Journal of Applied Botany and Food Quality 90, 306 - 314 (2017), DOI:10.5073/JABFQ.2017.090.038 1Faculty of Engineering, Chemical and Bioprocess Engineering Department 2Faculty of Agriculture and Forestry, Plant Science Department 3Faculty of Chemistry, Inorganic Chemistry Department, Pontificia Universidad Católica de Chile, Macul, Chile Relation between composition, antioxidant and antibacterial activities and botanical origin of multi-floral bee pollen P. Velásquez1, K. Rodríguez2, M. Retamal3, A. Giordano3, L.M. Valenzuela1, G. Montenegro2* (Received February 9, 2017; Accepted March 24, 2017) * Corresponding author Summary Harvested bee pollen is valuable for its nutritional value and healthy properties. This work relates the botanical origin of sixteen bee pollens from Chile with their phenolic, protein and carotenoid content, and antioxidant/antibacterial activities. Our results showed that the chemical properties of different bee pollens are associated with the plant’ species from which each one was derived from. Some correlations between chemical properties and botanical origin were observed. Bee pollen showed between 20.0-30.4% protein, 2.8-50.2 mg/kg carotenoids, 22.8-918.4 mg/kg phenolics, and 4.51-91.19 mmol Fe+2/kg pollen. Antibacterial activity was observed against all bacteria assayed even surpassing the activity of traditional antibiotics. Brassica sp. and Galega officinalis are an abundant source of antioxidants and antibacterial compounds. Other species such as those derived from fruit and endemic plants from Chile, although they occur less frequently, are also good source of these compounds. Some correlations between botanical origin and chemical, antioxidant and antibacterial properties were observed. Knowing the influence of plant species over the antioxidant or antibacterial properties of bee pollen, will allow selecting the best location for honeycombs and will allow beekeepers to differentiate and add value to their products. Keywords: Brassica sp., Galega officinalis, Trevoa quinquenervia, Prunus sp., Medicago sp., FRAP, diameter of inhibition, HPLC- DAD, protein, carotenoid. Introduction Bee pollen corresponds to microspores of spermatophytes and entomophilous plants with flowers collected and transported by bees in their last pair of legs as granules or pollen-loads. Once in the hive, bee adds salivary enzymes (e.g., amylase, catalase) to the pollen- loads and reserved them as nutrient resource for honeycomb. Bee pollen is valuable for its nutritional value and healthful properties and is considered by many beekeepers as a mean of diversifying and increasing their income. Bee pollen products are valuable for their nutritional value and healthful properties. As a nutrient, bee pollen provides to the human diet protein, fat and other components in lesser amount. Bee pollen presents all essential amino acids to the human diet and its content varies between 10 and 40% (Bogdanov, 2014). Dry bee pollen pre- sents an average protein content about 23.8% (almeida-muradian et al., 2005). Fatty acids are 3%, from which about half of them are oleic (omega-3), linoleic (omega-6) and linolenic acids (omega-3) (Bogdanov, 2014). Carbohydrates in bee pollen are mainly poly- saccharides such as starch and sugars, and represent between 13 and 55 g/100 g of sample. With respect to healthful characteristics, bee pollen has been described as anti-anemic, tonic and restorative, hormone regulator, intestinal regulator, vasoprotector, hepatopro- tective, anti-atheroscleorotic agent, antiallergic, anticarcinogenic, antioxidant, antibacterial and as antifungal (denisow and denisow- Pietrzyk, 2016; graikou et al., 2011). Phenolic acids, flavonoids and pigments as β-carotene, are mainly related to the healthy properties exhibited by bee pollen such as antioxidant and antibacterial (aloisi and ruPPel, 2014; alicic et al., 2014). Phenolic acids and flavonoid glycosides are present in the nectar of flowers visited by bees, which are hydrolyzed and transferred to the bee pollen. The number and variety of phenolic acids and flavonoids are highly variable, since beekeepers mix bee pollen from different botanical origins (morais et al., 2011; leja et al., 2007). The main group of pigments that compose bee pollen corresponds to carotenoids, especially β-carotene (17% of all carotenoids), whose concentration also depends on the botanical origin of the pollen (almeida-muradian et al., 2005). The type and concentration of the polyphenolic compounds in- fluence the antibacterial and antioxidant activity exhibited by bee pollen. The most important polyphenolic compounds related with these