2.3. Total Phenolic Content
Polyphenols represent a significant group of substances with various bioactive properties including antioxidative properties. The total phenolic content in BB delimits this potential in many fresh products as in this one. The free radical scavenging activity could be used to determine the validity of bee pollen and it was specie-dependent [25
]. Nevertheless, phenols and polyphenols are not the only chemicals responsible for this activity; in the present work the content of them was quantified using the Folin–Ciocaltau method and by HPLC/DAD. The amount of phenolic compound in ethanol extracts of BB obtained from the north of Romania was 6.30 ± 0.11 mg GAE/g (gallic acid equivalent/g BB sample) and in ethanol extract of Indian BB was similar, 6.41 ± 0.12 mg GAE/g (Table 3
), both correspond to 97% of Brassica
spp. (Figure 1
and Table 3
). The obtained results regarding the polyphenols content were similar to the data by [10
] from BB harvested in Columbia. The content of polyphenols in ethanol extracts [2
] is the highest with amounts of 24.60 mg GAE/g. The content of polyphenols in all extracts obtained is in line with other researches [5
]. The correlation with the taxon of provenience is not gave in the above-cited bibliography.
In the sample with a high quantity of Eucalyptus
sp. (India—June 2016) (Table 1
) the content is different from Brassica
sp. samples and this information can be used in the future for further studies in the standardization of this floral source (Table 3
Flavonoids are an important group of polyphenol components. In ethanol extracts, the content of flavonoids for the Romanian bee bread, in the samples, identified as Brassica
spp. origin (97%) amounted to 29.33 ± 0.34 mg QE/g and for the similar sample from Indian BB was 30.23 ± 0.06 mg QE/g. For the other samples, the values were different (Table 3
). In all extracts obtained, the content of flavonoids was in line with the results reported by Markiewicz-Zukowska et al. [2
] that found a total flavonoid content between 32.72–37.15 mg Qe/g. In contrast, in the study of Zuluaga et al. [10
] the total phenolic content from Columbian BB ranged from 2.5 to 13.7 mg GAE/g and total flavonoid content ranged from 1.9 to 4.5 mg Qe/g, but as pointed out before the taxa analyzed are not discussed.
It is expected that BB composition varies widely, being a fermented mixture of floral pollen collected by bees. The major variable in BB is the species composition of the pollen, which can be affected by differences in catchment area or season [20
]. So far, there are only a few papers detailing the biochemical composition of BB [14
In the study of Durán et al. [29
] the results indicate that in all samples of BB which analysed the amount of polyphenols vary both by the type of extraction and by their geographical location. Mǎrghitaş et al. [30
] also indicate that there is great variability in relation to the correspondence between antioxidant activity and total polyphenol content of pollen with different botanical origins what was previous discovery by Campos et al. [25
] given the special information that contributes to the taxon identification of the matrix under analysis.
In addition, these results also coincide with those presented by Carpes et al. [31
] who points out that the total amount of phenolic compounds not only varies with different extraction conditions, but also varies in different localities, which could be correlated with the floral source. Also Baltrušaityte et al. [15
], indicate that the concentration of polyphenols vary according to the origin of the pollen, which clearly depends on the flora and the geographical location of it.
It was reported that the composition in phenolics [16
], assessed by spectrophotometric methods, ranged between a minimum of 14 mg GAE/g BB and a maximum of 84 mg GAE/g. These values were higher than the ones reported by other authors [32
] for north-east Portuguese bee pollen, and may be explained due to the higher accessibility of compounds arising from the pollen structure degradation upon fermentation of bee bread, but as we verified in this work, probably the source of pollen origin was different given a high range of possibilities.
Regarding flavonoids, the content of flavones/flavonols in BB is much lower than the flavanones/di-hydroflavonols, ranging between 1 and 7 mg Qe/g and 10 to 89 mg Qe/g of extract, respectively. The poor flavone content was also reported by [15
] in BB from Lithuania. In general, flavones and flavonols are the main flavonoids in bee pollen with the predominance of the last ones [19
The literature data about Romanian BB total polyphenol content showed significant variability between samples from different floral species. Reported values ranged between 15.33–22.72 mg GAE/g [33
]. So far there are very few studies on the chemical composition and bioactive compounds of Romanian bee bread, and no study regarding the individual content of phenolic acids.
