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Article

Botanical Origin, Phytochemical Profile, and Antioxidant Activity of Bee Pollen from the Mila Region, Algeria

by
Nassiba Boulfous
1,*,
Hakima Belattar
1,
Roberto Ambra
2,
Gianni Pastore
2 and
Asma Ghorab
3
1
Laboratory of Natural Sciences and Materials, Institute of Natural Sciences and Life, University Center Abdelhafid Boussouf, Mila 43000, Algeria
2
CREA (Council for Agricultural Research and Economics), Research Centre for Food and Nutrition, 00178 Rome, Italy
3
Department of Vegetal Biology and Soil Sciences, Facultade de Ciencias, Universidade de Vigo, 32004 Ourense, Spain
*
Author to whom correspondence should be addressed.
Antioxidants 2025, 14(3), 291; https://doi.org/10.3390/antiox14030291
Submission received: 12 February 2025 / Revised: 25 February 2025 / Accepted: 26 February 2025 / Published: 28 February 2025
(This article belongs to the Special Issue Antioxidant and Antimicrobial Activity of Honey Bee Products—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
Bee pollen is a complex mixture of floral pollen, and nectar fused substances from bee saliva. It is well known for its high content of proteins, carbohydrates, lipids, vitamins, and phenolic compounds, among various other physiologically active components. Its composition varies significantly depending on its botanical sources and environmental conditions. This study investigates the relationship between the botanical origins, chemical compositions, and antioxidant activities of 15 bee pollen samples collected from different areas in the Mila region of northeastern Algeria. The botanical origins were identified using a palynological method, categorizing 11 samples as monofloral and the rest as polyfloral. The total phenolic and flavonoid contents were measured, and their antioxidant capacities were evaluated through DPPH radical scavenging assay, reducing power assay (FRAP), and total antioxidant capacity (TAC). HPLC analysis was conducted to measure 17 phenolic compounds. The data indicated that the total phenolic content (TPC) and flavonoid content (TFC) ranged from 7.72 ± 0.29 to 23.49 ± 1.48 mg GAE/g and from 1.48 ± 0.00 to 5.57 ± 0.27 mg QE/g of pollen, respectively. The variations in the concentration of bioactive compounds among samples led to significant differences in their antioxidant activities: DPPH (IC50: 1.12 ± 0.15 to 0.21 ± 0.00 mg/mL), FRAP (EC50: 0.06 ± 0.00 to 0.29 ± 0.00 mg/mL), and TAC (262.17 ± 3.41 to 677.14 ± 12.81 EAA mg/100 g of bee pollen), with the most active samples being monofloral types from Cistus type and Brassica type. A strong correlation was observed between TPC, TFC, and antioxidant activity. Among the 17 tested compounds, only coumaric acid, rutin, myricetin, naringenin, resveratrol, and kaempferol were detected. In conclusion, both monofloral and polyfloral bee pollen samples represent a rich source of polyphenols with significant antioxidant potential.

1. Introduction

Bee pollen, a widely recognized honeybee product, is valued for its biologically active compounds and use as a natural remedy. It results from bees collecting pollen from flowers and mixing it with nectar and their secretions [1]. Typically, bee pollen contains carbohydrates (40–85%), proteins (14–30%), and lipids (1–10%), along with vitamins, minerals, carotenoids, phenolics, and trace elements. Over 250 bioactive compounds have been identified in bee pollen [2].
In apitherapy, it is used for its medicinal properties, including antimicrobial, anti-inflammatory, antiviral, analgesic, immunostimulant, hepatoprotective, and antioxidant effects [2]. Studies emphasize its strong antioxidant properties, which contribute to cellular protection and overall health by reducing oxidative stress [3,4,5,6,7].
The quality and antioxidant power of bee pollen is influenced by factors such as the geographical location where it is collected (and the specific plants bees forage on), the time of year, weather conditions, and how the pollen is processed. Determining its botanical source is essential, and this is primarily performed through palynology, which involves microscopic analysis of pollen grains to trace their plant origins [8]. Flavonoids like isoquercetin, myricetin, tricetin, quercetin, luteolin, selagin, kaempferol, and isorhamnetin have been identified in bee pollen samples from Brazil [9].
Algeria, located in North Africa, has a Mediterranean climate and boasts a rich plant diversity with 4000 species, including 2500 in its northern region, 10% of which are endemic. Examples include Erysimum cheiri (Brassicaceae) and Hypochaeris saldensis (Asteraceae). Numerous plants, such as eucalyptus, orange trees, sunflowers, clover, French honeysuckle, rosemary, and thyme, as well as natural forests like pine, provide nectar and pollen for bees, enabling year-round honey production. The region’s diverse flora and government support have fostered the growth of beekeeping [10,11]. Honey and propolis have been extensively studied by researchers [12,13,14,15,16,17,18,19,20]. On the other hand, only a few studies have characterized the antioxidant properties of Algerian bee pollens.
In this context, the current work is devoted to determining the botanical origins, analyzing the antioxidant capacity and polyphenolic composition using HPLC of bee pollen collected from the Mila region of northeastern Algeria. By examining these aspects, the study seeks to understand the relationships between the botanical sources of the pollen, its phenolic content, and its antioxidant properties.

2. Materials and Methods

2.1. Collection of Samples

In this study, 15 bee pollen samples from the Mila region (northeastern Algeria) were analyzed. Bee pollen samples were collected by local beekeepers using pollen traps from Apis mellifera between 2022 and 2023 years from different locations, and stored in the freezer at 4 °C until further analysis (Table 1, Figure 1).

