Indonesian Journal of Pharmacy

VOL 31 (1) 2020: 1–10 | RESEARCH ARTICLE

Inhibition of Cell Cycle and Induction of Apoptosis y Ethanol Leaves

Extract of Chrysanthemum cinerariifolium (Trev.) In T47D Breast
Cancer Cells
Roihatul Mutiah1*, Alfiyah Laily Inayatin1, Rahmi Annisa1, Yen Yen Ari Indrawijaya1, Anik Listiyana2

1. Department of Pharmacy, Faculty of Medical and Health Sciences, Maulana Malik Ibrahim State Islamic
University of Malang Indonesia, East Java Indonesia 65144
2. Department of Medical, Faculty of Medical and Health Sciences, Maulana Malik Ibrahim State Islamic
University of Malang Indonesia, East Java Indonesia 65144

Info Article ABSTRACT

Submitted: 04-10-2019 Chrysanthemum cinerariifolium (C.cinerariifolium) is a plant of the
Revised: 29-02-2020 Asteraceae family, which has been applied by the community as an
Accepted: 20-04-2020 ornamental plant and traditional medicine. In this study, the effect of C.
cinerariifolium leaves extract on inhibition of cell cycle and induction of
*Corresponding author
Roihatul Mutiah
apoptosis in T47D breast cancer cells was tested and compared to the
standard chemotherapy agent. The citotoxic activity of C. cinerariifolium
Email: leaves extract against T47D cancer cells and Vero normal cells was tested by
roiha@farmasi.uin- MTT method. Profile of apoptosis and cell cycle were observed by flow
malang.ac.id cytometry method. Based on chemical compounds profil which is tested
used TLC showed that C.cinerariifolium leaves extracts contained flavonoid
and terpenoid chemical compounds. The result of cytotoxic test showed that
leaves extract of C. cinerariifolium was able to inhibit the growth of T47D
cancer cell at IC50 418.8μg/mL. Doxorubicin, extracted from Streptomyces
peucetius used as treatment in several cancers including breast cancer.
Doxorubicin could inhibit the growth of T47D cancer cells in 115.1μg/mL.
The results of cell cycle analysis showed that the C. cinerariifolium leaves
extract inhibited cell cycle in G0-G1 and S phase, whereas doxorubicin was
able to inhibit cell cycle in G0-G1 phase but experienced cell accumulation in
G2-M phase. The percentage of apoptosis in cycle was showed in M1 (sub
G1) and M5 (multinuclear) phase which treatment of C. cinerariifolium
leaves extract was higher than doxorubicin. Therefore, C. cinerariifolium
leaves extract has potential activity as anticancer agent causes inhibition of
cell cycle and induction apoptosis.
Keywords: Chrysanthemum cinerariifolium, apoptosis, cell cycle, T47D cells

INTRODUCTION
Breast cancer is a malignancy that occurs in breast cancer. However, the use of chemotherapy
cells contained in breast tissue, and both derived drugs is decreased because of the risk of side
from the components of the glands (ductal effects that often occur, such as resistance and
epithelial or globulus) and those derived from cardiomyopathy (Octavia et al., 2012). Therefore,
components other than glands such as tissue to minimize the occurrence of side effects used
nerves in the breast, fat tissue, and blood vessels traditional medicine using herbs. One of them is
(Sari et al., 2017). American Cancer Society chrysanthemum (Chrysanthemum cinerariifolium).
revealed in its latest data that by the year 2013, Crhysanthemum cinerariifolium (C.
every year there are about 39,620 women died cinerariifolium) is a plant of the Asteraceae family
caused by breast cancer (DeSantis et al., 2014). that has been used by the community as an
Breast cancer treatment efforts such as the ornamental plant because of its beautiful flowers.
use of chemotherapy agents, radiation, Various plant organs can utilized as drugs such as
radiotherapy has widely used. Doxorubicin is a antibacterial, anti-inflammatory, allergy, and also
chemotherapy drug widely used in the treatment of anticancer (Grdisa et al., 2009). Based on previous

Indonesian J Pharm 31(1), 2020, 1-10 | DOI: 10.14499/indonesianjpharm31iss1pp1 indonesianjpharm.farmasi.ugm.ac.id 1

