Chemical and biological characteristics of propolis from Apis mellifera caucasica from the Ardahan and Erzurum provinces of Turkey: a comparative study

All chemicals were purchased from Merck KGaA (Darmstadt, Germany) and its subsidiary Sigma-Aldrich (St. Louis, MO, USA) and included the following: certified reference material (CRM) BCR679 for mineral analysis, glucose, vanillic acid, sinapic acid, trans-ferulic acid, naringenin, sucrose, caffeic acid, p-coumaric acid, and sorbic acid standards (Sigma-Aldrich); vitamin C, benzoic acid, standard fatty acids CRM47885 (37 Component FAME Mix, Supelco, Bellefonte, PA), and standard fructose (Merck KGaA).

All the propolis samples were collected in October 2016, placed in clean plastic pouches, and stored at -80 °C until processing. Ardahan propolis samples were obtained from A. m. caucasica beehives located in Posof in Ardahan province, and Erzurum propolis samples were obtained from A. m. caucasica beehives located at the apiculture units of the Atatürk University.

To obtain biologically active compounds we used the ethanolic extraction method described elsewhere (18). Briefly, 2 g of solid propolis was mixed with 100 mL of 70 % ethanol. The mixture then sonicated in an ultrasonic bath (model 621.08.001, Isolab Laborgeräte GmbH, Eschau, Germany) at 300 W for 30 min. The solvent was removed in a rotary evaporator (Rotavapor R-210, Büchi Labortechnik AG, Flawil, Switzerland) at 16–17 kPa (160–170 mbar) and 60 °C. Extraction yields were 20.1 % (396.7/1973.6 w/w) and 23.75 % (486.7/2045.2 w/w) for the Ardahan and Erzurum samples, respectively. Dried matter was weighed and dissolved in 70 % ethanol to obtain a 20 mg/mL solution for further use, except for microbiological analysis (19). In our preliminary experiments, however, this ethanolic extract showed no antimicrobial activity against the tested microorganisms, which is why we used another extraction method to obtain propolis balsam for antimicrobial activity tests, as follows (20): 30 g of solid propolis was mixed with 300 mL of 95 % ethanol. The mixture was shaken in a shaker (SI-300, Lab Companion, Daejeon, South Korea) at 37 °C for 96 h, and the solvent evaporated in a rotary evaporator (RE100-Pro, SciLogex, Rocky Hill, CT, USA).

Because of lower mineral levels in the obtained propolis ethanolic extracts compared to raw samples reported elsewhere (21), we decided to determine the whole mineral content in raw material. Each raw propolis sample was divided in three samples, each analysed in triplicate to ensure statistical comparison. For this purpose, we used microwave-assisted digestion as described by Korn et al. (22) and analysed the samples for Co, Se, Li, Cd, As, Cr, Ni, Pb, Cu, Ca, Mg, K, Mn, Na, Zn, Fe, and Al content with inductively coupled plasma mass spectrometry (ICP-MS; NexION 350X, Perkin Elmer Inc., Waltham, MA, USA). Standard yttrium was also read to ensure the precision of the device (recovery interval was 97.8–119.3 %). Method accuracy was tested with the CRM BCR679 using the same protocol. Cd, Cu, and Ni were within the range of 95 % confidence interval (CI₉₅), while Zn was slightly above the CI₉₅ reported for the CRM.

N, C, and S content was determined with an elemental analyser (Flash 2000, Thermo Fisher Scientific Inc., Waltham, MA, USA). Approximately 2 mg of raw propolis were digested with oxygen at 950 °C using helium as mobile phase. The results are given as the percentage of the total mass.

Fatty acid content in raw propolis was analysed in a gas chromatograph equipped with a flame ionisation detector (FID) (GC QP2010 Plus, Shimadzu Corp., Kyoto, Japan). Lipid extraction followed the method described by Hara and Radin (23) with a minor modification as follows: 5 g of raw propolis was homogenised in 6 mL of 3/2 (v/v) hexane/isopropanol mixture for 30 s and the homogenate centrifuged at 4500 g for 10 min. Methyl ester forms were derivatised according to the method described by Christie (24). Methyl ester derivatives were then injected into the Rx-2330 column (60 m x 0.25 mm inner diameter, 0.1 μm film thickness, Restek Corp., Bellefonte, PA, USA) in split mode at 250 °C using helium as carrier gas at 113.1 mL/ min. The temperature program of the column was as follows: the initial temperature of 130 °C was held for 4 min, then increased at 3 °C/min to 230 °C and held for 10 min. The temperature of the FID was 255 °C. Standards were used to determine the retention time of fatty acids. The content of each fatty acid was calculated as the percentage of total peak area obtained from whole fatty acids in a propolis sample.

