Biochemical properties of different Algerian bee pollen samples

Abstract

Background

Bee pollen is a hive derived product formed from the agglomeration of flower pollen, nectar and bee saliva. It's hailed for its potential health benefits such as antioxidant, antibacterial and anti-inflammatory effect.

Objective

The present work aims to assess the Algerian bee pollen quality by highlighting their phenolic composition, their antioxidant, antimicrobial, enzymatic and antiviral activities, in addition to wound healing and Deoxyribonucleic acid (DNA) protection effect.

Methods

In this study, fourteen Algerian bee pollen 70% ethanol extracts samples were analyzed to determine their phenolic profils and biological activities. Thus, Reversed-phase high-performance liquid chromatography with Ultraviolet Detection RP-HPLC-UV is done, antioxidant, antibacterial, enzymatic, wound healing, DNA protection and anti-Herpes Simplex Virus 1 (anti- HSV-1) tests are carried out.

Results

The samples showed good antioxidant activity, variation in phenolic composition, selective antibacterial activity against Gram negative bacteria strains (Escherichia coli “E.coli”, Salmonella typhimurium and Yersinia pestis), inhibition to acetylcholinesterase and α-glucosidase and activation of Superoxide Dismutase (SOD). The samples P4, P7, P8 and P14 show high wound healing potential while P6 and P10 have DNA protection effect. Only P7 have anti-HSV-1 activity.

Conclusion

The different biological activities varied according to the phytochemical composition of each sample and not according to the harvest region. That is confirmed by Principal Component Analysis (PCA) and different correlations relationships between activities and bioactive compounds where it can be seen that the samples were grouped according to their composition and not by the region of the harvest.

Keywords

Algerian bee pollen phenolic compounds HPLC analysis biological activities antioxidant activity antibacterial activity enzymatic activity DNA protection anti-HSV-1

Introduction

Humain being has known hive products from antiquity and used them for various purposes. Among hive products, there is bee pollen which is obligatory for colony productivity and long-term survival, especially that pollen is the principal source of crucial nutrients for bees such as proteins, lipids, total carbohydrates, minerals and vitamins.^1–3

During foraging, the flowers pollen stick to the honey bees Apis mellifera thorax hairs and the latter transform it to bee pollen by adding saliva and nectar and secretions from the hypopharyngeal glands of honeybees such as β-glycosidase enzymes and finally it is rolled into pollen balls.⁴

Bee pollen is rich in carbohydrates, proteins, amino acids, enzymes, phytochemicals, considerable amounts of vitamins, minerals, fats, oligopeptides, anthocyanes and antioxidants agents such as phenolic substances and carotenoids.^4,5 Due to this rich composition, it has numerous virtues such as being antioxidant, antibacterial, antifungal, hepatoprotective and anti-inflammatory as well as other properties which makes it a huge health boosting product.^2,5,6 Bee pollen composition varies based on several factors such as the plant source, geographic origin, climatic conditions and soil type.⁷

Above all, bee pollen is a good source of very important and bioactive natural phenolic compounds known to be plants’ secondary metabolites produced to defend them against pathogens. Polyphenols are food antioxidants. Their presence in food is responsible for some characteristics like color and flavor and has a great impact on health.

Polyphenols cover a wide variety of molecules characterized from the chemical structure side by containing at least one aromatic ring with one or more hydroxyl groups in addition to other substituents which can give rise to various biological activities. For that, numerous research studies have been interested in studying them for their biological activities either by studying phenolic compounds individually or by studying the whole fruit, vegetable or plant.^8,9

During the last decades, problems like the resistance to antibiotics, enzymes inhibitors side effects and the viruses spread present major concern for public health^10,11 that's why it's essential to find new alternatives. In this context exactly, bee pollen can be a very promising product due to its biological activities underlined above. There were few studies investigating Algerian bee pollen like those made by¹² and¹³ but still it's insufficient because it is a product which is not consumed widely in Algeria and as a result there is lack of knowledge about its properties. For that, the present work has the aim to quantify and determine different antioxidant phenolic compounds existing within 14 bee pollen samples from different regions of Algeria using High Performance Liquid Chromatography (RP-HPLC) with UV detector, as well as studying different biological activities of the samples precisely antioxidant, antimicrobial, enzymatic, wound healing and antiviral (anti-HSV-1) activities.

Material and methods

The present study includes 14 bee pollen samples collected from different regions of Algeria in 2018 and stored at −20°C. After that, all the samples are ground to obtain a powder. The bee pollen samples used herein are not subjected to any treatment. Table 1 shows the harvesting region of each bee pollen sample as well as the abbreviation assigned to it. Figure 1 illustrates the distribution of harvest regions on the geographical map of northern Algeria.

Figure 1.

Geographical map of bee pollen samples harvesting regions.

