Bees produce propolis from beeswax, vegetable balsam, pollens, and resins to strengthen and disinfect their beehives (1), and its biological properties have shown potential for human use from ancient times. Current research has established that its chemical composition and antimicrobial, antioxidant, anti-inflammatory, and anticarcinogenic properties vary from one location to another (2, 3, 4, 5, 6, 7, 8), as they stem from plant origin, phenolic compounds, flavonoids and their esters (9, 10, 11), climate, season, time of collection, and bee race (12, 13). Another important factor to consider is contamination of the beehive location (14).
Spanning over different climatic and geographic regions Turkey has different honeybee races and ecotypes. The Erzurum province accommodates smaller, yellow-coloured local bee ecotype, too aggressive for keeping, and the more common genotypes
Considering that an earlier study singled out antimicrobial activities of propolis from
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,
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
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
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,
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-MAXTM 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-MAXTM 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 (EONTM, 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
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
As the fungal species and
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
Table 1 presents the mineral composition of propolis from both provinces. It was statistically similar except for Co, As, Cr, and Ca, which was higher in the Erzurum samples. The order of abundance in the Ardahan propolis samples was K>Fe=Al=Mg=Ca>Na>Zn>Mn>Cu>Ni= Cr=Pb>Li=Co=As>Se>Cd and in the Erzurum samples K >Mg = F e = C a = A l > N a > Z n >Mn >Cu = C r ≥Ni=Pb>Li=Co=As>Se>Cd. For comparison, reports on propolis mineral composition from Croatia (21) and Spain (41) single out Ca, Mg, K, Al, Fe, Na, and Zn as the most abundant. Spanish propolis was reported similar or higher Cd, Ni, Fe, and Zn levels as did Polish (42).
Comparison of mineral composition of
Element | Ardahan (mg/kg) | Erzurum (mg/kg) |
---|---|---|
Co | 0.14±0.04 |
0.25±0.06 |
Se | 0.038±0.016 | 0.025±0.006 |
Li | 0.20±0.04 | 0.22±0.04 |
Cd | 0.005±0.006 | 0.004±0.004 |
As | 0.13±0.02 |
0.22±0.049 |
Cr | 0.98±0.22 |
1.39±0.18 |
Ni | 1.08±0.23 | 1.53±0.34 |
Pb | 0.83±0.19 | 0.97±0.19 |
Cu | 2.45±0.16 | 2.01±0.79 |
Ca | 269.42±51.83 |
428.97±76.28 |
Mg | 376.17±85.91 | 560.21±171.45 |
K | 1156.06±278.58 | 2607.36±1468.87 |
Mn | 5.297±0.71 | 7.47±3.27 |
Na | 193.19±15.34 | 203.47±9.58 |
Zn | 30.05±7.30 | 41.77±19.65 |
Fe | 428.51±77.75 | 507.62±287.13 |
Al | 408.46±88.17 | 406.86±202.20 |
% of total mass | % of total mass | |
N | 0.42±0.09 | 0.35±0.02 |
C | 66.84±1.38 | 64.06±6.38 |
S | ND | ND |
Data are given as mean ± standard deviation (
According to Kruskal-Wallis
We believe that similarities in the mineral composition between the propolis from the two provinces is owed to their geographic vicinity. Similar reason may explain the highest Ca content in Turkey in propolis from these two adjacent provinces reported by Yozgat and Sivas (43).
Propolis from both our provinces had considerably lower Cd levels than reported in other Turkish provinces (44), most likely because these two provinces have little industry. However, Pb and Cr levels were much higher than in the rest of Turkey (44) and some regions of Croatia (21). Similar or lower levels than ours were reported in Polish and Spanish propolis (41, 42). While no data are available about soil mineral composition for both provinces, Erzurum is known for Cu, Pb, Zn, and Cr mining areas (44), especially near the Ardahan province border. This may partly explain the highest levels of Pb and Cr.
As and Al levels deserve special attention, those in the Erzurum propolis in particular. They are in the range reported in previous studies (21, 41) and are below the acceptable daily intake thresholds (300–1400 μg/day for Al, 20–514 μg/day for Pb, 20–250 μg/day for Cr, 10–60 μg/ day for Cd, and 12–25 μg/day for As) (45).
