Chemical characterization, antioxidant and cytotoxic activities of the methanolic extract of Hymenocrater longiflorus grown in Iraq

2.1 Reagents

Dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), trypan blue dye 0.4%, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Bradford reagent, bovine serum albumin, sodium-dodecyl sulfate (SDS), ammonium persulfate, isopropanol, bromophenol blue, tetra-ethylene-diamine, 4% paraformaldehyde, MOWIOL mounting medium, sulforhodamine101, camptothecin (CPT, C9911) and doxorubicin (D1515) were purchased from Sigma-Aldrich (Taufkirchen, Germany). DMEM media were purchased from Euroclone (Pero, Italy). Methanol, ethanol, and acetic acid were purchased from Carlo Erba (Cornaredo, Italy). Propidium iodide solution was obtained from Becton Dickinson (San Jose, CA, USA). Acetonitrile was purchased from DuPont (Wilmington, DE, USA). The anti-p53 monoclonal antibody (Clone sc-126), anti-p21 polyclonal antibody produced in rabbit (Clone sc-397), and anti-actinin monoclonal antibody (Clone sc-10750) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The rabbit monoclonal antibodies anti-P-p53-ser15 (Clone 9286) and anti-γH2AX (Clone 2577) were obtained from Cell Signaling Technology (Danvers, MA, USA). The mouse anti-parp monoclonal antibody (Clone sc-53643) and goat anti-actin monoclonal antibody (Clone sc-1616) were purchased from Santa Cruz Biotechnology. The anti-H2AX (SER 139) polyclonal antibody (JBW301, Cat.05-636) was purchased from Millipore (Molsheim, France). The anti-rabbit fluorescence monoclonal antibody (goat anti-rabbit Cy3) was purchased from Jackson Lab (Bar Harbor, ME, USA), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) from Sigma-Aldrich, and TRIS-base from BDH (London, UK).

2.2 Collection and extraction of H. longiflorus

Hymenocrater longiflorus Benth (Lamiaceae) was collected from the Hawraman Mountain (1739 m above sea level) above the Ballkha village in the Kurdistan Province of Iraq in May 2012. The plant material was identified by Dr. Saman Abdulrahman Ahmad, Department of Field Crop, College of Faculty of Agriculture Science, University of Sulaimani, Sulaimani, Iraq. The fresh aerial parts of the plant were washed thoroughly with tap water at room temperature to remove dirt prior to drying. The washed aerial parts were dried in the shade at room temperature for 7 d and then crushed into a fine powder. Approximately 100 g of powder were extracted once in a Soxhlet extractor with 70% (v/v) methanol in water (500 mL) 2 h at 40 °C. After filtering through Whatman filter paper no. 3, the extract was reduced to dryness in a rotary evaporator under reduced pressure at 45 °C. The residue (3 g) was stored, protected from light, at –20 °C in a glass container until use. For in vitro studies, the H. longiflorus extract was dissolved at a concentration of 10 mg mL^–1 in DMSO: water (2:1, v/v).

2.3 Qualitative analysis of H. longiflorus extract

The H. longiflorus sample (10 mg mL^–1 in DMSO: water) was diluted 1:100 (v/v) with acetonitrile: water (1:4 v/v). The qualitative analysis of the constituents was done with an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to a 1200 series capillary LC pump and auto-sampler (Agilent Technologies, Santa Clara, CA, USA). The ion source was a desorption electrospray ionization Omni spray (Prosolia, Indianapolis, IN, USA), which was used in nanoelectrospray mode for negative and positive ions. The HPLC separation was obtained with a Zorbax SB-C18 column (100 mm × 0.5 mm; 5 μm particle size), using an elution mixture composed of solvent A (0.05% acetic acid in water, v/v) and solvent B (acetonitrile). The injection volume was 1 μL, and the flow rate was 10 μL min^–1. The elution gradient ranged from 20% to 100% of solvent B within 40 min. The gradient was held at 100% for 6 min and re-equilibrated for 8 min at 20% of solvent B. Samples were directly injected into the HPLC column, which was directly coupled to the ion source spray capillary by a liquid junction. Data processing and calculations were done using the LTQ-Orbitrap Xcalibur 1.4 software [12]. The available reference standards were used (apigenin, caffeic acid and acacetin) for identification by direct comparison of the retention times and mass spectra acquired in the same analytical conditions. For other peaks, the measured accurate mass (or the most probable chemical formula that can be calculated from it) was compared with reference compounds available online at different chemical database websites (www.chemspider.com, www.chemfinder.com and pubchem.ncbi.nlm.nih.gov).

