2.1. Compositional Analysis of Cornelian Cherry (Cornus mas)
In the present study Cornus mas L. fruits of “Skinitsa” variety, cultivated in Veria area, Greece, were used. The Cornus mas L. fruits of full maturity were obtained from a commercial field and the year of sampling was the year when the project started (2012). Colour parameters were determined in healthy fruits, mineral nutrient, sugar and organic acid contents were measured in the flesh and juice, and the fatty acid composition in the kernel.
The Cornelian cherry fruit skin colour from healthy fruits was measured by the use of a portable Minolta colorimeter. Colour parameters
L*,
a*, and
b* were determined by the CIELAB colour space, defined by the International Commission on Illumination (CIE), where
L* indicates lightness and
a* and
b* are chromaticity coordinates;
a* and
b* are colour directions: +
a* is the red axis, −
a* is the green axis, +
b* is the yellow axis and −
b* is the blue axis. Water content was determined according to the official method of AOAC (Association of Official Agricultural Chemists) International [
11].
Preparation of fruits and fruit juice: Fruits collected in full maturity were washed, cut, the kernel was removed and the rest of the material, the pulp, was lyophilized and kept under −4 °C until use. In this lyophilized material, the mineral nutrient, sugar and organic acid contents were measured. The fruit juice was extracted from a different batch of fresh fruits. Cornelian cherry fruits were pressed through a laboratory press to obtain the fruit juice. Juice was kept frozen until it was further analysed for acidity, pH and Brix, as well as later for examining the antioxidant and antiproliferative activity.
Fat composition: Lyophilized fruits were used for fat extraction by the Soxhlet method.
Fatty acids analysis: The fatty acid analysis was done on dried fruit kernels. The fruit kernels were removed from the fruits, were grounded and their oil was extracted by the Soxhlet apparatus through the method of AOAC (991.31.1997) [
11]. The fatty acid composition and content (%) were analysed by gas chromatography-mass spectrometry (GC-MS) (Model Varian CP-3800, equipped with flame ionization detector, Thermo Fisher Scientific, Waltham, MA, USA), after methylation with boron trifluoride for fatty acid methyl esters (FAMEs) as described previously [
12]. The FAMEs were suspended in hexane and analysed by gas chromatography with an Agilent DB23 capillary column (Model No. 123-2332, 30.0 m × 0.32 mm × 0.25 μm, Agilent J and W Scientific, Santa Clara, CA, USA). Helium gas was used as a carrier gas with a column flow rate of 2.0 mL/min.
For the analysis, the following set-up conditions were used: Initial oven temperature was set at 150 °C, held for 18 min, subsequently rammed to 185 °C at a rate of 5 °C/min. Then the oven temperature was increased to 210 °C at a flow rate of 5 °C/min and held for 2 min, and then further increased to 240 °C at a rate of 10 °C/min. The injector and flame ionization detector temperatures were set at 260 and 270 °C, respectively. Individual fatty acids were identified by comparison of their retention times with external standard (Supelco 37 Component FAME Mix, CRM47885, Sigma-Aldrich, St. Louis, MO, USA) retention times. The amounts of individual fatty acid methyl esters identified were obtained from the chromatogram areas and expressed in percentage of the total fatty acids.
Organic acid analysis: Sugar and organic acid analyses were performed in the received juice. Organic acid analysis was also performed in the lyophilized fruits. Sugars and organic acids were analysed simultaneously by high performance liquid chromatography (HPLC model Agilent 1100, equipped with UV-MWD and RI detector, Agilent, Santa Clara, CA, USA). The separation was carried out on Aminex HPX-87H cation-exchange columns (300 × 7.8 mm I.D., Bio-Rad Laboratories, Hercules, CA, USA). The eluent was 4.5 mM sulphuric acid. Flow-rate was 0.7 mL/min, the column temperature was set at 65 °C and injection volume was 20 μL. The samples, after they were pitted, were lyophilized. Then they were diluted with water, centrifuged, filtered through 0.45 μm and injected into the HPLC system. The sugars and organic acids were determined using the external standard method.
2.2. Cell Lines and Cell Cultures
The human hepatocellular carcinoma HepG2, the human breast adenocarcinoma MCF-7, the human colon adenocarcinomas Caco2 and HT-29 were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Murine colon carcinoma CT-26 was kindly provided by V. Schirrmacher (DKFZ, Heidelberg). HepG2, MCF-7, HT-29 and CT-26 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), while Caco2 cells were grown and maintained in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), both supplemented with 10% fetal bovine serum (FBS) (Biosera, Boussens, France), penicillin (100 U/mL), and streptomycin (100 mg/mL) (Biosera, Boussens, France) and were incubated at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2. Stock cultures were passaged at 2 to 3-day intervals. For the Sulforhodamine B (SRB) assay, cells were seeded at a density of 5 × 103 cells/well in 96-well plates.
2.4. Determination of the In Vitro Antiproliferative Effect of Cornus mas L. Juice (Sulforhodamine B Assay)
The viability of cancer cells after treatment with the juice was determined using the Sulforhodamine B (SRB) assay, as previously described with few modifications [
14]. SRB is a dye that binds to basic amino acids of cellular proteins and, then, the number of viable cells is estimated with colorimetric evaluation [
15]. Cells were plated in 96-well plates and treated with different concentrations of the juice (0.0007–1%
v/
v). Then, the cells were fixed with the addition of 25 μL of 50% (
w/v) cold trichloroacetic acid (TCA) (Applichem, Darmstadt, Germany) to the growth medium and incubation of the plates at 4 °C for 1 h. The cells were washed five times with tap water and then stained with 50 μL of 0.4% (
w/v) SRB dye (Sigma-Aldrich, St Louis, MO, USA) in 1% (
v/
v) acetic acid (Scharlau, Barcelona, Spain) for 30 min at room temperature. Next, the cells were rinsed five times with 1% (
v/
v) acetic acid to remove the unbound dye. The fixed, stained plates were allowed to air-dry and afterwards the bound dye was solubilized by adding 100 μL of 10 mM Trizma base (Sigma-Aldrich, St Louis, MO, USA) for at least 5 minutes. Absorbance was measured at 570 nm using an ELISA plate reader (Sunrise, Tecan, Männedorf, Switzerland) and the percent cellular survival was calculated using the formula: