Medicinal plant materials were acquired from the wild in Kunming (Yunnan, China) during the summer of 2014 to prepare a phytochemical extract library, which was identified by Dr. Haizhou Li from the Faculty of Life Science and Technology of Kunming University of Science and Technology (Kunming, China). For the preparation of the phytochemical extracts, the plant materials, including branches and leaves, were washed, dried, and finely chopped and grinded. The samples were first extracted with 95% ethanol by an ultrasonic method (15), and were subsequently evaporated using a rotary evaporator (EYELA, Tokyo, Japan). Following this, the dried material was successively extracted using PE, and was subsequently treated with chloroform, ethyl acetate, n-butyl alcohol in a Soxhlet extractor (EYELA). Extracts were filtered and concentrated using a rotary evaporator to evaporate until they were dry. All the dried extracts were weighed and solved with 99.9% (v/v) DMSO (Beyotime Institute of Biotechnology, Haimen, China) to prepare stock solutions at concentration of 100 mg/ml. Subsequently, 100 µl of each phytochemical stock solution was allotted into each well of a 96-well microplate to form a phytochemical extract screening library.

Human non-small cell lung cancer A549 cell line was purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). A549 cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) and 100 U/ml penicillin and streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China), asnd were incubated at 37°C in a humidified incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% CO₂ supplementation.

A549 cells in 100 µl medium were seeded in a 96-well plate at a density of 5×10³ cells/well. Following 24 h, the cells were either treated with phytochemical extracts at different concentrations (3.91, 7.81, 15.63, 31.5, 62.5,125, 250 and 500 µg/ml) for 24, 48 and 72 h, respectively, or treated with 0.5% DMSO as controls. Subsequently, 5 mg/ml MTT (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) solution was added into each well and incubated for 4 h. Following this, the supernatant in each well was discarded and 100 µl DMSO was added. Optical density of each culture was measured at 490 nm using a microplate reader (Infinte-M200 Pro; Thermo Fisher Scientific, Inc.). The percentage of cell growth inhibition was calculated using the following formula: Percentage of cell growth inhibition = (C-T) / C × 100, where C denotes absorbance of control cells and T denotes absorbance of treatment cells. Data were presented in percentages of cell inhibition relative to the control. Percentage of cell growth inhibition was used to determine the IC₅₀ values of the anticancer activity of phytochemical extracts using Probit analysis with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).

A549 cells were plated in 6-well plates at a density of 200 cells/well. Each culture was mixed with a PE extract at concentrations of 0, 16.5, 31.5, 62.5, 125 and 250 µg/ml respectively. Following 12 days of incubation, the cell colonies formed in each well were stained with crystal violet (Beyotime Institute of Biotechnology) after fixation with formaldehyde, and the number of colony formed in each well was manually counted.

Morphology of A549 cells treated with a PE extract concentrations of 62.5 or 31.25 µg/ml, or with 0.5% DMSO control for 72 h was observed under a bright field using an inverted fluorescence microscope (Olympus Corp., Tokyo, Japan) at ×200 magnification.

Briefly, A549 cells were cultured in 24-well plates at 1×10⁴ cells/well and were analyzed following 24-h treatment with PE extract (31.25 and 62.5 µg/ml, respectively). Treated cells were fixed with cold 4.0% formaldehyde for 10 min, washed with phosphate-buffered saline (PBS), and incubated with 10 µM Hoechst 33342 (Sigma-Aldrich; Merck Millipore) at 37°C for 15 min. Subsequently, the cells were washed with PBS and the cell nuclei were observed under a fluorescence microscope (Olympus Corp.).

A549 cells at 50–60% confluence were treated with a phytochemical extract at concentrations of 31.25 and 62.5 µg/ml, respectively, for 24 h. Cells were subsequently harvested by trypsinization and washed twice with PBS. Afterwards, the cells were fixed with cold 70% ethanol for 24 h at 4°C and centrifuged at 1,000 × g for 5 min. Cell pellets were collected and washed with cold PBS. Finally, the cells were suspended in 500 µl staining buffer of 50 µg/ml propidium iodide (PI) with 100 µg/ml RNaseA (Beyotime Institute of Biotechnology), and incubated at 37°C for 30 min in the dark. Cell cycle progression was then analyzed by a flow cytometer (BD Biosciences, San Jose, CA, USA). A minimum of 10,000 cells were used for each assay and DNA content histograms were further analyzed by FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA) for cell cycle analysis.

