Recently, pollen has become a preferred nutritional supplement because of its complex composition. We examined the botanical origin, total phenolic/flavonoid content (TPC/TFC), antioxidant/antimicrobial activity, and element content of pollen samples collected from honeybees. This study also examined whether the elements contained in pollen, when consumed as food, posed a risk to human health.
Ten mixed pollen samples were randomly collected from honeybees in the apiaries of four different Turkish regions, which fall among the three phytogeographic regions of Türkiye.
We evaluated total flavonoid (TFC) and phenolic (TPC) contents; antioxidant activities (radical scavenging activity, hydrogen peroxide scavenging activity - HPSA, ferric reducing antioxidant power - FRAP, and ferrous ion chelating activity - FICA), element concentrations and antimicrobial activity.
According to the melissopalynological analysis, one sample was determined to be unifloral and nine samples were found to be multifloral. The values found ranged 271.42-601.85 mg GAE/100 g TPC, 23.53-34.50 mg CAE/100 g TFC, 22.19-23.78 μg/mL DPPH, 6.50-78.40 µg/mL ABTS, 20.43-150.94 μg/mL HPSA, 97.26-99.83% FRAP and 74.84-91.79% FICA. Rosmanic acid, p-coumaric acid, quercetin, apigenin, and naringin were identified in all samples, while catechin was detected only in S6 and S7. Element contents were found Mg > Fe > Mn > Zn > Cu > Se > Cr > Ni > Cd > Co. All the samples had high antibacterial activity against Bacillus cereus (MIC= 4.17-8.33 g/mL), and against Staphylococcus aureus (MIC= 8.33 g/mL), except S3 and S4.
Different levels and combinations of these components are efficient in the antioxidant and antibacterial activity of pollen.
(Tables S1-S2 and Figs. S1-S6) accompanies the paper on SJAR’s website.
Türkiye has a particularly rich biodiversity, with a characteristic climate, topography, and geomorphology, and is divided into three phytogeographic regions: Euro-Siberian, Irano-Turanian, and Mediterranean. This biodiversity enables the beekeepers to perform their beekeeping activities with both traditional and modern methods. Many melissopalynological studies have stated that plant species belonging to the families Fabaceae, Asteraceae, Lamiaceae, Rosaceae and Brassicaceae are important for beekeeping (De Souza et al., 2019; Lau et al., 2019). In Türkiye, there are 71 genera and 1013 species of which 400 are endemic in the Fabaceae family; 140 genera and 1186 species (446 endemic) in the Asteraceae family; 45 genera and 574 species (256 endemic) in the Lamiaceae family; 37 genera and 297 species (58 endemic) in the Rosaceae family; and 606 species (226 endemic) in the Brassicaceae family (Davis, 1965-1985; Erik & Tarıkahya, 2004; Mutlu & Karakuş, 2015).
Botanical richness enables beekeepers to produce bee products with various chemical and physical properties. Since ancient times, bee products have been among the most valuable products, which are indispensable for people because of their nutritional and therapeutic properties. Pollen, as one of these bee products, contains many bioactive components (phenolic and flavonoid compounds), vitamins, minerals, and pigments, as well as primary metabolites such as carbohydrates, proteins, and lipids (Denisow & Denisow-Pietrzyk, 2016). These phenolic and flavonoid components vary directly with the botanical origin, and indirectly depend on factors such as climate and soil (Komosinska-Vassev et al., 2015; Temizer et al., 2018). Additionally, the differences in the antioxidant activity properties of pollen are closely related to the chemical structure of these components (Rzepecka-Stojko et al., 2015). Furthermore, many researchers claimed that pollen has high antibacterial (Basim et al., 2006; Özkalp & Özcan, 2010), antifungal (Özcan, 2004), anti-inflammatory (Di Paola-Naranjo et al., 2004) and antimutagenic (Pascoal et al., 2014) effects.
The food supply needed due to the rapid increase in the world population, the use of synthetic chemical pesticides, mineral fertilizers, growth regulators and hormones in agricultural activities is increasing and as a result, pollen is also affected by the increasing environmental pollution. Many studies have emphasized that pollens are used as a bioindicator in environmental pollution (Temizer et al., 2018; Aldgini et al., 2019). The substances that are required at a minimum level to sustain the survival of living things, especially plants, are called trace elements. When the amounts of these elements exceed the tolerable limit values, they are defined as toxic contaminants. Essential and toxic elements that cause adverse effects for human health in pollen have been investigated in many countries, such as Türkiye, Bulgaria, Poland, Brazil, Romania, and Jordan (Dinkov & Stratev, 2016; Roman et al., 2016; Temizer et al., 2018; Aldgini et al., 2019).
The production and nutritional value of bee products other than honey such as pollen are ignored, although beekeeping is an important and common activity around the world. This study investigates the botanical origin, total phenolic/flavonoid components, antioxidant, and antimicrobial activities of the pollen samples. The levels of elements in the pollen were also determined to evaluate the health risks and environmental pollution.
Pollen samples were obtained at the end of the pollen flow season in 2018. In this study, each mixed pollen sample was randomly collected from different beekeepers. They were collected from four different locations: sample S1 from İspir-Erzurum, which has a semi-humid climate and is located in the Irano-Turanian phytogeographical region (40° 29’ 3.2424’’ N - 41° 0’ 11.0268’’ E); samples S2, S3, and S4 from Bingöl, which has a semi-humid climate and is located in the Irano Turanian phytogeographical region (38° 53’ 7.2564” N - 40° 29’ 53.8476” E); sample S5 from Bulancak-Giresun, which has a humid climate and is located in the Euro-Siberian phytogeographical region (40° 56’ 15.0396” N - 38° 13’ 55.3584” E); and samples S6, S7, S8, S9 and S10 from Fethiye-Muğla, which has semi-drought and humid climate and is located in the Mediterranean phytogeographical region (36° 37’ 34.04” N - 29° 6’ 33.23” E) (Davis, 1965-1985; TSMS, 2022).
All pollen samples (2 g) were dissolved in 50 mL of absolute ethanol and pollen slides were elaborated according to Wodehouse (1935). At least 500 pollen grains were counted on each slide to determine its composition (Freire et al., 2012).The description of pollens as form or type was conducted according to the method of Mateo & Bosch-Reig (1998). A Nikon Eclipse Ci model microscope was used for examinations, including taxon identification at x1000 magnification and counting at x400 magnification. Monofloral and heterofloral properties of pollen were classified according to Freitas et al. (2013).
Five grams of pollen sample were added to 60 mL of absolute ethanol and this suspension was stirred at room temperature for 24 h by using a magnetic stirrer. This extraction solution was then filtered through Whatman no: 4 filter paper and stored at 4 °C.
It has been reported that ethanol is the best solvent for pollen extraction for measuring TPC/TFC and antioxidant activities (Karkar et al., 2018). We used ethanol as a solvent in determining these parameters in the samples. TPC of pollen extracts was determined according to the Folin-Ciocalteu reagent and Slinkard & Singleton´s (1977) method. Absorbances of the compounds were measured at 760 nm. TPC of the samples was calculated using the gallic acid calibration curve, which was used as a standard (R2 = 0.9995).
The TFC of pollen extracts was determined according to the aluminium chloride colorimetric method (Chung et al., 2002). The absorbances were measured using a spectrophotometer (Optizen Pop UV/Vis Single Beam) at 415 nm. TFC of the samples were calculated using catechin’s calibration curve, which was used as a standard (R2 = 0.9979).
Fragmentation profiles for the identified phenolic compounds were performed in the Agilent 1260 High Performance Liquid Chromatography (HPLC) system, and these compounds were determined by matching standard peak retention times run under equal HPLC conditions. A reverse phase column, Inertsil ODS-2 GL Sciences Inc. 5 μm (4.6×250 mm) C18 and Shimadzu SPD-M10 Avp PDA detector (270 nm) was used during fragmentation in a Shimadzu Prominence HPLC (Liu et al., 1997). System flow was 1.5 mL/min and system temperature was 25ºC. The following segmented gradient elution was used: 0-6.5 min, 90% A; 6.5-7.5 min, 89% A; 7.5-9 min, 87% A; 9-15 min, 20% A; 15-17 min, 95% A; 17-35 min, B (Miura et al., 2002). The mobile phase includes A, which is 97.5 water: 2.5 phosphate (v:v), B is acetonitrile. The measurements were repeated at least twice.
— DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. The DPPH radical scavenging activity of pollen extracts was determined according to Blois (1958) with minor modifications. Sample solutions (3.0 mL) were mixed with 0.1 mM DPPH (1.0 mL) prepared by adding absolute ethanol. The mixture was incubated for 30 min at room temperature in a dark environment and the absorbance was measured at 517 nm using a spectrophotometer. The free radical scavenging activities of the reaction mixtures were calculated using the absorbance values after 30 min. The decline in absorbance is an indicator of the high rate of free radical scavenging activity in the samples, which was expressed in SC50 (μg/mL).
— ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity. The ABTS radical was dissolved in 7 mM potassium persulfate solution (2:1) and incubated for 16 h at room temperature in a dark environment. The prepared solution was diluted using 5 mM phosphate buffer (pH 7.4) so that the absorbance value reached 0.700 ± 0.020. Pollen extract samples (20 mL) were homogenized with 2 mL of the solution, kept for 5 min and measured in the spectrophotometer (Gökce et al., 2019).
— Hydrogen peroxide scavenging activity (HPSA). The HPSA of pollen samples (using 0.04 M phosphate buffer, pH 7.4) was performed according to Ruch et al. (1989). The HPSA of the samples was expressed in SC50 (μg/mL).
— Ferric reducing antioxidant power (FRAP). The FRAP of pollen extracts and standard antioxidant solutions prepared with absolute ethanol was determined according to Oyaizu (1986). The absorbance values of the samples were measured using a spectrophotometer at 700 nm. The increase in absorbance values in the reaction mixture is an indicator of the high level of reducing power in extracts and standard antioxidant substances (Eq. 1):
where As: extracts or standard materials absorbance values, Ac: control’s absorbance values.
— Ferrous ion chelating activity (FICA). The FICA of the solutions prepared with pollen extracts and standard antioxidant substances in absolute ethanol was performed according to Dinis et al. (1994). The absorbance values of the mixtures were measured at 562 nm. FICA of the extracts and standard antioxidant substances was calculated according to Eq. (2):
In this study, Mg (microelement), Fe, Zn, Mn, Ni and Cu (trace elements), Se, Cr and Co (ultra-trace elements), and Cd (toxic element) were examined by inductively coupled plasma mass spectrometry (ICP-MS) (Model Bruker 820-MS). The microwave digestion method was used for the preparation of pollen samples: 0.5 g of each sample was digested in CEM MARS for 5 min with HN03 (ultrapure) and in 2 mL of HCl (ultra-purification) for 15 min at 1600 Watt in 210 mL. These solutions, which were placed in a cooling sample lid falcon tube and completed to 50 mL with distilled water, were analysed by Bruker 820-MS ICP-MS by filtering through a membrane filter (0.45 µm). The calibration curve was obtained using a certified multi-element standard. An intermediate stock of 10 mg/L was prepared from the main stock solution and a calibration curve was plotted from standard stocks of 5, 10, 20, 50, 100, and 250 μg/L. Samples were prepared in triplicate and ten readings were taken on each parallel ICP-MS. The blank sample prepared in 1% HNO3 solution was measured 20 times. The standard slope, LOD (limit of detection) and LOQ (limit of quantification) were determined three, three and ten times, respectively (Temizer et al., 2018).
Antimicrobial activities of pollen extracts were tested against Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 10876) and Candida albicans (ATCC 10231) strains.
The strains were stored in a medium containing 15% glycerol and kept at -80 °C. Microbial cultures were incubated in sterile Mueller-Hinton Broth (MHB) for 6 h at 37 °C. Each culture was then transferred to a fresh MHB and incubated at 37 °C for 24 h. A medium containing 0.5X McFarland solution (approx. a culture density of 1.5 × 108 cells/mL) was prepared.
Antimicrobial activities of pollen extracts were studied using the agar diffusion test method (Rios et al., 1988). Pollen extracts were prepared at a concentration of 8.33 g/mL in ethanol. Ethanol was used as a negative control. Each strain was inoculated onto Muller-Hinton Agar (MHA) medium with sterile swabs. Each pollen extract solution (100 µL) and controls were dropped into 8 mm diameter wells opened in the medium. Petri plates were incubated at 37 °C for 24 h. After incubation, the diameter of the inhibition zone around each well was measured and recorded. Antimicrobial activity was expressed as the diameter of the regions of inhibition generated by the tested extract. Each test was run three times.
The minimum inhibitory concentration (MIC) was determined using the microplate method (Wiegand et al., 2008). Pollen extract (8.33 g/mL) was serially diluted to 50% with MHB medium and 50 µL of a microorganism culture solution was added to 12 wells of a 96-well microplate. The microplates were incubated at 37 °C for 24 h. The absorbance values of the samples after incubation were measured at 450 nm using an automated microplate reader (Multiskan FC, Thermo).
The intake of elements in the diet is considered 0.04 mg/day for adults and 0.02 mg/day for children (Zafeiraki et al., 2022). The equation below was used to get estimated daily intake (EDI):
where c refers to the element level in bee pollen; AFC is the amount of food consumption (mg/day), calculated as 0.04 mg for adult consumption and 0.02 mg for child consumption (Zafeiraki et al., 2022); and BW is the consumer´s mean body weight (70 kg for male adults, 60 kg for female adults, and 15 kg for children) (Azanu et al., 2018; Tutun et al., 2022; Zafeiraki et al., 2022).
This analysis investigated the potential health risks associated with consuming pollen as a food. As a result, only oral consumption was used to determine the THI (target hazard index) and SHI (sum of hazard index) evaluations; THI (Eq. 4) being used to describe the health hazards associated with just one chemical, and SHI (Eq. 5) the risk of exposure to two or more chemicals (Zafeiraki et al., 2022):
where CF is contact frequency (365 days/year); CD is contact duration (70 years for adults and 4 years for children); RfD (mg/kg·day) is the oral reference dose for which the health-based recommendation value; and TA is the mean lifetime (365 days/year 70 years). The RfD levels for Mn, Fe, Co, Ni, Cu, Zn, Cr, Cd are defined in Demir et al. (2020) and for Se in Singh et al. (2019). There is no noticeable health risk when the computed THI and SHI values (Eq. 5) are both lower than 1.
where N is the total number of distinct elements found in each sample of bee pollen.
Statistical evaluations were performed from several methodological perspectives to analyse the pollen samples in terms of their antioxidant activity, contents of the elements and phenolics they contain.
The linear relationships between element, antioxidant, and phenolic contents in the pollen samples were examined. The Pearson correlation coefficient was used for variables complying with the normality condition as tested by the Shapiro & Wilk (1965) test. For those not complying, the non-parametric Spearman’s Rank Correlation Coefficient (SRC) (Spearman, 1904) was used.
Pollen samples were submitted to complete-linkage clustering analysis to be classified in terms of element, phenolic, and antioxidant contents (Defays, 1977). The findings of the clustering analysis have been visualized in the form of a dendrogram.
PCA has been widely used as an exploratory tool for data analysis (Pearson, 1895; Hotelling, 1933). Principal component analysis (PCA) was also performed based on element, antioxidant, and phenolic contents to determine the patterns in the data structure. To determine the number of principal components, two different statistical perspectives such as eigenvalues and the ratio of explaining the total variance can be considered. In this study, both perspectives were evaluated to select components.