2.4. Phenolic and Polyphenolic Fingerprint by HPLC/DAD
The results obtained with the samples analyzed are in line with previous results carried out in bee pollen. Spectral data for all samples were accumulated in the range 220–400 nm using DAD. The structural identifications were made according to Campos and Markham [34
Comparing the results obtained with hand-collected pure pollen, bee pollen and BB the data remain the same. The proteins and polysaccharides present in the analyzed samples of BB did not affect the phenolic profiles obtained by HPLC/DAD analysis. With solvent programming the phenolic compounds eluted with retention times (RT) between 30 and 55 min. The first step in a tentative identification involves interpretation of the absorption spectrum to determine the compound type and compaction with reference spectra provides this information. In the range of BB examined, detection at 340 nm produced patterns comprising only flavonoids and cinnamic acid derivatives. The phenolic fingerprints of the analysed samples alone have proven to be sufficient to distinguish the pollen species represented in the present study.
and Figure 3
give the HPLC/DAD profile of BB from Romanian collect in July 2016 and from India collected in June 2016, corresponding both to 97% of Brassica
sp. pollen as the main taxon.
The respective UV-spectra of compounds 1
correspond to C3 glycosylated flavonols, both Kaempferol-3-O
-derivatives. All the other compounds are hydroxycinnamic acids derivatives (data in line with Campos and Markham [34
]) with a high probability of polymerized structures as spermidine polyamides.
It was identified in rape bee pollen three flavonols including quercetin-3-O
-glucopyranosyl)-glucopyranoside (in very low amounts), quercetin-3-O
-glucopyranosyl)-rhamnopyranoside and kaempferol-3-O
]. They also identified and N
-coumaroylspermidine in rape bee pollen crude extracts using high performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight-mass spectrometry (HPLC-ESI-QTOF-MS/MS). These last compounds are polyamines including the hydroxycinnamic acid, p
-coumaric linked in the N
of spermidine. Under UV analysis, only the structure of the phenolic acid is responsible for the absorption and the spectra obtained. The profile is not similar to the one that we present in this paper because the methodology applied was different. Rape bee pollen suffers a previous “wall-breaking treatment” and only after was extracted with 75% ethanol under reflux at a solid/liquid. The authors also prepare the final crude residue after a purification with petroleum ether to remove liposoluble impurity and, finally a water-saturated n-butanol clean-up. Nevertheless, in the results obtained by HPLC-ESI-QTOF-MS/MS the MS spectra of the three flavonols, does not correspond with the further identification.
In Figure 4
we show compounds 1
as quercetin and kaempferol glycosides, respectively, and as can be seen the UV spectra is different due to the hydroxylation in B-ring of flavonoid structure.
In Figure 4
, the HPLC/DAD profile corresponds to a Eucalyptus pollen source with the characteristic flavonoids, in this case two flavonols quercitine-3-O
-sophoroside (compound 1
) and myricetin (compound 4
) and the two flavones, tricetin (compound 5
) and luteolin (compound 6
). Tricetin is a biomarker for Myrtaceae [36
]. The kaempferol derivatives (compounds 2
) could be from citrus pollen. Nevertheless, until now we have not had access to this kind of pollen to confirm the identification.
As can be seen in Figure 5
, there is a huge similarity of the HPLC/DAD profile of phenolic/polypfenolic compounds from pure rape pollen samples analysed years ago and from Slovakia (data not published yet) with the BB profiles of samples from Romania and India in July and June 2016, respectively. These coincident fingerprints give the identification of the samples, as was previous proposed for bee pollens by Campos et al. [19
]. This paper gives for the first time some achievement to the taxon carried out previous only for bee pollens.