2.4 Stable free radical scavenging capacity

The free radical scavenging activities of the plant extract were measured with the DPPH assay [13]. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical has a deep violet color because of its unpaired electron; the radical scavenging capability can be determined spectrophotometrically by the absorbance loss at 517 nm, when the pale yellow non-radical form is produced. Equal volumes (0.5 mL) of DPPH (60 μM) and each plant extract (10, 20, 40, 100, 500, and 1000 μg mL^–1) were mixed in a cuvette and allowed to stand for 30 min at room temperature. The absorbance was read at 517 nm in a UV/VIS Lambda 19 spectrophotometer. The absorbance of the control (DPPH solution) was also read. The percentage of DPPH decoloration of the sample was calculated using the following formula:

Percentage of decoloration  = (Control absorbance–Sample absorbanceControl absorbance) × 100.

2.5 Cell growth and viability assay

The human osteosarcoma U2OS cell was procured from the cell bank of the Department of Biology, University of Naples Federico II, Naples, Italy. The cell line was cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 1% streptomycin/penicillin in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C in T 25 cm² tissue culture flasks. For cell growth and cytotoxicity assays, the cells were seeded in 12-well culture plates, at a density of 8 × 10⁴ cells mL^–1 and incubated for 24 h as above. Cells were treated with four concentrations (50, 100, 150, and 200 μg mL^–1) of plant extract for 24, 48, and 72 h. After each incubation period, cells were counted, and the viability was assessed using a cell counter [14]. The MTT assay was used for confirmation. Briefly, the cells were seeded at 10,000 cells per well (100 μL) in 96-well dishes and then incubated overnight as above. Different concentrations of plant extract (10, 20, 40, 80, 100, and 150 μg mL^–1) were used and incubated for 2, 4, 8, 24, 48, and 72 h. Subsequently, MTT (dissolved in phosphate buffered saline (PBS) at 5 mg mL^–1) was added to each well and incubated for 60 min in the dark. Exogenous MTT was removed, and 100 μL of acidified isopropanol (1 μL hydrochloric acid per 1 mL isopropanol) was added to each well to dissolve the formazan crystals. After 5 min incubation, absorbance was measured at 570 nm in a Biotex microplate reader. The intensity of color produced is directly proportional to the number of viable cells. Each experiment was performed in triplicate [15]. The relative cell viability (%) of the control wells containing the cell culture medium only as a vehicle was calculated as follows:

Percentage of cell viability (%) = (Sample  absorbanceControl  absorbance) × 100.

2.6 Flow cytometry

Analysis of the cell cycle was performed according to the method of Erba et al. [16], with slight modifications. The cells were counted and plated at a concentration of 1 × 10⁶ cells per 100 mm plate, allowed to grow for 24 h, and subjected to 50 or 100 μg mL^–1 plant extract. A negative control was run as well. The cells were harvested after 3 or 6 h of treatment by centrifugation at 400 g at 4 °C for 2 min. The supernatant was removed, and the cells were washed twice with PBS, fixed in cold methanol, and kept at –20 °C for at least 30 min. Approximately 500 μL of PBS was added, mixed well, and the cell suspension centrifuged at 240 g at 4 °C for 5 min. The cells were suspended in PBS, mixed, and incubated for 30 min in an ice bath. The cells were permeabilized for 20 min at room temperature by addition of 500 μL of hypotonic solution containing 0.1% Na-citrate, 50 μg RNase, and 50 μg propidium iodide (PI) in 1 mL of PBS. The fluorescence emitted (610 nm) from the PI-DNA complex was quantified after excitation (535 nm) of the fluorescent dye in the FACSCalibur flow cytometry system (BD Biosciences, San Jose, CA, USA). According to the distribution of the DNA content, the G1-, S-, and G2/M-phases were distinguished [17]. The data were analyzed with CellQuest and ModFit 3.0 software.

