Chaerophyllum macropodum Boiss. was collected in Diz Stream of Hakkari Cilo mountain at 1730 m in May. Scientific diagnoses of the plants were made by Mehmet FIRAT from University of Van YYU, Faculty of Education, Department of Biology.
C.macropodum samples were frozen at -80°C for a week and grounded by using a tissue lyser (Qiagen®, Germany) into powder at maximum speed for 5 minutes. Samples were incubated in ultra-pure distilled water at room temperature for 24 hours and supernatant were obtained by using centrifugation at 10.000 RPM for 3 minutes. The aqueous extracts were passed through a 0.40 µm membrane filter and lyophilized to obtain dried solutes. Extract quantities were determined via the use of an analytical balance (Shimadzu®, Japan).
The human fibroblast cell line (HDFa, ATCC® PCS-201-012™) was grown in DMEM medium with a 1% penicillin/streptomycin antibiotic mixture, 10% FBS, 5% CO2, and 37o C temperature until it reached %80 confluency.
Neuroblastoma cell line (SH-SY5Y, ATCC® CRL-2266™) was grown by using 1% penicillin/streptomycin, 10% fetal bovine serum in DMEM/F12 culture media. After the prepared medium was brought to 37°C, 5 ml was taken and transferred into a T25 flask and SHSY-5Y cells were cultivated in 37°C and 5% CO2 incubator. Cells were incubated until they reached 80% density in the flask.
Cytotoxicity and Cell Viability Analyses
Lactate dehydrogenase (LDH, Cayman Chemical Company®, USA) assay was used to analyze cell death ratios for the cell cultures in respect to manufacturer’s instruction. Briefly, cell cultures were seeded to 96-well plate and compounds were applied to the cultures in various concentrations (1.65 µg/m to 100 µg/m) for 24 hours. After the incubation period ends, 100 µL of culture supernatant were discarded into a fresh 96-well plate and 100 µL of the reaction mixture was added and immediately color intensity was investigated at 490 nm absorbance. Samples were incubated at room temperature for 30 minutes and again absorbance was monitored by using a microplate reader at 490 nm.
Cell viabilities for the fibroblast and the neuroblastoma cell cultures were investigated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For the assay, compounds were applied to the cell cultures at wide dose intervals (1.65 µg/m to 100 µg/m) and incubated for 24 hours. After the application period, 10 µL of MTT solution was added to each well and incubated for 3 hours at 37°C. Then, cell mediums were discarded and 100 µL of DMSO was added to each well to dissolve formazan crystals to yield blue color. Color intensities were measured by using a microplate reader at 570 nm wavelength.
Hoechst 33258 Fluorescent Staining and Nuclear Abnormality Analysis
Hoechst 33258 staining was used to detect abnormal nuclear structures. Compounds were applied to the fibroblast cell culture at 100 µg/ml concentrations and cell cultures were incubated for 24 hours to monitor the nuclear morphology. Then, cells were fixed by using phosphate-buffered saline with 4% paraformaldehyde at 4°C for 30 minutes. Cell cultures were washed two times with PBS and samples were incubated with 1 µM Hoechst 33258 fluorescent dye at room temperature for 5 minutes. Cells were observed and photographed under a fluorescent microscope (Leica® DM IL LED).
Statistical calculations were performed by using the GraphPad Prism (GraphPad Software®, USA, San Diego) statistical program and analyses were gathered via the use of one-way ANOVA, Dunnett comparison test. The statistical significance level was accepted as P <0.05.