Copyright © 2019 THE AUTHOR(S). This article is distributed under a Creative Commons Attribution-ShareAlike 4.0
International (CC BY-SA 4.0)
 Extract of Chrysanthemum cinerariifolium (Trev.) In T47D Breast Cancer Cells

research indicates that the extract of Sciences Maulana Malik Ibrahim State Islamic
Chrysanthemum zawadskii flower and leaves has University of Malang.
pharmacological activity, is as an anti-
inflammatory by inhibition of lipopolysaccharide Sample preparation
on RAW264.7 cell induced by nitrite oxide (Kim et Samples of C. cinerariifolium were harvested
al., 2012). by cutting on the leaves using scissors. Then begins
The previous study showed that by sorting each section, washed, dried under the
Chrysanthemum contain terpenoid compounds, sun, and final sorting. Dry samples are mashed up
flavonoids, and derivatives that are suspected with grinding machines and weighed C.
of having anticancer activity (Ukiya et al., 2002). cinerariifolium leaves powder.
Previous research has also reported that
C. cinerariifolium comprises a quercetin Extraction of C. cinerariifolium
compound (Jeong et al., 2012; Alviana et al., Leaves powder of C. cinerariifolium was put
2016). The quercetin compound is thought to have into the Erlenmeyer flask and 96% ethanol solvent
an anticancer effect through the induction of the was added with a ratio of 1: 20. Then extracted
p21 gene which is a CDK inhibitor, along with a using UAE (Ultrasonication Assisted Extraction) for
decline in Rb gene that inhibits cell cycle in G1 / S 2 min with three replications. The leaves filtrate C.
phase by inhibiting E2F (Yerlikaya et al., 2017). cinerariifolium of the UAE evaporated the solvent
Scientific evidence of anticancer activity of using a rotary evaporator at 50°C temperature to
chrysanthemum plants with C. cinerariifolium produce a crude extract. The sticky extract was
species has not widely performed in both apoptotic concentrated using an oven at 40°C temperature
and cell cycle testing. In this study reported the until the texture of the extract became
effects of C. cinerariifolium extract on apoptosis concentrated. Then calculated extracted yield using
induction and regulation of breast cancer cell cycle the formula:
T47D. Extract weight
Extracted yield = × 100%
Raw material weight
MATERIAL AND METHODS
The materials used in this study were C. Identification of compounds used Thin Layer
cinerariifolium leaves, 96% ethanol, distilled water, Chromatography (TLC)
n-Hexane p. a, ethyl acetate p.a, 10% H2SO4, T47D In the identification of compounds, the silica
cells and Vero cells obtained from Parasitology gel 60 F254 used as a stationary phase with the
Laboratory Faculty of Medicine Gajah Mada n-Hexane p. a (Merck, Berlin, Germany) and
University, Yogyakarta, Complete Medium (CM) ethyl acetate p.a (Merck, Berlin, Germany) (8: 2)
RPMI 1640 (Gibco, Invitrogen Canada), CM MI99 periods of motion. The stain used was 10% H2SO4.
(Gibco, Invitrogen Canada), PBS, Trypsin-EDTA, Identify stain compounds using Thin Layer
DMSO (EMSURE ACS, Japan), SDS (Merck, Berlin Chromatography (TLC) Visualizer.
Germany), doxorubicin HCL 50 mg, MTT solution
(Bio Basic Inc, Canada), trypsin-EDTA 0,25% Sample preparation for anticancer activity and
(Gibco, Invitrogen Canada), RNAse (Gibco, toxicity test
Invitrogen Canada), propidium iodide (Sigma- Leaves extract was weighed as much as
Aldrich, USA), triton-X (pro GC Merck, Berlin, 10mg, dissolved with 100μg/mL DMSO and made
Germany) and Annexin V (Sigma-Aldrich, USA). seven serial concentrations in T47D cells were
1000; 800; 600; 400; 200; 100; 50μg/mL and in
Plant determination Vero cells were 1000; 500; 250; 125; 62,5; 31,25;
C. cinerariifolium (Trev.) plants were 15.625µg/mL. While doxorubicin positive control
obtained from Nongkojajar, Pasuruan, East Java, was made seven serial concentrations, in T47D
Indonesia. Determination of C. cinerariifolium plant cells were 1087.04; 543.52; 271.76; 135.88; 67.94;
was conducted in Materia Medika Integrated 33.97; 16.985µg/mL and in Vero cells were 5435;
Service Unit Batu City, East Java, Indonesia. 2717.60; 1358.80; 679.40; 339.70; 169.85; 84,925
µg/mL.
Ethical approval
This study has received ethical approval No. Anticancer activity and toxicity test
002/EC/KEPK-FKIK/2018 from Medical Research An anticancer activity test conducted in
Ethics Committee of Faculty of Medicine and Health T47D cell culture with RPMI 1640 medium (Gibco,

