Collection of plant samples
P. lanceolata leaves were collected from a Usefia region south Baghdad at middle of Iraq. The plants were confirmed to be uninfected and healthy, and authenticated by the National Herbarium of Iraq who undertook the formal identification of the plant material used in our study, and a specimen of this material has been deposited in a National Herbarium of Iraq under the number (56432). The leaves were washed by clean tap water and distilled water to eliminate dust and other foreign particles, air-dried at room temperature, ground in a blender, and then weighed.
Plant extraction
Plant leaves (100 g) were ground into fine powder using a stainless-steel grinder and then extracted in 70% ethanol (200 ml) overnight. The fraction extracted by ethanol was isolated by using a muslin cloth and sterile filtered through a Whatman filter (No.02) paper. The filtered extract was then concentrated in a rotary evaporator.
Analysis of phenol and flavonoid content in P. lanceolata leaf extract
Detection of flavonoid was performed using a high-performance liquid chromatography (HPLC) system from Shimadzu, Japan, following previously described methods [19] at the Department of Chemistry, Ministry of Science and Technology. The column used was Shimpack C-18 (particle size of 3 μm; 50 × 4.6 mm 1.D). The mobile phase was 0.1% phosphoric acid-acetonitrile (52:24, v\v). UV detection was set at 285 nm. The flow rate was 1.5 ml/min, whereas the temperature was 25 °C. The concentration of each component was measured quantitatively by comparing the detected peak area of the samples with that of a standard according to the following equation:
$$ \mathrm{Sample}\ \mathrm{concentration}=\frac{\mathrm{area}\ \mathrm{of}\ \mathrm{the}\ \mathrm{sample}}{\mathrm{area}\ \mathrm{of}\ \mathrm{the}\ \mathrm{Standard}}\times \mathrm{standard}\ \mathrm{concentration}\times \mathrm{dilution}\ \mathrm{factor} $$
Maintenance of cell cultures
The human breast cancer cell lines AMJ13 [20], MCF7, MDAMB, and CAL51, as well as mouse embryo fibroblasts (MEF) were supplied by Cell Bank Unit. AMJ13 cells were cultured in RPMI-1640 medium (USbiological, USA) supplemented with 10% fetal bovine serum (FBS) (Capricorn Scientific, Germany), 100 units/mL penicillin, and 100 μg/mL streptomycin. MCF7, MDAB, and CAL51 cells were cultured in Minimum Essential medium (MEM) (USbiological) supplemented with 10% FBS (Capricorn- Scientific, Germany), 100 μg/mL streptomycin, and 100 units/mL penicillin. The cells were incubated at 37 °C in a humidified environment and 5% CO2.
Cytotoxicity assays
Crystal violet cell viability assay was employed to measure the cytotoxic effect of the plant extract. Human breast cancer cell lines (MDAMB, AMJ13, MCF7, and CAL51 cells) and normal mouse embryonic cells (MEF) were seeded at a density of 7000 cells/well in 96-well plates (Santa Cruz Biotechnology, USA). After 24 h of incubation or after a confluent monolayer was formed, the cells were treated with P. lanceolata leaf extract at 2-fold dilutions (4000, 2000, 1000, 500, 250, 125, 62.5, 31.25 μg/ml to 15 μg/ml) in culture media. The assay was performed in triplicate. Cell viability was determined after 72 h of treatment by cell staining with 50 μl of crystal violet (Sigma Aldrich, USA) followed by incubation at 37 °C for 2 h. The stain was aspirated, and PBS was used to wash the wells. A microplate reader (Biochrom, UK) was used to measure the absorbance at 492 nm. Results of the assay were shown as percentage of proliferation relative to control cells [21].
Morphology and quantitative image analyses
The treated and untreated cells were photographed at four haphazardly selected cultured fields using an inverted light microscope at × 200 magnification (Leica Microsystems, Germany) and digital color camera (Leica-microsystems, Germany). The images were examined using the ImageJ software (http://rsb.info.nih.gov/ij/). For statistical analysis, quantitative measurement of each picture was performed in triplicate. Percentage (%) of cells stained by crystal violet were calculated [22].
Clonogenic assay
The cells were seeded at a density of 5 × 105 cells/mL in 6-well tissue culture plates and incubated until confluency. Next, the cells were exposed to P. lanceolata leaf extract at different concentrations. The cells were then stained with crystal violet stain and examined under an inverted microscope [23].
Statistical analysis
The data are presented as means ± standard error of the mean. One-way analysis of variance was used for data comparison between treatment groups. Differences in data were considered statistically significant at P < 0.05. For this analysis, we used the GraphPad Prism 6 software (GraphPad Software, Inc. San Diego, California).