2.1. Extract preparation
2.1.1. Methanol crude extract
C. epithymum (Cuscutaceae) seeds were obtained from the Medicinal Plants Store (Tehran, April 2021). F.Mojab taxonomically identified the plant at the School of Pharmacy, Shahid Beheshti University of Medical Sciences, Iran, and registered with herbarium code SBMU-8236. The extraction process was performed by the maceration method. Briefly, the seeds were grounded and soaked for 24 hours in methanol (Merck, Darmstadt, Germany) as a solvent so that it completely covered the grounded seeds. After 24 hours, the top solvent was collected, and the new solvent was poured on the seeds. The procedure was repeated three times and during the procedure seeds and methanol were shaken. Finally, the collected solvent in three days was concentrated in an oven at 40 ℃.
2.1.2. Fractions of extract
C. epithymum seeds were macerated in n-hexane (Merck, Darmstadt, Germany) for 48 h at room temperature. The extraction procedure was entirely similar to crude extraction. After the third collection, the extraction was dried at room temperature for 24 hours to ensure that all the hexane had been completely removed. The residual plant was macerated further in two other solvents, dichloromethane (Merck, Darmstadt, Germany) and methanol, respectively, in the same manner as n-hexane. Using a rotary evaporator, the solvent was removed under a vacuum at 45 ℃. The fractions obtained from each solvent were kept at 4 ℃ for the subsequent study.
2.2. Total phenolic contents measurement
We used the Folin-Ciocalteu analysis method to evaluate the total phenolic content (TPC) in the crude methanolic extract and methanol, n-hexane, and dichloromethane fractions. Folin-Ciocalteu solution (Merck, Darmstadt, Germany) was utilized as a reagent, and gallic acid (Merck, Darmstadt, Germany) was the reference phenolic compound. 1 ml of different concentrations of gallic acid, including 0, 10, 20, 40, 60, 80, and 100 g/ml, was combined with 5 ml of diluted Folin-Ciocalteu and incubated at room temperature for 10 minutes. 4 ml of sodium carbonate solution (75 mg/mL) was added and incubated at room temperature for 30 minutes. A spectrophotometer was used to determine the absorbance of each standard and sample at 765 nm. For TPC evaluation in samples, 1 ml of the extract with a 400 µg/ml concentration was used, and by using the standard curve of gallic acid as a reference, TPC was determined. The data is presented as the average of three independently collected measurements.
2.3. Cell culture
C6 glial cells (rat glioma cell line) were purchased from Pastor Institute (Tehran, Iran) and cultured in 25-cm2 culture flasks in DMEM/F12 supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin-streptomycin (Biosera, France)in a humidified atmosphere of 5% CO2 at 37°C. Cells grown to 80–90% confluency were used to performing the following experiments.
2.3.1. Determination of L-glutamate cytotoxicity
The cells were sub-cultured in 96-well plates at a density of 8 × 103 cells/well and incubated for 24 h. Adherent cells were exposed to different concentrations of L-glutamate (0.312-20 mM) for 24 h. The morphology of C6 cells treated with various concentrations of L-glutamate was observed using an inverted light microscope (Olympus, Japan), and the cell viability was evaluated using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Sigma, Germany). Following 24 h incubation, cells were incubated at 37 °C for an additional 4 hours after adding MTT solution at a final concentration of 0.5 mg/ml. 100 μL of DMSO was added to dissolve the formazan crystals after removing the medium. The plate was shaken for 10 minutes, and the absorbances of the samples were measured at 570 nm by an ELISA reader (BioTek,USA). For all experiments, glutamate was dissolved in DMEM/F12 at final concentrations of 0.312, 0.625, 1.25, 2.5, 5, 10, and 20 mM.
Acridine Orange and Propidium Iodide (AOPI) staining was applied to determine the apoptosis percentage of C6 cells treated with various concentrations of L-glutamate (Merck, Darmstadt, Germany). For this purpose, 24 h after L-glutamate exposure, the medium was completely removed, and 30 µl of AOPI reagent (Nexcelom Bioscience, Massachusetts, United States) was added to each well. Cells were incubated with AOPI dye for 45 minutes at 37 °C and observed under a fluorescent microscope (Nikon digital camera, USA).
2.3.2. Determination of C. epithymum crude extract and fractions cytotoxicity
The cells were sub-cultured in 96-well plates at a density of 8×103 cells/well and incubated for 24 h. Adherent cells were exposed to different concentrations (9.37, 19, 37, 75, 150, and 300 µg/ml) of C. epithymum crude extract and fractions. The crude extract and fractions were diluted in 100 µL of ethanol (70%) and then in 5 ml of DMEM/F12 to prepare 300 µg/ml. The other concentrations were prepared from 300 µg/ml solution by serial dilution. After 24 h of adding C. epithymum crude extract and also fractions into plates, the MTT assay was performed as previously described. The remaining 96-well plates were incubated at 37°C to assess cell viability after 48 and 72 h. The morphology of cells was observed using an inverted microscope.