2.7 Determination of morphological aspects by fluorescence microscopy

U2OS cells were seeded at a density of 5 × 10⁴ cells in six multi-well plates containing a sterile cover slip on the bottom of each well and were allowed to adhere for 48 h. The cells were treated with 50 and 100 μg mL^–1, respectively, of H. longiflorus extract for 6 h. Subsequently, the medium was discarded by aspiration. The cells were washed with filtered PBS and stained with sulforhadamine101-DAPI (4,6-diamidine-2-phenylindole dihydrochloride) solution for 30–60 min. After staining, the slides were rinsed with distilled water, air-dried, and covered with a cover slip. Nuclei were observed with a fluorescence microscope by using a 365 nm filter [18].

2.8 Immunofluorescence of H2AX histone phosphorylation

U2OS cells were counted and seeded at a density of 5 × 10⁴ cells in 12 multi-well plates containing a sterile cover slip on the bottom of each well. The cells were allowed to adhere for 18 h in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C and subsequently treated for 4 h with H. longiflorus extract at 80 or 100 μg mL^–1. After treatment, the medium was removed, and the cells were washed with PBS (two to three times). The cells were then fixed with cooled 4% paraformaldehyde for 15 min. The cells were washed with 1 mL of filtered PBS, cold 70% ethanol was added, and the mixtures left standing for at least 6 h. Subsequently, the cells were re-hydrated and permeabilized with 0.5% Triton X-100 for 10 min–15 min at room temperature, washed with PBS, and subjected to the blocking solution TBS-T (5 g of milk powder in 100 mL of 20 mM Tris-HCl, 1.5 M NaCl, and 1% Tween 20, pH 8.0) for at least 30 min at room temperature. After blocking, the cells were incubated with 20 μL of anti-γ-H2AX (SER 139) polyclonal antibody, diluted 1:100, for 1 h in the dark at 37 °C. After washing the cells several times with 1 mL of TBS-T, the cells were incubated with 20 μL of anti-rabbit Cy3 conjugated secondary antibody for 45 min in the dark at room temperature. The slides were washed three times with TBS-T, then twice with PBS and stained with DAPI X 1 for 10 min at room temperature. Finally, the slides were mounted onto cover slips with the aid of MOWIOL and dried at room temperature. They were subsequently inspected under a fluorescence microscope (TE Eclipse 2000, Nikon). Images were acquired using Vico software.

2.9 Western blot analysis

For immunoblotting, U2OS cells were grown under the same conditions described above and treated with H. longiflorus extract, or with the positive reference compound camptothecin (CPT). Protein content was quantified by the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA) according to Amente et al. [19]. Equal amounts of proteins (50 μg each) were subjected to separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel. The gel was then equilibrated in transfer buffer, containing 10 mM Tris base, 200 mM glycine and 10% methanol, for 5 min before proteins were electroblotted onto a nitrocellulose membrane (Bio-Rad). The nitrocellulose membranes were immediately stained in 0.1% Ponceau S in 5% acetic acid and then photographed. The blots were then blocked with blocking solution (prepared as above) prior to incubation with the respective antibodies against actin, actinin, p53, p-p53, p21, parp, and γ-H2AX. Incubation of primary antibodies was performed overnight at 4 °C with slow constant shaking. This step was followed by 15 min washes with TBS-T prior to 1 and 0.5 h of incubation with the secondary antibody at room temperature. After extensive washes, results were visualized by enhanced chemiluminescence substrate (ECL, Amersham, Arlington Heights, IL, USA) by using a Bio-Rad Chemi-Doc MP Imager Station.

2.10 Statistical analysis

Data were obtained by tabulation in the statistical program GraphPad Prism version 5.01 (GraphPad Software, La Jolla, CA, USA) and presented as mean ± standard error. The significance of the difference between means was assessed by Duncan’s test, in which P ≤ 0.05 was considered significant.