

promising anticancer drugs (Petronelli, et al.,

2009)

3.2 Effect on Cell Cycle and Apoptosis

To assess the activity of nHF to increase cell death

by increasing cell cycle, we concentrated on it for

further studies using flow cytometry method. The

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

effect of nHF is given in Figure 1. Whereas

treatment of nHF in 15 µg/mL caused cell

accumulation at G

-M phase (37.47%) and for

control cell (30.11%). At S phase the accumulation

after nHF treatment (19.41%) and for control cell

(16.80%). This fact was to indicate that nHF can

inhibit cell grow that G

-M

and S phase. In the cell

cycle analysis, nHF was exhibited higher G

-M

and S phase accumulation compared to control

cells (Harahap, et al., 2018; Satria, 2015).

Figure 1: Cell cycle analysis using flowcytometry. T47D

cells were treated by nHF for 24h and stained using

propidium iodide. (a) control cells; (b) nHF 15 µg/mL.

nHF exhibited G

-M

and S phase.

Evaluation of apoptosis induction was

performed using flowcytometry method with

Annexin-V. as shown in Figure 2.

Figure 2. Apoptosis analysis using flowcytometry. T47D

cells were treated by nHF for 24h and stained using

Annexin-V. (a) control cells; (b) nHF 15 µg/mL.

As shown in Figure 2, the cells in the upper and

lower right quadrants represent late apoptotic/

necrotic and early apoptotic cells, respectively. The

percentage of control and nHF in early apoptotic

0.18% and 24.25%, in late apoptotic/early necrotic

0.06% and 4.26%. In apoptotic study, nHF

increased the cells go through apoptosis in early

apoptosis and late apoptosis if compared to control

T47D cell lines. Apoptosis is a mechanism of

programmed cell death with alterations on

morphology, membrane blebbing and chromatin

(Ruddin,et al., 1997).

4. CONCLUSIONS

The results suggest that n-hexane fraction of

Picria fel-terrae Lour. herbs may exhibit an

anticancer activity towards T47D cell lines

through cell cycle inhibition and induction

apoptosis.

ACKNOWLEDGEMENTS

We gratefully thank to DRPM Ministry of

Research Technology and High Education,

Indonesia through “Hibah Disertasi Doktor”

Research Grant 2018 for financial support in the

study.

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

Antiproliferative and Apoptotic Induction of n-Hexane Fraction of Picria fel-terrae lour. Herbs on T47D Cell Line

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