2.3.4. Effects of C. epithymum fractions and crude extract treatment in L-glutamate induced cytotoxicity
C6 cells were seeded in 96-well plates at 8×103 cell density in each well. C6 cells were pre-treated with various concentrations (9.37, 19, 37, 75, and 300 μg/mL) of C. epithymum crude extract for 24 h before exposure to L-glutamate. After 24 h, the medium was changed by a fresh culture medium containing L-glutamate (0.312, 2.5, and 10 mM) and incubated for 24 h. In the negative control and positive control groups, cells were treated with ethanol (70%) and L-glutamate, respectively. Finally, an MTT assay was performed. Each independent experiment was repeated three times. In parallel to this experiment, post-treatment of C6 cells with C. epithymum was performed. This way, C6 cells were first treated with L-glutamate (0.312, 2.5, and 10 mM) for 24 h. A fresh culture medium containing different concentrations of C. epithymum crude extract (9.37, 19, 37, 75, and 300 μg/mL) was added, and the cell viability was evaluated in the same procedure. Three concentrations (19, 37, and 75 μg/mL) were selected for next experiments to show the effects of C. epithymum treatment in L-glutamate-induced cytotoxicity.
2.3.5. Measurement of superoxide dismutases (SODs)activity
SODs are essential enzymes protecting cells against oxidative damage by reducing superoxide anion free radicals (O2-). SODs activity was measured in cells treated with L-glutamate and C. epithymum crude extract. C6 cells were grown in 25 cm 2 flasks for pre-treatment and post-treatment with 19, 37, and 75 g/ml crude extract and 10 mM glutamate to measure SODs activity. Using a rubber policeman, cells were removed from the flasks 24 hours after the last treatment. Two million cells were required for the test. The pellet was sonicated for 30 seconds at a medium power (10 s of sonication plus 10 s of rest; 3 times) before being centrifuged at 12000 g for 5 minutes at 4 ° C. The supernatant was separated and placed on ice. According to the kit protocol (TPR Innovative, Iran), samples and working solution (chromogenic reagent and SODS assay buffer) were added to 96-well plates. Eventually, the SODS enzyme solution was added and incubated for 20 minutes. After 20 minutes, a multi-mode reader read the absorbance at a 440–460 nm wavelength (BioTek, USA).
2.3.6. Malondialdehyde (MDA) assay
Malondialdehyde (MDA) is the most common reactive aldehyde produced during lipid peroxidation and an oxidative stress indicator. The thiobarbituric acid reactive substances (TBARS) were measured to determine the products of lipid peroxidation. 24 hours after the last treatment, the cells were trypsinized for the MDA test. According to the kit (TPR Innovative, Iran), one million cells were required in 1 ml of PBS buffer (pH: 7.8). First, 10 µL of butylated hydroxytoluene (BHT) was added to the cells. Cells were then placed on ice and sonicated with a medium power sonicator for 30 seconds. Detergent was added to all samples and incubated at 37 ˚C. Then, the chromogenic solution containing thiobarbituric acid, acetic acid, and alkali was added to the samples and placed in boiling water for an hour. After one hour, they were placed on ice for 10 minutes, then centrifuged at 10,000 g for 10 minutes at 4 °C. The supernatant was transferred to the 96-cell plate, and the absorbance of the tests was measured using a multi-mode reader at a wavelength of 530-540 nm. Protein concentrations were measured in samples by the multi-mode reader at 280 nm.
2.3.7. Effects of C. epithymum crude extract on the apoptosis of C6 cells in L-glutamate-induced damage condition
At the end of the final 24 h of cells incubation with C. epithymum crude extract and L-glutamate, C6 pre-treated and post-treated cells were harvested and centrifuged at 1500 rpm for 5 minutes. For determining the percentage of the apoptotic cells, Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) (Biolegend, USA) co-staining method was used. For this purpose, 500 µL of 1X binding buffer was added to the cell pellet, and the sample was divided into tubes. 5 µL of Annexin V-FITC and 3 µL of PI were added to labeled tubes and then incubated at 4 °C for 15 minutes in the dark. Then the specific fluorescence of cells was detected using a BD FACS Calibur (BD Biosciences, San Jose, CA, USA) and the outcomes were evaluated by FlowJo software.
2.3.8. Effects of C. epithymum crude extract on the C6 cell cycle in L-glutamate-induced damage condition
At the end of the final 24 h of cells incubation with C. epithymum crude extract and L-glutamate, C6 pre-treated and post-treated cells were harvested and centrifuged at 1500 rpm for 5 minutes. Then 50 µL of cold PBS was added to the cells and gently vortexed. The cells were resuspended, fixed with cold 70% ethanol, and vortexed gently. After fixation by ethanol, the cells were washed with PBS and centrifuged at 1500 rpm for 5 minutes. The pellet was resuspended in 1ml of PI master mix containing 40 µL of PI (1 mg/ml), 950 µL of PBS, and 10 µl of RNase (DNase free, 10 mg/ml) and incubated for a half-hour at room temperature. Finally, the cells were analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, USA), and the outcomes were evaluated by FlowJo software.
Statistical Analysis
Each experiment was conducted at least three times, and the data were presented as the mean ± standard deviation (SD). Statistical analysis for cell viability tests, SOD, and MDA were performed using one-way ANOVA and appropriate posthoc test in GraphPad Prism 8.4.3. Two-way ANOVA and appropriate posthoc test analyzed the apoptosis and cell cycle data. The P < 0.05 indicated that the result was statistically significant.