activities are vanillic acid, protocatechuic acid, gallic acid, p-coumaric acid, hesperidin, rutin, kaempferol, apigenin, luteolin, quercetin, and isorhamnetin (alicic et al., 2014). These compounds also serve as biochemical markers of bee pollen (tomás-BarBerán et al., 1989) related with the botanical origin. Bee pollen rich in these compounds has shown activity against specific pathogens such as Staphylococcus aureus (caBrera and montenegro, 2013), Escherichia coli (liBonatti et al., 2014; caBrera and monte- negro, 2013), Streptococcus viridians (camPos et al., 2010; Bisno and stevens, 1996), and Pseudomonas aeruginosa (aBouda et al., 2011; carPes et al., 2007). In order to establish relatioships between the botanical origin of multifloral Chilean bee pollen and their phenolic, protein and caro- tenoid content, and antioxidant/antibacterial activities, we present a characterization by HPLC-DAD of the phenolic compounds present in their extracts, their botanical origin, and a quantification of their total carotenoid and protein content. In vitro antioxidant and anti- bacterial activities were evaluated by Ferric Reducing Antioxidant Power (FRAP) and determining the zone of inhibition against E. coli, S. aureus, P. aeruginosa, and S. pyogenes, respectively. Cha- racterization on antioxidant and antibacterial properties present in samples of multiflora bee pollen will allow the beekeeping sector to add value to this product. Materials and methods Bee pollen Sixteen samples of commercial bee pollen were purchased from local beekeepers of Central Chile between December 2013 and February 2014. Samples were lyophilized and stored at -20 ºC. The determination of botanical origin was performed using palynological analysis method described at Chilean Regulation (NCh3255, 2011). Multi-floral bee pollen properties 307 Five grams of each type of bee pollen corbiculae were separated by color and each fraction was weighed. Then one corbiculae of each kind of bee pollen sample was crushed with alcohol to disperse the pollen grains. Several drops of red calberla were used to stain the grains allowing their observation under light microscope. To determine the botanical origin specific literature (marticorena and Quezada, 1985; Heusser, 1971) and the botanical palinoteca of Botanical laboratory at Pontificia Universidad Católica de Chile were consulted. Total protein content Protein determination was performed by Kjeldahl method based on standard AOAC (1984). One gram of sample was weighted and homogenized. In the digestion step organic nitrogen in the sample was decomposed by a solution of concentrated sulfuric acid, sodium sulfate, cuprum dioxide and applying a temperature cycle: 120 ºC for 15 min, 200 ºC for 2 min, 300 ºC for 2 min, and 402 ºC for 40 min, on a DK 6 Kjeldahl Digestion Unit (Velp Scientifica). Sodium hydro- xide was added and distillation on 3% boric acid was performed using a UDK 129 Kjeldahl Distillation Unit (Velp Scientifica). Titration was performed with 0.1 M hydrochloric acid. Conversion factor used was 6.25. Total carotenoid content Carotenoid extraction was performed weighting 4 g of bee pollen, milled and sonicated in an ultrasonic bath for 15 minutes with 20 mL of petroleum ether-acetone mixture (1:1 v/v). The extract was transferred to a separator funnel and washed with 60 mL of distilled water. The aqueous phase was discarded and the organic portion was passed through 2 g of anhydrous sodium sulfate. The whole process was repeated until the sample showed no coloration. Finally, the extract was evaporated to dryness under a stream of nitrogen, reconstituted in 2 mL of butanol and quantified by HPLC-DAD at 440 and 480 nm. Bee pollen extracts Bee pollen extraction process was based and adapted from leBlanc et al. (2009). Ten grams of multiflora bee pollen were mixed with 10 mL of distilled water and ultrasonicated for one hour. The mixture was centrifuged at 6000 rpm for 20 minutes and the supernatant was stored at 4 °C in darkness. This process was repeated 5 times. The collected supernatants were combined and filtered using qualitative paper (Whatman No. 2). Finally they were evaporated (rotary eva- porator Buchi R-210) and the dry extract was reconstituted with 10 mL of ultrapure water, filtered (EDLAB CA syringe filter 0.45 mm) and stored at -20 ºC. Total phenolic compounds Colorimetric determination of the phenolic content was evaluated by the Folin-Ciocalteu reaction (FC). Assays were performed on bee pollen extracts. The absorbance at 765 nm of the mixture of 200 μL extract, 50 μL Folin-Ciocalteu reagent, 150 mL of 20% w/v sodium carbonate solution (Na2CO3) and 600 mL of ultrapure water was measured in triplicate after 30 minutes of reaction. A calibration curve was constructed with gallic acid concentrations between 10 and 50 mg/mL. Antioxidant activity The antioxidant activity was determined by the Ferric Reducing Antioxidant Power (FRAP) assay. 200 μL of pollen extract w mixed with 1.8 mL of FRAP reagent, after 15 minutes in the dark the ab- sorbance was measured at 593 nm. FRAP reagent was prepared as follow: 25 mL of acetate buffer, 2.5 mL TPTZ solution (10 mmol/L of TPTZ in HCl 40 mmol/L) and 2.5 mL of 20 mM FeCl3 · 6H2O). A calibration curve was calculated with known solutions of FeSO4 · 7H2O in a concentration range of 20 and 100 μg/mL. In order to compare the antioxidant power of these samples, this test was performed to a blueberry sample, which is a recognized natural source of antioxidant compounds. Identification and quantification of flavonoids and phenolic acids The identification and quantification of flavonoids and phenolic acids on bee pollen extracts were performed by high performance liquid chromatography with a diode array detector based on Benzie and strain (1996). Elite Merck LaChrom HPLC Hitachi was used in a reverse phase column (LiChroCART RP-18) with a mobile phase of aqueous formic acid 5% (v/v) and methanol at constant solvent flow of 1 mL/min at 30 °C. Samples were injected manually. Chro- matograms were monitored at 290 and 340 nm. A calibration curve was made with high purity standards and area of peaks found with the EZChrom Elite v.3.3.1 (Scientific Software Inc. 1988-2005; Agilent 2005-2008) program. Antibacterial activity The antibacterial activity of bee pollen extracts was evaluated by diameter of inhibition against Escherichia coli ATCC-25922, Staphylococcus aureus ATCC-25923, Pseudomonas aeruginosa ATCC 27853 and Streptococcus pyogenes I.S.P. 364-00 (Supplied by Chilean Public Health Institute). Diameter of inhibition was determined using the standard reported by CLSI (2006): bacterial strains were inoculated on Mueller Hinton agar for 24 hours at 37 ºC. After that time, colonies were selected and diluted in saline solution to a concentration of 10-3 UFC by visual comparison with a standard of 0.5 McFarland (1.5×108, Becton & Dickinsson Company, USA). Once strains were swab on the agar, 6 mm diameter holes were made, and 100 μL of each extract were deposited in each hole. Petri dishes were incubated between 18 to 24 hours at 37 °C until measurements. The inhibition diameter that appeared around each hole was measured. Tetracycline, ampicillin and chloramphenicol were used as controls. Statistical analysis Statistical analysis of the results was performed using a one-way variance analysis (ANOVA) followed by Tukey HSD method with 95% (p<0.05) level of confidence and computed by STAT- GRAPHICS Centurion XV software 15.02.05. Samples were analyzed in triplicate. Correlations between results were made using the Pearson’s correlation coefficient (r) (p<0.05). Results and discussion The botanical origin described the presence of different plant sources used by bees to produce the bee pollen. This description permitted to classify them as native/non-native/mixed and unifloral/bifloral/ multiflora bee pollen (NCh 3255, 2011) (Tab. 1). Floral species found in the samples are closely related with the geographic location of hives. The analyzed samples of bee pollen were predominantly derived from non-native floral species and frequently from only one of them. The majority of samples analyzed corresponded to non-native unifloral (nine), followed by non-native multifloral, mixed multifloral, and non-native bifloral (two samples of each), and native unifloral (one). Among the samples analyzed, Galega officinalis predominated in thirteen samples and Brassica sp. was present in eleven samples. Brassica sp. accounted for 34% of the average weight of each sample, 308 P. Velásquez, K. Rodríguez, M. Retamal, A. Giordano, L.M. Valenzuela, G. Montenegro while Galega officinalis accounted for 40%. This indicates that Brassica sp. and Galega officinalis are important sources of pollen collection. The Asteraceae family was the least frequently detected in the samples analyzed, comprising only 2% of the average sample weight when present. Total protein content Total protein of the samples ranged between 20.0 and 30.4%, with an average of 25.4% (Fig. 1). The values observed are similar to the amounts reported in literature (almeida-muradian et al., 2005; Bogdanov, 2014; Balkanska and ignatova, 2012). This result Tab. 1: Botanical origin and classification of samples. Data represent analyses of 300 pollen grains counted in 5 