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, Indonesia

Keywords: Alkaloid Fractions, Cell cycle, HeLa, Litsea Cubeba Lour. Heartwood.

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 IC50 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 G2-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

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: 10.5220/0009844000002406

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

ISBN: 978-989-758-347-6

al., 2017; Dalimunthe, et al., 2018). The aim of this

study was 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%

) at 37

C. The inoculums seeded at 1 x

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 37

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).

2.5 Statistical Analysis

The results were presented as means ± SD.

2.5.1 Results

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.

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

Table 1. IC

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

3 times of replication)

Treatment

(µg/mL)

Ethanol Extract

156.24 ± 2.96

n-hexane Fraction

67.23 ± 0.63

Chloroform Fraction pH 3

175.92 ± 2.40

Chloroform Fraction pH 7

52.46 ± 0.34

Chloroform Fraction pH 9

94.81 ± 2.16

Cisplatin

24.01 ± 0.31

2.4 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 2: 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

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

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

3 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 alkaloids (+)-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

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.

REFERENCES

Berrington D and Lall N (2012). Anticancer activity

of certain herbs and spices on the cervical

epithelial carcinoma (HeLa) cell line. eCAM,

Article ID 564927.

Burgeiro Z, Gajate C, Dakir EH, Villa-Pulgar´ın JA,

Oliveira PJ. (2011). Involvement of

mitochondrial and B-RAF/ERK signaling

pathways in berberine-induced apoptosis in

human melanoma cells. Anti-Cancer Drugs,

22(6), 507–18.

Dalimunthe A, Achmad S, Satria D. (2016).

Phenolic, flavonoid content and antioxidant

activities of ethylacetate extract of litsea

cubeba (lour.) pers. barks. Der Pharma

Chemica, 8, 466-468.

Dalimunthe A, Hasibuan PAZ, Satria D. (2017).

Cell cycle arrest activity of Litsea cubeba

Lout. heartwood and fruit extracts against

T47D breast cancer cells. Asian J Pharm Clin

Resc, 10(11), 404-6.

Dalimunthe A, Hasibuan PAZ, Silalahi J, Sinaga

SF, Satria D. (2018). Antioxidant activity of

alkaloid compounds from Litsea cubeba Lour.

Orient J Chem, 34(2), 1149-52.

Eom KS, Kim HJ, So HS, Park R, Kim TY.

(2010). Berberine-induced apoptosis in human

glioblastoma T98G Cells Is mediated by

endoplasmic reticulum stress accompanying

reactive oxygen species and mitochondrial

dysfunction. Biol Pharm Bull, 33(10), 1644–9.

Feng T, Rong-Ting Z, Qin-Gang T, Xiang-Yun Z.

(2009). Two new isoquinoline alkaloids from

Litsea cubeba. Z Naturforsch, 64, 871-4.

Harahap U, Hasibuan PAZ, Sitorus P, Arfian N,

Satria D. (2018). Antimigration activity of an

ethylacetate fraction of Zanthoxylum

acanthopodium DC. fruits in 4T1 breast cancer

cells. Asian Pac J Cancer Prev, 19(2), 565-9.

Hasibuan PAZ, Harahap U, Sitorus P, Satria D

(2016). Ethylacetate extract of Zanthoxylum

acanthopodium DC. fruit against doxorubicin-

resistanced T47D cells. Der Pharma Chemica,

8(20), 172-174.

Hasibuan PAZ, Jessy C, Denny S (2015).

Combination effect of ethylacetate extracts of

Plectranthus ambonicius (Lour.) Spreng. with

doxorubicin againts T47D breast cancer cells. Int

J Pharm Pharm Sci, 7, 155-9.

Ho YT, Yang JS, Li TC. (2009). Berberine

suppresses in vitro migration and invasion of

human SCC-4 tongue squamous cancer cells

through the inhibitions of FAK, IKK, NF-κB, u-

PA and MMP-2 and -9. Cancer Letters, 279(2),

155–62.