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Roihatul Mutiah

Invitrogen Canada). Furthermore, toxicity test one and the cells are transferred to the conical.
performed in Vero cell culture used the M199 The wells are added 2mL of PBS to retrieve the
medium (Gibco, Invitrogen Canada). T47D cell remaining cells, then moved into the conical. The
cultures and Vero cells were grown on 96 well conical is centrifuged at 2000 rpm for 5min and the
plates and then incubated for 24h. After 24h the supernatant was removed. The each well was
media was removed and washed used PBS, then washed with 1mL PBS again and moved to the
each concentration of extracts was added into each conical. The conical is resuspended then moved to
well with three replications and incubated for 24h. microtube. The microtubes were recentrifuged at
After 24h the media was removed and washed used 2000rpm for 3min.
PBS, then added 100μL MTT reagents (Bio Basic In cell cycle analysis, the supernatant is
Inc, Canada) to each well, including media control removed by pouring and added 500μL 70% alcohol
(without cells), then re-incubated for 4h in the CO2 into the conical while shaking slowly. The conical is
incubator. kept at room temperature (37ᵒC) for 35min and
After 4h the cell condition was observed recentrifuged at 600rpm for 5min to removed the
under an inverted microscope, then a 100μL SDS added alcohol from the conical. The conical was
10% stopper was added and incubated at room added 500μL of PBS and centrifuged at 2000rpm
temperature overnight. Furthermore the for 3min. The washing was conducted twice used
absorbance value is read using ELISA reader and PBS and the conical is wrapped in aluminum foil
calculated cell viability using the following formula: and marked. For apoptotic analysis, the remained
(Ta – Mca) harvest cells was rinsed used PBS then it was
Viability cell = × 100% centrifuged again and the PBS was removed from
(Cca – Mca) the conical. The conical was added of Propidium
Ta = Treatment absorbance; Mca = Media control Iodide reagent and allowed to stand for 30min. The
absorbance; Cca = Control cell absorbance sludge was added PI-Annexin V reagents carefully
and immediately homogenized. The microtube
The result of viability cell obtained by IC50 containing the cell suspensions is wrapped in
analysis for anticancer activity and CC50 analysis for aluminum foil and incubated in a 37C water bath
normal cell toxicity using Microsoft Excel (Muti'ah, for 5min. The cells suspension is homogenized and
2017). transferred into a flow cytometer tube using a
nylon filter to test for cell cycle and cell apoptosis,
Flowcytometry test then ready for analysis with a flow cytometer
The cultured T47D cells as much as 5x105 (CCRC, 2009).
cells/well were grown in RPMI medium at 6-well
plate (for treatment and control cells) then RESULTS AND DISCUSSION
incubated for 24h. The cell condition was observed Identification of C. cinerariifolium leaves
in the microscope to see the cell distribution. extract chemical compound
Furthermore, the concentration of the sample and Chemical compounds identification was
doxorubicin was made to the level of IC50. The 6- conducted to analyze qualitative content and
well plate that already contain cells taken from the expect phenolic and terpenoid compounds before
incubator. Then, the media cell was removed by the cytotoxic and apoptosis to be tested. Thin
using a Pasteur pipette slowly and washed with Layer Chromatography (TLC) is a physicochemical
PBS. Furthermore, the treatment conducted by separation method based on two phases, which is a
inserting 2mL extract samples at the first well (for fluid phase as a mobile phase and a solid phase as a
cell cycle), 2mL doxorubicin at the second well, and stationary phase (Muti’ah et al., 2013). Based on the
2mL cell control at the third well and incubated for optimization results showed that the best mobile
24h. One conical was prepared for one type of phase were n-Hexane and ethyl acetate (8:2) as
treatment or one well. The medium is taken 1mL solvents. After elution and air drying of the plate,
from the well with micropipette and transferred to natural product reagent was sprayed using 10%
the conical. To the each well, 1mL PBL was added H2SO4 as universal staining. Furthermore, the plate
1mL PBS and moved into the conical. The conical was compared before and after to detection and
was added 200μL of trypsin-EDTA 0.25% and observed the spots characteristic. The result of TLC
incubated for 3min. Furthermore, the wells were visualizer identification used UV 366 rays shows
added 1mL of control media into each well and the difference of compound separation between
resuspension until the cells disenganged one by before and after spray (Figure 1).

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 Extract of Chrysanthemum cinerariifolium (Trev.) In T47D Breast Cancer Cells

Figure 1. Thin-layer chromatography (TLC) plates of C. cinerariifolium leaves extract stained with 10%
H2SO4 and visualized under TLC visualizer. A.) Before sprayed under UV 254 nm rays, B.) Before sprayed
under UV 366 nm rays, C.) After sprayed under white light.

