In our research we used the following materials: MTS from Promega (USA), Bradford reagent from Bio-Rad (Germany), z-VAD-fmk, Ac-DEVD-AFC, DTT from Bachem (Switzerland), FBS from Gibco (USA), antibiotics penicillium, streptomycin and glutamax from Gibco (USA), annexin V-PE and 7-AAD for flow cytometry were purchased from BD Biosciences (USA). Fluorescent organelle-specific probe Mitotracker CMXRos was of Molecular Probes (Eugene, OR, USA) and cellobiose, vitamin C, sulphur and selenium were from Calbiochem, Germany. All other reagents (RIPA buffer, caspase buffer, KDMEM, Tryple Select, PBS) were prepared at the institute according to standard procedures described [18, 19].
The cell line used in the experiments was human hepatocellular liver carcinoma cell line HepG2 acquired from ATCC. Before performing experiments the cells were tested for the cell line identification by isozyme typing and karyotyping. The cells were last tested one month before we started the experiments. Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 20 % bovine serum (FBS), 1 % penicilium/streptomycin and 1 % glutamax grown to obtain 90 % confluency. When cells were confluent and prepared for experiment with the plant extract, they were transferred in 96-well or 6-well plate, where they were grown in DMEM, free of any kind of supplement of animal origin.
Preparation of plant extract
Capsicum chinense and Allium sativum from the plant family Solanaceae were grown in a greenhouse. The seeds of above mentioned plants were provided by Sonnentor Kräuterhandels GmbH, Sprögnitz 10, 3910 Zwettl, Österreich, who also made the formal identification of the seeds (Capsicum chinense ZO354 AT-BIO-301 and Alium sativum ZO325 AT-BIO-301). Once the plants grown from above mentioned seeds in the greenhouse blossomed and ripened into fruits (seeds planted in April, harvest in September), the whole plants were collected and dried. Our calculation was as follows regarding to extrapolation of data in the cited reference and to generally known nutritional value: 100 g of Alium sativum contained 1 g cellobiose, 31.2 mg vitamin C, 14.2 μg of selenium and 10 μg of sulphur, while 100 g of Capsicum chinense contained 76.4 mg of vitamin C, 8.8 μg of selenium and 36.6 g of cellobiose . Dry plants were ground into a powder. The voucher of the specimen of the powder was not deposited in a publicly available herbarium. The powder of 500 mg was suspended in 50 mL of DMEM to which penicillium and streptomycin were added, together with glutamax (1 % v/v each of antibiotic and glutamax in the final mixture). The mixture was left at room temperature for 3 days and shaken several times in between. After 3 days the extract was filtered and diluted to reach target concentrations (1 μg/mL to 10 mg/mL) and by these means ready for the experiments and HPLC analysis.
We prepared the samples for the HPLC analysis by adding 10 g of the pulverized plant into 10 mL of distilled water. After three days of incubation at room temperature, we filtered the sample through 0.2 μm Sartorius filter for syringes. The filtrate was then mixed with the buffer 2 (1 % TFA (v/v) in 10 % acetonitrile (v/v)) to a final concentration of 10 mg/mL. For the preparation and flushing of the column C4 we used buffer 1 (1 % TFA (v/v) in water).
As a first step we analysed 600 μL of the sample (300 μL of the extract and 300 μL of buffer 2). HPLC analysis duration was 45 min and during this time 31 fractions with different elution times were acquired. The procedure was repeated with a larger sample volume of 1 mL (500 μL of the extract and 500 μL of buffer 2).
Further HPLC analysis was performed in the following manner. The separations were performed using a Finnigan Surveyor HPLC system equipped with a photodiode UV–VIS detector (Thermo Electron Corporation, San Jose, CA, USA) using a 50 mm light-pipe flow cell and data acquisition software ChromQuest version 3.1.6 (Thermo Electron Corporation). The column used was Hypercarb (100 % porous graphitic carbon phase) with dimensions of 100 mm × 3 mm i.d. (Thermo Electron Corporation) and its temperature was kept at 45 °C. UV detection was performed at 300 nm. The flow rate during analysis was constant with 1 mL/min and the injection volume was 10 μL. Mobile phase A consisted of ultrapure water with formic acid 1 % v/v, while mobile phase B consisted of acetonitrile with 1 % v/v formic acid.