Huang YC, Guh JH, Teng CM. (2004). Induction of

mitotic arrest and apoptosis by evodiamine in

human leukemic Tlymphocytes. Life Sciences,

75(1), 35-49.

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

Jemal A, Siegel R, Xu J, Ward E (2010). CA Cancer

J Clin,

Kan SF, Huang WJ, Lin LC, Wang PS. (2004).

Inhibitory effects of evodiamine on the growth

of human prostate cancer cell line LNCaP. Int J

Canc, 110(5), 641–51.

Liao CH, Pan SL, Guh JH. (2005). Antitumor

mechanism of evodiamine, a constituent from

Chinese herb Evodiae fructus, in human

multiple-drug resistant breast cancer NCI/ADR-

RES cells in vitro and in vivo. Carcinogenesis,

26(5), 968–75.

Nurrochmad A, Lukitaningsih E, Meiyanto E (2011).

Anti cancer activity of rodent tuber (Thyphonium

flagelliforme (lodd.) Blume on human breast

cancer t47d cells. International Journal of

Phytomedicine, 3, 138-146.

Piyapat T, Miyagawa K, Tamura H. (2013).

Induction of apoptosis in HeLa cells by methanol

extract of Litsea cubeba fruit residue from

essential oil extraction. J Life Sci. 7(9), 928-34.

Rosidah, Hasibuan PAZ, Haro G, Puteri M, Satria

D. (2018). Antioxidant activity of alkaloid

fractions of Zanthoxylum acanthopodium DC.

fruits with 1,1-diphenyl-2-picrylhydrazyl

assay. Asian J Pharm Clin Res, 11(1), 33-4.

Satria D, Furqan M, Hadisahputra S, Rosidah

(2015). Combinational effects of ethylacetate

extract of Picria fel-terrae Lour. and

doxorubicin on T47D breast cancer cells. Int J

Pharm Pharm Sci, 7, 73-6.

Satria D, Silalahi J, Haro G, Ilyas S, Hasibuan

PAZ. (2017). Antioxidant and antiproliferative

activities of an ethylacetate fraction of Picria

fel-terrae Lour. herbs. Asian Pac J Cancer

Prev, 18(2), 399-403.

Sharma J, Pitchaiah G, Satyawati D, Rao JV,

Kumar-Vikram HS (2011). In vitro anticancer

activity of methanolic extract of roots of

Glochidion zeylanicum Gaertn. Int J Res

Pharmaceut Biomed Sci, 2, 760-764.

Sun Y, Xun K, Wang Y, Chen X. (2009). A

systematic review of the anticancer properties of

berberine, a natural product from Chinese herbs.

Anti-Cancer Drugs, 20(9), 757–69.

Tang F, Wang D, Duan C. (2009). Berberine inhibits

metastasis of nasopharyngeal carcinoma 5-8F

cells by targeting rho kinase-mediated ezrin

phosphorylation at threonine 567. J Bio Chem,

284(40), 27456 – 66.

Trisonthi P, Akihiko S, Hisashi N, Hirotoshi T.

(2014). A new diterpene from Litsea cubeba

fruits: structure elucidation and capability to

induce apoptosis in HeLa cells. Molecules, 19,

6838-50.

Tyagi AK, Agarwal C, Chan DCF, Agarwal R

(2004). Oncology Reports, 11, 493-499.

Yadav VR, Sahdeo P, Bokyung S, Ramaswamy K,

Bharat BA (2010). Targetting inflammatory

pathways by triterpenoids for prevention and

treatment of cancer. Toxins, 2, 2428-66.

Zihlif M, Afifi F, Abu-Dahab R, Majid A, Somrain

H, Saleh M (2013). The antiangiogenic activities

of ethanolic crude extracts of four Salvia species.

Complementary & Alternative Medicine, 13,

358-65.

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