Table 1.

Bee pollen samples harvesting regions and their assigned abbreviations.

Bee pollen harvesting region	Bee pollen sample assigned abbreviation
Adekar	P1
Akbou	P2
Beni Ourtilane	P3
Blida	P4
Bordj	P5
Boukhlifa	P6
Boumerdès	P7
Feraoune	P8
Kherrata	P9
Melbou	P10
Sidi Aich	P11
Tazmalt	P12
Tizi Ouzou	P13
Toudja	P14

Bee pollen samples extraction

The extraction is made according to the method of Carpes et al.¹⁴ For that, a quantity of 0.5 g of each bee pollen sample is added to 70 ml of 70% ethanol and after stirring for 2 h; the extracts obtained are filtered and kept at 4°C (Figure 2).

Figure 2.

Bee pollen samples extraction diagram.

Antioxidant activities

Ferric reducing antioxidant power test (FRAP)

FRAP test is carried out following the protocol described by Benzie and Strain.¹⁵ Briefly, to prepare the FRAP reagent, 3 solutions are mixed: acetate buffer (300 mM, pH 3.6), 2, 4, 6-tripyridyls-triazine (TPTZ; 10 mM TPTZ in 40 mM HCl) and FeCl₃ (20 mM) with respective ratios of (10:1:1). A volume of 100 μl of bee pollen extract is mixed with 1 ml of the FRAP solution. After 5 min, the absorbance is measured at 593 nm. The concentrations are expressed in µmol equivalent of iron (II) sulfate per 1 g of bee pollen sample (µmol E FeSO₄ /1 g).

Reducing power

The evaluation of reducing power is made according to the protocol described by Beretta et al.¹⁶ A volume of 500 μl of bee pollen extract is homogenized with 1500 μl of phosphate buffer (0.2 M, pH 6,6) and 1500 μl of potassium ferricyannide [(K₃Fe (CN)₆] (1%). After incubation for 20 min at 50°C, 1500 μl of trichloroacetate (TCA, 10%) are added and 1250 μl is taken from the mixture to be diluted in 1250 μl of distilled water and 250 μl of ferric chloride (FeCl₃) (0.1%) are added. After 10 min, the absorbance is read at 700 nm. The concentrations are expressed as mg equivalent of gallic acid per 1 g of bee pollen sample (mg GAE / 1 g).

Anti-radical activity with DPPH

The evaluation of the synthetic 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging activity is determined using the method of Cuendet et al.¹⁷ A volume of 100 μl of bee pollen extract (7.14 mg/ml) is mixed with 2 ml of DPPH ethanolic solution. After incubation of 15 min; the absorbance is measured at 517 nm and scavenging percentage is calculated according to the following formula:

DPPH percentage scavenging (%) = [(A_{C} - As) / A_{C}] \times 100

A_C: Absorbance of control

As: Absorbance of sample

Antiradical activity with ABTS

The ABTS (2, 2-azobis ethylbenzothiazoline-6-sulphonic) radical method is based on the transformation and reduction of a cationic radical ABTS⁺ blue-green coloring in ABTS colorless by an antioxidant. A quantity of 100 μl of bee pollen extract (7.14 mg/ml) is mixed with 1 ml of 7 mM ABTS solution. The mixture is left for 7 min and the absorbance is read at 734 nm.¹⁸ Then, radical percentage reduction is calculated as following:

ABTS percentage reduction (%) = [(A_{C} - As) / A_{C}] \times 100

A_C: Absorbance of control

As: Absorbance of sample

High performance liquid chromatography (HPLC) analysis

Polyphenols extraction

The extraction is made according to the method described by Ribani et al.¹⁹ Thus, for 3 g of bee pollen sample, 35 ml of 70% ethanol are added. Then, the mixture is shaken for 24 h at room temperature. Samples are filtered with 0.45 micrometer filter and concentrated in a rotary evaporator at 40°C. After ethanol evaporation, residue of each sample is dissolved in 10 ml of acidified distilled water (pH = 2). Polyphenols are then extracted by adding diethyl ether and later ethyl acetate. Gradually after each stage of extraction, float is collected to be evaporated by rotary evaporator later. Samples are dilluted at 1/5 using methanol before HPLC analysis.

Analysis

HPLC (Elite LaChrom Hitachi, Japan) with a UV detector is used for qualitative and quantitative analysis of both standards and samples (280 nm wave length). The separation is done on column reversed- phase C18 (150 mm × 4.6 mm, 5 μm; Fortis), in gradient solvent systems A (2% acetic acid in water) and solvent B (70:30, acetonitrile/water) which is sonicated before stirring and continuously degassed by the built-in HPLC system. The gradient elution used for HPLC analysis is the one developed by.²⁰ A 20 μl sample is injected, with the column kept at 30°C and a flow rate of 0.75 ml/min. The solvent program began with linear gradient at 95% of solvent A, maintained for 3 min, then reducing to 80% A by 10 min, 60% A by 20 min, 20% A by 30 min, and finally back up to 95% A by 50 min (Table 2).

Table 2.

RP-HPLC-UV detector gradient program.

Time (min)	Solvent A (% 2 acetic acid, ultra pure water)	Solvent B% (70% acetonitril, 30% ultra pure water)
0.01	95.00	5.00
3.00	95.00	5.00
8.00	85.00	15.00
10.00	80.00	20.00
12.00	75.00	25.00
20.00	60.00	40.00
30.00	20.00	80.00
35.00	95.00	5.00
50.00	95.00	5.00

In this study, nineteen phenolic compounds standards are used: Gallic acid, Protocatechuic acid, Caffeic acid, Syringic acid, P-OH Benzoic acid, P- Coumaric acid, Ferulic acid, T-Cinnamic acid, Myrecetin, Rutin, Epicatechin, Catechin, Hesperetin, Pinocembrin, Resveratrol, Daidzein, Luteolin, Chrysin, Caffeic acid phenethyl ester (CAPE).

Calibration curves used for the quantitative determination of the individual phenolic components showed very high linearity, with correlation coefficients spanning the range of 0.998 to 1.000.

Antimicrobial activity

Bee pollen samples antimicrobial activity is tested against 8 bacteria and 2 yeast stains obtained from the Farabi medical microbiology laboratory in Trabzon, Turkey. The stains are namely: Bacillus subtilis ATCC 6633; Salmonella typhimurium ATCC 14028; Klebsiella pneumoniae ATCC 13883; Staphylococcus aureus ATCC 25923; Escherichia coli (E. coli) ATCC 25922; Chromobacterium violaceum ATCC 12472; Yersinia pestis ATCC 25924; Pseudomonas aeruginosa ATCC 22019; Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019.

Sensitivity test

The antimicrobial activity is assessed according to the CLSI standard methodology.^21,22 First, agar disk diffusion method is followed to measure inhibition zones diameters and later minimal inhibition concentrations (MICs) are calculated.

Bacteria cells suspensions of 1*10⁸ CFU/ml (0.5 McFarland density standard) and yeast suspensions of 3*10⁸ CFU/mL (1 McFarland density standard) are prepared from different overnight cultures in order to make the inoculums. Later, the different disks are put in each plate. Bee pollen samples are tested at a concentration of 142 μg /ml obtained from the dilution of the original extracts using distilled water.Gentamicine and ampicilline are used as positive control antibiotics for gram positive and gram negative bacteria, respectively at a concentration of 100 μg/ml for both. The diameters of the inhibition zones (IZD) are measured in millimiters (mm) and the samples are then classified according to the estimated scale of antimicrobial activity as following: less than 8 mm it's non sensitive, from 8 to 14 mm it's sensitive, from 15 to 19 mm it's very sensitive and more than 20 mm it's extremely sensitive.²³

Minimum inhibitory concentration (MIC) determination

Minimum inhibitory concentrations (MICs) are determined as the lowest concentrations preventing visible growth. This test is only realized with the sensitive bacterial strains having shown inhibition zone in previous sensitivity test according to the CLSI standard methodology.²⁴

A suspension of 0.5 Mc is prepared from an overnight culture for each strain. Then, every suspension is diluted to 1/100 and 100 µl is put in every well of the 96 well plates. For all assays, stock solutions of bee pollen samples extracts are prepared at 10 mg/ml, after that 100 µl of appropriate extract is added to the first well and serial microdilutions are prepared. MHB alone and MHB with bacteria are used as positive and negative controls respectively. The MIC results are done visually by observing bacterial growing aspect and by spectrophotometric absorbance measurement at 620 nm.

Enzymatic activities

In this work, the enzymatic activity of bee pollen extracts is studied, particularly anti- acetylcholinesterase (anti-AChE), anti- α-glucosidase and SOD activity. The IC₅₀ values are calculated using GraphPad Prism 5 software.

Anti- acetylcholinesterase (anti-AChE) activity

In this test, Ingkaninan's method using a 96-well microplate reader is followed to study AChE inhibition effects.²⁵ For that, Tris-HCl buffer at pH 8 (50 μl, 50 mM), 5,5-dithio-bis (2 nitrobenzoic) 190 acid (DTNB) (125 μl, 3 mM), AChE (25 μl, 0.2 U/ml) and the bee pollen extracts (from 50 to 1000 μg/ml) are added in the microplate and incubated for 15 min at room temperature. Galantamine here is used as a positive control. Later, the enzymatic reaction is started by adding 25 μl of 15 mM of the substrate (ACh). The absorbance is read at 412 nm using microplate reader.

AChE inhibition percentage is calculated using the formula (1) then IC₅₀ is calculated. Formula (1):

% Inhibition = ((A - B) / A) \times 100

A: The enzyme activity without compound

B: The enzyme activity with compound.

Anti- α-glucosidase activity

α-Glucosidase inhibition assay is made following the method of Şöhretoğlu et al.²⁶ Acarbose is used as a positive control. The extracts at different concentrations (from 50 µl to 1000 µg/ml) in phosphate buffer at pH 6.9 are added to α-glucosidase (100 µl, 0.5 U/ml) and the mixture is left to incubate for 15 min in a microplate.

After incubation, p-nitrophenyl-α-glucopyranoside (4-pNPG) (50 µl, 5 mM) is added and the mixture is incubated again for 15 min at room temperature. The absorbance is then measured at 405 nm with a 96-well microplate reader and α-glucosidase inhibition percentages are calculated using formula 1 and later IC₅₀ values are determined.

SOD activity

This test is conducted according to the method of Sabuncuoğlu & Şöhretoğlu²⁷ and the inhibition of Nitroblue Tetrazolium Test (NBT) reduction by SOD is estimated.

For this experiment, a reaction mixture containing 250 mM of phosphate buffer (pH 7.8), EDTA (0.1 mM), NBT (75 μM), methionine (13 mM) and riboflavin (2 μM) is combined with different concentrations of bee pollen ethanolic extracts and incubated at room temperature for 15 min. Absorbance is then measured at 560 nm.

Generated superoxide anion inhibition percentage is calculated according to the following formula:

% inhibition of NBT reduction by SOD = (control OD - treatment OD / control) \times 100

Cell viability test by micro culture tetrazolium technique (MTT)

The cytotoxicity of bee pollen samples is tested by MTT method described by Takenouchi and Munekata.^28,29

After 24 h of incubation (37°C; 5% CO₂), HaCaT, vero and HeLa cells are cultured in 96 well plate using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% of Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin in such a way as to get 10⁴ cells per well and incubated again for other 24 h. Next day, the density of cells is checked under microscope and cytotoxicity test started. For every bee pollen sample, a filtration using 0.22 µm syringe filter is made and different concentrations are prepared: 0; 12.5; 25; 50; 75; 100 and 150 µg/ml. At this moment, the 96 well plates containing different cells (HaCaT, vero and HeLa) are taken from incubation oven and old medium are replaced by bee pollen extracts at different concentrations prepared in DMEM and incubated for 24 h. After 24 h, the medium containing bee pollen extract is removed from all the wells and 100 µl of fresh DMEM medium supplemented with 10 µl of MTT is put in every well before letting in incubation for 3 h. Later, every well is emptied and 50 µl of DMSO is put inside. After incubation for 30 min, absorbances are measured at 570 nm. The appearance of purple color in the wells indicates the viable cells and the colorless ones indicate the dead cells. Cell viability is calculated according to the following formula: Cells viability % = (sample absorbance/control absorbance) ×100.

Wound healing test (Scratch assay)

The samples P4, P7, P8 and P14 previously selected according to MTT test results at a concentration of 100 µg/ml are tested for their wounds healing ability according to the method described by Şahin et al.³⁰ For that, HaCaT cells are seeded in 6 well plates with a density of 10⁶ cells/well in DMEM medium supplemented with 10% FBS. Every well is scratched using 1000 μl sterile pipette tip and cells are washed by PBS buffer (pH 7.4) before putting in every well the appropriate extract. The visual aspect of the scratches is checked under microscope before incubating plates (37°C; 5% CO₂). Pictures are taken in the same place of the scratch for every sample at different timings of incubation 0, 24, 48 and 72 h using an invert microscope with a digital camera. Inverted microscope with Zen 3.0 software linked to AxioCam ICc1 is used to take photos of cell cultures.

Antiviral activity

The antiviral activity is evaluated according to the method outlined by Chiamenti et al.³¹ with few adaptations.

To perform antiviral activity assay, Vero cells are seeded for 24 h (37°C; 5% CO₂) in DMEM medium with 10% FBS in order to have monolayer of 240 000 cells in each well. After that, the cells are infected by Herpes Simplex Virus-1 (HSV-1) suspension. After incubation for 1 h, the HSV-1 suspension is removed and replaced by bee pollen extracts (P4, P7, P8 and P14) at 100 µg/ml. For P7 sample, different concentrations are later studied (50, 100, 200, 400, 600, 800 and 1000 µg/ml). The different cultures are made in 24 well plates and they are observed at 0, 24 and 48 h of the experiment to assess the formation of plaques which is the infection index. The different wells are examined using an inverted microscope and photos are taken.

In this test, negative control contains only medium while positive control contains acyclovir at 100 µg/ml. At the end, MTT test is made for the samples showing the best results in order to confirm the vitality of the cells. Inverted microscope with Zen 3.0 software linked to AxioCam ICc1 is used to take photos of cell cultures.

DNA protection activity

Supercoiled plasmid pR322 DNA nuclease effects of extracts

The nuclease impacts of the extracts demonstrating the strongest antioxidant properties on the supercoiled plasmid pBR322 DNA are evaluated using agarose gel electrophoresis on the supercoiled pBR322 plasmid DNA, following the methodology outlined in reference.³² The supercoiled pBR322 plasmid DNA is incubated with increasing concentrations of bee pollen extracts (500, 1000 and 2000 µg/ml) in a buffer containing 50 mM Tris-HCl at pH 7.0. All samples are then incubated at 37°C for 1 h. Subsequently, a loading buffer (containing bromophenol blue, xylene cyanol, glycerol, EDTA and SDS) is added, and the mixtures are loaded onto an agarose gel (0.8%) containing ethidium bromide stain, in a Tris-acetate-EDTA (TAE) buffer. Electrophoresis is carried out at 100 V for 90 min, and the results are visualized using a BioRad Gel Doc XR system and analyzed using Image Lab Version 5.0.1 software.

Supercoiled pBR322 plasmid DNA protection experiments

The protective effects of extracts P6 and P10 on the supercoiled pBR322 plasmid DNA against Fenton reagents are investigated using agarose gel electrophoresis, following the protocol described in reference.³³ In this work, the mixture containing 50 mM Tris-HCl buffer (pH 7.0), plasmid DNA, FeSO₄ (1 mM), H₂O₂ (2%) and extracts at concentrations of 500, 1000 and 2000 µg/ml are incubated at 37°C for 1 h. The electrophoresis experiments are carried out according to the detailed method outlined above. Image Lab Version 5.0.1 Software program is used to visualize DNA aspect.

Statistical analysis

Statistical analysis is performed by the STATISTICA 7.1 software by applying an analysis of one variance (A.N.O.V.A) for all parameters. Correlation matrixes and PCA (principal component analysis) are used in order to study the correlation between the different parameters. The results are expressed as the average of three trials. The results are ranked in descending order a > b > c > d > e > f > g > h > i > j. Values with the same letter have no significant difference.

Results

Antioxidant activities

The results of assessment of antioxidant activities are shown in Table 3.

Table 3.

Antioxidant activity results.

	Antioxidant activity
Samples	DPPH (%)	ABTS (%)	Reducing power (mg GAE / 1 g)	FRAP(µmol E FeSO₄ /1 g)
P1	84.85 ± 2.09 ^a	89.71 ± 0.001 ^ab	76.56 ± 6.69 ^abc	84.92 ± 0.00 ^e
P2	82.15 ± 3.34 ^ab	89.65 ± 0.19 ^ab	53.69 ± 3.00 ^bc	62.10 ± 2.07 ^f
P3	77.98 ± 0.64 ^cd	89.94 ± 0.98 ^a	80.27 ± 8.61 ^ab	97.91 ± 0.92 ^d
P4	70.38 ± 3.31 ^e	88.45 ± 0.19 ^cd	51.27 ± 7.01^c	34.17 ± 5.92 ^j
P5	72.71 ± 0.38 ^e	89.71 ± 0 ^ab	75.46 ± 7.66 ^abc	53.45 ± 0.23^g
P6	81.11 ± 0.56 ^bc	89.54 ± 0 ^abc	78.15 ± 12.95 ^ab	238.02 ± 8.26 ^a
P7	71.42 ± 0.42 ^e	89.23 ± 0.06 ^abcd	82.79 ± 16.27 ^a	121.93 ± 4.29^c
P8	58.55 ± 4.05 ^g	89.48 ± 0.19 ^abc	81.30 ± 19.45 ^a	88.92 ± 4.13 ^e
P9	64.80 ± 0.69 ^f	88.19 ± 1.98 ^d	83.12 ± 14.40 ^a	37.54 ± 1.45 ^ij
P10	76.64 ± 1.10 ^d	88.62 ± 1.10 ^bcd	83.45 ± 15.63 ^a	142.71 ± 3.06 ^b
P11	71.73 ± 0.82 ^e	89.54 ± 0.45 ^abc	80.40 ± 21.15 ^ab	47.59 ± 0.95 ^h
P12	77.25 ± 0.64 ^d	89.52 ± 0.03 ^abc	77.58 ± 27.56 ^abc	85.25 ± 4.74 ^e
P13	76.45 ± 0.97 ^d	89.48 ± 0.39 ^abc	84.45 ± 20.76 ^a	35.74 ± 0.81 ^j
P14	69.46 ± 3.54 ^e	90 ± 0.19 ^a	96.11 ± 21.45 ^a	41.54 ± 0.82 ⁱ

Values are mean ± standard deviation. Means followed by the same letter in each column are not different according to ANOVA (Analysis of Variance).

FRAP test

The FRAP test shows that the reducing capacity varies between 34.17 µmol equivalent FeSO₄ /1 g (P4) and 238.02 µmol equivalent FeSO₄ / 1 g (P6). The statistical analysis revealed significant difference (p < 0.05) between all samples except between three groups: P1, P8 and P12; P9 and P14 and P4; P9 and P13.

Reducing power

From the results, it can be seen that all bee pollen samples have reducing power activity.

The best one is displayed by P14 (96.11 mg EAG/1 g) in opposition to P4 with lowest capacity (51.27 mg EAG / 1 g). According to statistical analysis at p < 0.05, the samples are divided into 3 groups; the first is formed by P1, P3, P5, P6, P7, P8, P9, P10, P11, P4, P12, P13 and P14; the second contains: P1, P2, P3, P5, P6, P11 and P12 and for the third is: P1, P2, P4, P5and P12. The difference between all groups is significant.

Anti-radical activity with DPPH

The highest DPPH scavenging activity is recorded by sample P1 (84.85%) in contrary P8 is the lowest in term of antiradical activity (58.55%). Samples are divided into 7 groups according to statistical analysis (p < 0.05) with significant difference between all groups.

Antiradical activity with ABTS

In this test, ABTS radical reducing results range from 88.19 for P9 to 90% for P14.

HPLC analysis

The results of HPLC analysis are shown in Table 4. Bee pollen is a highly biologically active product well known for its phenolic constituents; it may also possess biological active properties.

Table 4.

Phenolic components of the 14 bee pollen samples detected by HPLC (µg/g). (ND: not detected).

Phenoliccompounds	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12	P13	P14
Phenolic acids:
Gallic acid	33.34	8.48	27.34	11.92	43.74	19.91	14.78	31.60	11.34	17.31	18.11	6.17	19.46	28.80
Proto-catequic acid	ND	ND	8.14	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Caffeic acid	49.27	53.14	37.60	7.16	20.28	38.66	2.74	ND	5.55	3.93	13.98	7.87	16.71	N.D
Syringic acid	15.31	2.24	ND	3.48	11.36	8.78	5.28	5.35	2.75	1.34	ND	3.83	2.29	2.96
p-OH benzoic acid	4.22	3.62	3.76	ND	ND	4.02	5.45	2.39	3.14	1.53	ND	2.74	2.90	3.72
p-coumaricacid	1.93	3.95	29.91	9.61	19.80	9.36	2.69	22.67	6.17	2.01	3.69	8.28	13.02	5.78
Ferulic acid	2.32	ND	ND	ND	ND	2.78	ND	ND	ND	1.43	8.00	ND	2.05	3.54
t-cinnamic acid	26.53	58.17	8.89	ND	41.57	28.00	31.33	77.26	48.07	27.27	54.75	6.82	30.67	208.82
Flavonols
Myerecetin	97.18	530.15	ND	351.67	116.47	25.32	106.88	766.07	243.13	72.87	162.23	199.14	297.91	170.38
Rutin	ND	17.23	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	44.27
Flavan-3-ol
Epicatechin	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Catechin	3.85	ND	5.01	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
Flavanones
Hesperetin	30.26	33.22	10.32	37.69	54.74	8.29	5.91	19.57	30.54	10.83	63.18	28.79	32.38	67.35
Pinocembrin	ND	ND	ND	ND	ND	ND	ND	ND	ND	53.285	ND	ND	10.45	ND
Stilbenes
Resveratrol	278.70	293.83	205.12	91.18	243.26	394.69	373.12	212.77	89.87	80.26	69.82	42.32	108.13	89.58
Isoflavones
Daidzein	ND	ND	ND	ND	ND	12.595	ND	ND	30.52	3.06	12.85	16.96	ND	ND
Flavones
Luteolin	ND	ND	ND	ND	36.26	30.37	15.73	21.19	17.13	ND	71.78	41.62	ND	47.39
Chryrisin	13.69	ND	6.33	ND	ND	7.16	9.57	ND	36.30	ND	65.34	ND	14.27	21.35
Other
CAPE	9.42	68.04	8.89	271.27	69.60	11.54	5.75	10.31	42.32	ND	175.89	255.01	278.32	57.63

There are numerous distinct phenolic compounds present in any natural sample, and it can be challenging to measure each one individually.^34,35 Consequently, only 19 of the phenolic substances have been detected by HPLC and total phenolic content has been determined by a spectrophotometric method in this study. The chromatograms of standard phenolic compounds are presented in Figure 3.

Figure 3.

Standards chromatogram: 1. Gallic acid, 2. Protocatechuic acid, 3. P-OH Benzoic acid, 4. Catechin, 5. Caffeic acid, 6. Syringic acid, 7. Epicatechin, 8. P- Coumaric acid, 9. Ferulic acid, 10. Rutin, 11. Myrecetin, 12. Resveratrol, 13. Daidzein, 14. Luteolin, 15. T-Cinnamic acid, 16. Hesperetin, 17.Chrysin, 18. Pinocembrin, 19. CAPE.

The amounts of the phenolic compounds are given in Table 4 as µg/g of sample.

Most of the phenolic compounds exhibit absorption maxima in the ultraviolet (UV) absorption spectra at a wavelength of 280 nm. They were identified by comparing their retention times (through peak normalization) with those of authentic standard compounds.³⁶

For phenolic profile of bee pollen, the most used HPLC detection systems are: UV-VIS detector,³⁷ PDA (photodiode array) detector and MS (mass spectrometry) detector.³⁸

In the analytical conditions selection, it is important to consider the diversity of the phenolic compounds group, given their different chemical structures, as well as the varying sensitivity of the compounds to pre-treatment processes.³⁹

Among the 19 studied phenolic compounds previously detailed, 4 are found in all samples (gallic acid, p-coumaric acid, resveratrol and hesperetin). Epicatechin is not found in any sample. For the other compounds, their amounts differ from sample to another.

Antimicrobial activity

According to antimicrobial study, it seems that bee pollen samples are more effective against Gram negative stains especially E. coli, Salmonella typhimurium and Yersinia pestis (Table 5).

Table 5.

Antimicrobial activity results (-: no activity detected).

	Antibacterial activity
Samples	E. Coli		Salmonella typhimurium		Yersinia pestis
Samples	IZD (mm)	MIC (mg/ml)	IZD (mm)	MIC (mg/ml)	IZD (mm)	MIC (mg/ml)
P1	–	–	–	–	–	–
P2	–	–	–	–	14	5
P3	–	–	–	–	–	–
P4	16	5	–	–	14	5
P5
P6	18	2.5	–	–	13	5
P7	–	–	12	5	–	–
P8	18	2.5	–	–	–	–
P9	16	2.5	–	–	–	–
P10	–	–	15	5	15	5
P11
P12	15	5	–	–	15	5
P13	17	2.5	–	–	–	–
P14	–	–	–	–	–	–

E. coli strain is sensitive to 5 monofloral samples (P4, P6, P8, P9 and P12) and one polyfloral sample (P13). The inhibition diameters range from 15 to 18 mm for samples and reach 20 mm for gentamicine.

The highest inhibition is got with P6 and P8 against E. coli with 18 mm of inhibition zone diameter both.

For Salmonella typhimurium strain, it's only sensitive to two bee pollen samples namely P7 and P10 with inhibition zone diameter of 12 and 15 mm, respectively.

Yersinia pestis strain is sensitive to 5 samples (P2, P4, P6, P10 and P12). MIC values for the three stains E. coli, Salmonella typhimurium and Yersinia pestis in our study range from 2.5 mg/ml to 5 mg/ml.

Enzymatic activities

Enzymatic activities results are given as IC₅₀ values for different tested enzymes AChE, α- glucosidase and SOD (µg/ml) in Table 6.

Table 6.

Enzymatic activity results (-: no activity detected).

	Enzymatic activity
Samples	AChE IC50(µg/ml)	α glucosidase IC 50(µg/ml)	SOD IC50(µg/ml)
P1	3203.50	892.75	–
P2	2501.50	550.20	–
P3	2735.50	317.95	–
P4	1506	280.30	205.13
P5	1351	300.70	–
P6	1735.50	257.40	–
P7	868.50	382.45	515.85
P8	1040.70	217.35	103.31
P9	798.25	173.35	–
P10	1022.85	214.40	–
P11	1341	309.40	–
P12	552.90	134	–
P13	1343.50	337.05	–
P14	928.30	239.65	15.97
Positive control	Galantamin:	Acarbose :	–
Positive control	8.42	190.45

Anti-AChE activity

The sample P12 shows the highest AChE inhibitory effect with lowest IC₅₀ of 552.90 µg/ml followed by P9 and P7 with 798.25 and 868.5 µg/ml, respectively.

The different samples show a very highly statistical difference (p˂0.001) between each other except P11and P13 which form the same group.

The AChE IC₅₀ values are in a very highly significant correlation (p˂0.001) with p-OH benzoic acid (r = - 0.94).

Anti-α-glucosidase activity

α-Glucosidase inhibition IC₅₀ values range from 134 µg/ml (P12) to 892.75 µg/ml (P1), so P12 has the best activity against α-glucosidase. The statistical analysis display that there is a very highly significant difference (p˂0.001) between all the samples.

