Fitri Meiriani - PPT Iccsp

The Effect Of Treatment Of Lime (Citrus Aurantifolia)
Essential Oil On The Type Of T47d Breast Cancer Cells

With Double Staining Method
Fatma Sri Wahyuni, Fitri Meiriani, Dachriyanus, Dira Hefni
Department of Pharmacology and Clinical

Pharmacy,Faculty of Pharmacy, Andalas University
INTRODUCTION
Breast cancer is a malignancy
Cancer is a disease in which the in breast tissue that can
Cancer is still the leading cause
irregularity of the cell cycle in the originate from the ductal
of death in the world
process of division cells normally. epithelium or lobules in the
breast.
One of the characteristics of 2 mechanisms of cell death:

Lime peel essential oil (Citrus cancer cells is to avoid death by • Apoptotis
aurantifolia) showed toxicity to apoptosis. • Necrosis
T47D breast cancer cells with an
IC50 value of 7.89 µg/mL.
The Effect Of Treatment Of Lime (Citrus

Aurantifolia) Essential Oil On The Type Of T47d
Breast Cancer Cells With Double Staining Method
Citrus aurantifolia (Christm.) Swingle Essential oil
The major compounds contained in the

essential oil of lime peel are limonene,
linalool, citronellal, and citronellol.
Plant classification
Kingdom : Plantea
Division : Magnoliophyta
Class : Magnoliposida
Ordo : Sapindales
Family : Rutaceae
Genus : Citrus
Species : Citrus aurantifolia (Christm.) swingle
MATERIALS AND METHODS
Materials
The materials used for cell apoptosis test by double staining method were T47D
breast cancer cells, stock solution of lime peel essential oil (Citrus aurantifolia)
available in the cell culture laboratory of the Faculty of Pharmacy, Andalas
University, dimethyl sulfoxide (DMSO), 70% ethanol, Roswell Park Memorial
Institute (RPMI) medium, Fetal Bovine Serum (FBS), Penicillin Streptomycin,
doxorubicin, Trypsin-EDTA, Phosphate buffered Saline (PBS), acridine orange and
propidium iodide.
T47D Cell Setup
 Taken from Nitrogen Tank (-130°C)

T47D Breast Cancer cell  Warmed in a water bath (37°C, <1 menit)
 Transferred to a conical tube containing 3 ml of RPMI
medium, then homogenized
 Centrifugation at 3000 rpm for 5 minutes

 Discard the supernatant, leaving only the cell pellet
Conical tube
 Add 4 ml of RPMI medium and homogenize
 Transfer to 2 T25 flasks
 Observe the condition of the cells under an inverted

Flask T25 microscope
 Incubation for 24 hours in a 5% CO2 incubator at 37°C
 Observe cell growth with an inverted microscope
 When the number of cells is almost full of the flask, do
a cell harvest
Harvesting of T47D cells and preparation of T47D cells forcell death
mechanism test
Cells that have been

Add 2 ml of RPMI medium to
attached to the bottom of Do trypsination
inactivate trypsin
the flask
The cell pellets Transfer to a conical tube, then

obtained were centrifuge at 3000 rpm for 5 Observe under an inverted
calculated with a minutes microscope
hemacytometer
Prepare coverslips, put them in 3 wells on the well plate 6. Add 3 ml of

complete RPMI medium to each well, incubate in a 5% CO2 incubator
at 37°C for 24 hours so that the cells adhere to the coverslip.
Testing the mechanism of cell death by
double staining method
Take the well plate that Immediately make a test (+) 3 ml of positive control into 1 well
(+) 3 ml of test sample into 1 well
already contains the cells solution at IC50 7.89 µg/mL and
(+) 3 ml negative control containing only cells
and coverslip that has been a positive control doxorubicin and medium
incubated with a concentration of 0.125 Mark the wells, incubate in the incubator for
µg/ml 48 hours
(+) 10 µg/ml orange After incubation is complete, remove the

Take the cover slip, place it on the
acridine reagent well plate from the incubator. Observe the
cover glass
(+) 10 µg/ml propidium
iodide reagent condition of the cells under an inverted
microscope
Observe under an inverted Perform cell morphology observations, cell

microscope with fluorescence calculations and data analysis.Minimum
setting calculation for 200 cells
RESULTS
Description :
a. positive control
doksorubicin group 0,125
µg/ml
b. Negative control group
c. Treatment group
a b withessential oil 0,789
µg/ml
: Living cell
: apoptotic cells
: necrosis cell
c
Percentage of viable cells, apoptosis and necrosis.
Groups Repetation Observation
% viable % apoptotic % necrosis
Positive control 1 17,47 79,85 3,34
2 18,77 78,31 3,71
3 33,48 72,14 3,69
Average ± Deviation 23,24 ± 8,89 76,76 ± 4,08 3,58 ± 0,2
standard
negative control 1 96,7 2,47 0,82
2 97,2 1,77 1,01
3 99,3 0,69 0
standard
Treatment 1 43,13 56,20 0,65
2 53,67 45,32 0,95
3 52,86 45,79 1,34

CONCLUSION
1. The percentage of T47D breast cancer cells that died with the type of apoptotic death by giving
essential oil of lime peel was 49.10% ± 6.1, the percentage of T47D breast cancer cells that died with
the type of necrosis death was 0.98% ± 0, 34 and the percentage of living/viable T47D breast cancer
cells was 49.8% ± 5.86.
2. Administration of lime peel essential oil has an effect on the death of T47D breast cancer cells
with apoptotic type of death with an average percentage of cell death of 49.10% ± 6.1.

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The Effect Of Treatment Of Lime (Citrus Aurantifolia)

Essential Oil On The Type Of T47d Breast Cancer Cells

Department of Pharmacology and Clinical

One of the characteristics of 2 mechanisms of cell death:

The Effect Of Treatment Of Lime (Citrus

The major compounds contained in the

 Taken from Nitrogen Tank (-130°C)

 Centrifugation at 3000 rpm for 5 minutes

 Observe the condition of the cells under an inverted

Cells that have been

The cell pellets Transfer to a conical tube, then

Prepare coverslips, put them in 3 wells on the well plate 6. Add 3 ml of

(+) 10 µg/ml orange After incubation is complete, remove the

Observe under an inverted Perform cell morphology observations, cell

Groups Repetation Observation

% viable % apoptotic % necrosis

Positive control 1 17,47 79,85 3,34

2 18,77 78,31 3,71

3 33,48 72,14 3,69

Average ± Deviation 23,24 ± 8,89 76,76 ± 4,08 3,58 ± 0,2

negative control 1 96,7 2,47 0,82

2 97,2 1,77 1,01

Average ± Deviation 97,73 ± 1,37 1,64 ± 0,89 0,61 ± 0,53

Treatment 1 43,13 56,20 0,65

2 53,67 45,32 0,95

3 52,86 45,79 1,34

Average ± Deviation 49,88 ± 5,86 49,10 ± 6,1 0,98 ± 0,34

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