Analysis was performed on HPLC instrument Hewlett Packard Series 1100. The instrument was equipped with 1000 μL injection loop, fluorescent detector and ultraviolet detector and integrator. Fluorescence: excitation 280 nm and emission 325 nm, UV detector: 215 nm. As a mobile phase we used one liter of the buffer consisting of 40 % v/v acetonitrile and 60 % v/v distilled water miliQ with the addition of TFA in the final percentage of 1 % v/v. Flow rate was 1.5 mL/min. After the analysis was concluded, we obtained 93 fractions.
All cells were grown in 10 cm Petri dishes (N = 3) to obtain 90 % confluency and afterwards washed with 1 × PBS and treated with Tryple Select 5–10 min. In the next step the cells were transferred into 96-well plate (N = 1 for 96-well plate, each concentration in 96 well-plate was represented by N = 3 repeats) and 6-well plates (N = 1 for 6-well plate, each concentration in 96 well-plate was represented by N = 3 repeats) at a ratio of 1 × 105 cells/well (HepG2) and grown overnight. The next day the cells were treated with either the HPLC fraction 10 containing cellobiose sulphur, selenium and vitamin C, solution of pure cellobiose itself or with cellobiose in the combination with added vitamin C, sulphur and selenium in the concentration range of 0.00 l–1000 mg/mL (raising concentrations by a factor of 10; N = 3 for each concentration) and incubated for 24 h. The controls are represented by the same cell line (HepG2) maintained in culture in two different ways, with and without antibiotics. All experiments were repeated three times.
Determination of cell death
After the incubation period as described above under the section Cell culture, we estimated cell morphology using light microscopy (Olympus IX71, Japan, magnification 40 and 60). The number of viable cells in proliferation or dead cells were measured by the CellTiter 96R AQueous One Solution Reagent, which contains a novel tetrazolium compound. The MTS tetrazolium compound (Owen’s reagent) is bioreduced by cells into a colored formazan product that is soluble in tissure culture medium. Therefore in the end we then added 20 μL of MTS into 100 μL samples in the 96-well plate (N = 3 for each concentration). After 45 min of incubation we measured the absorbance at 490 nm with a 96-well plate reader (TECAN XFLUOR4 version V 4.51). The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in the culture. We calculated the average absorbance of three parallels for control cells, dead cells and individual samples. The percentages of dead cells were calculated according to established formula: (average aborbance of sample - average absorbance of dead cells)/(average absorbance of control cells - average absorbance of dead cells) in percentage. For further analysis of caspase activity in treated HepG2 cells we had chosen the concentrations of plant extracts that induced 20–50 % loss of viability, as determined by the MTS assay (N = 3).
Caspase (DEVD-ase) activation determination
Cells were cultured and treated as described above. Then, to prepare the cell extracts for caspase activity detection, cells were collected in three parallels, pelleted by centrifugation at 1000 rpm for 5 min and washed twice with PBS. Whole-cell extracts were prepared in 35 μl RIPA buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 0.1 % (w/v) SDS, 1 % (v/v) Nonidet P-40 0.5 % w/v deoxycholic acid, 1 mM EDTA) in three parallels. After 7 min of incubation on ice cells were again subjected to centrifugation this time 5 min on 14,000 rpm. In white 96-well-plate we transferred 40 μl of each samples and added 50 μl of 2 × caspase buffer/ 1 M DTT and 10 μl of DEVD substrate. We gathered 40 μg of protein from cells, untreated and treated with the plant extract in the presence or absence of inhibitor z-VAD-fmk, to determine caspase activity and by measuring the proteolytic cleavage of the fluorogenic substrate Ac-DEVD-AFC (Bachem) as also shown in standard protocols . Before absorbance measurement the plate was incubated at 37 °C in the incubator with 5 % CO2. The absorbance was measured by TECAN xfloor4 program at 400–505 nm and DEVD-ase activity calculated in RFU .