distinct optical areas in three different samples. Sample Predominant pollen Secondary pollen Important minor pollen Minor pollen Classification (>45%) (16 - 45%) (3 - 15%) (<3%) Specie % Specie % Specie % Specie % 1 Brassica sp. 57.8 Galega officinalis 42.2 Non-native unifloral 2 Eschscholzia cali- 34.8 Medicago sativa 12.2 Apiaceae 2.0 Non-native fornica Schinus sp. 10.2 Fungal spores 2.0 bifloral Brassica sp. 30.6 Olea europaea 8.2 3 Schinus sp. 37.5 Mutisia sp. 2.5 Mixed Brassica sp. 22.9 Medicago poly- 0.4 multifloral morpha Medicago sativa 0.4 4 Galega officinalis 50.0 Asteraceae 26.0 Non-native raphanus sp. 24.0 unifloral 5 Medicago sativa 58.0 Galega officinalis 39.6 Hypochaeris/ 2.4 Non-native Taraxacum unifloral 6 Brassica sp. 36.5 Galega officinalis 15.6 Non-native Medicago poly- 27.1 multiflora morpha Convolvulus sp. 20.8 7 Brassica sp. 51.0 Adesmia sp. 2.0 Non-native Galega officinalis 46.9 unifloral 8 Prunus sp. 52.2 Maytenus boaria 2.2 Native Trevoa 45.7 unifloral quinquenervia 9 Brassica sp. 58.3 Sonchus sp. 22.9 Oxalis sp. 12.5 Asteraceae 2.1 Non-native Papilonaceae 4.2 unifloral 10 Brassica sp. 32.7 Medicago sativa 8.2 Trifolium sp. 2.0 Non-native Galega officinalis 26.5 Hypochaeris/ 8.2 Asteraceae 2.0 multifloral Dysopsis sp. 20.4 Taraxacum 11 Brassica sp. 72.9 Actinidia deliciosa 12.5 Galega officinalis 2.1 Non-native Fabaceae 4.2 Asteraceae 2.1 unifloral Hypochaeris/ 2.1 Taraxacum Quillaja saponaria 2.1 Vicia sp. 2.1 12 Galega officinalis 74.5 Brassica sp. 19.6 Fern spores 2.0 Non-native Trifolium repens 2.0 unifloral Fabaceae 2.0 13 Convolvulus sp. 43.2 Chenopodiaceae 2.3 Non-native Convolvulus sp. 38.6 bifloral Brassica sp. 16.0 14 Chenopodiaceae 58.0 Convolvulus sp. 24.0 Brassica sp. 12.0 Chenopodiaceae 2.0 Non-native Clarkia tenella 4.0 unifloral 15 Cactaceae 51.2 Galega officinalis 34.1 Amaranthaceae 12.2 Tecophilaceae 2.4 Mixed multifloral 16 Medicago sp. 62.5 Asteraceae 10.4 Non-native Mirtaceae 10.4 unifloral Galega officinalis 8.3 Malvaceae 8.3 Multi-floral bee pollen properties 309 Fig. 1: Total protein and carotenoid content of samples (mean ± SD; n = 3). In each column different letters imply significant differences (p<0.05). confirms that bee pollen could be a good source of vegetable protein replacing dietary animal sources such as meat (20% protein content, scHmidt et al., 1985), that currently are highly criticized for causing or increasing the likelihood of developing diseases (WHO, 2015; Bernstein et al., 2010). Bee pollen is even a better vegetable protein than quinoa (12-23% protein content, james, 2009). The samples composed by Prunus sp. 52.2% / Trevoa quinquener- via 45.7% (sample 8) and by Medicago sativa 58.0% / Galega offici- nalis 39.6% (sample 5) have the highest protein contents. These results are in agreement with vanderPlanck et al. (2014), whom reported 25.8% of protein in Prunus sp. bee pollen. There is not reported protein content of bee pollen from Trevoa quinquenervia. andrada and tellería (2005) reported that Medicago sativa has 22% of protein content and according to Peiretti and gai (2006) Galega officinalis has 20%. Moreover, samples such as Eschscholzia californica 34.8% / Brassica sp. 30.6% / Medicago sativa 12.2% / Schinus sp. 10.2% / Olea europaea 8.2% (sample 2), and Brassica sp. 58.3% / Sonchus sp. 22.9% / Oxalis sp. 12.5% (sample 9) samples have poorest content. These results are also expected since Forcone et al. (2013) reported 21.1% of protein content in bee pollen from Eschscholzia californica. Regarding the correlation of the protein content with the botanical origin, no relation was found with a confidence level of 95% (p <0.05). This indicates that the protein content is not dependent on any particular species. It is also observed that the bee pollen samples did not present significant difference in protein content between them (p <0.05). Total carotenoid content Carotenoids were observed in nine bee pollen samples, which ranged between 2.8 and 50.2 mg/kg of pollen with 12.0 mg/kg of pollen in average (Fig. 1). The values obtained in our samples were lowers than those ranged between 10 - 200 mg/kg reported in the literature from other multiflora bee pollen samples (almeida-muradian, 2005; Mărgăoan et al., 2010). This difference can be explained by the wide difference in carotenoid content between genus, families and species. Significant differences (p<0.05) were observed in total carotenoid content, where the samples of Galega officinalis 50% / Asteraceae 26.0% / Raphanus sp. 24.0% (sample 4) and Brassica sp. 57.8% / Galega officinalis 42.2% (sample 1) presented the highest carotenoid content. Meanwhile the sample composed by Cactaceae 51.2% / Galega officinalis 34.1% / Amaranthaceae 12.2% / Tecophilaceae 2.4% (sample 15) showed the lowest content that indicates that bee pollen from these species are poor as carotenoid sources. It is possible that the high content present in sample 4 was due to the presence of bee pollen from Galega officinalis since bee pollen from Brassica sp has been reported as very poor in carotenoids (stanciu et al., 2016) but also exists a high variation in carotenoid content inside species that compose this genus (jaHangir et al., 2009). oliveira et al. (2009) and BoBis (2014), has been reported that Asteraceae and Raphanus sp. are sources of high carotenoid content. There is no information about carotenoid contents of bee pollen from Cactaceae, Amaranthaceae and Tecophilaeaceae families but probably have lower contents. A positive correlation was found between carotenoid content and Asteraceae (r=0.92; n=5; samples 4, 9, 10, 11, 16) and Raphanus sp. (r=0.95; n=1; sample 4). A moderate interdependence between carotenoid content and Galega officinalis was observed (r=0.45; n=13; samples 1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, and 16). However, it is not possible to affirm that the presence of these species really correlated with the carotenoid content since there is no information about these parameters in samples of bee pollen from these species. Identification and quantification of flavonoids and phenolic acids Six phenolic acids (p-coumaric, chlorogenic/caffeic, ferulic, sinapic, and cinnamic acids) and two flavonoids (kaempferol, luteolin) were identified by liquid chromatography with diode array (Tab. 2). p-Coumaric acid was presented in all samples and chlorogenic/ caffeic and ferulic acids were presented in ten samples. The most frequent flavonoid was kaempferol and the least frequent was luteolin. Kaempferol was presented in six samples and luteolin in only one. Samples with the highest polyphenolics concentrations (i.e., phenolic acid +flavonoids) were Medicago sp. 62.5% (sample 16, 918.42 mg/kg) and Brassica sp. 51.0% / Galega officinalis 46.9% (sample 7, 638.63 mg/kg). These results differ from phenolic acids and flavonoids concentration previously reported for similar botanical origin bee pollen. The presence of kaempferol in bee pollen derived from Brassica sp. has been previously reported (Fatrcová-Šramková et al., 2013). It has been previously observed that bee pollen from Brassica napus subsp. napus L. contains luteolin (Fatrcová-Šramková et al., 2013). However, only one of the samples in this study that contain this botanical origin (Brassica sp. 51.0% / Galega officinalis 46.9% / Adesmia sp. 2%) presents luteolin, with a concentration 10 times higher than that reported by Fatrcová-Šramková et al. (2013). Apigenin, a common flavonoid present in bee pollen with a biological activity was not found in any sample. This variability in bee pollen 310 P. Velásquez, K. Rodríguez, M. Retamal, A. Giordano, L.M. Valenzuela, G. Montenegro is derived by the variability of phenolic compounds produced by plants, which depends on the stress conditions, geographic location and vegetation around the apiaries, which conditioned the flowering (morais et al., 2011; leja et al., 2007). Regarding the correlation between phenolic acids/flavonoids and botanical origin several dependences were observed. Medicago showed a positive correlation with p-coumaric acid (r=0.67; n=16) that indicates high concentrations of this compound in samples that include it (i.e., samples 2, 3, 5, 6, 10, and 16). Bee pollen from Eschscholzia californica and Olea europaea has a positive correlation with cinnamic acid content (r= 0.54, 0.54, respectively) that is in agreement with high content of this compound at samples with these kind of bee pollen (i.e., sample 2). Samples with bee pollen from Malvaceae has a high correlation with chlorogenic/caffeic acid and p-coumaric acid (r=0.67, 0.87, respectively) that indicates a high content of this compounds at samples that have bee pollen from these species (i.e., sample 16). Samples with Mirtaceae bee pollen also have a high correlation with chlorogenic/caffeic acid and p-coumaric acid (r=0.67, 0.87, respectively) that is present in sample 16. Bee pollen from Sonchus sp., Oxalis sp and Papilonaceae have a high correlation with kaempferol (r=0.97, 0.97, 0.97, respectively; n=16) that is in agreement with the high content of kaempferol at sample 9. Sample 10, composed by bee pollen from Dysopsis sp. has a positive correlation with ferulic and sinapic acid (r=0.57, 0.52) that indicates a high content of this compound. Finally, bee