Materials. Catharanthus Roseus plants were provided from Dharmapuri District. The plants were cleaned with H2O to eliminate dust and unwanted materials and it was grinned with filtered.
Nano-size plant powder preparation. The green synthesis of the plant under the photo-induced method was used for the solvent extraction procedure. About 200 ml of plant dispersion was added in 1000 ml of deionized water for 25 days under light irradiation. The resulted product was dried and annealed at 110 °C for 60 min. Characterizations Techniques: XRD patterns of the samples were monitored on a Bruker D8 Advance powder X-ray diffractometer with Cu-Kα (λ = 1.5406 Å). Elemental compositions were measured by EDX analysis (Hitachi S-4800). The morphology of NPs was ascertained via TEM (JEM-2100F). Fourier transforms infrared (FTIR) spectra of the NPs were recorded using the KBr pellet technique (Bruker, Tensor 27 spectrometer). Particle size and Z-average of samples were characterized using DLS analysis (Horiba). UV-vis Diffuse reflectance spectroscopy (UV-DRS) was obtained with a Perkin Elmer Lambda 25 spectrometer.
GC/MS analysis. GC/MS (Clarus 680 GC) was used to elucidate the existence of active constituents and the chemical composition of CR-NPs. GC/MS was accomplished employing a fused silica column consists of Elite-5MS with 95% dimethylpolysiloxane, 5% biphenyl, and 30 m × 0.25 mm ID × 250μm df. The Helium was used as carrier gas to separate the components with 1 ml/min flow. The operation temperature was maintained at 260 °C during the chromatographic process. The plant material (1μL) was applied into the device and the temperature rate was set at: 60 °C (120 s); 300 °C at the rate of 10 °C min−1; and 300 °C. The mass detector was operated at: 230 °C transfer line temperature; 230 °C ion source temperature; and ionization mode electron impact at 70 eV, a scan time of 0.2 s and scan interval of 0.1 s. The spectrum of the component was compared with the data of the of known component kept in the GC-MS NIST (2008) library.
In vitro cytotoxicity of the CR-NPs Procedure. The MCF 7 cancerous and VERO normal cells were provided from National Centre for Cells Sciences. The cells were supplemented 10% fetal bovine serum (FBS) in Eagle’s MEM. The samples were attained at 37ºC with 5% carbon dioxide and 95% air conditions. The culture medium was checked and maintained frequently as well as replaced twice a week.
Cells treatment protocol. The cultured cells were separated with trypsin ethylene diamine tetra acetic-acid, then viable cells were measured employing a diluted hemo-cytometer possess 5% FBS (1x105 cells/ml). Afterward, a 0.1 ml of cells solutions were added into 96-well plate at a density of 10,000 cells/well. The cells were incubated at 37 ºC with 5% carbon dioxide and 95% air. After one day of incubation, the suspensions were modified with serial contents of the prepared NPs. The samples were dispersed in dimethylsulfoxide and sample dispersion was diluted twice and the required final test amount with serum free medium. Another four serial dilutions were performed to provide a total of five sample concentrations. A solutions of 0.1 ml of the diluted samples were inserted to the suitable wells, which having 0.1 ml of cultured media. The plates were maintained for two days at 37 ºC with similar conditions. The medium-only sample was acted as reference and triplicate was attained for all contents.
MTT assay. The mitochondrial enzymes in organ cell called succinatedehydrogenase cleave the tetrazolium and dissolving the MTT to an insoluble purple formazan. Thus, the produced formazan is precisely proportional to the viable cells number. After 2 days, 15 µl of MTT (5 mg/ml) in phosphate buffered saline was seeded to each well and kept at 37 ºC for 4 h. Then, the media with MTT were flicked off and the developed formazans were added in 100 µl of DMSO. Finally, the absorbance was recorded at 570 nm utilizing micro-plate reader.28, 29