Cell Cycle Arrest Activity of Alkaloid Fraction of

Litsea cubeba Lour. Heartwoods towards HeLa Cancer Cell

Aminah Dalimunthe

, Poppy Anjelisa Zaitun Hasibuan

, Denny Satria

Department of Pharmacology,

Department of Pharmaceutical Biology

Faculty of Pharmacy, University of Sumatera Utara, Medan, 20155

*Presenting author

Keywords: Cell cycle, Litsea cubeba Lour. heartwood, alkaloid fractions, HeLa.

Abstract: Cervical cancer therapy with chemotherapeutic agents is limited because of drug resistance problem and

toxic effect on normal tissue leads to immunosuppression and cardiotoxicity. This study was to investigated

cell cycle arrest activity towards HeLa cell lines of Litsea cubeba Lour. heartwood alkaloid fraction. Litsea

cubeba Lour. heartwood powder was extracted by maceration method with ethanol 96% and fractionated

with n-hexane and chloroform at pH 3,7 and 9. The cytotoxic study was using MTT method and analysis cell

cycle was using flow cytometry method. The IC

of ethanol extract, n-hexane and chloroform fractions at

pH 3,7 and 9 at were 156.24 ± 2.96; 67.23 ± 0.63; 175.92 ± 2.40; 52.46 ± 0.34; and 94.81 ± 2.16 µg/mL

respectively. The chloroform fractions at pH 7 concentration 25 and 10 µg/mL were caused accumulation in

-M phase (33.84 and 29.08%). The results reveal that Litsea cubeba Lour. heartwood alkaloid fraction

provides effective as cell cycle arrest. Our further study is to assess the mechanism of alkaloid fraction in

inhibit metastasis in cervical cancer.

1 INTRODUCTION

Cancer is one of the high incidence dangerous

diseases in human and presently there is a

considerable number of new anticancer agents from

natural products (Sharma, et al., 2011). According to

WHO data, cancer is one of the leading cause of

death worldwide especially cervical cancer

(Berrington and Lall, 2012). Cervical cancer

theraphy with chemotherapeutic agents is limited

cause of drug resistance and toxic side effect on

normal tissue leads to some effects such as

immunosuppression and cardiotoxicity (Jemal, et al.,

2010; Tyagi, et al., 2004).

Attarasa (Litsea cubeba (Lour,) is a plant from

Lauraceae family which contain many essential oils

which used as antideppressants, antiinflammation,

antioxidant, pesticide, antimicrobial, anticancer on

breast cancer and neuro pharmacology. The

methanol extract from attarasa fruits showed to be

active on HeLa cell lines which cause apoptosis

through activation of caspase 3/7 (Trisonthi, et al.,

2014; Piyapat, et al., 2013). There are more than

forty isoquinoline alkaloids that contained in Litsea

genus which are active as antibacterial agents

against Staphylococcus aureus (Feng, et al., 2009).

The heartwoods of Litsea cubeba contained high

level of phenolic and flavonoid and found to be

active as antioxidant and has anti breast cancer

activity which causes cell cycle inhibition. Alkaloids

compound which isolated from heartwood have

antioxidant activity with DPPH and ABTS methods

(Dalimunthe, et al., 2016; Dalimunthe, et al., 2017;

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

174

Dalimunthe, A., Zaitun Hasibuan, P. and Satria, D.

Cell Cycle Arrest Activity of Alkaloid Fraction of Litsea cubeba Lour. Heartwoods Towards HeLa Cancer Cell.

DOI: 10.5220/0008359401740177

In Proceedings of BROMO Conference (BROMO 2018), pages 174-177

ISBN: 978-989-758-347-6

to assess cell cycle arrest activity of alkaloid fraction

of Litsea cubeba Lour. heartwoods on HeLa cells.

2 MATERIALS AND METHODS

2.1 Fractions Preparation

Fresh heartwoods of Litsea cubeba Lour. was

collected from Balige subdistrict, Sumatera Utara

province, Indonesia. The air-dried and powdered

heartwoods of Litsea cubeba (Lour,) (1 kg) were

repeatedly macerated with ethanol 96% (3x3 d, 7.5

L), The filtrate was evaporated to give a viscous

extract. Viscous extract was fractionated with n-

hexane and continue with chloroform at pH 3,7 and

9 (Rosidah, et al., 2018; Dalimunthe, et al., 2018;

Satria, et al., 2015).

