Collection of plant materials and preparation of extracts
T. vulgaris seeds were purchased from PlantiCo (Poland) and implanted for six months. O. syriacum were obtained from Al-Breem plantation (Khanyounus) and authenticated by Dr. Mohammad Abo-Oda (PhD plant taxonomy, Alaqsa University, Gaza). Aerial parts were collected, washed, air-dried under shade for 3 weeks, and then completely dried by oven at 40°C for 1 to 1.5 hour.
Both plant extracts were prepared by soaking the dried and grinded aerial parts in ethanol at room temperature with occasional mixing (7 days, 40% ethanol for O. syriacum; and 4 days, 90% ethanol for T. vulgaris) in a weight/solvent volume ratio of 1/10. Different ethanol concentrations were used according to the solubility preferences of the literature detailed effective compounds of each plant. The extracts were filtered under vacuum for three times, and dried by rotary evaporator at 40°C for T. vulgaris and 45°C for O. syriacum.
O. syriacum stock solution was finally prepared at a concentration of 200 mg of the dry extract/mL of RPMI-1640 medium with 10% fetal bovine serum (FBS). T. vulgaris stock solution was prepared at the same concentration in dimethyl sulfoxide (DMSO). DMSO was used because T. vulgaris dry extract contains high percentage of oils and does not dissolve in the aqueous RPMI-1640 medium. The stock solutions were stored at -20°C until used.
Cell line culturing and maintenance
The THP-1 cell line was derived from the peripheral blood of a 1 year old male with acute monocytic leukemia. The cells were cultured in modified RPMI-1640 complete medium with 2.05 mM L-glutamine and 25 mM HEPES, supplemented with 10% heat inactivated FBS at 37°C in a humidified atmosphere with 5% CO2. Cell counts and viability estimation by trypan blue dye exclusion test were performed regularly. Throughout the study procedures, THP-1 cells were maintained in a logarithmic growth phase at a concentration between 105-106 cells/mL. Media feeding was performed periodically every 2–4 days.
Isolation of human Peripheral Blood Mononuclear Cells (PBMCs)
PBMCs were isolated from sodium heparin anticoagulated venous blood of healthy donors using Sigma’s Histopaque-1077 as recommended. The donors’ consents were obtained before sample collection, and the procedure was approved by the local Helsinki committee of the Palestinian Health Research Council, according to the World Medical Association Declaration of Helsinki (approval reference No. 1-12/2012). The cells were suspended at a concentration of 106 cells/mL in modified RPMI-1640 medium, with 2.05 mM L-glutamine and 25 mM HEPES, supplemented with 10% FBS, 5 μg/ mL phytohemagglutinin (PHA), 100 μg/mL streptomycin, and 100 U/mL penicillin.
Cells preparation and extracts treatment
THP-1 cells in the exponential growth phase and viability of at least 95% and freshly isolated PBMCs with viability of at least 98% were used for the viability assays. The cells were seeded in 96-well plates at a density of 104 THP-1 cells/well and 105 PBMCs cells/well in 100 μL of the culture medium. Another 100 μL of the proper extract working concentration were added to the corresponding wells in triplicates, and the cells were incubated for 48 ± 1 hours in a humidified CO2 incubator at 37°C and 5% CO2.
The various extract working concentrations were prepared by dilution of the stock solutions in culture medium and filter-sterilization with 0.22 μm Millex-GP syringe filters. Corresponding DMSO concentrations were similarly prepared as vehicle controls for T. vulgaris extract.
Cell viability assay
The THP-1 cells and PBMCs previously incubated with the proper extract or DMSO concentration were washed several times with 150 μL phosphate buffered saline (PBS) (pH = 7.2 - 7.6) and plate-centrifugation, to remove any residual extract color that may interfere with the colorimetric assay. The extract-free cells were finally suspended in 100 μL media with 10% heat inactivated FBS, and assayed for viability using the colorimetric MTT based in vitro toxicology assay kit following the manufacturer instructions. The assay measures the amount of the blue-colored formazan accumulated intracellularly following cleavage of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent by mitochondrial dehydrogenases of the viable cells. The absorbance of the produced intracellular formazan which is proportional to the number of viable cells present was determined at 550 nm. Absorbance for background correction was determined at 620 nm. Absorbance of wells filled with media alone was used as a blank and untreated control wells were seeded with cells that incubated without any extracts. The percentage of cell survival was calculated after background absorbance correction and blank absorbance subtraction as follows: % Cell viability = 100 Χ Experimental well absorbance / untreated control well absorbance.
Cytotoxicity assay
The THP-1 cells were treated with various extracts or DMSO concentrations. Cells in the positive control wells were treated with 1% Triton X-100 solution, and negative control wells cells were incubated in culture media alone. Blank wells contained the corresponding extract concentrations or Triton X-100 solution or media without cells. The lactate dehydrogenase (LDH) based in vitro toxicology assay kit was used to assay for cytotoxicity following the manufacturer instructions. The assay measures membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. LDH reduces NAD into NADH, which is utilized in the reduction of a tetrazolium dye to colored formazan. The amount of formazan which is proportional to the amount of LDH release from dead cells was measured colorimetrically at 450 nm. Absorbance for background correction was determined at 620 nm.
The percentage of cell viability was calculated as follows: % Cell viability = 100 –% cell cytotoxicity. The % cell cytotoxicity = 100 X (experimental well absorbance –negative control well absorbance) / (positive control well absorbance –negative control well absorbance). All calculations were performed after background absorbance correction and blank absorbance subtraction.
Statistical analysis
All experiments were performed in triplicates and each experiment was performed for two times. The results were expressed as a mean viability percentage ± standard deviation (SD). Dose response curves were prepared with Microsoft Office Excel 2007 software.
The data were analyzed using the GraphPad Prism software (Version. 6). The log (inhibitor) vs. response curve equation was used to determine the best fit and to determine the IC50 and LC50 values, the concentration that provokes a response half way between the maximal and the maximally inhibited cell viability. The obtained curves on leukemic and normal cells for each extract were compared with respect to their IC50/LC50 and slope and the P-value was determined with 95% confidence intervals. Two-regression curves comparison was performed by the F-test and a significant difference was obtained at P-values < 0.05.