In contrast to the specie-specific data show above from samples with high percentage of one specie, in Figure 6
the HPLC/DAD profile obtained with BB from Romania collected in April 2016 corresponds to a complex mixture of Salicaceae—Salix
sp. (61%), Rosaceae—Prunus
sp. (26%) and Fagaceae—Quercus
sp. (13%). The UV-spectra correspond to 8-methylherbacetin-3-O
-derivative (Compound 1
-derivative (Compound 2
-derivative (Compound 3
), compounds 4
-derivative (Markham and Campos [37
]). The additional compounds are phenolic acid derivatives (data in line with Campos and Markham [34
]). In this case it was not possible to find a correct correspondence to a high provenance of pollen because, in fact, it does not exist. For pollen from Romania, June 2016, shown in Figure 7
, the correspondence was even more difficult for some reason, as explained above.
As shown, through the results from various samples, different polyphenolic profiles were obtained with BB from Romania and India. Firstly we compared them and then compared them with pure pollen samples, hand collected (data from other essays that belong to the data base of the Pharmacognosy laboratory—University of Coimbra, Portugal, some of them already published [19
]). The BB samples analyzed were almost pure in taxon
(samples collected in Romania July 2016 and India June 2016). It contained pollen from Brassica napus
almost entirely, 97%, Romanian bee bread, respectively 98% Indian bee bread. In order to confirm the botanical origin of the bee bread, microscopic analysis was carried out as well as the HPLC/DAD analyses of floral pollen collected directly from Brassica
plants collected in Portugal.
BB samples that had Brassica majority pollen (>90%) have been analyzed as well as pollen collected directly from Brassica family. An attempt was made to find out by means of chromatographic comparisons of natural plant pollen, bee pollen and bee bread, if bee saliva and the fermentation of bee pollen affects the phenolic composition by enzymic action. From the data obtained, the profile was maintained in the three samples, from Romania, India and even from Slovakia. Similar HPLC/DAD profiles were obtained with bee pollen samples from Sultanate of Oman (in 2010) and from Brazil (in 2012) (data not shown).
In the tested samples of Brassica
bee bread, five Hydroxycinnamic acid derivatives, as well as two flavonoids, Kaempferol-3-O
-glucoside, were found. The quantitative analysis was performed based on calibration curves plotted for the reference substances: Hydroxycinnamic acids (R = 0.997) and rutin (R = 0.998). For all calibration curves, the level of significance p
was lower than 0.001 (p
< 0.001). The content of the compounds measured in BB extracts is presented in Table 4
Hydroxycinnamic acid derivatives were present in these samples in the greatest amounts while flavonoids, such as Kaempferol-3-O-
glycosides, were present in a lower proportion. Strong antioxidative properties of hydroxycinnamic acids derivates were described by other authors [39
], and for these samples the potential could be similar. This will be explored in further studies. The proportion of the two flavonoids in the samples was similar, and corroborated the identification of the same taxon.
It was reported for the first time that this phenolic profile remains unchanged in the case of floral pollen (hand collected), bee pollen and bee bread. Despite the biochemical transformation that occurs during the fermentation of bee bread, it seems that these phenolic compounds are not affected and remain unchanged. Also, variables such as soil and climate do not seem to influence these compounds for the kind of samples under study.
Honeybee pollen seems to accumulate a species-specific selection of flavonoids [40
]. Using HPLC/DAD, Campos et al. [19
] pointed out that each botanical species studied gave a unique fingerprint of polyphenolic compounds (flavonoids plus other phenolics such as hydroxycinnamic acid derivatives). Flavonoids are generally recognized as reliable chemotaxonomic markers of plants [41
]. Evidence obtained by Campos et al. [19
] suggests that this also applies for bee pollen flavonoids. It was found that pollen from Ulex europaeus
plants from the Bay of Plenty (New Zealand) has the same polyphenolic profile as did the plants growing in Lower Hutt (New Zealand) 400 km away and even from plants growing in Portugal. The same situation was reported for Eucalyptus globulus
when pollen analysis from plants grown in different geographic areas showed an identical phenolic profile. In the main pollen samples, flavonoid profiles identified from pollen extracts are characterized by the presence of flavonoid glycosides mainly from quercetin, kaempferol, isorhamnetin and/or myricetin [19
It is known that phenolic composition of the plants species depends on its chemotype and on various environmental factors; therefore, it would be reasonable to assert that the phenolic composition of BB depends on its floral origin [15
]. The authors reported that seven samples of BB, from the nine tested, contained significant amounts of p
-coumaric acid. BB also contained higher amount of kaempferol than honey. Instead, chrysin and apigenin were present in BB in trace levels. The reported results clearly demonstrated that HPLC peak areas of the polyphenolic compounds in honey and BB extracts and, therefore, the quantity of phenolic compounds, varied in a very wide range.
