Antiproliferative Effect of Ethanolic Extract Eugenia Uniflora Lam. Leaves On T47D Cells
Antiproliferative Effect of Ethanolic Extract Eugenia Uniflora Lam. Leaves On T47D Cells
Antiproliferative Effect of Ethanolic Extract Eugenia Uniflora Lam. Leaves On T47D Cells
ISSN: 2088–0197
e-ISSN: 2355-8989
Abstract
Eugenia uniflora Lam. is one of herbal products developed for anticancer. The aim of
the present study was to identify the antiproliferative effect of ethanolic extract of Eugenia
Uniflora Lam. leaves (EEU) on breast cancer cell line T47D. This Research was initiated by
extracting the active contents of Eugenia uniflora Lam. leaves by maceration with ethanol
96%. The extract was then analyzed by thin layer chromatography (TLC). Cytotoxic assay of
EEU was carried out by using MTT assay. Apoptosis phenomenon was observed with double
staining using acridine orange-ethidium bromide. EEU showed cytotoxic effect on T47D cells
with IC50 value of 65 µg/ml. Moreover, EEU 50µg/ml and 100µg/ml induced apoptosis. TLC
examination showed that EEU used in this study contain phenolic, flavonoid, and saponin
compounds which were suggested to be responsible for antiproliferative effect. Further
molecular mechanism underlying EEU antiproliferative effect needs to be done.
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Indones. J. Cancer Chemoprevent., 3(2), 370-375
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Indones. J. Cancer Chemoprevent., 3(2), 370-375
100
80
% viabiity
60
40
20
0
0 100 200 300 400 500 600
concentration (μg/ml)
(E)
Figure 1. Cytotoxic Effect of EEU on T47D cells. T47D cells (1x104 cells/well) were seeded in 96-well
plate and treated with EEU 25, 50, 100, 250, and 500 μg/ml. Cell treated with EEU (A) none
(control); (B) 25 μg/ml; (C) 100 μg/ml; and (D) 500 μg/ml, observed under light microscope
with 400x magnification. Arrows ( ) indicationg viable cells and dotted arrows ( )
indicating death cells. Cells viability was then measured by using MTT assay as described in
the methods and EEU showed cytotoxic effect on T47D cells in dose dependent
phenomenon as shown in graph (E).
Effect of EEU on T47D Cells correlated with cell viability at these times.
Proliferation Cells were treated with EEU 25, 50, and 75
Cell proliferation was also measured µg/ml. Proliferation kinetics showed in graph
by using MTT assay. The absorbance at between incubation time versus cell
certain incubation times was measured and absorbance (Fig.2).
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0.6
0.5 Control
Vehicle
0.4
25 µg/ml
abosrbance
0.3 * 50 µg/ml
75 µg/ml
0.2 * *
*
*
0.1
*
0
0 20 40 60 80
hours
Figure 2. Effect of EEU on T47D Cells Proliferation. T47D cells (1x104 cells/well) were seeded in 96-
well plate and treated with EEU 25, 50, and 75 μg/ml. Cells viability was determined by
using MTT assay and the cells absorbance were measured at 24, 48, and 72 hour of
incubation. Absorbance then were plotted in the graph and analyzed statistically by using
paired sample t-test (p<0.05). (*) indicating significant difference between treated cells and
control cells at the same time of incubation.
Data obtained were then analyzed by regression between times of incubation versus
using paired sample t-test with p<0.05. EEU 25 absorbance (Table I). The higher concentration
g/ml decreased T47D cells proliferation of EEU, the smaller the slope obtained. EEU 75
significantly only at 48 hours of incubation; g/ml gave the smallest slope, meaning the
EEU 50 g/ml decreased cells proliferation highest cells proliferation inhibition. Based on
significantly at 48 and 72 hours of incubation; the data, EEU inhibited cells proliferation in
while EEU 75 g/ml decreased cells dose and time dependent manner. The decrease
proliferation at all incubation time (24, 48, and of cells viability could be attributed with cell
72 hours). Inhibition of cells proliferation also cycle arrest, apoptosis, necrosis, or combination
could be observed from the slope value of linier of them.
Effect of EEU on T47D Cells Apoptosis early apoptotic cells (McGahon et al., 1995).
Double staining using acridine orange- Moreover, EEU 100 µg/ml caused a lot of cells
ethidium bromide was done to evaluate the became orange with fragmented DNA, which
effect of EEU on apoptosis. EEU 50 µg/ml indicating the late apoptotic cells.
caused cells undergoing green with lighter
nuclear (Fig.3). This phenomenon indicated
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Figure 3. Effect of EEU on T47D Cells Apoptosis. T47D cells (5x104) were seeded at coverslip in 24-
wellplate, treated with EEU, and stained with acridine orange-ethidium bromide as
described in methods. Cells were then observed under fluorescent microscope with 100x
magnification. Cells treated with (A) none (control), (B) EEU 50 µg/ml, and (C) EEU 100
µg/ml. White arrows ( ) showed viable cells, yellow arrows ( ) showed early
apoptotic cells, and red arrows ( ) showed late apoptotic cells.
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