2.2 Cytotoxicity Assay

Extract and alkaloid fractions were submitted for

cytotoxicity test. In that way, HeLa cell line was

grown in RPMI 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 1 x 10

cells/mL at an optimal

volume of 0.1 mL per well. After 24 h incubation,

the medium was discharged and treated by fractions.

After incubation for 24 h, the cells were incubated

with 0.5 mg/mL MTT for 4 h at 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 and Nurrochmad, et al., 2014).

2.3 Cell Cycle Inhibition Assay

HeLa cells (7.5x10

cells/well) were seeded into 6-

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

cells were treated and then incubated for 24 h. Both

floating and adherent cells were collected in conical

tube using trypsin 0.025%. The cells were washed

thrice with cold PBS and centrifuged at 2500 rpm

for 5 min. The supernatant was separated, while the

sediment was collected and fixed in cold 70%

ethanol in PBS at 4

C for 1 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

C for 30 min. The samples were analyzed using

FACScan flow cytometer. Based on DNA content,

percentage of cells in each of stage in cell cycle (G1,

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

(Harahap, et al., 2018 and Satria, et al., 2017).

2.4 Statistical Analysis

The results were presented as means ± SD.

3 RESULTS

3.1 Inhibitory Concentration 50% (IC

)

MTT method was used to determine cell viability

after incubation for 24 h. In every treatment extract

and alkaloid fractions were shown in Table 1.

Table 1. IC

value of extract and alkaloid fractions of Litsea cubeba heartwood with MTT assay (Mean ± SD, 3 times of

replication)

Treatment IC

(µg/mL)

Ethanol Extract 156.24 ± 2.96

n-hexane Fraction 67.23 ± 0.63

Chloroform Fraction

H 3 175.92 ± 2.40

Chloroform Fraction

H 7 52.46 ± 0.34

Chloroform Fraction

H 9 94.81 ± 2.16

Cisplatin 24.01 ± 0.31

Cell Cycle Arrest Activity of Alkaloid Fraction of Litsea cubeba Lour. Heartwoods Towards HeLa Cancer Cell

175

3.2 Effect on Cell Cycle

To evaluate the effect of chloroform fraction at pH 7

(CF-7) to increase cell death by modulating cell

cycle, we concentrated on it for further studies using

flow cytometry method.

The effect of CF-7 at 25 and 10 µg/mL is given in

Figure 1. Whereas treatment of CF-7 at 25 and 10

µg/mL caused cell accumulation at G

/M phase

(33.84% and 29.08%) and for control cell (17.78%).

Figure 1. Percentage of cell cycle phase of HeLa cells were treated for 24h. (a) Control cell, (b) 25 µg/mL (1/2 IC

), (c) 10

µg/mL (1/5 IC

4 DISCUSSION

The cytotoxicity estimate of herbal is correlated to

content of active compound in these plants including

Litsea cubeba Lour. Alkaloids as major compound

have main role in cytotoxicity effect (Yadav, et al.,

2010). Litsea genus is rich in isoquinoline alkaloids

and for Litsea cubeba Lour has been found two

alkaloid (+)-N-(methoxy-carbonyl) N-

norlauroscholtzine and (+)-N-(methoxy-carbonyl)-

N-norglaucine (Feng, et al., 2009). Alkaloids are the

compound which potentially in inhibits the cancer

proliferation for the example berberine is an

isoquinoline alkaloid which inhibits proliferation of

multiple cancer cell line by inducing cell cycle arrest

at G

or G

/M phases and by apoptosis (Sun, et

al., 2009; Eom, et al., 2010; Burgeiro, et al., 2011).

Inhibition of tumor invasion and metastasis is the

mechanism of action of berberine (Tang, et al.,

2009; Ho, et al., 2009). Evodiamine is a quinolone

alkaloid inhibits topoisomerase enzyme, induces

DNA damage, exhibit G

/M phase arrest (Liao, et

al., 2005; Kan, et al., 2004; Huang, et al., 2004).

ACKNOWLEDGEMENTS

We gratefully thank to Research Center University

of Sumatera Utara through Hibah Talenta ““Hibah

Penelitian Dasar” Research Grant 2018 “No:2590/

UN5.1.R/PPM/2018” for financial support in the

study.

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