The Activity of Alkaloid Fraction of Litsea cubeba Lour. Heartwoods

on Down Regulation Cyclin D1 Expression against HeLa Cell Line

Aminah Dalimunthe

Poppy Anjelisa Zaitun Hasibuan

, Denny Satria

Department of Pharmacology, University of Sumatera Utara, Jl.Tri Darma, Medan, Indonesia

Doctoral Programme, University of Sumatera Utara, Jl.Tri Darma, Medan, Indonesia

Keywords : Anticancer, Litsea Cubeba Lour. heartwood, Alkaloid, Cervic Cancer.

Abstract : Objective: This study was to determined the activity of Litsea cubeba Lour. heartwood alkaloid fraction

on down regulation cyclin D1 expression towards HeLa cell line. Methods: The heartwood powder of

Litsea cubeba Lour. was macerated by ethanol 96% and fractionated with n-hexane and chloroform at pH

3,7 and 9. Antiproliferation study was using MTT assay and cyclin D1 expression analysis was using flow

cytometry method from chloroform fraction at pH 7. Results: The IC

of chloroform fractions at pH 7

was 52.46 ± 0.34 µg/mL. The fractions of chloroform at pH 7 in concentration of 25 and 10 µg/mL were

as antiproliferation assay with viable cells at 53.57 ± 0.28% and 68.43 ± 0.48% respectively after 72 h

incubation and decreased cyclin D1 expression (69.10 and 67.63%). Conclusion: The results reveal that

Litsea cubeba Lour. heartwood alkaloid fractions provide effective as anticancer with mechanism through

down regulation of cyclin D1. Our further study is to isolate alkaloid compounds and assess its moleculer

mechanism.

1 INTRODUCTION

Cancer is a serious condition in human and nowadays

there is a high amount of novel anti-cancer drugs out

of natural subtances (Sharma, 2011). Based on

World Health Organization report, cancer is one of

the high lead of death global especially cervical

cancer

(Berington and Lall 2012). Cervical cancer

treatment with chemotherapy drugs has limited

because of resistance problem and toxic impact on

normal tissue which leads to cardiotoxicity and cause

of immunosuppression (Tyagi, 2004) and (Jemal,

2010)

Attarasa or Litsea cubeba (Lour,) is a plant

which contain volatile oils used as antimicrobial,

antideppresant, antioxidant, anticancer on breast

cancer, pesticide, antiinflammation and neuro

pharmacology. Methanol extract of attrasa fruit of

indicate active on HeLa cell lines (cervical cancer)

promote apoptosis with induction of caspase 3 and 7

(Piyapat, 2013), (Trisonthi, 2014). Litsea genus

contain over than 40 isoquinoline alkaloids in which

are active as antibacterial activity (Feng, 2009). The

Litsea cubeba heartwood is conceived superior

amount of flavonoid, phenolic and active as

antioxidant and has anti breast cancer effect through

cell cylce inhibition. Alkaloids which isolated from

Litsea cubeba heartwood have radical scavenging

activity with ABTS and DPPH (Dalimunthe, 2018),

(Dalimunthe, 2016) and (Dalimunthe, 2017) The aim

of this reseach was to evaluate antiproliferative

activity and cyclin D1xpression of alkaloid fraction

of Attarasa heartwood towards HeLa cells

2 MATERIAL AND METHOD

2.1 Extraction and Fractination

Extract and fractions were obtained as previously

describe (Dalimunthe, 2018), (Hasibuan, 2016),

(Rosidah, 2018) dan (Satria, 2015).

790

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

The Activity of Alkaloid Fraction of Litsea cubeba Lour. Heartwoods on Down Regulation Cyclin D1 Expression against HeLa Cell Line.

DOI: 10.5220/0010091107900793

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

790-793

ISBN: 978-989-758-449-7

2.2 Cytotoxic Activity

The inoculums seeded on a 96 well plate (1 x

cells/mL) and incubated at 37

C for 24 hours.

