Monofloral Corn Poppy Bee-Collected Pollen—A Detailed Insight into Its Phytochemical Composition and Antioxidant Properties

The aim of this study was to compile a detailed phytochemical profile and assess the antioxidant properties of bee-collected pollen (PBP) obtained from corn poppy (Papaver rhoeas L.) plants. To achieve this, a lipid fraction was prepared for quantifying fatty acids using GC-FID. Extractable and alkaline-hydrolysable PBP fractions (obtained from a defatted sample) were used to determine the qualitative and quantitative profiles of phenolic compounds, phenylamides and alkaloids using UHPLC/Q-ToF-MS. Additionally, various spectrophotometric assays (TAC, FRP, CUPRAC, DPPH⦁) were conducted to evaluate the antioxidant properties. Phenolic compounds were more present in the extractable fraction than in the alkaline-hydrolysable fraction. Luteolin was the predominant compound in the extractable fraction, followed by tricetin and various derivatives of kaempferol. This study presents one of the first reports on the quantification of tricetin aglycone outside the Myrtaceae plant family. The alkaline-hydrolysable fraction exhibited a different phenolic profile, with a significantly lower amount of phenolics. Kaempferol/derivatives, specific compounds like ferulic and 5-carboxyvanillic acids, and (epi)catechin 3-O-gallate were the predominant compounds in this fraction. Regarding phenylamides, the extractable fraction demonstrated a diverse range of these bioactive compounds, with a notable abundance of different spermine derivatives. In contrast, the hydrolysable fraction contained six spermine derivatives and one spermidine derivative. The examined fractions also revealed the presence of seventeen different alkaloids, belonging to the benzylisoquinoline, berberine and isoquinoline classes. The fatty-acid profile confirmed the prevalence of unsaturated fatty acids. Furthermore, both fractions exhibited significant antioxidant activity, with the extractable fraction showing particularly high activity. Among the assays conducted, the CUPRAC assay highlighted the exceptional ability of PBP’s bioactive compounds to reduce cupric ions.


Introduction
Nutrition is one of the most important aspects of our lives, but it is also one of the most delicate. Modern consumers are increasingly seeking high-quality food from natural sources. Consequently, food production has become a rapidly growing and demanding The PBP extraction procedure ( Figure 2) followed the method detailed in our vious research [26] with one modification-the ultrasound-assisted extra pre-treatment was extended to 1 h instead of 30 min. Prolonged ultrasound-assiste traction should have a positive effect on the improved extraction of bioactive compon via the additional destruction of pollen membranes-exine and intine. As a result, different extracts were obtained and used: (1) lipid PBP extract was used for the d mination of fatty-acid composition; (2) extractable PBP phenolic fraction was us determine the composition of phenolic compounds, phenylamides and alkaloids; ( kaline-hydrolysable PBP extract (referred to as bound PBP fraction) was applied t termine phenolic, phenylamide, and alkaloid composition; and (4) PBP extract in acetone was used to determine the total carotenoid content. The PBP extraction procedure ( Figure 2) followed the method detailed in our previous research [26] with one modification-the ultrasound-assisted extraction pre-treatment was extended to 1 h instead of 30 min. Prolonged ultrasound-assisted extraction should have a positive effect on the improved extraction of bioactive components via the additional destruction of pollen membranes-exine and intine. As a result, four different extracts were obtained and used: (1) lipid PBP extract was used for the determination of fatty-acid composition; (2) extractable PBP phenolic fraction was used to determine the composition of phenolic compounds, phenylamides and alkaloids; (3) alkaline-hydrolysable PBP extract (referred to as bound PBP fraction) was applied to determine phenolic, phenylamide, and alkaloid composition; and (4) PBP extract in 80% acetone was used to determine the total carotenoid content.

General Phytochemical Characterization
Corn poppy bee-collected pollen (PBP) extracts obtained for extractable and bound phenolic fractions were examined and characterized as previously described [26] to determine the following general phytochemical parameters: (1) total phenolic content (TPC); (2) total flavonoid content (TFC); (3) total hydroxycinnamic-acid-derivative content (HCA); and (4) total carotenoid content (TCC). All results are expressed as mg of adequate equivalents per g of dry weight (dw) sample except for TCC (µ g/g dw) [26].