Figure 2. Comparison of inhibitory effect of T47D cell growth and Vero cell after treated with extract C.
Cinerariifolium and Doxorubicin after 24 hours incubation; (A) Control cells; (B) 135.88 µg/ml Doxorubicin
as positive controls; (C) Treated cells used 600 µg/ml leaves extract. Cell morphology was observed under
an inverted microscope with magnification 400 times. Alive cells () and dead cells (- ->). Each treatment
is repeated three times as triplicate.

It seen in the Rf results obtained, the Rf wavelength 341-389nm after being sprayed
compounds on the TLC plate after spraying more showed the flavonol group compound, and the
than the Rf value on the TLC plate before spraying. purple, red color showed terpenoid group
The number of Rf TLC plate on the leaves extract compounds (Harborne, 1987). Meanwhile, purple
after sprayed is 8.0. The yellow color with

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Figure 3. The viability cell using MTT method: Percentage graphic of cell viability in T47D cells after
treatment of C. cinerariifolium leaves extract (left) and doxorubicin treatment (right). Percentage graphic
of cell viability of Vero cells after treatment of C. cinerariifolium leaves extract (left) and doxorubicin
treatment (right). This data is repeated three times as triplicate and point of each graphic represented the
average concentrations.

stains are suspected as sesquiterpenes compounds viable Vero cells was polygonal and flat (Goncalves,
(Mutiah et al., 2013). Compounds of flavonoids and 2012). The treatment of C. cinerariifolium leaves
terpenoid groups play a major role in health, one of extract possesed cytotoxic activity against human
which is having anticancer activity in breast cancer cancer cell lines T47D but does exert damage to
(Bishayee et al., 2011; Weeb and Ebeler, 2004). normal vero cells. To determine the viability of
cancer cells due to the treatment of C.
Cytotoxic test of C. cinerariifolium leaves extract cinerariifolium extract, then tested the cytotoxicity
The anticancer activity test of C. of T47D cells and Vero cells used the MTT method.
cinerariifolium leaves extracts known by The intensity of the purple color that formed is
decreasing living cells percentage based on proportional to the number of living cells (Doyle
50% Inhibitor Concentration (IC50) value. Cell and Griffiths, 2000). The higher the intensity of the
morphological observations conducted under an purple color indicates a more significant amount of
inverted microscope after treatment on cells with living cells (CCRC, 2009). Percentage of cell viability
each extract. Comparison of the inhibitory effect of in T47D and Vero cells due to the treatment of
T47D cell growth and Vero cell after treated with doxorubicin and leaves extract (Figure 3).
extract C. cinerariifolium (Figure 2). Morphological
change between T47D control cells using the Cell cycle due to treatment of C. cinerariifolium
treatment of C. cinerariifolium leaves extract and leaves extract
doxorubicin positive control (Figure 2). The form of Inhibition in the phase of the cell cycle
viable T47D cells was elongated, while the dead that observed was conducted using flow
T47D cell was shaped rounded shrink (Iin et al., cytometry method. The flow cytometry
2014). In Vero cells, there was no apparent cell method was a method that can detect every phase
death due to the treatment of C. cinerariifolium in the cell cycle based on the number of
leaves extract compared to control. Morphology of chromosomes on each phase (G1, S, and G2/M).

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 Extract of Chrysanthemum cinerariifolium (Trev.) In T47D Breast Cancer Cells

Figure 4. The results of flowcytometry analysis of cell distribution at each phase of cell cycle, A.) control
cell, B.) treatment of doxorubicin against T47D cells, C.) treatment of Chrysanthemum cinerariifolium leaves
extract on T47D cells D.) graphic of cell cycle distribution.

Through this flow cytometry (Figure 4) method, the obtained from cells that emit epi-fluorescence due
distribution of cells at each phase in the cell cycle to Annexin V or PI bonds which are then captured
after treatment could be known. Furthermore, the by UV rays (Indradmojo, 2015).
pathway inhibition of C. cinerariifolium leaves Anticancer activity on T47D and Vero cell
extracts in blocking the cell cycle could be and the effects of C. cinerariifolium leaves extract
estimated. on apoptosis induction and regulation of breast
cancer cycle T47D conducted as the purpose of this
Apoptosis induction after treating of C. study. IC50 result obtained from C. cinerariifolium
cinerariifolium leaves extract leaves extract was 418.8μg/mL, while IC50
In flow cytometry test of C. cinerariifolium doxorubicin result was 115.1μg/mL (Figure 3).
leaves extracted, the IC50 was 418.8μg/mL and The results of IC50 obtained showed that 96%
compared with doxorubicin at IC50 115.1μg/mL. ethanol leaves extract of C. cinerariifolium to
The results of the apoptotic flowcytometry test have anticancer activity against breast cancer
against T47D cells (Figure 5). The colors formed in (T47D). An extract has high anticancer activity
the cell dispersion data were analyzed using a Cell if IC50 <500μg/mL and has weak activity if
Quest program so that the colors formed can be IC50> 500μg/mL (Costa et al., 2017). The IC50
separated according to the population. Living cells result in positive control equal to 115.1μg/mL and
indicated by green, early apoptotic cells exhibited these result obtained was close to IC50 researchers
by yellow, late apoptosis indicated by pink, that IC50 doxorubicin value against T47D cells is
and necrosis indicated by red. The resulting colors 250nM or 135.9μg/mL (Abdolmohammadi, 2008).