We were surprised to see that propolis from neither province contained any sulphur, especially as it is an important component of various biological compounds. Similar absence of sulphur was reported in other Turkish provinces (43).
Table 2 shows that propolis fatty acid content differed between the provinces. Such variation has also been found in the rest of Turkey (46) and other countries of the world, such as Algeria (47), Brazil (48), and New Zealand (49).
Comparison of fatty acid content in
Fatty Acid | Retention time (min) | Ardahan (%) | Erzurum (%) |
---|---|---|---|
Caproic acid | 6.54 | – | 8.85 |
Caprylic acid | 9.24 | 0.26 | – |
Decanoic acid (capric acid) | 13.85 | 1.14–1.47 | 0.08 |
Undecanoic acid | 17.80 | 0.67 | – |
Dodecanoic acid (lauric acid) | 21.53 | 24.39–36.95 | 49.62–54.27 |
Tridecanoic acid | 25.38 | 5.00–6.59 | 6.29–8.10 |
Tetradecanoic acid (myristic acid) | 30.81 | 4.68–5.86 | 8.66 |
Pentadecanoic acid | 34.13 | 6.97–8.85 | – |
Hexadecanoic acid (palmitic acid) | 36.89 | 0.84–0.89 | 1.53 |
Heptadecanoic acid (margaric acid) | 41.10 | 1.03–1.64 | 0.54–1.12 |
Octadecanoic acid (stearic acid) | 44.19 | 1.86 | 1.48–2.12 |
Eicosanoic acid (arachidic acid) | 49.47 | – | 3.13–4.43 |
Heneicosanoic acid | 54.98 | 1.21 | 1.57–2.05 |
Docosanoic acid (behenic acid) | 56.66 | 0.54 | 0.78–1.35 |
Tricosanoic acid | 59.78 | 0.31–2.44 | 0.26 |
Lignoceric acid | 62.16 | 0.45–0.57 | 0.22–0.45 |
9-tetradecenoic acid (myristoleic acid) | 32.80 | – | 0.49 |
36.57 | 3.36–4.70 | 0.88–1.69 | |
39.52 | 1.48–2.61 | 0.32–2.83 | |
42.58 | 3.18–3.35 | – | |
45.97 | – | 0.59–1.73 | |
46.98 | 1.75–1.92 | 0.56–1.15 | |
51.98 | 1.14 | 1.61–2.28 | |
57.64 | 0.66 | – | |
65.02 | 0.54–0.79 | 0.16–0.26 | |
9,12-octadecadienoic acid (linolelaidic acid) | 47.80 | – | 1.04–1.09 |
Linoleic acid | 48.20 | 1.42–1.89 | 7.89–11.46 |
Gamma-linolenic acid | 50.96 | 0.61–1.26 | 1.10–1.39 |
Linolenic acid | 53.33 | 0.65–0.79 | 0.14–0.28 |
55.09 | 1.44 | 0.26 | |
57.22 | 16.30–22.14 | 0.16 | |
58.10 | 2.00–2.14 | 1.04 | |
59.28 | 0.94–2.69 | 0.66–0.92 | |
60.93 | 0.65–0.72 | – | |
64.00 | 0.79–2.16 | 0.07–0.40 | |
66.92 | 0.60–0.75 | 0.22–0.45 | |
Of the 36 fatty acids determined in the samples, six were found only in the Ardahan propolis and five only in the Erzurum propolis. The Ardahan propolis had higher mono- and polyunsaturated fatty acid content than the Erzurum propolis (12.1–15.2 % vs 4.6–10.4 % and 25.4– 36.0 % vs 12.6–17.5 %, respectively). The Erzurum propolis, in turn, had much higher saturated fatty acid content (83.0–93.3 % vs 49.4–69.8 %).
While our findings show the highest content of lauric acid in the
The saturated fatty acid content of the Ardahan propolis was between the one reported for Algerian (41 %) (47) and Romanian (71 %) (51) propolis samples, while the Erzurum propolis had much higher saturated fatty acid content. Judging by earlier reports from Erzurum (17) and Brazil (48, 52), not only did forage on several botanical species but also genetic differences influence hydrocarbon chemistry of propolis samples.