2.2. Determination of Botanical Origin

Botanical origin was determined using a melissopalynological procedure [21]. First, the original samples were homogenized, then 1 g was weighed and placed in separate vials. Subsequently, a colorimetric separation using a white light and a black background was carried out to obtain different subsamples, which were weighted and dissolved in distilled water (15 mL). The solutions were shaken for 10 min and at 4500 rpm for 5 min centrifuged. An aliquot of 100 μL was taken from the sediment to prepare the slides. An optical microscope was used to identify the botanical origin of the different subsamples. The pollen spectra of the samples were determined considering the weight of each subsample and its botanical origin. The results were expressed in percentages.
Bee pollen samples were categorized microscopically into four sections based on the abundance of pollen grains: dominant pollen (≥45%), secondary pollen (16–45%), significant minor pollen (>3–15%), and minor pollen (<3%). The dominant pollen indicates the botanical origin of the bee pollen. However, identifying pollen grains from the same genus or family can be challenging. In such cases, only the genus or family name is specified, such as Anthemis sp., Taraxacum sp., or Asteraceae. According to Louveaux et al. [21], a sample is classified as monofloral if the percentage of a specific pollen type exceeds 45% of the total.

2.3. Preparation of Bee Pollen Extracts

Ethanolic extracts (EEPs) of the bee pollen samples were prepared according to the method of Almaraz-Abarca et al. [22], with minor modifications. The EEP was prepared by mixing 10 g of crushed bee pollen, with a precision of 0.01 g in 100 mL final volume 80% (v/v) aqueous ethanol for 24 h at room temperature. The sample was filtered under reduced atmosphere pressure using a water pump. The filtrate obtained was centrifuged at 10,000 rpm for 10 min, and then it was evaporated under reduced pressure in a rotary vacuum evaporator. The evaporated extract was dried in a laboratory incubator at 38 °C until a solid mass was obtained, and stored in the freezer at 4 °C until further analysis.

2.4. Phytochemical Analysis of Samples

2.4.1. Assessment of Total Phenol Content

The determination of the total phenolic content (TPC) was carried out based on the method developed by Singleton and Rossi [23], adapted to bee pollen. Briefly, 0.2 mL of the extract (2 mg/mL) was mixed with Folin–Ciocalteu’s reagent (1 mL). After 3 min, 0.8 mL of 7.5% sodium carbonate was added to the mixture. The solution was kept in the dark at room temperature for 2 h, and the absorbance was measured at 765 nm. The total phenolic content was expressed as mg gallic acid equivalents (GAE) per g bee pollen. All measurements were performed in triplicate.

2.4.2. Assessment of Total Flavonoid Content

The total flavonoid content (TFC) was determined using a modified version of the method described by Arvouet-Grand et al. [24]. Specifically, 0.75 mL of bee pollen extract (2 mg/mL) was mixed with 0.75 mL of a 2% aluminum chloride solution. This mixture was then incubated for 15 min at room temperature. Subsequently, the absorbance of the solution was measured at 430 nm. A calibration curve was generated using quercetin as a standard, and the flavonoid content of the bee pollen extract was determined by applying the linear regression equation of the calibration curve. The results were expressed as milligrams of quercetin equivalents (QE) per gram of bee pollen. All measurements were performed in triplicate.

2.4.3. DPPH Radical Scavenging Assay

The ability of bee pollen extracts to scavenge free radicals was evaluated using the DPPH method, which measures the antioxidant’s capacity to neutralize the stable 2,2-diphenyl-1-picrylhydrazyl radical using a modified method described by Tomás et al. [25]. Specifically, 0.75 mL of phenolic extracts, with concentrations ranging from 3 to 0.43 mg/mL, were mixed with 0.75 mL of DPPH solution (0.04 mg/mL). The absorbance of the resulting mixture was measured at 515 nm. The percentage of radical inhibition was calculated using the following equation:
% Inhibition = [(ADPPH − ASample)/ADPPH] × 100
The EC50 value, representing the concentration of the extract required to inhibit 50% of DPPH radicals, was determined by plotting the percentage of radical inhibition against the extract concentration.

2.4.4. Reducing Power (FRAP)

The FRAP method, which relies on the reduction in the Fe3⁺–TPTZ (ferric tripyridyltriazine) complex to its ferrous form under acidic conditions, was conducted following a previously described method [26]. A 0.25 mL aliquot of bee pollen extract (with concentrations ranging from 3 to 0.43 mg/mL) was combined with 1.25 mL of sodium phosphate buffer (pH 6.6). To this, 1.25 mL of 1% potassium ferricyanide was added, and the mixture was incubated at 50 °C for 20 min. Next, 1.25 mL of 10% trichloroacetic acid was added. The solution was then centrifuged at 3000× g for 10 min, and 1.25 mL of the supernatant was transferred to a new tube. Afterward, 1.25 mL of water and 0.25 mL of 0.1% ferric chloride were added, and the absorbance was measured at 700 nm. Extract concentration providing 0.5 of absorbance (EC50) was calculated from the graph of absorbance against extract concentration in the solution. Ascorbic acid was used as positive control. Tests were conducted in triplicate, and the results were given as mean ± SD.

2.4.5. Total Antioxidant Capacity

The total antioxidant capacity (TAC) was measured using the ammonium molybdate colorimetric method described by Prieto et al. [27]. This method is based on the reduction of molybdenum (VI) to molybdenum (V) by antioxidants present in the extracts, leading to the formation of green phosphate/Mo (V) complexes, which can be quantitatively measured. In brief, 0.1 mL of the ethanolic bee pollen extract was combined with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The mixture was sealed and incubated in a thermal block at 95 °C for 90 min. The absorbance of the reaction mixture was then measured at 695 nm, using a blank for reference. Ascorbic acid was used as the standard for calibration, and the results were expressed as milligrams of ascorbic acid equivalent per 100 g of bee pollen.