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Roihatul Mutiah

Figure 5. Result of flowcytometry of apoptotic test against T47D cell A.) control cell, B.) treatment used
doxorubicin, C.) C. cinerariifolium leaves extract, D.) Comparison graphic of T47D cell population using
LSD analysis.

Doxorubicin has a low IC50 result because it has viability to 77.78%, an increase in apoptosis by
high activity against breast cancer cells (Anjarsari, 17.13%, and an increase in the number of necrosis
2013). The results of cytotoxic activity test on by 5.18% when compared with control cells.
Vero cell (Figure 3) that obtained from IC50 showed However, based on the percentage of cell viability
that 96% ethanol leaves extract of C. cinerariifolium when compared with the rate of apoptosis and
has low toxicity to normal cells. The result of necrosis cells, the percentage of living cells still
IC50 leaves extract of C. cinerariifolium in Vero cell more significant than dead cells. It might because
was 676.182μg/mL, while IC50 doxorubicin doxorubicin was able to increase the activity of
value was 1234.5μg/mL. The low toxicity of phosphorylation of P13K/ Akt then activate Bcl
normal cells in vitro tests correlates with high protein which is an antiapoptosis protein and could
levels of safety against normal cells (Mutiah et al., activate Bad protein which is a protein trigger
2017). apoptosis (Setiawati, 2011).
Percentage analysis of the viability of In the treatment of ethanol leaves extract
living cells, apoptosis, and necrosis showed of C. cinerariifolium showed that T47D cell
untreated cells (cell control) had a cell viability apoptosis induction was 49.88% and necrosis
percentage of 94.47%, 3% apoptotic cells, was 47.29%, and the living cell was 2.83%
and 1.82% necrosis cells (Figure 5). While (Figure 4). The result of statistical analysis of
doxorubicin-treated cells showed a decrease in cell three treatments showed a significant difference

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 Extract of Chrysanthemum cinerariifolium (Trev.) In T47D Breast Cancer Cells

between apoptosis percentage on cell control, after apoptosis at IC50 418.8μg/mL concentration, and
treatment of doxorubicin, and after treatment of could inhibit cell cycle in phase G0-G1 and S phase
C.cinerariifolium leaves extract with the and increased cell number in phase M1. The
considerable p-value (p<0.01). The results of the flavonoid compounds that content in C.
statistical analysis of the percentage of cell necrosis cinerariifolium supposed could induce apoptosis
also showed similar results. The flow cytometry through p53 pathways. The ability of C.
analysis conducted used cell quest program (Figure cinerariifolium ethanol leaves extracts to induce
5). Cell distribution in each phase of the cell cycle apoptosis to suggest that it could be a new
was colored used PI reagents as it was able to candidate of anticancer therapy.
interact with DNA (Putri, 2014). Based on Figure 5
there was changed in the cell cycle between the C. ACKNOWLEDGEMENT
cinerariifolium leaves extract and control cells that This work supported by The Directorate
indicated by the reduction in cell numbers in the General of Islamic Higher Education (DIKTIS) of
G0-G1 phase and S phase. These reductions meant Interdisciplinary Basic Research Grant numbers
the cessation of the cell. While in phase M1, the 3209/Un.3/HK.00.5/05/2018.
control cell was increased which indicates the
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Compositae plants III. Anti-tumor promoting

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effects and cytotoxic activity against human Compounds: Structural Determinates of

cancer cell lines of triterpene diols and triols Activity. Biochem J. 527-541.
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Letters, 177(1):7-12 2017. Breast Cancer and Flavonoids as
Weeb MR, Ebeler SE. 2004. Comparative Analysis of Treatment Strategy. From Biology to
Topoisomerase IB Inhibition and DNA Medicine, 2:305-326.
Intercalation by Flavonoids and Similiar

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