pollen from Actinidia deliciosa has a positive correlation with sinapic acid (r=0.68) that is in agree with the higher content at sample 11. Determination of total phenolic content and ferric reducing antioxidant power (FRAP) Total phenolic content and ferric reducing antioxidant power (FRAP) for bee pollen samples are showed in Fig. 2. The total phenolic con- tent of samples ranged between 6.86 and 52.99 g GAE/kg of pollen, with an average value of 12.64 g GAE/kg of pollen. These contents are higher than other reports such as values published by Pascoal et al. (2014) and Feás et al. (2012) which showed ranges between 4.96 and 19.80 g GAE/kg, respectively. The sample of Prunus sp. 52.2% /Trevoa quinquenervia 45.7% / Maytenus boaria 2.2% (sample 8) and Brassica sp. 58.3% / Sonchus sp. 22.9% / Oxalis sp. 12.5% (sample 9) have the highest values (33.34 and 52.99 g GAE/ kg, respectively). Mărgăoan et al. (2013) and stanciu et al. (2016) reported a content of 8.87g GAE/kg and 7.57g GAE/kg on average respectively for bee pollen from Prunus sp. In addition, stanciu et al. (2016) reports that the content of bee pollen from Brassica sp. is 11.62 g GAE/kg and 5.46 g GAE/kg for Oxalis sp. bee pollen. All values being higher than the bee pollen of another species. As for the phenolic content of bee pollen from Sonchus sp., Trevoa quinquenervia and Maytenus boaria, there are no reports, however they may have a high content considering the total content of the samples. The high phenolic value of Brassica sp. 58.3% / Sonchus sp. 22.9% / Oxalis sp. 12.5% / Papilonaceae 4.2% / Asteraceae 2.1% (Sample 9) may be due to the high content reported at Tab. 2. Brassica sp. has been reported with a high content of kaempferol associated with its antioxidant activity (Fatrcová-Šramková et al., 2013; li et al., 2016). There are no reports of the presence of kaempferol in bee pollen of the other species present in the sample 8 and 9. FRAP values ranged between 4.51 and 91.19 mmol Fe+2/kg pollen, with an average of 35.95 mmol Fe+2/kg pollen (Fig. 3). The sample of Prunus sp. 52.2% / Trevoa quinquenervia 45.7% / Maytenus boaria 2.2% (sample 8) has the highest FRAP value with 91.19 mmol Fe+2/ kg pollen. This value was higher than the FRAP value of blueberry, a very well known natural antioxidant (between 58.99 and 63.41 Fe+2 mmol/kg). However, compared to the FRAP values found in literature (5.36 mM Fe+2/g, margHitas et al., 2009 and 21 mM eq. Fe+2/g, montenegro et al., 2013), our results are much smaller (less Tab. 2: HPLC-DAD profile of bee pollen samples evaluated (mean ± SD; n = 3). In each column different letters imply significant differences (p<0.05). Nd: Non-detected (under detection threshold). Sample Chlorogenic + Ferulic acid Sinapic acid p-Coumaric acid Cinnamic acid Kaempferol Luteolin Caffeic acid (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) tR (min) 6.90 + 7.06 11.74 9.09 8.42 11.74 12.67 11.96 Λ(nm) 340 290 340 290 340 340 340 1 18.16 ± 0.73ab Nd Nd 4.02 ± 0.16a Nd Nd Nd 2 18.07 ± 0.72ab 5.66 ± 0.23b Nd 92.01 ± 3.68bc 8.93 ± 0.36d Nd Nd 3 Nd Nd Nd 287.37±11.50g Nd Nd Nd 4 Nd Nd Nd 109.91 ± 4.40c Nd Nd Nd 5 Nd Nd Nd 458.52 ±18.34i Nd Nd Nd 6 Nd Nd Nd 337.05±13.48h Nd Nd Nd 7 Nd 9.97 ± 0.40d Nd 255.17±10.21ef Nd 57.49 ± 2.29c 316.00±2.64a 8 26.94 ± 1.08bc Nd Nd 71.55 ± 2.86b Nd Nd Nd 9 Nd 13.35±0.53e Nd 4.02 ± 0.16a Nd 344.20±13.76d Nd 10 29.79 ± 1.19c 26.33 ±1.05h 72.95±2.92a 286.21±11.45fg Nd 17.11 ± 0.68b Nd 11 111.60± 4.46f 22.61±0.90g 89.67±3.59b 169.60 ± 6.78d Nd 63.68 ± 2.55c Nd 12 43.37 ± 1.73d 4.09 ± 0.16a 22.28±0.89c 82.19 ± 3.29bc Nd Nd Nd 13 86.11 ± 3.44e 5.48 ± 0.22b Nd 73.28 ± 2.93b 7.30 ± 0.29b 5.33 ± 0.21a Nd 14 45.16 ± 1.81d 7.50 ± 0.30c 9.12 ± 0.36d 191.91 ± 7.68d 6.49 ± 0.26a Nd Nd 15 11.29 ± 0.45a 6.72 ±0.27bc 51.84±2.07e 243.21 ± 9.73e Nd 19.23 ± 0.77b Nd 16 258.92±10.36g 20.58±0.82f Nd 630.92 ±25.24j 8.00 ± 0.32c Nd Nd Multi-floral bee pollen properties 311 Fig. 3: Antibacterial activity showed by bee pollen extracts against Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. Dashed line indicate diameter of hole with bee pollen extracts (mean ± SD; n = 4). Fig. 2: Total phenolic content and antioxidant activity of samples (mean ± SD; n = 3). than 0.1 mmol Fe+2/g). This may be due to the fact that our extracts were obtained using water instead of ethanol or methanol (do et al., 2014). As expected from similar studies (ulusoy and kolayli, 2014; Borycka et al., 2016), there is a positive correlation coefficient between total phenolic content and FRAP: Pearson’s correlation coefficient of 0.59 (Fig. 3). Thus, the antioxidant power showed by bee pollen samples can