2.5. Determination of Phenolic Compounds by HPLC

The analysis of phenolic acid compounds in ethanolic pollen extracts was performed using a Thermo Scientific Vanquish HPLC system, equipped with a Diode Array Detector (DAD) set to 280, 300, 320, and 350 nm. Spectra were continuously recorded in the range of 210–600 nm, with a data collection rate of 10.0 Hz. The system also included a Vanquish Autosampler maintained at 6 °C. A 250 × 4.6 mm C18 Luna column (Phenomenex, Torrance, CA, USA) was used, kept at 25 °C. The mobile phase consisted of 0.5% formic acid in water (phase A) and acetonitrile (phase B). The gradient used for analysis was as follows: 95% A for 1 min; 90% A for 4 min; 90% A for 5 min; 85% A for 47 min; 75% A for 6 min; 48% A for 15 min, followed by 13 min of re-equilibration with 95% A between runs. Standards were prepared at approximately 1 mg/mL, then diluted to create calibration curves (ranging from 0 to 50 µg/mL) for various phenolic acids, including gallic acid, trans-cinnamic acid, caffeic acid, syringic acid, ferulic acid, p-coumaric acid, hydroxycinnamic acid, vanillic acid, chlorogenic acid, kaempferol, epicatechin, naringenin, resveratrol, quercetin, myricetin, rutin, and catechin. The standards used for HPLC analysis were obtained from Sigma-Aldrich (Merk Life Science S.r.l., Milan, Italy). The limits of detection (LOD) and quantification (LOQ) values are provided in Table 2. The sample was injected at room temperature with a volume of 20 μL.

2.6. Statistical Analysis

All experiments were conducted in triplicate, and the results were expressed as mean ± standard deviation (SD). The data were processed using R-4.4.2.2 (2024). A one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, was performed to determine statistical significance, with p-values less than 0.05 considered significant. Furthermore, Pearson’s correlation coefficients were calculated to evaluate the relationships between the analyzed parameters.

3. Results

3.1. Botanical Originof Bee Pollen

The results of the palynological analysis of bee pollen samples are summarized in Table 3 and Figure 2.
Among the analyzed samples, 30 pollen types from 17 families were identified. The most diverse families were Asteraceae, with 7 types, and Fabaceae, with 6 types. The remaining families were represented by only one or two types. The most frequently occurring pollen types were Cistus, Brassica, and various Asteraceae, appearing in nearly all samples.
The analysis classified eleven samples as monofloral and four samples as polyfloral. Samples P5, P8, P14, and P15 exhibited pollen from various botanical origins at different relative frequencies, as shown in Table 3. The monofloral samples were identified as originating from the following types: Cistus (P1, P2, P3, and P11), Brassica (P4, P6, P7, and P12), Pimpinella anisum (P9), Erica sp., (P10), and Aster (P13).

3.2. Concentration of Total Phenols, Flavonoids, and Antioxidant Capacity of Bee Pollen Extracts

Table 4 provides a summary of the total phenolic content, flavonoid levels, and antioxidant capacity of bee pollen.
The TPC of the tested samples ranged from 7.72 ± 0.29 to 23.49 ± 1.48 mg GAE/g. The highest TPC was recorded in sample P2, followed by P3 and P4, with values of 19.69 ± 0.69 GAE/g and 19.05 ± 1.90 GAE/g, respectively. The lowest TPC was observed in sample P13.
The TFC ranged from 1.48 ± 0.00 to 5.57 ± 0.27 mg QE/g of pollen. Sample P9 had the highest flavonoid content (5.57 ± 0.27 mg QE/g of pollen), followed closely by samples P14, P5, and P10, with values of 4.98 ± 0.09, 4.74 ± 0.24, and 4.28 ± 0.31 mg QE/g, respectively. The lowest TFC was found in sample P13, accordingly with the lowest. Interestingly, a contrasting trend was observed in samples P2, P3, and P4, where higher TPC was accompanied by lower TFC.
Different assays were employed to evaluate the antioxidant potential of bee pollen extracts, including the DPPH free radical scavenging activity, the reducing power assay (FRAP), and the molybdate ion reduction assay. The results are shown in Table 4.
DPPH radical scavenging activities ranged from IC50 1.12 ± 0.15 mg/mL to IC50 0.21 ± 0.00 mg/mL. The lowest IC50 value indicates high radical scavenging activity. The monofloral samples (Brassica type) P12 and P4 exhibited the highest antioxidant activity (0.21 mg/mL, 0.25 mg/mL, respectively) together with Cistus type monofloral bee pollen (P1), followed by P2 (0.34 ± 0.04 mg/mL), P8 (0.38 ± 0.01 mg/mL), P6 (0.42 ± 0.00 mg/mL), and P7 (0.50 ± 0.04 mg/mL).
The reducing power method revealed that sample P3 demonstrated the highest inhibition (EC50 0.06 ± 0.00 mg/mL), while P11 showed the lowest inhibition (EC50 0.29 ± 0.00 mg/mL). The antioxidant capacity of the samples decreased in the following order: P13 > P15 > P14 > P6, P8, P10 > P1, P7, P9 > P5 > P4, P2 > P12.
The total antioxidant capacity (TAC) values ranged from 262.17 ± 3.41 to 677.14 ± 12.81 EAA mg/100 g. Sample P12 exhibited the highest antioxidant activity (677.14 ± 12.81 EAA mg/100 g), followed by P5 (672.36 ± 3.41 EAA mg/100 g), P4 (612.54 ± 5.12 EAA mg/100 g), and P7 (595.96 ± 6.83 EAA mg/100 g). Conversely, P6 and P2, representing monofloral Brassica and Cistus types, respectively, showed the lowest activity. Significant differences were found between the mean values for all the variables analyzed in the bee pollen samples (p < 0.05).