The medium was discharged and treated with CF-7

and incubation for 24 h, the further procedure were

followed as previously describe (Nurrochmad, 2011),

(Satria, 2017) and (Hasibuan, 2015).

2.3 Antiproliferative Activity

Alkaloid fraction was submitted for antiproliferative

activity. In that way, HeLa cell line (2.5 x

cells/mL) was grown in RPMI complete medium.

After 24; 48 and 72 h treatment, MTT assay was

performed and cell viability was counted to calculate

the antiproliferative activity (Zihlif, 2013), (Harahap,

2018).

2.4 Cyclin D1 Expression

HeLa cells (750.000 cells/well) were seeded into 6-

well plate and incubated for 24 h in incubator CO2

5% . for the treatment , harvested and analysis of cells

with flow cytometer were followed procedure from

(Satria, 2017) with added cylin D1 antibody which

labelled with FITC.

3 RESULTS

3.1 Inhibitory Concentration 50% (IC

)

The result of treatment CF-7 can be seen in the Table

Table 1. IC

value of alkaloid fraction at pH 7 of Litsea

cubeba heartwood with MTT assay

Treatment

(µg/mL)(n=3)

Chloroform Fraction pH 7

(CF-7)

25.88 ± 0.16

3.2 Antiproliferative Activity

To evaluate the effect of CF-7 to decrease the number

of cells by inhibiting cell proliferation. The

percentage of viable cells after treatment with

alkaloid fraction in 25 µg/mL and 10 µg/mL and

incubation for 24, 48 and 72 h (79.08 ± 0.45 and 86.05

± 0.31; 64.24 ± 0.41 and 77.27 ± 0.28; 53.57 ± 0.28

and 68.43 ± 0.48) respectively showed the inhibition

effect of alkaloid fraction towards proliferation of

HeLa cells. The effect of CF-7 is given in Figure 2

Figure 2. Percentage of viable cells of HeLa cells were treated by alkaloid fraction at pH 7 for 24; 48 and 72 h.

100

24 48 72

ViableCells

Hours

12.5µg/mL 5µg/mL

The Activity of Alkaloid Fraction of Litsea cubeba Lour. Heartwoods on Down Regulation Cyclin D1 Expression against HeLa Cell Line

791

3.3 Analysis of Cyclin D1 Expression

Figure 3. Analysis of cyclin D1 with flow cytometry.

HeLa cells were treated by CF-7.(a) control cells

unstaining; (b) control cells; (c) CF-7 25 µg/mL; (c)

CF-7 10 µg/mL.

To evaluate the effect of CF-7 to decrease cyclin

D1 expression, we concentrated on it for further

studies using the flow ytometrymethod. The effect of

CF-7 is given in Figure 3. Whiles treatment of CF-7

in 25 and 10 µg/mL caused cell accumulation in M1

area (14.60% and 13.06%) and for control cell.

4 DISCUSSION

Alkaloids have cytotoxic activity through

various pathway (Sun, 2009). Litsea genus

contain many of isoquinoline alkaloids (Feng,

2009). Alkaloids are the main compound which

potentially in prevents the proliferation of cancer

which inhibits proliferation of multiple cancer

cell line by apoptosis stimulation and inducing

cell cycle arrest at G

or G

/M phases (Tang,

2009), (Burgeiro, 2011) and (Eom, 2010).

Interdiction of invasion and metastasis in tumor

is the one of mechanism of action of isoquinoline

alkaloids in inhibits cancer growth (Ho, 2009)

and (Liao, 2005). The other mechanism of

quinolone alkaloid are induces DNA damage,

inhibits topoisomerase enzyme, exhibit G

phase arrest (Huang, 2004) and (Kans, 2004)

ACKNOWLEDGEMENTS

This research finance was supported by

Universitas Sumatera Utara through “Hibah

Penelitian Dasar 2018”.

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