Chemicals and reagents
Thermo Fisher Scientific (Massachusetts, USA) provided the acetonitrile, and DiKMA Technologies provided the formic acid (Beijing, China). Water was produced via a Milli-Q® integral water purification system (Merck, Germany).
Lichen material
Ramalina specimens were collected in the Hyrcanian forest in Mazandaran Provinces (Iran) in May 2018, and Ramalina Sinensis (R. sinensis) was selected for this study. Lichen materials were collected in accordance with the relevant guidelines and regulations of the Plant Varieties Protection, Environmental Protection Organization of Iran. The Museum of Iranian Lichens in the Iranian Research Organization for Science and Technology (Tehran, Iran) provided the principal keys to identifying species (Figure 1). The lichen was identified by Dr. Mohammad Sohrabi, lichenologist from the Iranian Research Organization for Science and Technology. The voucher specimen was deposited in the Museum of Iranian Lichens, Iranian Research Organization for Science and Technology (IROST), Iran (https://irost.org/museum/). The cleaned lichen thallus was grounded to a fine powder using a mortar and pestle under a small amount of liquid nitrogen.
Cell lines and culture
The human oral epidermal carcinoma cell line (KB cell line, NCBI Code: C152) were obtained from the Pasteur Institute of Iran (Tehran, Iran) (Supplementary File). Cancer cell lines were cultured in DMEM with 10% FBS and 1% PS at 37°C, 5.0% CO2 and 95.0% humidity. Cells were cultured in a humidified atmosphere of 95.0% air, and 5.0% CO2 at 37°C.
Cell viability
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine viability, which is based on the reduction of MTT by mitochondrial dehydrogenase in intact cells into an insoluble purple formazan product. In 96-well plates, cells (1*104/well) were seeded, and after 24 hours, the cells were treated with R. sinensis extract at various doses (200, 100, 50, 25, 12.5, and 6.25 μg/ml) and then R. sinensis -containing medium were carefully removed after the treatment. Cells were washed twice with PBS before each well received 100 mL media containing 0.5 mg/ml MTT in PBS, then the plate was incubated at 37°C for 4 hours. The medium was then completely removed, 200 mL of Tris-DMSO solution was added to each well, and the plate was vibrated for 30 minutes. Using an ELISA plate reader, the absorbance, as proportional to cell viability, was then measured in at least three independent measurements using a microplate reader (Bio-Rad Corp, California, USA) at 570 nm in each well and Gen 5 Version 2.07.17 software (BioTek, Winooski, USA) for data analysis. The average of measurements was reported for each well.
Flow cytometry
Flow cytometry analysis was performed on apoptotic cells using a Beckman Coulter (EPICS XL). The ramalina-treated and untreated cells were collected, twice-washed in PBS, fixed in 70.0% ethanol at 4°C for at least 12 hours, centrifuged, and then incubated for 30 minutes at 37°C in the dark with 0.1% Triton X-100, 200 mg/ml RNase A, and 50 mg/ml propidium iodide (PI) in PBS. More than 3*104/well cells were counted in each sample, and cells with lower DNA content than those in the G0/G1 phase were labeled as apoptotic.
Scratch assay
After 24 hour of cultivation as confluent monolayers in the complete medium, cells were wounded by removing cells across the well with a standard 200 μL pipette tip [13]. To eliminate the non-adherent cells, the wounded monolayers were washed twice. When the wound was made, and 24 hours later, it was observed using an inverted phase contrast microscope (Leica, Wetzlar, Germany). This 24 hour was selected since it is less than the doubling time in control. Four separate fields from each sample were evaluated for quantitative measurement of the distance between the borderlines, while four different equidistant spots in each picture were measured to get a better approximation of the true breadth of the wounded region. The migration rate was estimated as the ratio of the mean distance between both borderlines generated by scratching to the distance that remained cell-free after re-growing and is represented as a percentage of the control. In quadruplicates, two separate sets of tests were carried out.
Metabolic profiling of R. sinensis
The chemical composition of R. sinensis was investigated according to Norouzi et al. briefly (13); lichenochemicals were separated by Waters Alliance e2695 separation module (Milford, MA, USA) and Atlantis T3 C18 column (2.1mm × 100 mm, 3 μm; Milford, MA, USA) and Column temperature set to 30℃. Samples were dissolved in methanol, and after filtration (PTFE membrane filters, 0.45 μm, Simplepure, China), 10 μL of each sample was injected into the separation module. Lichenochemicals were eluted within 25 min as follows: elution began by 95% (water + 0.1% formic acid, v/v) and gradually decreased to 5% within 20 min. The Elution process went on for another 5 min by 95% B (acetonitrile). The flow rate of eluents was adjusted to be 0.25 ml min-1. A Quattro micro API mass spectrometer (Milford, MA, USA) was used for tandem mass analysis. The MS/MS parameters applied were as follows: source temperature and desolvation temperature were set to be 120 ℃ and 300 ℃, respectively; capillary voltage, cone voltage, and collision energy were regulated at 3.5 kV, 30 V, and 30 eV, respectively; for both nebulizing and drying the gas, N2 was used. MassLynx 4.1 and MZmine 2.53 were used for data acquiring and analysis. Initial annotation of detected compounds was carried out based on the lichen spectral database (LDB) provided in GNPS public spectral libraries (14). Chemical structures were sketched using ChemDraw Ultra 12.0.
Statistical analysis
SPSS software (version 22; SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.2.1 (GraphPad Software, San Diego, CA) were used for statistical analysis. All results are given as as mean ± standard deviation (SD), with p ≤ 0.05 considered statistically significant.