Here too we used raw propolis samples because of higher solubility of carbohydrates and vitamin C in water than in ethanol. 2 g of raw propolis was vortexed and then sonicated in a 2 mL of 95/05 methanol/water mixture (pH 3.0) for vitamin C analysis. 50 μL of this extract was injected into a high-performance liquid chromatograph (HPLC) (Prominence LC-20A, Shimadzu Corp.) equipped with a C18 ODS3 column (150 x 4.6 mm, 5 μm, Inertsil, GL Sciences Inc., Tokyo, Japan). The injection volume, pressure, flow rate in isocratic mode, and temperature were 50 μL, 200 bar, 1 mL/min, and 40 °C, respectively. Vitamin C content was determined with the photo-diode array detector (SPD-M20A, Shimadzu Corp.) at 242 nm. The mobile phase was methanol/water mixture (5/95 v/v, pH 3). Vitamin C concentration in propolis was calculated using the standard curve.

For carbohydrate analysis, raw propolis (5 g) was mixed with 80 mL of ultra-pure water and then with 20 mL acetonitrile. Fructose, glucose, and sucrose in this extract were analysed with an HPLC (Prominence LC-20A, Shimadzu Corp.) equipped with an NH₂ column (250 x 4.6 mm, 5 μm, Inertsil, GL Sciences Inc.) and a refractive index detector (RID-20A, Shimadzu Corp.). The mobile phase was acetonitrile/water mixture (80/20 v/v). The injection volume, column pressure, flow rate in isocratic mode, and temperature were 20 μL, 200 bar, 1.3 mL/min, and 30 °C, respectively. Standard curves prepared with fructose, glucose, and sucrose were used to calculate the amount of these components in propolis samples.

Alkaloids, organic acid, and flavonoids were determined in ethanolic propolis extracts with a gas chromatograph (7890, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a mass spectrometer (5975C, Agilent Technologies Inc.). Silylation followed the method described by Proestos and Komaitis (25). The injection temperature was 280 °C, split ratio 40:1, flow rate 1 mL/ min, and the run time 35 min. The composition of the eluates was matched with the NIST MS Search 2.0 library (National Institute of Standards and Technology, Gaithersburg, MD, USA).

Phenolic acid content was analysed in ethanolic propolis extracts with an HPLC (Prominence LC-20A, Shimadzu Corp.) equipped with an ODS-3 column (250 x 4.6 mm, 5 μm, Inertsil, GL Sciences Inc.). The injection volume was 20 μL, column pressure 200 bar, flow rate in gradient mode 0.7 mL/min, and temperature 25 °C. Eluent A was a mixture of methanol, water, and acetic acid (10/89/1 v/v/v) and eluent B a mixture of methanol and acetic acid (99/1 v/v). The gradient program started with 100 % of solvent A to gradually reduce it to 95, 80, 75, 70, 60, 50, 40, and 0 % at 3, 18, 30, 35, 40, 55, 65, and 68 min, respectively. Diode array detector (SPD-M20A, Shimadzu Corp.) was used at 226 nm wavelength for benzoic acid and at 254 nm wavelength for vanillic acid, sinapic acid, trans-ferulic acid, naringenin, caffeic acid, p-coumaric acid, and sorbic acid. Standard curves were prepared to determine their amounts in the samples.

Total phenolic and flavonoid contents were determined spectrophotometrically (S1205, Unico Science, Dayton, NJ, USA) in 20 mg/mL propolis extracts through total antioxidant capacity determination based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) reducing potential and ferric reducing antioxidant power (FRAP).

For total phenolic content determination, we used the Folin-Ciocalteu method as described elsewhere (26). 0.5 mL of propolis extract was mixed with 2.5 mL of 0.2 eq/L Folin-Ciocalteu’s reagent and 2 mL of 75 g/L sodium carbonate, and the mixture was incubated at room temperature for 2 h. The absorbance of the final solution was measured at 760 nm and converted to mass fraction using the standard graphic prepared with gallic acid (in the range of 0–250 mg/L). All the assays were done in triplicate, and the results presented as mg of gallic acid equivalent (GAE) per gram of propolis.