Table 3 shows vitamin C and carbohydrate content in propolis from both provinces. Vitamin C content was higher in the Ardahan propolis and was comparable to the one reported in India (53). Ardahan propolis also had higher fructose and glucose but lower sucrose content than the Erzurum propolis. According to reports from Egypt (54), Canary Islands (55), and South East England (56), propolis fructose, glucose, and sucrose content very much depended on plant origin and geographic region.
Comparison of vitamin C and carbohydrate content in
Ardahan (μg/g dry weight) | Erzurum (μg/g dry weight) | |
---|---|---|
Vitamin C | 40.31±2.97 |
16.18±1.48 |
Fructose | 1.58±0.30 |
0.86±0.18 |
Glucose | 0.98±0.20 |
0.39±0.05 |
Sucrose | 0.15±0.04 |
0.69±0.19 |
Data are given as mean±standard deviation (
Tables 4 and 5 show differences in alkaloid, organic acid, flavonoid, and phenolic content in propolis between the two provinces. The Ardahan samples had 17 and Erzurum 16 alkaloid, organic acid, and flavonoid compounds. Gallic acid, vanillic acid, benzoic acid, sorbic acid, naringenin, and myricetin were not found in samples from either province with HPLC analysis, but GC-MS revealed benzoic acid and naringenin peaks in the Ardahan propolis and naringenin peak in the Erzurum propolis. Ferulic acid in the Erzurum propolis was detected with both HPLC and GC-MS analysis, but only with HPLC in the Ardahan samples. This inconsistency points to the limitations of silylation, or characteristics of a particular column, as already observed in the study of García-Viguera et al. (57).
Alkaloid, organic acid, and flavonoid content in ethanolic extracts of
Peak | Retention Time (min) | Quantity in the sample (%) | Name ARDAHAN | Retention Time (min.) | Quantity in the sample (%) | Name ERZURUM | ج |
---|---|---|---|---|---|---|---|
7.41 | 0.70 | Benzyl alcohol | 8.43 | 1.59 | 2-Phenylethanol (Benzeneethanol) | ||
8.13 | 1.18 | Guaiacol | 9.28 | 1.35 | 1,2-dihydroxybenzene (pyrocatechol) | ||
9.02 | 3.85 | Benzoic acid | 9.52 | 0.62 | 2,3-dihydrobenzofuran (coumaran) | ||
9.53 | 7.64 | 2, 3-dihydrobenzofuran (coumaran) | 15.61 | 0.73 | 5-phenylpenta-2,4-dienoic acid (cinnamylidene acetic acid) | ||
10.71 | 19.15 | 4-hydroxy-3-methoxystyrene ( |
18.35 | 2.71 | 3-hydroxy-4-methoxycinnamic acid (isoferulic acid) | ||
11.64 | 0.61 | Iso-vanillin or vanillin | 18.89 | 0.74 | Palmitic acid | ||
15.91 | 0.60 | β-caryophyllene | 20.41 | 1.02 | 2-nonadecanone | ||
16.77 | 2.25 | Benzylbenzoate | 20.73 | 1.03 | Elaidic acid | ||
17.39 | 1.11 | [1S,3S,(+)]-l-methyl-3-isopropenyl-4- cyclohexene | 21.95 | 1.48 | Methyl 4-hydroxycinnamate ( |
||
20.54 | 13.22 | Benzyl cinnamate | 22.08 | 0.82 | 4-hydroxy-3- methoxycinnamic acid (ferulic acid) | ||
22.19 | 1.70 | Tricosane or eicosane | 22.19 | 2.14 | Tricosane or eicosane or |
||
23.34 | 0.59 | Corydaldme | 23.11 | 2.89 | 5-methylisophthalic acid | ||
23.57 | 0.79 | 6-methoxy-1,3-benzodioxole-5-carbaldehyde (6-metlioxy piperonal) | 23.62 | 1.73 | 1,2-dimethylcyclopropene | ||
23.78 | 1.42 | (E)-l-(2,6-dihydroxy-4-methoxyphenyl)-3- phenylprop-2-en-l-on (pinostrobin chalcone) | 23.82 | 9.64 | (E)-l-(2,6-dihydroxy-4-methoxyphenyl)-3-phenylprop-2-en-1-one (pinostrobin chalcone) | ||