A Detailed Profiling of Obtained Extracts
Corn poppy bee-collected pollen (PBP) extracts obtained for lipid, extractable and bound phenolic fractions were studied to determine detailed PBP profiles. For fattyacid composition determination, the previously described method was applied [27]. The obtained results are expressed as % of total fatty acids determined in the sample.
The following procedure was applied for the detailed profiling of phenolics, phenylamides and alkaloids present in PBP. The analyses were carried out using an Agilent 1290 Infinity ultra-high-performance liquid chromatography (UHPLC) system coupled with quadrupole time-of-flight mass spectrometry (6530C Q-ToF-MS) from Agilent Technologies, Inc., CA, USA. The chromatographic separation was conducted at 40 • C on a Zorbax C18 column (2.1 × 50 mm, 1.8 µm) from Agilent Technologies, Inc., CA, USA. The mobile phase consisted of a mixture of (A) ultrapure water and (B) acetonitrile (MS grade), both A and B containing 0.1% HCOOH (MS grade). The flow rate was kept constant at 0.3 mL/min, and the injection volume was 5 µL. The gradient elution program began with 2% solvent B for the first 2 min, which then reached 98% B over the next 17 min, and over the next 5 min the gradient returned to its initial state (2% B) to re-equilibrate the column. The QToF-MS system was equipped with a Dual Agilent Jet Stream electrospray ionization (ESI) source operating in both positive (ESI + ) and negative (ESI − ) ionization modes. The operation parameters for ESI were set as follows: nebulizer pressure of 45 psi, a drying gas temperature of 225 • C and a flow rate of 8 L/min, sheath gas temperature of 300 • C and sheath gas flow 10L/min, capillary voltage of 2500 V, fragmentor energy of 175 V, skimmer voltage of 65 V, octopole RF Peak at 750 V. The QToF-MS system recorded the spectra over the m/z range 50-1700, with a scan rate of 2 Hz. Data-dependent acquisition (DDA) was employed for suspect screening using the Auto MS/MS acquisition mode with collision energy at 30 eV. Parameters for the Auto MS/MS mode were as follows: m/z = 50-1700, scan rate 1 spectra/sec. Agilent MassHunter software was used for data evaluation and analysis. Phenolics were identified based on their monoisotopic mass and MS fragmentation, while they were quantified via direct comparison with available standards. Due to the lack of some specific standards, the quantities of the individual phenolic derivatives were quantified using available standards and expressed as µg/kg of the sample DW. Table S1 shows a list of phenolic compounds used for quantification, together with their equation parameters and correlation coefficient (r 2 ). Anthocyanin, phenylamide derivatives and alkaloids were identified based on their monoisotopic mass and MS 2 fragmentation and confirmed using previously reported data found in the literature. Accurate masses of components were calculated using ChemDraw software (version 12.0, CambridgeSoft, Cambridge, MA, USA). All the results for the content of phenolic compounds are given as µg/kg dw of the sample. Equations used for quantification of identified phenolic compounds are given in the supplementary file (Table S1).

Antioxidant Properties of PBP
Corn poppy bee-collected pollen extracts, containing extractable and bound fractions of bioactive compounds, were subjected to the examination of antioxidant properties by applying four different assays: in vitro phosphomollybdenum total antioxidant capacity (TAC), DPPH (α, α-diphenyl-β-picrylhydrazyl) radical scavenging activity, Cupric-Reducing Antioxidant Capacity (CUPRAC) and Ferric-Reducing Power (FRP), following the method detailed in our previous research [26].

Statistical Analysis
Results for general phytochemical characterization, antioxidant properties and fattyacid composition were performed in triplicate and presented as means ± standard deviation (SD). For evaluation of statistical differences between the means, we applied t-tests (p < 0.05) (Statistica software version 12.0, StatSoft Co., Tulsa, OK, USA).

General Phytochemical Composition
Based on different spectrophotometric methods, it is possible to determine the general and outline content for different groups of phytochemicals. In this case, the results obtained for the PBP content of the determined phytochemicals are presented in Table 1. In all cases, phenolic compounds were predominantly present in the extractable fraction compared with the bound fraction. The extractable fraction exhibited the highest content of total phenolics (11.6 mg/g GAE dw) and flavonoids (12.8 mg/g QE dw). On the other hand, hydroxycinnamic acid derivatives were the least represented subclass of phenolics in both fractions, ranging from 1.14 to 5.96 mg/g CGAE dw. Interestingly, the spectrophotometric assay failed to quantify the total flavonoids in the bound fraction. In addition, the total carotenoid content determined for corn poppy PBP was 65.05 µg/g dw.

UHPLC Phenolic Profile of PBP Extracts
The obtained results derived from UHPLC-QToF-MS analysis are presented in Table 2. According to the obtained results, it can be observed that phenolics belonging to four subclasses were identified and quantified: phenolic acids and derivatives, flavones, flavanones and derivatives and flavonols and derivatives. In total, fifty metabolites (fortyeight phenolics and two organic acids) were identified and quantified: thirteen phenolic acids/derivatives, five flavones, two flavanones, one ester of (epi)catechin and four anthocyanin derivatives. However, among phenolics, flavones, flavonols and derivatives were strongly predominant, with a remarkable diversity of twenty-three different compounds. Interestingly, (epi)catechin 3-O-gallate was identified and quantified only in the bound fraction (50.81 mg/kg dw), as well as two simple organic acids: citric (in both fractions) and gluconic (in the extractable fraction). The highest content in the extractable fraction was observed for two flavones, luteolin (4398.1 mg/kg dw) and tricetin (3048.97 mg/kg dw), followed by kaempferol (414.85 mg/kg dw) and derivatives: kaempferol 3-O-(2 -pentosyl)hexoside (885.46 mg/kg dw) and kaempferol 3-O-(6 -pentosyl)hexoside (714.93 mg/kg dw). Furthermore, two quercetin derivatives (487.11-577.59 mg/kg dw) were quantified in significant quantities, as well as isorhamnetin as an aglycone (321.30 mg/kg dw). Once again, this confirmed the great predominance of flavones and flavonols and, in particular, their glycosides as phenolic compounds present in bee-collected pollen. Unlike the extractable fractions, the bound fractions contained a significantly lower amount of different phenolics with much lower diversity. Among them all, kaempferol 3-O-(2 -pentosyl)hexoside (700.17 mg/kg dw) and its aglycone (364.72 mg/kg dw) were predominant. Moreover, some phenolic acids were present only in the bound fraction, like ferulic acid (125.32 mg/kg dw) and 5-carboxyvanillic acid (610.81 mg/kg dw), which were probably liberated from the cell-wall component due to the breaking of the chemical bonds caused by strong alkaline conditions [28][29][30]. Curiously, none of the quercetin and most of the isorhamnetin derivatives were present in the bound fraction. Due to the lack of appropriate standards, anthocyanin derivatives were only identified (Table 3).  It is worth noting that different anthocyanin derivatives were identified in the fractions. While the extractable fraction contained cyanidin 3-O-(6 -pentosyl)hexoside isomer I and delphinidin 3-O-(6 -O-hexosyl)hexoside, the bound fraction was characterized by the presence of cyanidin 3-O-(6 -pentosyl)hexoside isomer II and cyanidin 3-O-glucoside. These compounds (most probably delphinidin derivative) are likely responsible for the purple color of the extractable fraction prepared for analysis ( Figure 1).

UHPLC Phenylamide (Derivatives) Profile of PBP Extracts
The obtained results derived from UHPLC-QtoF-MS analysis are presented in Table 4. In total, twenty-seven phenylamide derivatives were identified, while two were partially identified as coumaroyl derivatives. Among them, phenyl derivatives of spermine were predominant (eighteen compounds), followed by five phenyl derivatives of putrescine and four compounds originating from spermidine. Based on the phenolic moiety present in these phenylamides, a significant predominance was observed for different coumaroyl derivatives, followed by an acetyl, caffeoyl, feruloyl and benzoyl structural unit or in combination with them.

UHPLC Alkaloid Profile of PBP Extracts
The obtained results derived from UHPLC-QToF-MS analysis are presented in Table 5. In total, seventeen different alkaloids were identified in both fractions belonging to three distinct alkaloid subclasses: benzylisoquinoline, berberine and isoquinoline types. All compounds were detected in the extractable fraction, whereas the bound fraction contained twelve different alkaloids.

Fatty-Acid Profile of PBP Extract
The obtained results derived from GC-FID analysis are presented in Table 6. In total, nine different fatty acids (FAs) were identified and quantified. Based on the obtained results, it can be observed that the most abundant FA in lipid PBP extract was docosahexaenoic (DHA) acid (25.26%), followed by α-linolenic (22.98%) and stearic (13.72%) acids. A significant predominance of unsaturated fatty acids was observed, since almost 75% of all the FAs were different UFAs.
As can be seen from the given results, the extractable fraction exhibited significantly higher antioxidant activity in all applied assays. Both fractions showed a high ability to reduce Cu 2+ ions, as determined by the CUPRAC assay. However, the bound fraction had a low total antioxidant capacity (0.92 mg/g AAE dw) as well as a low ability to reduce ferric ions (0.35 mg/g AAE dw).

General Phytochemical Composition
Based on the results obtained for carotenoid content, it can be concluded, in line with the literature, that PBP possessed a significantly higher content of carotenoids compared with a monofloral coconut sample from Brazil (2.17-3.55 µg/g dw) [31] or an artichoke bee-collected pollen sample from Serbia (5.00 µg/g dw) [26]. This difference may be related to the lighter color intensity observed in both coconut and artichoke bee-collected pollen samples [26,31] compared with that in PBP. Among the different phenolic subclasses, spectrophotometric analyses showed a significantly higher content for all phenolics in the extractable fraction compared with the bound fraction. The obtained TFC (12.82 mg/g QE dw) in the extractable fraction was high, while in the bound fraction, total flavonoids were below the limit of detection. The obtained value was significantly higher or in line with previously published data on pollen samples from Brazil and Serbia, where the results for the extractable fractions ranged from 0.3 to 11 mg/g QE [10,26,32]. The only sample with a higher TFC value was the monofloral Myrcia bee-collected sample (17.5 mg/g QE) from the Brazilian state Rio Grande de Sul [32]. According to TPC results, the PBP extractable fraction contained a significantly higher amount of total phenolics compared with a sample with similar botanical origin-P. somniferum bee-collected pollen from Slovakia (0.82 mg/g GAE) [23]. However, the TPC result was in line with several beecollected pollen samples from Brazil [32], particularly with monofloral Mimosa caesalpiniifolia bee-collected pollen (12.1 mg/g GAE dw), while it was significantly higher compared with that of coconut (~2 mg/g GAE dw) [31], sunflower (extractable-2.9-3.8 GAE dw) [10] and artichoke (extractable-5.3 mg/kg GAE dw; bound-0.5 mg/g GAE dw) [26] monofloral bee-collected pollen samples from Brazil and Serbia, respectively. Moreover, a beecollected pollen sample from Morocco (Coriandum sativum + Cistaceae) showed a similar TPC value-13.73 mg/g GAE [33]. Unlike TPC and TFC assays, results for the HCA content in bee-collected pollen samples are quite rare. It was reported that artichoke bee-collected pollen contained 1.06 mg/g CGAE dw in the extractable fraction, while these derivatives were not observed in the bound fraction [26]. In both cases, the results were significantly lower compared with PBP. The presented comparative literature analysis once again confirms the importance of both the botanical and geographical origin of pollen samples for their content of different bioactive compounds.