The floral origin of bee pollen samples (n = 10) was determined, and the taxa found are given in
| Taxa | Pollen samples | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | |
| Acacia sp. | 2.1 | |||||||||
| Cistaceae | 10.3 | 14 | ||||||||
| Rosaceae | 98.4 | 80.3 | 2.7 | 15.9 | 2.3 | |||||
| Pistacia sp. | 57.5 | 3.7 | 50.5 | |||||||
| Brassicaceae | 62.1 | 2.4 | 11.5 | 64.3 | 3.1 | |||||
| Thalictrum sp. | 0.8 | 0.7 | ||||||||
| Moraceae | 0.7 | |||||||||
| Taraxacum sp. | 1.4 | |||||||||
| Lamiaceae (3-colpate type) | 8.2 | |||||||||
| Geraniaceae | 4.5 | 1.4 | 1.2 | 26.2 | 37.4 | |||||
| Lamiaceae (6-colpate type) | 26.3 | 0.7 | 26.8 | 0.8 | ||||||
| Asteraceae (echinatetype) | 1.6 | 17.9 | 12.3 | 0.7 | 1.2 | 1.5 | ||||
| Apiaceae | 53.8 | |||||||||
| Epilobium sp. | 9.2 | |||||||||
| Boraginaceae | 2.3 | 14.6 | 3.1 | 4.7 | ||||||
| Centaurea sp. | 1.4 | 2.4 | 3.1 | |||||||
| Onobrychis sp. | 86.4 | 34.1 | ||||||||
| Chenopodiaceae | 2.4 | |||||||||
| Trifolium sp. | 48.2 | |||||||||
| Daucus sp. | 2.6 | |||||||||
| Sanguisorba sp. | 9.6 | |||||||||
| Myosotis sp. | 0.9 | 8 | ||||||||
| Lotus sp. | ||||||||||
| Fabaceae | 26.3 | 4.7 | ||||||||
| Morus sp. | 2.8 | |||||||||
| Ericaceaae | ||||||||||
| Asteraceae (lacunate type) | 2.3 | 2.1 | ||||||||
| Betula sp. | 0.5 | |||||||||
| Campanula sp. | 0.8 | |||||||||
The Apiaceae family was the predominant type of pollen in S1; Rosaceae family was the predominant type of pollen in S2 and S3; Fabaceae family was predominant type of pollen in S4 (Trifolium genus) and S5 (Onobrychis genus). The Pistacia genus was dominant in S6, S7, and S10 samples collected in Muğla. Many studies have reported that these taxa are crucial for the survival of bees (Freire et al., 2012; Freitas et al., 2013; Temizer et al., 2019). This vegetation also affects the phenolic compound, antioxidant, and antimicrobial activities of bee pollen.
TPC, TFC and antioxidant activities of the pollens are given in
| Samples | TPC[a] | TFC[b] | FICA[c] | HPSA[d] | ABTS[e] | DPPH[f] | FRAP[g] |
|---|---|---|---|---|---|---|---|
| 1 | 316.57 | 32.27 | 90.19 | 20.43 | 64.98 | 22.99 | 98.65 |
| 2 | 271.42 | 25.86 | 91.61 | 150.94 | 60.48 | 22.71 | 97.26 |
| 3 | 545.41 | 23.53 | 74.84 | 20.35 | 74.99 | 22.57 | 98.36 |
| 4 | 601.85 | 24.66 | 83.34 | 21.77 | 72.65 | 22.74 | 98.74 |
| 5 | 276.55 | 26.44 | 88.28 | 24.17 | 78.4 | 23.78 | 99.45 |
| 6 | 412.01 | 29.42 | 91.79 | 21.37 | 75.1 | 22.47 | 99.75 |
| 7 | 368.91 | 30.65 | 91.44 | 22.04 | 62.17 | 22.83 | 99.83 |
| 8 | 507.44 | 34.5 | 84.64 | 23.57 | 73.8 | 22.19 | 99.83 |
| 9 | 302.21 | 32.36 | 79.03 | 23.14 | 66.78 | 23.34 | 99.54 |
| 10 | 392.51 | 28.87 | 91.23 | 22.23 | 64.56 | 22.94 | 99.75 |
[a] TPC: total phenolic content, mg GAE/100 g. [b] TFC: total flavonoid content, mg CAE/100 g. [c] FICA: ferrous ion chelating activity, % (at the 100 μg/mL concentration). [d] HPSA: hydrogen peroxide scavenging activity, SC50 (µg/mL). [e] ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid. [f] DPPH: 2,2-diphenyl-1-picrylhydrazyl. [g] FRAP: ferric reducing antioxidant power.
TPC and TFC of the pollen samples ranged 271.42-601.85 mg GAE/100 g and 23.53-34.50 mg CAE/100 g, respectively (
Pollen samples were classified by a hierarchical clustering method in terms of phenolic contents. The dendrogram indicated that the pollen samples can be split into three main clusters (Fig. S1 [suppl]). PCA was also performed (Fig. S2a [suppl]) to determine the patterns in the data structure based on phenolic contents. Fig. S2b [suppl] represents the score plot of samples for PC1 and PC2, where S2 significantly dissociates from all other samples in terms of phenolic contents, parallel to the clustering analysis results.
HPSA and DPPH radical scavenging activities of pollen samples are ranged 20.43-150.94 and 22.19-23.78 μg/mL, respectively (
The FICA and FRAP of the pollen samples varied between 74.84-91.79% and 97.26-99.83%, respectively (
| Samples | Gallic acid | Catechin | Chlorogenic acid | p-coumaric acid | Hesperidin |
|---|---|---|---|---|---|
| S1 | ND | ND | 0.0051±0.00 | 0.0036±0.00 | 0.0383±0.01 |
| S2 | 0.0076±0.01 | ND | ND | 0.0019±0.00 | 0.0077±0.00 |
| S3 | 0.0021±0.00 | ND | 0.0010±0.00 | 0.0702±0.00 | 0.0087±0.00 |
| S4 | 0.0102±0.00 | ND | ND | 0.0884±0.00 | 0.0061±0.00 |
| S5 | 0.0226±0.00 | ND | ND | 0.0019±0.00 | ND |
| S6 | 0.0034±0.00 | 0.0113±0.00 | 0.0027±0.00 | 0.0060±0.00 | 0.0127±0.00 |
| S7 | 0.0030±0.00 | 0.0039±0.00 | 0.0023±0.00 | 0.0059±0.00 | 0.0098±0.01 |
| S8 | ND | ND | 0.0026±0.00 | 0.0039±0.00 | 0.0283±0.00 |
| S9 | 0.0010±0.00 | ND | 0.0021±0.00 | 0.0023±0.00 | 0.0128±0.00 |
| S10 | 0.0022±0.00 | ND | 0.0688±0.00 | 0.0043±0.00 | 0.0305±0.00 |
| Rosmanic acid | Quercetin | Apigenin | Naringin | Quinic acid | |
| S1 | 0.0278±0.00 | 0.451±0.01 | 0.0047±0.00 | 0.0688±0.00 | ND |
| S2 | 0.187±0.00 | 0.064±0.00 | 0.0005±0.00 | 0.5643±0.00 | ND |
| S3 | 0.0239±0.00 | 0.177±0.00 | 0.0002±0.00 | 0.0009±0.00 | ND |
| S4 | 0.0259±0.00 | 0.2100±0.00 | 0.0007±0.00 | 0.0182±0.00 | ND |
| S5 | 0.0101±0.00 | 0.0688±0.00 | 0.0018±0.00 | 0.0006±0.00 | ND |
| S6 | 0.0593±0.00 | 0.0367±0.00 | 0.0002±0.00 | 0.0044±0.00 | ND |
| S7 | 0.0529±0.03 | 0.4137±0.00 | 0.0005±0.00 | 0.0478±0.00 | ND |
| S8 | 0.0357±0.00 | 0.1727±0.00 | 0.0006±0.00 | 0.0075±0.00 | ND |
| S9 | 0.1407±0.00 | 0.0712±0.00 | 0.0304±0.00 | 0.0060±0.00 | ND |
| S10 | 0.2045±0.00 | 0.0594±0.00 | 0.0022±0.00 | 0.0285±0.00 | ND |
ABTS radical scavenging activities ranged 6.50-78.40 µg/mL. Freire et al. (2012) reported ABTS radical scavenging activities of pollen samples in Brazil as 6.0-97.20 µg/mL, quite similar to our study. Sun et al. (2017) determined ABTS radical scavenging activities as 3.19-3.85 µg/mL, lower than the values in our study. In our study, ABTS radical scavenging activity of S1 was higher than that of the other pollen samples (
A hierarchical clustering method was used to classify different locations according to the antioxidant contents of pollen samples. Fig. S3 [suppl] shows a dendrogram which illustrates the relationships among all pollen samples from the perspective of antioxidant content based on the pre-determined measure of similarity. Fig. S4a [suppl] shows a map for the antioxidant contents (PCA, PC1 and PC2). Fig. S4a [suppl] indicates that the positive part of PC1 was related to TPC, ABTS, FRAP and TFC, while the positive part of PC2 was related to HPSA, TPC and ABTS. For both PCs, the negative parts were related with these elements. Fig. S4b [suppl] shows that S2 significantly dissociates from the rest of samples in terms of antioxidant contents, parallel to the clustering analysis results. These findings demonstrate that the antioxidant contents can be used to classify these pollen samples.
The linear relationships between the antioxidant and phenolic contents of the pollen samples are shown in Table S1 [suppl]. Chlorogenic acid had a significant positive linear correlation with hesperidin (r=0.91; p<0.001) and TFC (r=0.57; p=0.085), at 0.05 and 0.10 significance levels, respectively; p-coumaric acid, at 0.05 significance level, had a significant positive linear correlation with TPC (r=0.91; p<0.001) and a significant negative linear correlation with HPSA (r=-0.74; p=0.013); hesperidin and TFC had a significant positive linear correlation (r=0.65; p=0.042), at 0.05 significance level.
| Samples | Mg | Cr | Mn | Fe | Co | Ni | Cu | Zn | Se | Cd |
|---|---|---|---|---|---|---|---|---|---|---|
| S1 | 613.76 | 1.534 | 21.628 | 123.1 | ND | 0.451 | 6.234 | 26.6 | 6.386 | 0.157 |
| S2 | 633.28 | 4.423 | 32.752 | 156.25 | ND | 1.954 | 8.724 | 38.044 | 6.216 | 0.24 |
| S3 | 899.16 | 3.396 | 29.029 | 183.92 | ND | 0.855 | 3.907 | 37.181 | 7.898 | 0.155 |
| S4 | 128.47 | 1.722 | 38.832 | 192.21 | ND | 2.551 | 8.545 | 52.497 | 6.592 | 0.137 |
| S5 | 807.83 | 2.349 | 36.707 | 87.104 | ND | 1.858 | 13.851 | 32.976 | 7.497 | 0.122 |
| S6 | 700.63 | 3.633 | 20.707 | 63.591 | 0.006 | 3.269 | 6.164 | 27.82 | 5.942 | 0.163 |
| S7 | 714.6 | 3.771 | 32.935 | 12.181 | ND | 3.392 | 6.936 | 33.086 | 6.173 | 0.138 |
| S8 | 885.36 | 2.197 | 27.794 | 12.721 | ND | 2.463 | 6.734 | 29.406 | 6.317 | 0.153 |
| S9 | 525.32 | 1.338 | 18.118 | 16.753 | ND | 2.475 | 5.968 | 32.658 | 6.416 | 0.193 |
| S10 | 820.15 | 3.206 | 22.5 | 154.085 | ND | 1.486 | 6.467 | 30.781 | 6.04 | 0.112 |
The findings related to the relationships between elements are shown in Table S2 [suppl]. The linear correlation between Cr and Co (r=0.588; p=0.074), and between Fe and Co (r=-0.583; p=0.077) showed a significant value at 0.10 significance level, in a positive and negative way respectively. Moreover, both Cu (r=0.758; p=0.011) and Zn (r=0.745; p=0.013) had a significant positive linear correlation with Mn at 0.05 significance level.
Fig. S5 [suppl.] shows the results of the clustering analysis with a dendrogram, which illustrates the relationships among all pollen sample locations based on element contents according to Euclidean distance. The dendrogram explained that the pollen samples can be split into three main clusters. S4 dissociated from all other samples and forms the third cluster alone. Fig. S6a [suppl] illustrates the element map. The positive part of PC1 was related to Cu, Zn, Mn, Fe, and Se, while the positive part of PC2 was related to Co, Cr, Ni, Cd, Cu, Zn, Mn. For each PC, the negative parts were related with these elements. For each PC, the negative parts were related with these elements. Fig. 6Sb [suppl] represents the score plot of samples for PC1 and PC2 and shows that S4 significantly dissociated from all other samples for element contains, parallel to the clustering analysis results. Besides, for PC1 and PC2, the proximity of the positions of S6, S7, and S9 on the plot indicates that these samples had a similar structure in terms of their element content.
The antimicrobial activity of ten pollen samples was investigated for three bacteria and one yeast. Different levels of antimicrobial activity in pollen samples were determined against B. cereus ATCC 10876 and S. aureus ATCC 29213 (
| Samples | Microorganisms | Zone of inhibition (mm) | MIC value (g/mL) |
|---|---|---|---|
| S1 | B.cereus | 13 | 8.33 |
| S.aureus | 12 | 8.33 | |
| S2 | B.cereus | 13 | 8.33 |
| S.aureus | 13 | 8.33 | |
| S3 | B.cereus | 12 | 8.33 |
| S4 | B.cereus | 12 | 8.33 |
| S5 | B.cereus | 13 | 8.33 |
| S.aureus | 12 | 8.33 | |
| S6 | B.cereus | 13 | 8.33 |
| S.aureus | 12 | 8.33 | |
| S7 | B.cereus | 13 | 8.33 |
| S.aureus | 12 | 8.33 | |
| S8 | B.cereus | 12 | 8.33 |
| S.aureus | 12 | 8.33 | |
| S9 | B.cereus | 14 | 4.17 |
| S.aureus | 12 | 8.33 | |
| S10 | B.cereus | 12 | 8.33 |
| S.aureus | 11 | 8.33 |
All pollen samples had the same characteristics and properties regarding MIC and inhibition zones of C. albicans, B. cereus and E. coli. When S. aureus is considered, all samples exhibited similar behaviour except for S3 and S4, which could not eliminate the bacteria. These findings can be statistically supported by a cluster analysis (Fig. S5 [suppl]), where S3 and S4 formed a cluster (70.97% similarity), and the other samples formed separate clusters.
The risk to human health was assessed based on bee pollen intake, considering the RfD values together with EDI (
| Samples | Cr (III) | Mn | Fe | Co | Ni | Cu | Zn | Se | Cd | |
|---|---|---|---|---|---|---|---|---|---|---|
| S1 | M | 0.00088 | 0.01200 | 0.07000 | ND | 0.00026 | 0.00300 | 0.01500 | 0.00400 | 0.00009 |
| W | 0.00100 | 0.01400 | 0.08200 | ND | 0.00030 | 0.00400 | 0.01800 | 0.00400 | 0.00011 | |
| C | 0.00200 | 0.02800 | 0.16400 | ND | 0.00060 | 0.00800 | 0.03500 | 0.00800 | <0.00001 | |
| S2 | M | 0.00300 | 0.01800 | 0.08900 | ND | 0.00100 | 0.00500 | 0.02200 | 0.00300 | <0.00001 |
| W | 0.00200 | 0.01900 | 0.12200 | ND | 0.00057 | 0.00300 | 0.02500 | 0.00500 | 0.00010 | |
| C | 0.00600 | 0.04300 | 0.20800 | ND | 0.00300 | 0.01200 | 0.05000 | 0.00800 | <0.00001 | |
| S3 | M | 0.00200 | 0.01600 | 0.10500 | ND | 0.00049 | 0.00200 | 0.02100 | 0.00500 | 0.00009 |
| W | 0.00200 | 0.01900 | 0.12200 | ND | 0.00057 | 0.00300 | 0.02500 | 0.01000 | 0.00010 | |
| C | 0.00500 | 0.03900 | 0.24500 | ND | 0.00100 | 0.00500 | 0.05000 | 0.01000 | 0.00021 | |
| S4 | M | 0.00100 | 0.02200 | 0.11000 | ND | 0.00100 | 0.00500 | 0.03000 | 0.00400 | 0.00008 |
| W | 0.00100 | 0.02600 | 0.12800 | ND | 0.00200 | 0.00600 | 0.03500 | 0.00400 | 0.00009 | |
| C | 0.00200 | 0.05200 | 0.25600 | ND | 0.00300 | 0.01100 | 0.07000 | 0.00900 | 0.00018 | |
| S5 | M | 0.00100 | 0.02100 | 0.05000 | ND | 0.00100 | 0.00800 | 0.01900 | 0.00400 | 0.00007 |
| W | 0.00200 | 0.02400 | 0.05800 | ND | 0.00100 | 0.00900 | 0.02200 | 0.00500 | 0.00008 | |
| C | 0.00300 | 0.04900 | 0.11600 | ND | 0.00200 | 0.01800 | 0.04400 | 0.01000 | 0.00016 | |
| S6 | M | 0.00200 | 0.01200 | 0.03600 | <0.00001 | 0.00200 | 0.00300 | 0.01600 | 0.00300 | 0.00009 |
| W | 0.00200 | 0.01400 | 0.04200 | <0.00001 | 0.00200 | 0.00400 | 0.01900 | 0.00400 | 0.00001 | |
| C | 0.00500 | 0.02800 | 0.08500 | 0.00001 | 0.00400 | 0.00800 | 0.03700 | 0.00800 | 0.00022 | |
| S7 | M | 0.00200 | 0.01900 | 0.00700 | ND | 0.00200 | 0.00400 | 0.01900 | 0.00300 | 0.00008 |
| W | 0.00300 | 0.02200 | 0.00800 | ND | 0.00200 | 0.00500 | 0.02200 | 0.00400 | <0.00001 | |
| C | 0.00500 | 0.04400 | 0.01600 | ND | 0.00400 | 0.00900 | 0.04400 | 0.00800 | 0.00018 | |
| S8 | M | 0.00100 | 0.01600 | 0.00700 | ND | 0.00100 | 0.00400 | 0.01700 | 0.00400 | 0.00009 |
| W | 0.00100 | 0.01900 | 0.00850 | ND | 0.00100 | 0.00400 | 0.02000 | 0.00400 | <0.00001 | |
| C | 0.00200 | 0.03700 | 0.01700 | ND | 0.00300 | 0.00900 | 0.04000 | 0.00800 | 0.00020 | |
| S9 | M | 0.00100 | 0.01000 | 0.00900 | ND | 0.00100 | 0.00300 | 0.01900 | 0.00400 | 0.00011 |
| W | 0.00100 | 0.01200 | 0.01100 | ND | 0.00100 | 0.00400 | 0.02200 | 0.00400 | 0.00013 | |
| C | 0.00200 | 0.02400 | 0.02200 | ND | 0.00300 | 0.00800 | 0.04300 | 0.00800 | 0.00026 | |
| S10 | M | 0.00200 | 0.01300 | 0.08800 | ND | 0.00085 | 0.00400 | 0.01700 | 0.00300 | 0.00006 |
| W | 0.00200 | 0.01500 | 0.10300 | ND | 0.00099 | 0.00400 | 0.02100 | 0.00400 | 0.00007 | |
| C | 0.00400 | 0.03000 | 0.20500 | ND | 0.00200 | 0.00900 | 0.04100 | 0.00800 | 0.00015 | |
| Cr (III) | Mn | Fe | Co | Cu | Zn | Se | Cd | Ni | SHI | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| S1 | M | 0.00058 | 0.08800 | 0.10049 | ND | 0.09000 | 0.05000 | 0.73000 | 0.09000 | 0.01300 | 1.16100 |
| W | 0.00068 | 0.11724 | ND | 0.10400 | 0.06000 | 0.85000 | 0.10400 | 0.01500 | 1.34000 | ||
| C | 0.00100 | 0.20600 | 0.23448 | ND | 0.20800 | 0.12000 | 1.70300 | 0.20900 | 0.03000 | 2.68000 | |
| S2 | M | 0.00200 | 0.13400 | 0.12755 | ND | 0.12500 | 0.07200 | 0.71000 | 0.13700 | 0.05600 | 1.30700 |
| W | 0.00100 | 0.13800 | 0.17516 | ND | 0.06500 | 0.08200 | 1.05300 | 0.10300 | 0.02800 | 1.62000 | |
| C | 0.00400 | 0.31200 | 0.29762 | ND | 0.29100 | 0.17000 | 1.65700 | 0.32000 | 0.13000 | 3.05000 | |
