## 2 Experimental
### In Vitro Cytotoxicity
#### 2.3.3 MTT Assay on A549 Cells and Morphological Changes  
This assay is a colorimetric technique to determine the viable cell counts, which is based on the reduction of MTT by the cells to produce a soluble product (formazan, soluble in the DMEM and detected through UV spectrophotometry). Through predominantly mitochondria-mediated apoptosis, this assay approximates the viability of the cells. Here, it was applied to determine the anticancer potential of F1 and F3 in comparison to GEF pure. Around 150 \(\upmu\)L cultured A549 cells (\(\approx\)3 \(\times\) 10\({}^{3}\) cells/well density) from the exponential growth phase was seeded in 96-well flat-bottomed plates (Corning Inc., New York, NY, USA) and left untouched for 24 h. Thereafter, the cells in the wells were washed two times with phosphate-buffered saline (PBS) and replaced with DMEM medium (supplemented by 10% FBS) with varying concentrations of GEF pure, F1 and F3. The control wells where only DMEM medium (with drug) was added were maintained to check the survival and viable cells in the culture medium.  
The treated and untreated (control) cells were then incubated at 37 \({}^{\circ}\)C in CO\({}_{2}\) incubator for 72 h. After incubation, 10 \(\upmu\)L of MTT (5 mg/mL) was added into each well and incubated further at 37 \({}^{\circ}\)C for 4 h. The viable cells reduced the MTT (yellow color) to formazan crystals (dark color). The medium was pipetted out carefully from each well to avoid disruption of the monolayer and discarded. Then, DMSO (150 \(\upmu\)L) was added into the wells and the plate was allowed to be shaken slowly at ambient temperature for 10 min to dissolve the formazan crystals [31]. The optical density (OD) of the appeared dark color was measured using a Synergy HT spectrophotometric microplate reader (BioTek Instruments, Winooski, VT, USA) at 570 nm [30; 31; 32; 33]. Each concentration was poured in six wells and the experiment was performed in triplicate. The proliferation or relative cell viability (%) was articulated as percentage of growth in comparison to the control (untreated cells) by Equation (4):  
\[\textit{Cell Viability}\left(\%\right)=\frac{\left(\text{OD of GEF test samples}-\text{OD of blank}\right)}{\left(\text{OD of control}-\text{OD of blank}\right)}\times 100 \tag{4}\]  
The data were plotted as percentage cell viability versus log concentration of GEF, normalized relative to the viable control cells (assuming as 100%). The IC\({}_{50}\) values of each GEF product were determined against the A549 cells. With the aid of GraphPad Prism-5 (GraphPad Software, San Diego, CA, USA), the data were represented graphically. The curves were fitted by non-linear regression model symbolized as \"\(Y\)\" as expressed in Equation (5) with the sigmoidal dose responses.  
\[\textit{Regressed equation}\left(Y\right)=\frac{100}{\left(1+10(\left(\text{ LogIC}_{50}-X\right)\times\text{HillSlope}\right)\right)} \tag{5}\]  
The phase contrast inverted microscope (Olympus CLX 41) was used to observe the morphological changes in the treated A549 cells. The A549 cells (\(\approx\)3 \(\times\) 10\({}^{3}\) cells/well density) per well were seeded in 24-well plates with DMEM and incubated for 24 h at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\) supply. Thereafter, cells were treated with the GEF formulations (considering 3.135 \(\upmu\)M GEF concentration for each). All the treated and untreated cells were incubated for 72 h at above-mentioned conditions. After 72 h of incubation, microscopic observation was done and the photographs were captured to see any apoptotic morphological changes [31; 32].