In fact, the content in phenolic compounds of BB has been very poorly studied and has been reported by few authors who have used different methods of identification and quantification [2
]. Most existing literature only mentions the total phenolics content measured by the Folin–Ciocalteu colorimetric assay [10
] and do not provide detailed characterization of individual phenolic compounds. A few studies of BB samples from Poland, Georgia and Portugal identify some individual phenolic compounds, but all of them have used different analytical approaches. The first to present data on the biochemical composition of BB was Isidorov et al. [28
]. In this study, the gas chromatography with mass spectrometry (GC-MS) method was used, and six flavonoids were identified (kaempferol, chrysin, naringenin, isorhamnetin and apigenin) and four phenolic acids (4-hydroxybenzoic acid, p
-coumaric, ferulic and caffeic acid). Markiewicz-Zukowska et al. [2
] identified, using the GC-MS method, kaempferol and apigenin. The presence of the flavonoids as naringin, rutin and quercetin, was reported in Georgian BB samples, using high performance liquid chromatography with UV-VIS detector (HPLC-UV-VIS) [42
]. Rutin [quercetin-3-O
-rhamnosyl (1–6) glucoside] is often confused with quercetin-3-O
-rhamnosyl (1–2) glucoside. In terms of chromatography is impossible to determine the configuration of the sugars linkage among them. As was published before, the main sugar linkage in flavonoid sugars found in pollen is 1–2 [37
In BB from Portugal, up to 32 different flavonoids, using HPLC-DAD-ESI/MS were identified [14
]. The phenolic compounds found in BB were mainly flavonol derivatives, such as quercetin, kaempferol, myricetin, isorhamnetin and herbacetin glycosides. They found several compounds and classified various as rutinosides, despite the situation cited above. Quercetin-3-O
-glucoside were found in all the studied samples, while myricetin-3-O
-rutinoside and isorhamnetin-3-O
-glucoside were only detected in some BB samples. In this study, all these compounds were positively identified depending to their retention time, mass and ultraviolet-visible (UV-vis) characteristics in comparison with commercial standards. Once again, using this methodology it is impossible to classify the sugar linkage. It is correct to state that this compound had rhamnose and glucose as glycosidic moiety but is not classified as rutinose [rhamnosyl (1–6) glucoside] because in this case the 1–6 linkage is implicit.
Bees foraging preference is still being discussed; recent studies shows that honey bees prefer diets that reflect the proper ratio needed for optimal survival and homeostasis. The nutritional quality of pollen, mainly linked to the protein and amino acid content, is only in part the main influencing factor, and other non-nutritional ones are cited as involved, such as plant resource availability, color, odor and morphology of flowers [43
]. In effect, honeybees do collect and consume pollen and pollen-like substances with little or no nutritive value [44
]. Honeybee pollen generally shows characteristic quantities of total polyphenols due to its different botanical and geographical origin [45
]. Flavonoids are minor compounds, but of great importance in bee pollen and thus bee bread. These compounds influence the aspect of the pollen grain (pigmentation) and taste (astringency and bitter taste) [46
]. The most common flavonoids founded in bee pollen are the flavones and flavonols which occur almost exclusively as glycosides. Aglycones have in general simple structures such as quercetin or kaempferol, but their glycosylation patterns have structures that are more complex than those found in the vegetative plant parts [47
] as discussed above.