24.26 | 3.06 | Pentacosane or eicosane | 24.27 | 2.14 | Pentacosane | ||
24.43 | 5.29 | Benzyl 4-acetylbenzoate | 24.79 | 20.74 | Pinocembrin | ||
24.75 | 9.42 | Pinocembrin | 26.45 | 5.31 | 5-hydroxy-7-methoxyflavone (tectochrysin) | ||
25.83 | 9.52 | 2,5-bis dimethylanino-3,9-dimethyl-3H-1,3,4,6-tetrasacyclopentazulene | 27.02 | 3.38 | Eicosane | ||
26.40 | 0.72 | 5-hydroxy-7-methoxyflavone (tectochrysin) | 27.34 | 10.02 | Naringenin or chrysophanic acid | ||
27.01 | 4.94 | Eicosane or heptacosane | 27.85 | 11.98 | Chrysin | ||
27.27 | 1.23 | Naringenin or chrysophanic acid | 28.44 | 0.93 | 4',5-dihydroxy-7-methoxyflavone (an apigenin derivative) | ||
27.74 | 2.68 | Chrysin | 28.76 | 6.92 | Galangin | ||
28.67 | 0.77 | Galangin | 29.20 | 0.94 | Sakuranetin | ||
29.18 | 1.11 | Sakuranetin | |||||
31.03 | 0.94 | Nonacosane | |||||
33.64 | 0.83 | 7,3',4'-trmethoxyflavone |
Comparison of phenolic acid content in
Phenolic acids | Ardahan (mg/kg) | Erzurum (mg/kg) |
---|---|---|
3,4,5-trihydroxybenzoic acid (gallic acid) | ND | ND |
4-hydroxy-3-methoxybenzoic acid (vanillic acid) | ND | ND |
3,4-dihydroxycinnamic acid (caffeic acid) | 0.033 | 0.046 |
4-hydroxycinnamic acid ( |
0.042 | 0.008 |
4-hydroxy-3,5-dimethoxycinnamic acid (sinapic acid) | ND | ND |
4-hydroxy-3-methoxycinnamic acid ( |
0.079 | 0.005 |
Quercetin | 0.018 | 0.054 |
Benzoic acid | ND | ND |
2,4-hexadienoic acid (sorbic acid) | ND | ND |
Naringenin | ND | ND |
Myricetin | ND | ND |
ND – not detected
The Erzurum propolis had higher pinostrobin chalcone, pinocembrin, tectochrysin, naringenin, chrysin, galangin, caffeic acid, and quercetin flavonoid and phenolic content than the Ardahan propolis. All these compounds are known anticarcinogens with or without antioxidant properties. Some have already been reported in the propolis of the three bee races from Erzurum studied earlier (17, 58), but our study is the first to report pyrocatechol, isoferulic acid,
Of the compounds with known antioxidant and anticarcinogenic potential Ardahan propolis had higher levels of sakuranetin,
Table 6 shows total flavonoid and phenolic content and total antioxidant capacity of propolis samples from both provinces. The Ardahan propolis had 2.18 times higher total phenolic and 1.79 times higher total flavonoid content. As a result, it had significantly higher antioxidant activity, as determined by the FRAP test. We think that the FRAP test is more convenient for this purpose, because the DPPH test showed no significant difference. Our flavonoid and phenolic content findings in the Ardahan propolis were similar to or higher than reported elsewhere for Ardahan and higher than reported for the Turkish province of Ankara (63). In addition, both Ardahan and Erzurum propolis had higher flavonoid and phenolic content than propolis collected from Greece (64), Ireland, Germany (59), and Argentina (65) but lower than Ethiopian propolis (66). Most of this variability may be depend on poplar forage (67) but also on the extraction method (68) and climate (62).