Total flavonoid content was spectrophotometrically determined following the methods described elsewhere (27, 28). 0.5 mL of the propolis extract was incubated with 1.5 mL of 95 % ethanol, 0.1 mL of 10 % of AlCl₃, 0.1 mL of 1 mol/L potassium acetate, and 2.8 mL of distilled water at room temperature for 30 min. Absorbance was measured at 415 nm and converted to mass fraction using the standard graphic prepared with quercetin (in the range of 0–700 mg/L). Data were presented as μg of quercetin equivalent (QE) per gram of propolis.

Total antioxidant capacity of ethanolic propolis extracts was determined with two methods: DPPH and FRAP. For the DPPH determination we mixed 1.5 mL of extract with 1.5 mL of 0.1 mmol/L DPPH and incubated the mixture in a dark place at room temperature for 50 min. Absorbance was measured at 517 nm and converted to concentration expressed as percentage of control using the formula provided by Molyneux (29).

For the FRAP analysis we mixed 100 μL of extract with 3 mL of freshly prepared FRAP solution and incubated it at 37 °C for 4 min. Absorbance was measured at 595 nm and converted to the concentration using the standard graphic prepared with FeSO₄ (in the range of 0.1– 10 mmol/L). The analysis was done in triplicate and data presented as μmol/L of FeSO₄ equivalent per gram of propolis (30).

For cytotoxicity tests we used human peripheral lymphocytes from blood samples donated by healthy volunteers (two men and two women), whose participation was approved by the Ethics Committee of the Kafkas University Faculty of Medicine (approval no. 80576354050-99/158). 10 mL of whole blood was collected with a sterile syringe from each donor. Mitomycin C (MMC) was used a cytotoxic agent (positive control) in the concentration of 0.25 μg/mL (0.74 μmol/L), which was based on our preliminary tests and an investigation by Kocaman and Topaktaş (31), who used mitotic index as toxicological endpoint. Lymphocyte viability was tested with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which determines cellular metabolic activity (32). The lymphocytes were first cultured in a PB-MAX^TM karyotyping medium (ThermoFisher Scientific Inc., Waltham, MA, USA) and then isolated from whole blood with a Histopaque^®-1077 solution (Sigma-Aldrich). Isolated cells were counted, placed into the PB-MAX^TM karyotyping medium (75 cells/μL of the medium), and incubated in a 5 % CO₂ Forma incubator (ThermoFisher Scientific Inc.) at 37 °C for 72 h. Propolis extracts were added at different concentrations either 24 h or 48 h after incubation started to see their 48 or 24 h effect on the lymphocytes. The concentrations used were based on preliminary 24 h LC₅₀ test as follows: 500 μg/mL (LC₅₀), 250 μg/mL (1/2 LC₅₀), 125 μg/mL (1/4 LC₅₀), and 62.5 μg/mL (1/8 LC₅₀). They were added to cultures with or without 0.25 μg/mL (0.74 μmol/L) of MMC. Solvent control consisted of cultures with added ethanol as solvent (10 μL/mL) and negative control of untreated cultures in the medium. Four tubes were prepared for each propolis concentration and controls. After 24 h or 48 h of exposure to propolis, three measurements were done to evaluate cell viability using the MTT cell proliferation assay kit (Vybrant^®, ThermoFisher Scientific Inc.) according to the kit protocol. The cells were seeded into wells of a microplate and 10 μL of 12 mmol/L MTT was added to each well. The microplate was incubated at 37 °C at 5 % CO₂ for 4 h. Absorbance was measured at 540 nm on a microplate reader (EON^TM, BioTek, Winooski, VT, USA), and cell viability calculated using the formula provided by Cheki et al. (33).