3.3. Phenolic Compounds by HPLC Analysis

In this study, phenolic compounds in ethanolic bee pollen extracts were analyzed using Thermo Scientific Vanquish HPLC. Figure 3A,B, illustrate chromatograms for phenolic standards and the phenolic profile of bee pollen extracts, respectively, using sample P9 as an example. Table 5 outlines the phenolic compounds (µg/g) identified in the extracts.
Out of 17 tested molecules, only p-coumaric acid, rutin, myricetin, naringenin, resveratrol and kaempferol were detected. Notably, high levels of rutin (11.4–1052.7 µg/g), myricetin (10.5–1418.1 µg/g), and naringenin (7.6–1418.1 µg/g) were found. p-coumaric Acid was absent in samples P1, P2, P3, P7, P11, P14, and P15. Sample P9 had the highest amounts of rutin (1052.7 µg/g) and naringenin (1418.1 µg/g) and the second-highest level of myricetin (1062.1 µg/g), correlating with its high flavonoid content (5.57 ± 0.27 mg/100 g).

3.4. Correlation Among Phenolic Compounds and Antioxidant Activity

Using Pearson’s correlation (Figure 4), this study identified a strong positive correlation between total phenolic content (TPC) and kaempferol (r = 0.368), TPC and myricetin (r = 0.298), total flavonoid content (TFC), and total antioxidant capacity (TAC) (r = 0.472), TFC and rutin (r = 0.482), TFC and myricetin (r = 0.417), TFC and naringenin (r = 0.428). These correlations are explained by the fact that rutin, myricetin, and naringenin belong to the flavonoid family. Additionally, DPPH radical scavenging activity correlated positively with FRAP. A positive correlation was also observed between TAC and specific phenolic compounds, such as coumaric acid and kaempferol. There was further evidence of relationships between coumaric acid with resveratrol and kaempferol, as well as between rutin with myricetin and naringenin. However, a negative correlation was observed between TPC and both DPPH and FRAP (r = −0.594 and r = −0.734, respectively), which was attributed to the EC50 expression of these assays, where lower values indicate higher activity. Negative correlations were also noted between DPPH and TAC, resveratrol, and kaempferol, as well as between FRAP and TAC, coumaric acid, myricetin, and resveratrol. Among phenolic compounds, only naringenin and resveratrol showed a weak negative correlation. Pollen sample P12, belonging to the Brassica type with average TPC and TFC levels, showed the highest activity in both DPPH and TAC assays and ranked second in FRAP. Conversely, monofloral bee pollen P13 (Aster type) with low TPC and TFC exhibited the lowest FRAP activity.