be attributed to their phenolic content. A positive correlation was also found between total phenolic content and Prunus sp. (n=16; r=0.50), Trevoa quinquenervia (n=16; r=0.50), Oxalis sp. (n=16; r=0.79), Papilonaceae (n=16; r=0.79) and Sonchus sp. (n=16; r=0.79), and between FRAP with Prunus sp. (n=16; r=0.76) and Trevoa quinquenervia (n=16; r=0.76). Furthermore a high dependence between total phenolic content and kaempferol (n=16; r=0.78) was observed. These results indicate that phenolic content of samples that includes bee pollen from Prunus sp., Trevoa quinquenervia or Sonchus sp. are result of kaempferol content. Antibacterial activity Antibacterial assays showed that bee pollen is more active against Gram-positive (i.e., S. aureus and S. pyogenes) than Gram-negative bacteria (i.e., P. aeruginosa and E. coli) (Fig. 3). Fifteen samples inhibited S. pyogenes, fifteen samples inhibited S. aureus, five samples inhibited P. aeruginosa and only one showed inhibition against E. coli. These results show that Gram-positive bacteria are better controlled by the bee pollen than Gram-negative bacteria, which showed more resistance. This tendency was also reported in another study from our research group (caBrera and montenegro, 2013). Gram-negative bacteria have a double cell wall, composed by lipopolysaccharides and proteins, which hinders the antibacterial action of bee pollen thus E. coli and P. aeruginosa because are more resistant therefore were less controlled (taFur et al., 2008). Fifteen out of sixteen samples showed control against Streptococcus pyogenes. The range of inhibition was observed between 9.3 and 31.3 mm, similar to the range of between 9 and 28 mm reported in the literature (aBouda et al., 2011; caBrera and montenegro, 2013). The highest diameter of inhibition was observed in sample 13 (31.3 mm), higher than tetracycline and closer to ampicillin. This sample was composed by Convolvulus arvensis 43.2% / Galega officinalis 38.6% / Brassica sp. 16%. There are no reports indica- ting that Galega officinalis and Convolvulus arvensis bee pollen have antibacterial activity against S. pyogenes. caBrera and montenegro (2013) report one sample that contains 30% of Brassica sp. bee pollen which controlled S. pyogenes. There is a positive correlation between Chenopodiaceae (r=0.65; 312 P. Velásquez, K. Rodríguez, M. Retamal, A. Giordano, L.M. Valenzuela, G. Montenegro n=16) and Convolvulus sp. (r=0.58; n=16) with the inhibition activity against S. pyogenes, and a negative correlation with Eschscholzia californica (r=-0.58; n=16) and Olea europaea (r=-0.57; n=16). These correlations would indicate that some species enhance the antibacterial activity of bee pollen while others decrease it when are present. These species could contain phenolic/flavonoid compounds that would not necessarily be poor antibacterials since they could compete for sites of action with phenolic/flavonoid compounds from other species or results in antagonistic effects (kumar and Pandey, 2013; mandalari et al., 2010; PalaFox-carlos et al., 2012). It is also possible that samples containing bee pollen from Chenopodiaceae and Convolvulus sp. contain compounds that inhibit the bacterial growth of S. pyogenes, however do not exist in the literature reports on this. Since Eschscholzia californica, Olea europaea and Chenopodiaceae correlates with cinnamic acid (r=0.54, 0.54 and 0.56, respectively) it suggest a relation with antibacterial exerted against S. pyogenes. However 15 samples inhibited S. pyogenes and only 4 samples have cinnamic acid. Therefore no correlation between botanical origin and antibacterial activity against S. pyogenes. Fifteen out of sixteen samples showed growth inhibition against Staphylococcus aureus. Sample 5 showed the highest inhibition diameter with 18.7 mm, similar to half of the ampicillin and tetracycline diameters. Medicago sativa (58.0%) and Galega offici- nalis (39.6%) predominate in sample 5. There is no data indicating that the bee pollen obtained from these species has antibacterial activity. However, there are many studies indicating that some of these plants have antibacterial activity against S. aureus, which has been attributed to certain flavonoids, saponins and peptides (rodrigues et al., 2013; karakas et al., 2012; erturk, 2010). There is a positive correlation between antibacterial activity of bee pollen samples against S. aureus and Medicago sativa (n=16; r=0.50) and a negative correlation with Malvaceae (n=16; r=-0.72), Medicago sp. (n=16; r=-0.81) and Mirtaceae (n=16; r=-0.81). The antibacterial activity in this case is mainly a result of chlorogenic/ caffeic and