Prepared A549 cells were cultured in 6-well plates at a density of 5×10⁵ cells/ml and were treated with a PE extract at concentrations of 31.25 and 62.5 µg/ml, respectively, for 24 h. Following treatment, the cells were collected and washed with 1 ml cold PBS, and were resuspended with 250 µl staining buffer (Beyotime Institute of Biotechnology) with Annexin V/fluorescein isothiocyanate (5 µl) and PI (10 µl, 20 µg/ml). Cells were incubated at 37°C in the dark for 15 min. Finally, the stained cells were analyzed using a flow cytometer. Data were analyzed by FlowJo 7.6 software.

All data were presented as the mean ± standard deviation. Student's t-tests were performed to analyze the significant difference between treatment and control data. P<0.05 was considered to indicate a statistically significant difference.

Cytotoxic activities against A549 cell growth following treatment with phytochemical extracts of PE, chloroform, ethyl acetate, and n-butyl alcohol of C. album L. at concentrations of 3.91, 7.81, 15.63, 31.25, 62.5,125, 250 and 500 µg/ml were screened and measured respectively for 72 h. Gemcitabine treatment was used as a positive control for cytotoxicity (Table I). The IC₅₀ values of these extracts of C. album L. toward A549 cell growth were calculated and the PE extract of C. album L. exhibited the strongest cell growth inhibitory effect with the lowest IC₅₀ value of 33.31±2.79 µg/ml among the extracts screened (Table I). Dose effect assays showed the PE extract of C. album L. repressed A549 cell growth in a dose-dependent manner (Fig. 1A). Time effect assays demonstrated that the PE extract of C. album L. inhibited A549 cell growth in a time-dependent manner at the various extract concentrations tested (Fig. 1B). These results demonstrated the PE extract of C. album L. had a potent and specific growth inhibitory effect on A549 cells.

The capability of cell colony formation may represent cell viability after cell inoculation and indicate how cell growth depends on the cell population and the ability of cell propagation. When A549 cells were treated with increasing concentrations of the PE extract of C. album L. from 7.81, 15.63, 31.25 and 62.5 to 125 µg/ml, the number of the cell colonies formed was reduced in a dose-dependent manner (Fig. 2). These results demonstrated that the colony formation and cell propagation abilities of A549 cells were sensitive to the treatment of PE extract of C. album L.

When comparing the morphological properties of control A549 cells treated with 0.5% DMSO and the PE-treated A549 cells, the morphologies of A549 cells treated with the PE extract of C. album L. at concentrations of 31.25 and 62.5 µg/ml for 24 h exhibited apoptotic-associated cellular phenotypes, including cell roundness and shrinkage (Fig. 3). PE extract treatment induced the nuclear compaction of A549 cells, whereas the control cells treated with 0.5% DMSO showed normal nuclear morphology (Fig. 4). These cellular phonotypical results indicated that treatment with the PE extract of C. album L. may have induced A549 cell apoptosis.

To investigate the mechanism of the cell growth inhibitory effect induced by the PE extract on A549, the cell cycle of A549 cells was assessed following treatment with 31.25 and 62.5 µg/ml PE extract for 24 h. The results showed that these phytochemical treatments significantly increased the ratio of the G1 population of the cells (Fig. 5) in a concentration-dependent manner (untreated, 59.37%; 31.25 µg/ml, 69.74%, P<0.01; 62.5 µg/ml, 76.93%, P<0.001).

In addition, the effect of PE extract of C. album L. on cell apoptosis was assessed by measuring the ratio of apoptotic cells in the cell population following different PE extract treatments. PE extract-treated A549 cells were subjected to cell apoptosis analysis using a flow cytometer after the cells were stained by Annexin V-FICT/PI. The results showed that A549 cells treated with either 31.25 or 62.5 µg/ml of the PE extract for 24 h exhibited significant increases in the ratio of apoptotic cells in the cell population (untreated, 0.775%; 31.25 µg/ml, 11.9%, P<0.001; 62.5 µg/ml, 22.3%, P<0.001; Fig. 6). These findings indicated that A549 cell growth inhibition following treatment with the PE extract of C. album L. may be associated with the induction of cell cycle G1 phase arrest and apoptosis.