4. Discussion

Bee pollen is a highly nutritious natural product, rich in antioxidants, carbohydrates, proteins, and essential amino. It also contains unsaturated fatty acids, vitamins, and minerals. Due to its nutritional and bioactive properties, bee pollen is widely used in dietary supplements, functional foods, and nutraceuticals for its health-promoting benefits. Its bioactive compounds, especially phenolic acids and flavonoids, contribute to their biological activities. Since pollen is derived from various plant species, its quality, safety, and characterization are determined by its botanical and geographical origins [28,29,30].
The botanical analysis of the bee pollen samples from this research highlights the rich floral diversity of the Mila region (northeastern Algeria), influenced by its Mediterranean climate, orography, and human activity. Most identified pollen types originate from spontaneous species with high melliferous potential, supporting the sustainable growth of beekeeping in the region [10,16,31]. With the exception of the Brassicaceae family, all the mentioned families are recognized as excellent pollen sources. However, most species within the Brassicaceae and Asteraceae families are primarily classified as nectar-producing. The Fabaceae family is known for its strong nectar and pollen production potential [32]. The dominant pollen types in the samples were Cistus and Brassica, suggesting that honeybees in the Mila region primarily collect these types for sustenance. Almost all samples were monofloral (11 samples). Carpes et al. [33] highlighted that the nutritional quality of certain pollen types makes bees favor a single floral source. Similarly, Aličić et al. [34] noted that bees typically visit the same plant species when collecting pollen, resulting in predominantly monofloral pollen with minor contributions from other species. There is limited data on the characterization of Algerian pollen in the literature. Bakchiche et al. [35] investigated pollen from the Laghouat region and identified taxa from families such as Asteraceae, Betulaceae, Boraginaceae, Brassicaceae, Cistaceae, Ericaceae, Fabaceae, Liliaceae, Myrtaceae, Salicaceae, and Rosaceae, with Brassicaceae being dominant. Mokhtari et al. [11] analyzed eight bee pollen samples from different Algerian regions and classified them as polyfloral, identifying 40 pollen types across 22 botanical families. The most diverse families included Asteraceae (8 types), Brassicaceae (4 types), and Cistaceae, Fabaceae, and Fagaceae (3 types each). These pollen types were also noted in other Algerian bee products, especially honey [16,20,36,37]. Similarly to our findings, El Ghouizi et al. [38] attributed the botanical origin of eight monofloral bee pollen samples to the Apiaceae, Fabaceae, Resedaceae, Rosaceae, and Lamiaceae families. Significant variation in pollen composition was observed, likely due to the diversity of environmental flora at different altitudes where beehives are located. Furthermore, bee pollen samples collected from the same or nearby areas can reflect different plant sources. This variation may depend on the dominant plant species in the vicinity of the hives and the foraging preferences of honeybees [26].
Several studies across various countries have investigated the total bioactive components of bee pollen. The TPC and TFC of Algerian bee pollen were similar to those found in samples from Marocco by El Ghouizi et al. [38], who reported TPC in monofloral bee pollen ranging between 8.070 ± 1.037 mg GAE/g and 32.387 ± 0.148 mg GAE/g, and TFC between 0.202 ± 0.044 mg QE/g and 6.30 ± 0.37 mg QE/g. The TPC values in our samples are consistent also with the results reported by Ilie et al. [39] for Romanian bee pollen. However, our TFC values are notably higher. In addition, we obtained higher levels of TPC and TFC when compared with the findings of Aylanc et al. [26]. A recent study in Algeria by Hemmami et al. [40] showed TPC values ranging from 3.79 to 9.17 mg GAE/g and TFC values between 2.07 and 5.50 mg QE/g. Compared to other studies involving bee pollen samples from Algeria, Brazil, Northwest Spain, Turkey, and India [5,7,11,18,28,41], our extracts exhibited lower amounts of total phenols and flavonoids. A high phenolic content does not necessarily correspond to elevated flavonoid levels, a finding consistent with previous research [26]. This discrepancy was observed in certain samples.
Bee pollen exhibits strong antioxidant potential due to its rich polyphenolic content. Various methods are used to assess its antioxidant activity, as different compounds require specific treatments and yield varying responses [29]. According to the literature, our DPPH radical scavenging assay results are consistent with those reported for Brazilian bee pollen, where the highest antioxidant activity was observed in the ethanolic extract of Brassica napus pollen [42]. However, our findings exceed the previously documented range of 7.77 to 1.68 mg/mL [6]. In addition, the methanolic extract of coriander bee pollen from India was revealed as a potent free radicals inhibitor (93.75 ± 0.05%) due to a minimum IC50 value (0.006 ± 0.21 mg/mL) [43]. The IC50 values of Greek pollen [4] and pollen from Portugal and New Zealand [44] were 0.181 ± 1.70 mg/mL and 0.04–0.5 mg/mL, respectively, which are within the range of our Algerian bee pollen samples from several regions. When comparing the FRAP assay results with those of other researchers, we observe notable similarities and differences, providing valuable insights into antioxidant capacity variations. Studies evaluating the antioxidant capacity of bee pollen extracts from various floral regions in Morocco [38] reported EC50 values ranging from 0.133 ± 0.036 to 0.790 ± 0.175 mg/mL. Similarly, analyses of ethanolic pollen extracts from apiaries in Bahia, Brazil [41] showed antioxidant activity values between 1.45 ± 0.03 and 8.77 ± 0.23 mg/mL. These findings suggest that the antioxidant activity of Moroccan and Brazilian bee pollen is generally lower than that of Algerian samples. The antioxidant capacity of bee pollen extracts has been extensively evaluated using the FRAP method, though results often differ in their expression. Alimoglu et al. [5] investigated 11 bee pollen samples from Turkey, reporting FRAP values between 3.42 ± 0.07 mg Trolox/g and 16.03 ± 0.25 mg Trolox/g. Similarly, Ulusoy and Kolayli [45] analyzed 13 Anzer bee pollen samples, and Karkar et al. [46], examined 10 samples from Turkey, finding FRAP values ranging from 11.77 ± 0.63 mmol Trolox/g to 4.75 ± 0.02 mmol Trolox/g and from 1.53 ± 0.06 mg Trolox/g to 4.75 ± 0.02 mg Trolox/g, respectively. Additionally, coriander bee pollen extracts demonstrated a wide range of FRAP values, from 32.54 ± 0.57 to 103.98 ± 0.82 mmol Fe2⁺/g, with the highest values [7]. These variations highlight the diverse antioxidant potential of bee pollen across different geographical and botanical origins. Limited data exists on total antioxidant capacity using the phosphomolybdenum method. In this study, pollen samples exhibited lower TAC compared to Moroccan samples (3.98 ± 0.16 mg EAA/g to 9.69 ± 0.34 mg EAA/g) [38] and Algerian samples (808.2–3311 mg/100 g) [40], but higher values than those reported by Rebiai and Lanez [47]. Significant variations in TPC, TFC, and antioxidant capacities of bee pollen have been observed worldwide. These differences are influenced by factors such as the choice of standards (e.g., gallic acid, pinocembrin, or chlorogenic acid), the extraction solvent (e.g., methanol, ethanol, or dichloromethane), and the extraction method. Additionally, the botanical and geographical origins of bee pollen, along with environmental factors like soil type, climate conditions, and beekeeping practices, play a crucial role in determining its composition [30,38,46].
Bee pollen has garnered increased attention for its phenolic profile, which has been fairly well reported in the literature [48]. In this study, the phenolic compounds identified in Algerian bee pollen samples align with the findings of Bayram et al. [49], who reported rutin as the dominant compound in Turkish bee pollen. Additionally, resveratrol and kaempferol were present in considerable amounts, whereas coumaric acid was detected in lower concentrations and was absent in samples P1, P2, P3, P7, P14, and P15. However, research on Algerian bee pollen remains scarce, highlighting the need for further studies. Hemmami et al. [40], analyzed 13 Algerian bee pollen, identifying high levels of rutin (6751.996 ± 168.799 µg/g), quercetin (20,749.05 ± 518.726 µg/g), and gallic acid (648.558 ± 12.971 µg/g). All 13 Hemmami and coworker’s samples contained gallic acid, p-coumaric acid, and quercetin. However, different from our results specific compounds, i.e., vanillin, chlorogenic acid, naringin, vanillic acid, rutin, and caffeic acid, were completely lacking in several samples, and only one sample contained all nine studied phenolic compounds. Aylanc et al. [26] detected caffeic acid and p-coumaric acid only in one Moroccan sample, with concentrations of 150 µg/g and 200 µg/g, respectively. Similarly, Kahraman et al. [30] found kaempferol (3264.5 µg/g) in Turkish bee pollen but did not identify epicatechin, syringic acid, vanillic acid, or catechin. For instance, Karkar et al. [46] did not detect gallic acid in ethanolic extracts of Turkish chestnut bee pollen but found low levels of syringic acid, kaempferol, and myricetin. Alimoglu et al. [5] reported different profiles using ethyl acetate and dichloromethane extracts, demonstrating that solvent selection significantly affects phenolic extraction. Thus, bee pollen extracts contain phenolic compounds, whose profiles differ markedly, as clearly documented in the literature.
Numerous studies have explored the relationship between phenolic content and the antioxidant capacity of bee pollen [3,50,51], and recent findings highlight a strong positive association [26,28,29,47]. Limmongkon et al. [52] reported a significant correlation (r = 0.773) between the DPPH and FRAP assays, both of which measure the electron transfer potential of antioxidants. Our results are consistent with this observation, further supporting the link between phenolic compounds and their antioxidant efficacy.
Certain bee pollen samples present higher TPC values and lower DPPH IC50. The presence of specific phenolic compounds with high radical-scavenging efficiency may explain this relationship. This trend is expected, as phenolic compounds are known for their ability to scavenge free radicals [53]. The strong correlations may be due to the presence of flavonols such as rutin, kaempferol, and myricetin derivatives. Their planar structures, influenced by hydroxyl groups at position 3, facilitate radical capture through conjugation and electron delocalization. Moreover, the high number of hydroxyl groups contributes significantly to their potency as electron scavengers. Despite this, several studies have concluded that antioxidant capacity is not necessarily linked to total phenolic content [46,54,55,56]. In this study, the sample with the highest TPC (P2) showed the lowest TAC. This indicates that the presence of specific phenolic compounds, rather than their concentration, may play a more critical role. Synergistic and antagonistic effects among phenolic compounds and interactions with other phytochemicals in the extract further influence antioxidant activity [52]. Environmental factors, such as soil conditions and plant physiology, also impact free radical reactions and the antioxidant capacity of bee pollen [34,41]. Furthermore, the collection period significantly affects antioxidant activity, with higher values observed during UV-intense periods, particularly from early to late summer [3]. Consequently, proper management practices and the timing of collection are crucial for optimizing the chemical and functional properties of bee pollen.
While this study provides valuable insights into the botanical origin, phytochemical profile, and antioxidant activity of bee pollen from the Mila region, certain limitations should be acknowledged. The sample size, though representative, could be expanded to improve the generalizability of the findings. Additionally, while three antioxidant assays (DPPH, FRAP, and TAC) were employed, incorporating a broader range of assays would provide a more comprehensive evaluation of the antioxidant potential. Further, the HPLC analysis, which identified key phenolic compounds, could be extended to include a wider range of bioactive compounds for a more detailed phytochemical characterization. Despite these limitations, the findings of this study contribute to the growing body of knowledge on natural antioxidants and highlight the need for further investigations to overcome these challenges.