UHPLC Phenolic Profile of PBP
There is a lack of data about the phenolic profiles of both poppy (P. somniferum) and corn poppy bee-collected pollen. To the best of our knowledge, the only available data are provided by Kačaniova et al. [34] for the P. somniferum sample collected in Slovakia. The authors reported the presence of four different aglycone flavonoids (luteolin, kaempferol, apigenin, quercetin) according to HPLC analysis, with a great prevalence of luteolin (1390.67 mg/kg dw). This is consistent with the data from the present research, as luteolin and other identified/quantified flavonoids were present in the PBP sample in the form of aglycone or some glycoside derivatives, with luteolin found in a concentration four times higher than the most prevalent compound. However, what is interesting is the presence of tricetin, another flavone found as the second most prevalent phenolic compound in the extractable fraction. Until know, this compound has been recognized as a taxonomic marker for Myrtaceae pollen samples [35] as well as for honey samples originating from the same botanical family [36]. However, a recent review article clearly stated that there is a strong possibility that this aglycone is occasionally overlooked in different samples due to the lack of modern and more precise techniques [36] such as QToF. In line with this and the results of the present study, recent research has provided detailed data on the phenolic profile of the Perilla frutescens L. (Lamiaceae) plant, confirming the presence of tricetin as an aglycone [37]. Additionally, it should be mentioned that there is a strong possibility that this aglycone is actually a precursor for the biosynthesis of alkaloids in Papaver plants [38]. Moreover, detailed phenolic profiles of different pollen samples originating from different areas and plants have revealed an astonishing diversity of different phenolic compounds/derivatives [19,32,39,40]. What is common in all the research studies is the predominant representation of flavonols, flavones and derivatives. Some of the compounds found in the current research have already been identified in different samples. For instance, p-coumaric (0.20 mg/g) and caffeic (0.15 mg/g) acids were determined in monofloral Moroccan bee-collected pollen samples originating from Apiaceae and Fabaceae plants, respectively [41]. Furthermore, similar to the current research, the authors found different quercetin and kaempferol glycosides (ranging from 1.19 to 1.77 mg/g) as predominant compounds [41]. In a polyfloral sample consisting mostly of Cytisus stratius and Eucalyptus sp. pollen from north-east Portugal, quercetin-3-O-rhamnoside and luteolin were identified as predominant phenolics [19]. Similarly, in bee-collected pollen samples from Brazil, isorhamnetin-3-O-(2 -O-rhamnosyl)glucoside, the same compound found in the PBP sample, was identified as one of phenolics in monofloral Cocos nucifera L. beecollected samples [40]. Additionally, quercetin-3-O-arabinoside, identified in seven floral pollen samples originating from São Paolo, a Brazilian state [39], could respond to quercetin-3-O-pentoside, identified and quantified in the current research. In the same study, the presence of kaempferol-3-O-glucoside was also confirmed in all examined samples [39]. Interestingly, the most recent publication provided data on monofloral Castanea bee-collected pollen from the Iberian Peninsula (Galicia and northern Portugal) containing exclusively isorhamnetin derivatives (five compounds) as well as naringenin among the phenolics [42]. However, significant differences in other specific compounds found in PBP compared with the literature data can be attributed to the botanical origin of the samples. When comparing the extractable and bound phenolic fractions, it is clear that most phenolics in PBP are present in the extractable fractions, as a significant number of compounds were absent in the bound fraction or quantified in decreased amounts. Similarly, a lower amount of bound phenolics was observed in artichoke monofloral bee-collected pollen [26]. However, some of the compounds, particularly certain phenolic acids and (epi)catechin-3-O-gallate, were identified and quantified only in the bound fraction. One possible reason for this is their release from complex biomacromolecules such as sugars, proteins and polymers present in cell walls under strong alkaline conditions. This phenomenon is strongly related to the plant's origin. For instance, it is well-known that in the case of cereals and wheat, the bound phenolic fraction can be predominant, accounting for up to 99% in cereal brans [29]. Regarding the identified anthocyanins in PBP, there are no available data on the presence of these phenolic subgroups in corn poppy pollen. In fact, there is a significant shortage of data on specific anthocyanins in pollen in general. Nevertheless, two independent studies have reported that anthocyanins are predominant compounds in the overall phytochemical composition (53.44-77.37 mg/L cyanidin-3-glucoside equivalents) of Castanea, Cistus and Rubus bee-collected pollen from Tuscany, Italy [43,44].