Our findings have also confirmed earlier reports (58, 64) that total phenolic and flavonoid content positively correlates with total antioxidant capacity, and propolis rich with phenols and flavonoids could replace commercial preparations of butylated hydroxytoluene and butylated hydroxyanisole used in food and medicinal preparations (58).
Total flavonoid and phenolic content and antioxidant capacity of
Ardahan | Erzurum | |
---|---|---|
Total flavonoid content (μg quercetin equivalent of total flavonoids/g) | 591.5±26.2 |
271.7±2.9 |
Total phenolic content (mg gallic acid equivalent/g) | 235.5±5.3 |
131.3±3.1 |
DPPH (% of control) | 94.9±0.3 |
94.6±0.7 |
FRAP (μM FeSO4 equivalent/g) | 4017.7±16.4 |
3813.2±3.6 |
Data are presented as mean ± standard deviation (
Table 7 shows the protective effects of ethanolic propolis extracts from the Ardahan and Erzurum provinces against MMC toxicity in human lymphocytes. Interestingly, the 0.74 μmol/L concentration of MMC used as positive control was cytotoxic only after the first 24 h of exposure to lymphocytes tested with the Ardahan propolis samples. Even this cytotoxicity might actually have originated from exposure to solvent and not MMC. We think that the difference in toxicological endpoints used, namely mitotic index and MTT, may explain this anomaly. We selected the MMC concentration based on our preliminary test with mitotic index, and a cytotoxicity report on MMC against human peripheral lymphocytes from an earlier study, also based on mitotic index (31). However, tested with the MTT test, MMC turned out not to be as cytotoxic. Similar was observed in other studies (69, 70, 71).
Effects of different concentrations of propolis extracts obtained from Ardahan and Erzurum on the viability of human lymphocytes
Cell viability (% of control) | ||||
---|---|---|---|---|
Ardahan | Erzurum | |||
24 h | 48 h | 24 h | 48 h | |
Control | 100.0±0.0 |
100.0±0.0 | 100.0±0.0 | 100.0±0.0 |
Solvent control | 97.3±1.3 |
102.2±8.5 | 96.5±13.3 | 89.8±14.9 |
MMC (0.74 μmol/L) | 97.3±2.6 |
97.7±1.2 | 92.4±13.4 | 89.1±14.0 |
62.5 μg/mL | 98.0±2.8 |
102.8±11.4 | 99.7±13.5 | 93.9±13.9 |
125 μg/mL | 102.9±5.7 |
105.2±7.9 | 104.0±19.6 | 100.7±19.5 |
250 μg/mL | 107.0±1.8 |
102.9±2.1 | 118.1±17.8 | 113.3±20.3 |
500 μg/mL | 116.5±6.6 |
108.8±3.9 | 139.9±22.5 | 127.0±20.0 |
62.5 μg/mL+MMC | 99.5±3.2 |
101.1±7.7 | 98.9±19.2 | 94.7±16.7 |
125 μg/mL+MMC | 99.6±1.9 |
99.3±7.6 | 116.4±41.1 | 109.1±10.3 |
250 μg/mL+MMC | 100.5±6.1 |
101.6±6.0 | 117.2±16.2 | 119.6±11.7 |
500 μg/mL+MMC | 112.5±2.8 |
113.0±5.5 | 138.9±23.8 | 133.5±19.6 |
Data are presented as mean ± standard deviation (
Even though MMC cytotoxicity was low, the Erzurum and Ardahan propolis extracts showed their protective effects against MMC and solvent (Table 7) by restoring cell viability to normal (control) there were it was reduced. Their protective (proliferative) effect was concentration-dependent and independent of the presence of MMC. Similar effects of propolis components were reported in a Brazilian green propolis water extract (72).
Table 8 shows the protective effects of propolis from both provinces against lipid peroxidation caused by MMC. Like with human peripheral lymphocytes, MMC did not cause lipid peroxidation in the MCF-7 breast cancer cell line, which confirms earlier reports that MMC may have a low oxidative potential against cancer cells (73) and erythrocytes in Sprague-Dawley rats (74).