5. Conclusions

This study investigated bee pollen samples from the Mila region in northeastern Algeria. Bee pollen is recognized as a valuable functional food due to its high levels of phenolics and strong antioxidant properties. These compounds are known to offer various health benefits. The study aimed to determine the botanical origin of the pollen samples, analyze their antioxidant capacity using different methods (DPPH, FRAP, and TAC), and characterize their phenolic composition using HPLC. The results showed significant variations in phenolic content and antioxidant activity among pollen samples from different plant sources. Phenolic compounds were identified as the primary contributors to the antioxidant properties of the pollen. HPLC analysis revealed the presence of six phenolic compounds across all samples, with rutin, myricetin, and naringenin being the most abundant. Moreover, the results suggest that bee pollen from the Mila region could contribute to human health as a powerful natural antioxidant and should be applied to various fields of food and pharmaceutical industries. However, the study emphasizes the need and opportunity for further research, increasing such as the number of samples analyzed, expanding the research to other regions of Algeria, and exploring other research approaches to gain a deeper understanding of the therapeutic and economic value of Algerian bee pollen.

Author Contributions

N.B. and H.B. conceived and designed the methodology. N.B., R.A. and G.P. carried out the experiments. A.G. determined the botanical origin of bee pollen. N.B. and H.B. analyzed and interpreted data. N.B. writing—original draft preparation. H.B., R.A., G.P. and A.G. writing—review and editing. H.B. supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Acknowledgments

The authors would like to thank the beekeepers and for their collaboration in this study and CREA (Council for Agricultural Research and Economics), Research Centre for Food and Nutrition, 00178 Rome, Italy; under the administration of Fausta Natella for HPLC analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