UHPLC Phenylamide Profile of PBP
This group of phenyl derivatives began to be examined in the last decade in beecollected pollen samples, and the data have been published in several reports [15,19,[39][40][41]45]. Similar to this current work, Zhang et al. [45] found a significant predominance of coumaroyl derivatives (thirty compounds) of spermine, spermidine and putrescine in monofloral samples from China, followed by six caffeoyl and four feruloyl derivatives. It was found that apricot monofloral bee-collected pollen contained predominantly different coumaroyl phenylamides, as well as sunflower pollen (but in a lower quantity), while rose beecollected pollen was also characterized by a significant abundance of caffeoyl derivatives. On the other hand, the camellia sample contained a higher amount of feruloyl derivatives compared with the other samples. Bee-collected pollen obtained from Quercus mongolica was characterized by four different, specific, polyamine derivatives-mangolicine A, and mangolidine A, B and C [15]. Corresponding to the present study, different isomers of tricoumaroyl spermidine were identified in Castanea bee-collected pollen samples [42], although a prevalence of caffeoyl spermidine derivatives was observed, unlike PBP. Coconut bee-collected pollen from Brazil [40] also contained tricoumaroyl spermidine, as well as six additional spermidine derivatives different from PBP. What was specific for PBP was the significant presence of spermine derivatives as opposed to previous research studies, as well as the presence of an acetyl moiety in these structures. Related to this, tetracoumaroyl spermine (isomer) was identified and quantified (3.34 mg/g) as the most predominant bioactive compound in the bee-collected pollen sample, containing, with great predominance, grains of two plant species/genera-Crepis capillaris (Smooth hawksbeard) and Plantago sp. [19]. These isomers were also identified and quantified in polyfloral Cystaceae and Asteraceae samples from Morocco [41]. Clearly, a great diversity of these compounds can be linked to botanical origin. With some additional statistical analyses of all known data, there is a possibility that some of them can be used as chemotaxonomic markers. In the case of PBP, acetyl derivatives should be of particular interest since there is only one report about their presence in Q. mongolica bee-collected pollen [15]. In the case of PBP, a great diversity has been observed: acetyl spermine, five coumaroyl acetyl spermine isomers, caffeoyl acetyl spermine, three coumaroyl diacetyl spermine isomers, dicoumaroyl acetyl spermine, coumaroyl feruloyl acetyl spermine, dicoumaroyl diacetyl spermine, tricoumaroyl acetyl spermine and dicoumaroyl acetyl feruloyl spermine were identified. Interestingly, there is a significant absence of phenylamides in the bound fraction, as only seven spermine derivatives were identified, while all putrescine and spermidine derivatives were deficient, probably due to the strong alkaline conditions applied for compound liberations from chemical bonds. It was reported that, depending on the amide structure, alkaline conditions can enhance amide bond hydrolysis [46].

UHPLC Alkaloid Profile of PBP
In the case of PBP extracts, the presence of alkaloids is strictly related to their botanical origin and corn poppy's chemical characteristics as alkaloid-rich plants. Specifically, different benzylizoquinoline alkaloids are present in corn poppy, with papaverine being the most important one [47]. In addition to papaverine, papaveraldine, a carbonyl derivative of papaverine, as well as papaverrubine E were also identified as specific corn poppy alkaloids in this study. Among pollen samples, the presence of alkaloids is not expected, except in the case of pyrrolozidine alkaloids [4,48]. They are predominantly specific for the pollen of Echium, Senecio and Eupatorium plants [48], representing undesirable components of pollen due to their toxicity. However, unlike these, other alkaloids also possess healthpromoting properties such as antiviral activities [49]. In particular, the alkaloids detected in PBP extracts can actually exert beneficial effects for humans through consumption. For instance, isoquinoline alkaloids possess anticancer properties [50], while papaverine, per se, has been recognized as a miracle compound with different health benefits [51]. It has been demonstrated that local application of papaverine on patients with end-stage renal disease can induce a reduction in arteriovenous fistula maturation without any additional difficulties [52]. This literature evidence indicates that the presence of alkaloids in PBP does not have to be harmful in itself in the event of eventual consumption as a functional food ingredient. Of course, in this case, the question of dosage is quite important.