p-coumaric acids since correlation were found between these compound and bee pollen from these species (see section above: Identification and quantification of flavonoids and phenolic acids). The samples with a higher concentration of p-coumaric acid than chlorogenic/caffeic acids (i.e., samples 2, 3, 5, 10, 16) showed a higher antibacterial activity. The bacterial growth inhibition exerted by bee pollen extracts against P. aeruginosa was less effective than the other bacteria assayed. Only 5 out of sixteen samples showed positive results. The sample composed by Medicago sativa 58.0% / Galega officinalis 39.6% / Hypochaeris-Taraxacum 2.4% (sample 5) showed the high- est diameter of inhibition (11.3 mm). This result was similar to tetracycline and higher than ampicillin that did not have inhibition against P. aeruginosa. The inhibition diameters observed were found to be similar to that reported by aBouda et al. (2011). There is a positive correlation between antibacterial inhibition showed against P. aeruginosa and Hypochaeris-Taraxacum sp. (r=0.52; n=16). Since a moderate dependence were found between Hypochaeris- Taraxacum sp. and ferulic and sinapic acids, it suggests a relation of these compounds with antibacterial effect exerted against S. pyogenes. However, only two out of five samples that inhibited S. pyogenes have ferulic and sinapic acids in their composition. Therefore no correlation between botanical origin and antibacterial activity against P. aeruginosa. Escherichia coli was controlled only by one sample (sample 12) that formed an inhibitory diameter of 10.0 mm. However, zones of inhibition were reported in the literature ranging from 15 to 40 mm (kHider et al., 2013). This difference could be explained by the different botanical origin of bee pollen sample compared with kHider et al. (2013) and the extractant used (i.e., methanol/ hexane vs. water). The botanical origin of sample 12 was composed mainly by Galega officinalis 74.5% / Brassica sp. 19.6%, which have shown inhibition against Gram-negative bacteria, so they should be further investigated for the responsible compounds of their activity (erturk, 2010). Inhibition activity against E. coli showed a positive and moderate dependence the presence of bee pollen from Galega officinalis (r=0.52; n=16). However, the antibacterial activity observed cannot be attributed to Galega officinalis since is also presented in other samples that no showed this control against E. coli. Conclusion s We reported for the first time the relationship bet ween botanical origins of bee-pollen from Chile and their phenolic, protein and carotenoid content, and antioxidant/antibacterial activities. It was demonstrated that several plant species contribute these parameters, mainly Brassica sp. and Galega officinalis. Less frequent species such as fruit and endemic species as Medicago sativa, Prunus sp., Trevoa quinquenervia, Prunus sp., and Convolvulus arvensis contribute to differences between composition and antioxidant/antibacterial activities of bee pollen samples. In addition species present in lower concentrations also help to accentuate these differences. Samples with high protein content are composed by bee pollen from Prunus sp., Trevoa quinquenervia, Medicago sativa and Galega officinalis. Samples with high carotenoid content are composed by bee pollen from Galega officinalis, Asteraceae, Raphanus sp. The samples with higher content of polyphenols are composed by Medicago, Brassica sp. and Galega officinalis bee pollen. Six phenolic acids (p-coumaric, chlorogenic / caffeic, ferulic, synapic and cinnamic acids) and two flavonoids (kaempferol and luteolin) were identified in the samples. The highest content of phenolics was presented in samples composed by Prunus sp. and Brassica sp. bee pollen. While those with higher antioxidant power (FRAP) presented Prunus sp. and Trevoa quinquenervia bee pollen. Samples composed by Con- volvulus arvensis, Galega officinalis and Brassica sp. showed inhibitory activity against S. pyogenes; those which contain Medi- cago sativa and Galega officinalis bee pollen inhibited S. aureus and P. aeruginosa; E. coli was controlled by samples with Galega officinalis and Brassica sp. bee pollen. Botanical origin analysis of bee pollen permits correlation with some chemical, antioxidant and antibacterial properties, suggesting new resources of bioactive compounds. Further studies with monofloral bee pollen loads are needed in order to provide more accurate correlations between botanical origin and composition and antioxidant/antibacterial acti- vities. 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