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Antioxidants 14 00291 g001
Figure 1. Geographic distribution origin by municipality (in blue) of the bee pollen samples collected the Mila region (northeastern Algeria). Created with ArcGIS 10.8.
Figure 1. Geographic distribution origin by municipality (in blue) of the bee pollen samples collected the Mila region (northeastern Algeria). Created with ArcGIS 10.8.
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Figure 2. Microscopic appearance of the different pollen types found in Algerian pollen samples. (a) Brassica type. (b) Cistus type. (c) Hedysarum coronarium. (d) Erica sp. (e) Eucalyptus sp. (f) Apiaceae. (g) Pimpinella anisum. (h) Liliaceae. (i) Aster type. (j) Helianthus annuus. (k) Carduus type. (l) Taraxacum type. (m) Tamarix sp. (n) Rubus sp. (o) Quercus sp. (p) Olea europaea.
Figure 2. Microscopic appearance of the different pollen types found in Algerian pollen samples. (a) Brassica type. (b) Cistus type. (c) Hedysarum coronarium. (d) Erica sp. (e) Eucalyptus sp. (f) Apiaceae. (g) Pimpinella anisum. (h) Liliaceae. (i) Aster type. (j) Helianthus annuus. (k) Carduus type. (l) Taraxacum type. (m) Tamarix sp. (n) Rubus sp. (o) Quercus sp. (p) Olea europaea.
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Figure 3. Chromatogram of the standards (A). As an example, the chromatogram of sample 9, zoomed with RT > 40 min (B).
Figure 3. Chromatogram of the standards (A). As an example, the chromatogram of sample 9, zoomed with RT > 40 min (B).
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Figure 4. Pearson Correlation shown by the parameters studied in bee pollen samples from different botanical origins.
Figure 4. Pearson Correlation shown by the parameters studied in bee pollen samples from different botanical origins.
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Table 1. Geographical location and harvesting period of bee pollen samples.
Table 1. Geographical location and harvesting period of bee pollen samples.
Sample Date of HarvestGeographical OriginLatitude (°N)Longitude (°E)Altitude (m)
P1Spring 2023Tessala Lamtai36.45006.2667600
P2Spring 2023Tassadane Haddada36.36676.2500700
P3Spring 2023Grarem Gouga36.40006.5667500
P4Spring 2023Derradji bousselah36.38336.3333550
P5Spring 2023Rouached36.46676.1167650
P6Spring 2023Zeghaia36.45006.4000450
P7Spring 2023Hamala36.36676.4167600
P8Spring 2023Rouached36.46676.1167650
P9Spring 2023Minar Zarza36.31676.2000750
P10Summer 2023Amira Arres36.30006.2833800
P11Spring 2023Tessala Lamtai36.45006.2667600
P12Spring 2023Oued Althmenia36.46676.3667450
P13Spring 2023Tassala Lemtai36.45006.2667600
P14Spring 2023Mila36.45006.2667710
P15Spring 2023Tassadane Haddada36.36676.2500700
Table 2. Retention times (Rt), detection, and quantification the phenolic standards.
Table 2. Retention times (Rt), detection, and quantification the phenolic standards.
Time (min)RT (min)LOD (µg/mL)LOQ (µg/mL)
Gallic Acid7.330.030.09
Hidroxycinnamic acid17.000.030.09
Chlorogenic acid21.330.020.06
Catechin22.940.040.13
Vanillic acid25.080.010.04
Caffeic acid27.250.020.06
Syringic acid28.000.030.10
Epicatechin33.000.040.13
Trans cinnamic acid38.350.110.33
p-Coumaric acid44.740.040.12
Ferulic acid53.660.090.28
Rutin65.610.080.24
Myricetin71.640.020.06
Resveratrol74.550.040.13
Quercetin79.100.010.04
Naringenin84.950.050.16
Kaempferol86.500.040.12
Table 3. Palynological analysis of bee pollen samples.
Table 3. Palynological analysis of bee pollen samples.
SamplePredominant Pollen Grains (>45%)Secondary Pollen Grains (16–45%)Important Minor Pollen
(3–15%)		Minor Pollen (<3%)Classification
P1Cistus type a (63%)Brassica type b (16%)Trifolium repens j (11%), Onobruchis sp. j (7%)Anthemis type e,
Castanea sativa i,
Centaurea sp. e, Fabaceae j, Rosaceae k		Monofloral
P2Cistus type a (82%)/Olea europaea l (14%)Quercus sp. iMonofloral
P3Cistus type a (65%)Erica sp. c (25%)Quercus sp. i (7%)Oxalis sp. mMonofloral
P4Brassica type b (82%)/Meliotus type j (15%)Rosaceae k, Prunus sp. k,
Cistus type a, Carduus type e, Apiaceae d		Monofloral
P5/Brassica type b (39%),
vitex sp. g (41%)		Melilotus type j (10%),
Erica sp. c (7%)		Cistus type aPolyfloral
P6Brassica type b (50%)Erica sp. c (36%)Rubus sp. k (6%),
Aster type e (4%),
Taraxacum type e (4%)		Capparis spinosa n,
Cistus type a		Monofloral
P7Brassica type b (55%)Cistus type a (29%),Melilotus type j (8%),
Lotus sp. j ( 5%)		Rubus sp. k, Anthemis type eMonofloral
P8/Brassica type b (43%), vitex sp. g (42%)Melilotus type j (7%),
Erica sp. c (5%)		Cistus type aPolyfloral
P9Pimpinella anisum d (49%)/Hedysarum coronariumj (10%), Taraxacum type e (15%),
Anthemis type e (12%),
Quercus sp. i (11%)		Rubus sp. k, Eucalyptus sp., Carduus type e,
Trifolium type j, Cistus type a		Monofloral
P10Erica sp. c (53%)Melilotus sp. j (32%)Asreraceae e (7%),