GC-FID Fatty-Acid Profile of PBP
Fatty acids are one of the most important nutrients present in bee-collected pollen, with great diversity observed depending on the botanical origin of the samples [31,[53][54][55]. However, to the best of our knowledge, there are no data for the fatty-acid profile of corn poppy pollen, although there are extensive data on the lipid composition of poppy seeds and some other corn poppy plant parts. Based on the obtained results, it can observed that the lipid fraction of PBP is in line with the available data for poppy seeds, showing significant predominance of unsaturated FAs [56][57][58][59]. Nevertheless, the distribution of single acids differs in bee-collected pollen compared with poppy-seed composition. For instance, linoleic acid was predominantly found in seed samples [56][57][58], whereas in the case of PBP, docosahexaenoic acid (DHA) followed by α-linolenic acid were the main FAs. It has also been documented that α-linolenic acid is one of the predominant FAs in the leaves of corn poppy plants [60]. The significant presence of ω-3 FAs makes them an excellent source of these acids, which are important for balanced nutrition intake, along with ω-6 FAs, in the human diet. They are recommended to prevent the development of obesity [61]. Additionally, the significant share of DHA is extremely important since this FA has been recognized as one of the most important ω-3 FAs that can benefit both children and adults. It is documented to be important for brain development, the prevention of premature birth, cardiovascular diseases, as well as the improvement of cognitive function and vision in older people [62].

Antioxidant Properties of PBP Phenolic Extracts
The antioxidant properties of some pollen samples are mostly related to the content of different bioactive compounds, particularly phenolics. However, the importance of polyamines and their phenyl derivatives, which are identified in significant amounts in PBP extracts, should not be overlooked. It is well-known that polyamines can play a crucial role in a plant's fight against oxidative stress by activating several antioxidant enzymes [12]. Therefore, expanded research should focus more attention on phenylamides as potential antioxidants. The examined PBP-extractable fraction exhibited significant antioxidant properties based on the results of all applied assays. According to the TAC value for the extractable fraction, it showed significantly higher total antioxidant capacity (28.92 mg/g AAE) compared with several samples from Morocco, where TAC values ranged from 3.98 to 9.03 mg/g AAE [33]. Possible reasons for the observed differences include different geographical and botanical origins (the Moroccan samples were all polyfloral) as well as a different extraction solvent (ethanol). The results of the FRP assay for the extractable PBP fraction were consistent with the results for a polyfloral sample from Portugal (5.0 mg/g GAE) predominantly originating from C. striatus and Plantago sp. [19]. However, the PBP-extractable fraction exhibited strong antioxidant activity in the CUPRAC assay (69.00 mg/g AAE), comparable to similar results for a polyfloral bee-collected pollen sample from Turkey-85.59 mg/g TE [63]. The bound PBP fraction also exhibited CUPRAC activity (22.78 mg/g GAE), unlike all other assays. Moreover, for several commercial pollen samples from Turkey, the authors reported significant CUPRAC results with the extractable fraction (6.25-64.88 µmol/g TE), lower than those with the hydrolysable fraction (69. .96 µmol/g TE) [64]. It should be noted that the authors prepared the hydrolysable fraction differently, without strong alkaline conditions but with acidic hydrolysis. It is well-known that these conditions are favorable for CUPRAC assays [65]. Furthermore, it should be pointed out that significantly higher results for the CUPRAC compared with the FRP assay may be attributed to the fact that the CUPRAC assay measures both the actions of lipophilic and hydrophilic antioxidants, while FRP/FRAP assays can only detect hydrophilic compounds [66]. Finally, the PBP-extractable fraction showed a good ability to quench DPPH radicals (16.71 µmol/g TE), with results that are fully consistent with the extractable fraction for Turkish commercial bee-collected pollen samples (mean value 15.17 µmol/g TE) [64]. On the other hand, the bound fraction exhibited a significantly lower capability to neutralize free radicals compared with the Turkish samples.