# br served and documented using phase contrast

served and documented using phase contrast microscope at 100 mag-
3. Results

nification then harvested from monolayer and proceed to cell quanti-

fication using trypan blue dye exclusion assay (Fouz, Amid, and
3.1. The IC50 of IL-GFE against MCF-7 and VERO cell lines

Hashim, 2014). The number of viable cells was calculated as the

equation below:

To investigate the half maximal inhibitory concentration (IC50) of

by plotting graph of the percentage of cell viability (%) versus diﬀerent

where n is the average cell number, 2 is the dilution factor, and 104 is

growth kinetics graph and calculate the cell generation number using
respectively. This result shows that the proliferation of MCF-7 cells has

the following equations:

fi

dramatically and signi cantly inhibited after the addition of IL-GFE in a

dose-dependent manner.

Moreover, MTT assays of IL-GFE against the normal VERO cell lines

(Fig. 3) did not show any IC50 value compared to the tumor cell lines.

Log10 N = Log10 N0 + μt
(4)
This signifies that the IL-GFE is more cytotoxic against cancer cells than

the normal cell lines and will not aﬀect the growth of healthy cells, even

with a high concentration of 100 μg/mL. This result confirms those

μ

obtained in the previous study (Dai et al., 2011). Thus, the Graviola

where X is the number of generations; N is the final cells number; N0 is
fruit serves as is a promising alternative or complementary supplement

towards
reducing breast
cancer
growth generally
and MCF-7

the initial cells number; μ is the specific growth rate (slope); t is the

duration of treatment; td is the doubling time.

Firstly, the flow cytometry analysis was applied to detect changes in VH298 distribution induced by IL-GFE treatment at an IC50 value in a time-dependent manner. In brief, MCF-7 cells (5 × 104 cells/mm) were treated with IL-GFE at 4.75 μg/mL then incubated for 24, 48 and 72 h, respectively. The cells were harvested and washed with 1x phosphate buﬀered saline (PBS), then collected and centrifuged at 1500 rpm for 5 min. After that, cells were fixed in 70% ethanol at 4 °C for 1 h then centrifuged at 4000 rpm for 10 min, at ambient temperature, removing the supernatant and wash twice with PBS. Later, cells were stained with 500 μL of RNase A/PI solution containing 0.05 mg/mL of propidium iodide (PI) and 0.05 mg/mL of RNase A in PBS (Magadi et al., 2015). The percentages of cell cycle distribution of the treated and untreated cells were determined using CytoFLEX S flow cytometry (Beckman

Fig. 1. The percentage of cell viability vs. concentrations of IL-GFE treated on MCF-7 cells.

D. Daddiouaissa, et al.

Fig. 2. The percentage of cell viability vs. concentrations of Taxol treated on MCF-7 cells.

Fig. 3. Graph of the percentage cell viability vs. concentrations of IL-GFE against VERO cell lines.

specifically.

3.2. Eﬀect of IL-GFE on MCF-7 cell growth kinetics

The growth kinetics of untreated (control) and treated MCF-7 with IL-GFE (4.75 μg/mL) and Taxol (0.99 μg/mL) was studied and com-pared. Fig. 4 shows the growth behaviour and the changes of MCF-7 cells after treatment. The number of cells was initially inoculated at 2 × 105 cells/mL. The lag phase was similar for the untreated and the treated MCF-7 cells at first 16 h. Then the cells started proliferation and showed clear log phase after 24 h. However, the treated cells did not show a clear log phase and took 88 h to reach the late log phase. The taxol showed clear log phase but produced less total cell number. It can be observed that the IL-GFE and Taxol treated MCF-7 shows a great reduction in the number of cell proliferation compared to the control. The highest number of cells obtained at the stationary phase for the IL-GFE and Taxol-treated cells were only 6.4 × 105 and 9.1 × 105 cells/ mL, respectively, as compared to the control 26.3 × 105.

Based on the fourth equation that was obtained from the growth curve (Fig. 4), cell growth at exponential phase and death phase (Fig. 5) and the cell generation number (Table 1) were obtained for all treat-ments.

The number of cell generation was reduced from 3.71 generations in the untreated cells to 1.67 generations in IL-GFE treated cells. While Taxol reduced cell generation from 3.71 to 2.18 generations, this output
Journal of Ethnopharmacology 236 (2019) 466–473

Fig. 5. MCF-7 Cells Growth at A: Exponential Phase and B: Death Phase, for untreated MCF-7 and the treated MCF-7 with IL-GFE and Taxol.

is significantly (p ≤ 0.05) deferent when the cell generations number were compared between diﬀerent treatments and the control.

The exemplary photographs of the untreated and the IL-GFE-treated MCF-7 at 24, 72 and 120 h were presented in Fig. 6. The longer the treatment time, the less density of MCF-7 observed. This probably due to the antiproliferative eﬀect of IL-GFE toward MCF-7 cells growth.