Brassica type b (5%),		Apiaceae d, Rosaceae k,
Carduus type e,
Asparagus type p,
Alluim sp. h, Rubus sp. k		Monofloral
P11Cistus type a (47%)Liliaceae (44%)Carduus type e (9%)/Monofloral
P12Brassica type b (71%)Apiaceae d (26%)/Carduus type eMonofloral
P13Aster type e (52%)Cistus type a (23%)Erica sp. c (12%),
Quercus sp. i (10%)		Asparagus type p,
Anthemis type e,
Taraxacum type e, Fabaceae j		Monofloral
P14/Oleaceae l (39%),
Tamarix sp. f (29%)		Quercus sp. i (8%),
Lamiaceae g (9%),
Brassica type b (5%),
Centaurea sp. e (7%)		Carduus type e,
Taraxacum type e,
Plantago sp. q,
Trifolium pratanse j,
Eucalyptus sp. o,
Genista type j,
Melilotus sp. j		Polyfloral
P15/Artemesia sp. e (37%), Brassica type b (21%),
Helianthus annuus e (41%)		Alluim sp. h (4%)Trifolium repens j,
Cistus type a, Rosaceae k,
Tamarix sp. f,
Chenopodiaceae,
Apiaceae d		Polyfloral
Different letters (a–q) indicate the families: a Cistaceae, b Brassicaceae, c Ericaceae, d Apiaceae, e Asteraceae, f Tamaricaceae, g Lamiaceae, h Alliaceae, i Fagaceae, j Fabaceae, k Rosaceae, l Oleaceae, m Oxalidaceae, n Capparaceae, o Myrtaceae, p Asparagaceae, and q Plantaginaceae.
Table 4. Antioxidant content and activities of the ethanolic extract of bee pollen.
Table 4. Antioxidant content and activities of the ethanolic extract of bee pollen.
SampleTotal Phenolic Content (TPC) (mgGAE/g)Total Flavonoid
Content (TFC)
(mg QE/g)		DPPH Radical
Scavenging Assay (IC50) (mg/mL)		Total Antioxidant Capacity (TAC)
(EAA mg/100 g)		Reducing Power (FRAP)
(EC 50) (mg/mL)
P112.37 ± 0.19 b,c2.80 ± 0.33 c,d0.26 ± 0.01 a,b401.52 ± 1.13 d,e0.12 ± 0.02 c,d
P223.49 ± 1.48 i2.99 ± 0.02 d0.34 ± 0.04 a,b,c465.56 ± 25.63 g0.09 ± 0.00 a,b
P319.69 ± 0.69 i2.95 ± 0.07 d0.52 ± 0.01 d,e,f262.17 ± 3.41 a0.06 ± 0.00 a
P419.05 ± 1.90 h,i1.70 ± 0.05 a,b0.25 ± 0.03 a,b612.54 ± 5.12 i0.09 ± 0.00 a,b
P515.91 ± 0.64 e,f,g4.74 ± 0.24 f,g0.67 ± 0.05 f672.36 ± 3.41 j0.10 ± 0.00 b,c
P613.69 ± 0.50 c,d,e2.43 ± 0.05 c,d0.42 ± 0.00 b,c,d,e246.56 ± 10.25 a0.13 ± 0.00 c,d
P717.09 ± 0.32 g,h3.92 ± 0.24 e0.50 ± 0.04 b,c,d,e,f595.96 ± 6.83 i0.12 ± 0.00 a,b
P817.18 ± 0.27 f,g1.77 ± 0.21 a,b0.38 ± 0.01 a,b,c373.26 ± 5.12 c,d0.13 ± 0.01 c,d
P914.70 ± 0.37 c,d,e,f5.57 ± 0.27 h0.53 ± 0.00 d,e,f551.92 ± 1.13 h0.12 ± 0.00 b,c,d
P1014.87 ± 1.02 d,e,f4.28 ± 0.31 e,f0.58 ± 0.05 e,f592.03 ± 8.54 i0.13 ± 0.01 d
P118.28 ± 0.67 a2.94 ± 0.10 d1.12 ± 0.15 g354.46 ± 6.83 b,c0.29 ± 0.00 f
P1213.42 ± 0.30 c,d3.74 ± 0.33 e0.21 ± 0.00 a677.14 ± 12.81 j0.07 ± 0.00 a
P137.72 ± 0.29 a1.48 ± 0.00 a0.67 ± 0.07 f421.12 ± 8.54 e,f0.20 ± 0.00 e
P1411.05 ± 0.08 b4.98 ± 0.09 g,h0.63 ± 0.01 f433.08 ± 17.09 f0.15 ± 0.01 d
P158.52 ± 0.08 a2.28 ± 0.03 b,c0.99 ± 0.08 g330.19 ± 5.98 b0.18 ± 0.01 e
Average14.323.260.54476.240.12
We compared differences between the resulting TPC, TFC, DPPH, TAC, and FRAP of all BPEs using one-way ANOVA and Tukey’s multiple comparisons tests. Within the same column, different letters (a–j) indicate significant differences (p < 0.05).
Table 5. HPLC analysis of phenolic compounds of bee pollens (µg/g).
Table 5. HPLC analysis of phenolic compounds of bee pollens (µg/g).
Samplep-Coumaric Acid Rutin Myricetin ResveratrolNaringeninKaempferol
P1Not detected124.8995.4186.2177.137.0
P2Not detected 103.8532.159.684.539.5
P3Not detected488.12081.7377.77.662.0
P489.048.1255.1291.2161.0347.6
P577.638.336.1453.4488.711.5
P640.9613.189.9255.2112.15.4
P7Not detected187.0977.269.7102.14.6
P823.212.956.7160.6625.990.8
P932.81052.72062.148.41418.1172.4
P1064.823.852.8156.3220.821.8
P11Not detected32.210.58.4705.96.6
P12130.3496.3883.4413.1205.667.0
P133.971.1163.2122.7192.517.2
P14Not detected361.2696.2149.3193.322.7
P15Not detected11.426.7149.297.813.7
Average31.3303.3700.1191.5354.653.5
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Boulfous, N.; Belattar, H.; Ambra, R.; Pastore, G.; Ghorab, A. Botanical Origin, Phytochemical Profile, and Antioxidant Activity of Bee Pollen from the Mila Region, Algeria. Antioxidants 2025, 14, 291. https://doi.org/10.3390/antiox14030291

AMA Style

Boulfous N, Belattar H, Ambra R, Pastore G, Ghorab A. Botanical Origin, Phytochemical Profile, and Antioxidant Activity of Bee Pollen from the Mila Region, Algeria. Antioxidants. 2025; 14(3):291. https://doi.org/10.3390/antiox14030291

Chicago/Turabian Style

Boulfous, Nassiba, Hakima Belattar, Roberto Ambra, Gianni Pastore, and Asma Ghorab. 2025. "Botanical Origin, Phytochemical Profile, and Antioxidant Activity of Bee Pollen from the Mila Region, Algeria" Antioxidants 14, no. 3: 291. https://doi.org/10.3390/antiox14030291

APA Style

Boulfous, N., Belattar, H., Ambra, R., Pastore, G., & Ghorab, A. (2025). Botanical Origin, Phytochemical Profile, and Antioxidant Activity of Bee Pollen from the Mila Region, Algeria. Antioxidants, 14(3), 291. https://doi.org/10.3390/antiox14030291

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