Protective effects of the Ardahan and Erzurum propolis against oxidation in MCF-7 cells exposed to mitomycin C
TBARS (nmol/mL) | ||
---|---|---|
Control | 0.40±0.02 |
|
Solvent control | 0.41±0.02 |
|
MMC (0.74 μmol/L) | 0.40±0.05 |
|
Propolis + MMC | Ardahan | Erzurum |
32.5 μg/mL | 0.03±0.02 |
0.03±0.02 |
65 μg/mL | 0.10±0.02 |
0.05±0.01 |
125 μg/mL | 0.06±0.02 |
0.07±0.02 |
250 μg/mL | 0.09±0.01 |
0.10±0.03 |
500 μg/mL | 0.15±0.04 |
0.16±0.04 |
Data are presented as mean ± standard error (
Propolis extracts from both provinces were the most effective at lowering lipid peroxide levels in MCF-7 cells at their lowest concentrations, and this effect generally weakened as propolis concentrations increased, especially with the Erzurum propolis (Table 8). Similar was observed in a Croatian study (75) in which propolis at 100 mg/kg showed better effect at lowering lipid peroxide levels in female CBA/Hr mice plasma than at 300 mg/kg. Antioxidants in propolis such as phenols and especially flavonoids can become oxidants as their concentrations increase (76). For example, galangin, chrysin, and pinocembrin may start to behave as electron-carriers in the presence of metals such as iron and increase oxidative stress on the cell, as reported in human gastric and lung adenocarcinoma cell lines exposed to a New Zealand propolis extract (77).
Figures 1 and 2 show the anticarcinogenic potential of
Apoptotic effects of
Apoptotic effects of
Considering, however, that the TUNEL assay can only determine apoptotic cells, further mechanistic studies should determine the fate of cells exposed to propolis concentrations higher than 125 μg/mL, test our assumption of a necrosis pathway, and give a more comprehensive idea about the anticarcinogenic effects of propolis.
Tables 9 and 10 show that the Ardahan propolis was more potent against
Antimicrobial activities of the ethanolic extracts of A.
Ardahan | (in millimetres) | Erzurum | (in millimetres) | |||||||
---|---|---|---|---|---|---|---|---|---|---|
Microorganism | 50 μg | 100 μg | 150 μg | 50 μg | 100 Mg | 150 μg | Gen | Amp | Ery | Pen |
8.07±1.22 | 9.00±1.29 | 9.38±1.28 | 7.94±0.36 | 8.52±0.47 | 9.56±0.97 | 24.25±1.20 | 9.22±0.67 | 31.64±1.56 | 9.62±0.36 | |
8.10±0.23 | 10.84±2.36 | 12.46±3.42 | - | 8.90±1.41 | 9.80±1.11 | 23.83±0.15 | 10.68±1.03 | 30.99±0.70 | 10.54±1.22 | |
7.52±0.49 | 9.44±0.59 | 11.96±1.22 | - | 7.72±0.80 | 9.16±1.20 | 24.73±1.41 | 10.03±0.78 | 31.07±1.19 | 10.35±0.50 | |
- | - | - | - | - | - | 28.02±0.11 | 13.92±0.68 | 26.83±0.79 | 13.92±0.94 | |
- | - | - | - | - | - | - | - | - | - | |
- | - | - | - | - | - | - | - | - | - |
Data are presented as mean±standard deviation. Amp - ampicillin; Ery - erythromycin; Gen - gentamicin; Pen - penicillin
Minimal inhibition concentrations of the ethanolic extracts of
Microorganism | Ardahan (μg/mL) | Erzurum (μg/mL) |
---|---|---|
30.71 | 43.37 | |
30.71 | 43.37 | |
30.71 | 43.37 |
In conclusion, our findings confirm that geographical differences are important for the chemical composition of propolis and the related biological activity. Both the Ardahan and Erzurum propolis samples were produced by the same Caucasian bee race yet showed different fatty acid, phenolic, flavonoid, and other organic content. Higher sugar, flavonoid, and phenolic content of the Ardahan propolis may have contributed to its higher antioxidant and antibacterial properties. In turn, the Erzurum propolis showed higher anticarcinogenic potential, but this aspect requires further investigation to include the cell necrosis pathway.