MCF-7 human breast cancer cells [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured aseptically in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Thermo Fisher Scientific Inc.) containing 10 % foetal bovine serum (FBS; PAA Laboratories, GE Healthcare, Chicago, IL, USA), 1 % antibiotic solution (100 IU/mL penicillin and 0.1 mg/mL streptomycin) (Sigma-Aldrich) at 37 °C and 5 % CO₂. The medium was removed at 80 % cell growth, and trypsin-EDTA solution (Gibco, ThermoFisher Scientific Inc.) was added until it covered the plate surface (8–10 mL). Separated cells were collected and dissolved in complete-DMEM after removing the trypsin solution. Oxidative stress was tested in MCF-7 cells treated with 0.25 μg/mL of MMC (0.74 μmol/L) and propolis extracts from both provinces in one of the following concentrations: 32.5, 65, 125, 250, or 500 μg/mL. Antioxidative activity of propolis was established as reduction in respect to positive control treated with MMC alone. For negative control we used MCF-7 cells in culture and for solvent control MCF-7 cells treated with 10 μL/mL of ethanol. Oxidative stress was determined by measuring thiobarbituric acid reactive substances (TBARS) using a method described by Jain (34) and modified by Do et al. (35). 400 μL methanol containing 0.01 % butylated hydroxytoluene and 500 μL 1 % thiobarbituric acid dissolved in 1 % sulphuric acid was mixed with 100 μL supernatant. The mixture was vortexed and then incubated at 100 °C for 15 min. The absorbance of the resulting supernatant was obtained at 532 and 600 nm and converted to nmol/mg protein using a standard curve prepared with 1,1’,3,3’-tetramethoxypropane. Total protein was measured using a modified Lowry method as described elsewhere (36).

MCF-7 cells were cultured and treated as described in the above subsection. Cells were counted in a Thoma counting chamber (Isolab Laborgeräte GmbH) under a microscope (Olympus Corp., Tokyo, Japan) at 10x magnification and seeded onto a 6-well plate (4500 cells per well). The plates were incubated at 37 °C and 5 % CO₂ for 24 h, treated and incubated for another 24 h.

Apoptotic cells were counted using the Roche In Situ Cell Death Detection Kit according to the producer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). Treated cells were incubated with 0.002 % Triton X-100 for 10 min, added 50 μL of terminal deoxynucleotidyl transferase (dUTP) nick end labelling (TUNEL) master mix per well, and incubated at 37 °C and 5 % CO₂ for another 80 min. Red-stained TUNEL-positive cells were visualised under a fluorescent microscope (BX43 equipped with DP74 camera, Olympus Corp.) at 20x magnification and quantified with Image Processing and Analysis in Java version 1.51h software (ImageJ; National Institutes of Health, Bethesda, MD).

Propolis extracts were sterilised through 0.22 μm Millipore filters and their antimicrobial potential tested by disc diffusion or broth microdilution (37, 38) against known bacterial and fungal pathogens, cultured in Mueller-Hinton agar and 2 % Sabouraud dextrose agar, respectively. These included gram-positive bacteria Bacillus megaterium (Kahramanmaraş Sütçü İmam University Department of Biology, Kahramanmaraş, Turkey) and Staphylococcus aureus (ATCC 6538), gram-negative Pseudomonas aeruginosa (ATCC 9027) and Klebsiella pneumoniae, and fungi Yarrowia lipolytica and Candida albicans (Kahramanmaraş Sütçü İmam University). Gentamycin (Gen), ampicillin (Amp), erythromycin (Ery), and penicillin (Pen) were used as positive controls for the disc diffusion method. After 48 h of treatment, inhibition zones were measured with a digital calliper. All the tests were triplicated, and the results presented as means ± standard deviations.

As the fungal species and P. aeruginosa showed no inhibition zone with broth microdilution method in our study, we tested antimicrobial activity of propolis against these species only with the disc diffusion method (39).

For broth microdilution method we diluted the propolis extracts to 0.67–173.5 μg/mL for the Erzurum and to 0.95–245.7 μg/mL for the Ardahan samples according to our preliminary experiments. After incubation at 37 °C for 18 h, 0.5 % 2,3,5-triphenyltetrazolium chloride (TTC) was added, and incubation continued at 37 °C for another 30 min. Plate wells without colour change were considered containing minimal inhibition concentrations (MIC) of propolis (40).

All statistical analyses were run on SPSS Statistics for Windows, Version 17.0 (SPSS Inc., Chicago, IL, USA). Normality of distribution was assessed with the Kolmogorov-Smirnov test, except for the MTT assay, for which we used the Shapiro-Wilk test. For the data that did not show normal distribution we ran the non-parametric Kruskal-Wallis test, followed by the post-hoc Mann-Whitney U-test. Data with normal distribution were analysed with one-way analysis of variance (ANOVA). Homogeneity of variance was tested with Levene’s test. Duncan post-hoc test was applied for homogeneous subsets, while Dunnett’s C test was used for non-homogeneous ones. All data are presented as means ± standard deviations. Significance (P-value) was set at 0.05.