| S3 | M | 0.00100 | 0.11800 | 0.15014 | ND | 0.05600 | 0.07000 | 0.90300 | 0.08800 | 0.02400 | 1.38800 |
| W | 0.00100 | 0.13800 | 0.17516 | ND | 0.06500 | 0.08200 | 1.05300 | 0.10300 | 0.02800 | 1.62000 | |
| C | 0.00300 | 0.27600 | 0.35032 | ND | 0.13000 | 0.16500 | 2.10600 | 0.20700 | 0.05700 | 3.23800 | |
| S4 | M | 0.00066 | 0.15800 | 0.15691 | ND | 0.12200 | 0.10000 | 0.75300 | 0.07800 | 0.07300 | 1.36900 |
| W | 0.00077 | 0.18500 | 0.18306 | ND | 0.14200 | 0.10000 | 0.87900 | 0.09100 | 0.08500 | 1.59800 | |
| C | 0.00100 | 0.37000 | 0.36611 | ND | 0.28500 | 0.20000 | 1.75800 | 0.18200 | 0.17000 | 3.19600 | |
| S5 | M | 0.00090 | 0.15000 | 0.07111 | ND | 0.19800 | 0.06200 | 0.85700 | 0.07000 | 0.05300 | 1.40900 |
| W | 0.00100 | 0.17500 | 0.08296 | ND | 0.23100 | 0.07300 | 0.10000 | 0.08100 | 0.06200 | 1.64300 | |
| C | 0.00200 | 0.35000 | 0.16591 | ND | 0.46200 | 0.14600 | 2.00000 | 0.16300 | 0.12400 | 3.28700 | |
| S6 | M | 0.00100 | 0.08500 | 0.05191 | 0.01100 | 0.08800 | 0.05300 | 0.68000 | 0.18600 | 0.09300 | 1.15500 |
| W | 0.00100 | 0.09900 | 0.06056 | 0.01300 | 0.10300 | 0.06200 | 0.79200 | 0.10900 | 0.10900 | 1.23900 | |
| C | 0.00300 | 0.19700 | 0.12113 | 0.02700 | 0.20500 | 0.12400 | 1.58400 | 0.21700 | 0.21800 | 2.47900 | |
| S7 | M | 0.00100 | 0.13400 | 0.00994 | ND | 0.10000 | 0.06300 | 0.70500 | 0.07900 | 0.09700 | 1.09200 |
| W | 0.00100 | 0.15700 | 0.01160 | ND | 0.11500 | 0.07400 | 0.82300 | 0.09200 | 0.11300 | 1.27400 | |
| C | 0.00300 | 0.31300 | 0.02320 | ND | 0.23100 | 0.14700 | 1.64600 | 0.18400 | 0.22600 | 2.54800 | |
| S8 | M | 0.00100 | 0.11300 | 0.01038 | ND | 0.09600 | 0.05600 | 0.72200 | 0.08700 | 0.07000 | 1.08600 |
| W | 0.00100 | 0.13200 | 0.01212 | ND | 0.11200 | 0.06500 | 0.84200 | 0.10200 | 0.08200 | ||
| C | 0.00200 | 0.26500 | 0.02423 | ND | 0.22400 | 0.13100 | 1.68500 | 0.20400 | 0.16400 | 2.53400 | |
| S9 | M | 0.00051 | 0.07400 | 0.01368 | ND | 0.08500 | 0.06200 | 0.73300 | 0.11000 | 0.07000 | 1.07900 |
| W | 0.00060 | 0.08600 | 0.01596 | ND | 0.10000 | 0.07300 | 0.85500 | 0.12900 | 0.08200 | 1.25900 | |
| C | 0.00100 | 0.17200 | 0.03191 | ND | 0.20000 | 0.14500 | 1.71100 | 0.25700 | 0.16500 | 2.51800 | |
| S10 | M | 0.00100 | 0.09200 | 0.12578 | ND | 0.10000 | 0.05900 | 0.69000 | 0.06400 | 0.04200 | 1.12400 |
| W | 0.00100 | 0.10700 | 0.14675 | ND | 0.10000 | 0.06800 | 0.80500 | 0.07400 | 0.05000 | 1.31100 | |
| C | 0.00200 | 0.21000 | 0.29350 | ND | 0.20000 | 0.13700 | 1.61000 | 0.14900 | 0.01000 | 2.62300 | |
Türkiye has suitable ecological conditions for beekeeping activities because it is at the intersection of three phytogeographical regions and has its own topographic and climatic conditions, which cause the blooms of plants located in different geographic locations to occur at different periods of the year. The taxa detected in pollen samples were found to belong to many plants regardless of whether the flowering period was long or short in this study. The antioxidant activities of pollen samples were evaluated using methods with different principles. The chemical structure of phenolic compounds was effective on antioxidant activity, although the total phenol and flavonoid levels in the pollen did not directly affect the antioxidant activity in our study. Our results showed as well that the amount and combinations of these compounds were effective on the antioxidant and antimicrobial activities of pollen.
Pollen samples were found to be rich in the essential element (Se); however, all pollen samples had toxic Cd contamination. According to the health risk assessment, if adults consume daily 0.04 mg of pollen and children 0.02 mg, there may be a possibility of toxic effects due to Se. Pollens have potential bioactive properties, its content needs to be studied extensively to be used for cosmetic, pharmacy, and food industry.