Apoptosis detection by Annexin V and 7-AAD staining
Early apoptotic and dead cells were quantified by flow cytometry measurements of phosphatidylserine exposure and 7-AAD incorporation. For this purpose, the cells were cultured and treated as described in the sections above. Afterwards the cells were washed twice with cold PBS and then resuspended in 1X Binding Buffer at a concentration 1 × 106 cells/mL. Briefly, 100 μL aliquots of cells were labelled with 5 μl annexin V-PE and 5 μl 7-AAD according to the manufacturer’s instructions and transferred into 5 mL tube for each concentration of plant extract, pure cellobiose or combination of molecules separately. The cells were gently vortexed and incubated for 15 min at room temperature (25 °C) in the dark. In the end 400 μl of 1X binding buffer was added to each tube. The cells were then subjected to flow cytometry analysis using FACScalibur flow cytometer (BD Biosciences) and analyzed with the CellQuest software within one hour.
Determination of apoptotic pathway
To determine by which apoptotic pathway (intrinsic, extrinsic) the plant extract triggers apoptosis we used Mitotracker Red CMXRos to assess and monitor the integrity of mitochondria. Cells were cultured and treated as previously described, and after the incubation period, cells were washed twice with cold PBS and then resuspended in Mitotracker CMXRos, which was added to the cells at a final concentration of 20 nM. Following 30 min incubation at 37 °C the cells were washed in PBS and again re-suspended in PBS for the measurement on a flow cytometer. Afterwards we measured red fluorescence of 5000 or 10,000 cells per sample, corresponding to mitochondria using the FL3 channel on a flow cytometer.
Mass spectrometric analysis
Mass spectrometric analysis measurements of the Solanaceae plant extract were run on a hybrid quadrupole time of flight mass spectrometer (Q-TOF) provided with an orthogonal Z-spray ESI interface (Waters Micromass, Manchester, UK). Mass spectrometer was interfaced to an ultra performance liquid chromatography (UPLC) system based on a Waters Acquity (Waters, Milford, USA) binary pump with a BEH C18 column (1.7 μm, 50 × 2.1 mm i.d). The mobile phases consisted of water and acetonitrile with a mixture of 0.1 % of formic acid in water. Compressed nitrogen (99.99 %, Messer Slovenia) was used as both the drying and the nebulising gas. The nebulizer gas flow rate was set to approximately 20 L/h and the desolvation gas flow rate to 600 L/h. A cone voltage of 30 V and a capillary voltage of 2.7 kV were used in a positive ion mode. The desolvation temperature was set to 250 °C and the source temperature to 150 °C. The mass resolution of approximately 9500 (fwhm) was used for determination of elemental composition with TOF mass spectrometer. MS and MS/MS spectra were acquired in centroid mode over an m/z range of 50–1000 in a scan time of 0.25 s and inter scan time of 0.05 s. For MS/MS experiments, argon (99.995 %, Messer Slovenia) was used as collision gas at a pressure of approximately 2 × 105 mbar in the collision cell. Product ion spectra were generated at collision energies profile: 10–30 V. The detector potential was set to 2300 V. Reproducible and accurate mass measurements at 10,000 mass resolution were obtained using an electrospray dual sprayer with leucine encephalin ([MH] + = 556.2771) as a reference compound, introduced into the mass spectrometer alternating with a sample solution. The data station operating software was Mass Lynx v. 4.1 (Micromass, Manchester). Several chromatographic peaks were identified with mass spectrometric detection.
1H-1H and 1H-13C correlation NMR spectra of plant extract were acquired on a Agilent (Varian) VNMRS 800 MHz NMR spectrometer equipped with a cold probe. 1H-1H TOCSY spectra were recorded with mixing times of 20, 40, 60 and 80 ms on an Agilent (Varian) Unity Inova 300 MHz NMR spectrometer equipped with ID probe. All data were recorded in 2H2O at 298 K. 1H chemical shifts were referenced to the residual solvent signal of 2H2O at δ 4.80 ppm. 13C chemical shifts have been determined on the basis of 1H-13C correlations in HSQC spectrum and are reported relative to TMS (δ = 0 ppm).
Analysis and statistics
The percentage of apoptotic and dead cells were reported as mean ± standard error. Data were analyzed using the two-way analysis of variance (ANOVA). Differences between experimental and control groups and among different exposure groups were regarded as statistically significant at the level of * p < 0.05, **p < 0.001 and *** p < 0.0001.