SOD activity

In this study, the best activity for SOD goes for P14 with IC₅₀= 15.97 µg/ml. All the samples present a very highly significant difference between each other (p˂0.001).

Cell viability test by micro culture tetrazolium technique (MTT)

The bee pollen cytotoxicity is evaluated by MTT assay and cells viability is calculated for different cells HaCaT, Vero and HeLa. From the results it can be seen that, for all samples, the cells viability percentage is very high and higher to 80% where the lowest one was for P8 with 82.01% at 150 µg/ml with HaCaT cells. Vero and HeLa cells both show very high cells viability percentages with all the samples.

Wound healing test

Wound healing test serves to assess the degree of scratch closure and therefore the eventual healing power of the tested samples.⁴⁰ In the present study, 4 bee pollen samples: P4, P7, P8 and p14 are tested for their wound healing effect on a scratch created in a cell monolayer. After a repair period of 72 h, the observation under microscope shows HaCaT cells proliferation on both sides of the scratch and migration to the damaged area with the P14 sample by comparison to a negative control that contains only medium and didn’t show any change (Figure 4).

Figure 4.

Wound healing test results. The newly formed cell layer is indicated by the red arrow in the case of P14 bee pollen sample. This layer of cells leads to the closure of the scratch previously created which is an indication of the healing power of bee pollen. The red arrow points to the healing site for P14 sample.

Antiviral activity

The effect of bee pollen was evaluated by comparison to negative and positive control (acyclovir) (Figure5). The Vero cells seeded without acyclovir or bee pollen showed cytopathologic changes, such as rounding of the cells, aggregation and nuclear enlargement disruption of membrane integrity.

Figure 5.

Antiviral activity (anti-HSV-1) results. Cell layer density is an index of anti-HSV-1 activity. With medium only (negative control), the cells are not protected from viral infection which leads to the formation of plaques. Acyclovir is a positive control which protects cells from infection; therefore the number of plaques formed is very limited. With P7 bee pollen sample at 600 µg/ml, it can be noticed that the plaques formed are limited which means that P7 gives protection anti-HSV-1.

The four bee pollen samples behave in different way towards the HSV-1 infection. The sample P4 was efficient until 48 h of incubation, after 48 h its efficiency decrease and the infection plaques became numerous. The P8 sample behavior is average and stable along the experiment where it gave a good cell vitality signs at 48 h and 72 h of the experiment such as the cells adhesion to the well even after 72 h of incubation. P14 is the least efficient against HSV-1 and shows the formation of highest number of infection plaques in addition to the cells detachment from the wells which is a clear infection sign. The sample P7 is the most effective one at 100 µg/ml, it shows a very good vitality cells aspect at 48 h and average efficiency at 72 h, that's why the decision to test it at different concentrations is made. From the seven tested, the three concentrations 200, 400 and 600 µg/ml showed the best antiviral activity with 6.97; 7.63 and 20.49% cells vitality percentages respectively and IC₅₀ of 64.79 µg/ml. This IC₅₀ belongs to IC₅₀ values interval recommended for plant extracts against infectious diseases which must be lower than 100 μg/ml.⁴¹

The statistical analysis reveals that P7 at 600 µg/ml possess efficiency different from 200 and 400 µg/ml in a very highly significant way (p ˂0.001).

DNA protection activity

Supercoiled plasmid pR322 DNA nuclease effects of extracts

The hydrolytic nuclease activity of extracts is investigated using agarose gel electrophoresis on supercoiled pBR322 plasmid DNA in order to determine extracts’ potential to cause DNA damage. It is known that Form I (supercoiled form), Form II (nicked form) and Form III (linear form) bands can be observed on agarose gel after electrophoresis. In this study, the percentages of Form I is similar in the absence and presence of extracts. These findings indicate that no DNA nuclease effect is observed at these concentrations of extracts (Figure 6(a)).

Figure 6.

(a) DNA protection activity. Supercoiled pBR322 plasmid DNA damage effects of extracts. Lane 1: DNA control; lane 2-4: DNA + P6 (500, 1000, and 2000 µg/mL); lane 5-7: DNA + P10 (500, 1000, and 2000 µg/mL). (b) DNA protection activity. Supercoiled pBR322 plasmid DNA protective effects of extracts against Fenton reagent. Lane 1: DNA control; lane 2: DNA + 1 mM FeSO₄+ 2% H₂O₂ ; Lane 3-5: DNA + 1 mM FeSO₄+ 2% H₂O₂+ P6 (500, 1000, and 2000 µg/mL); Lane 6-8: DNA + 1 mM FeSO₄+ 2% H₂O₂+ P10 (500, 1000, and 2000 µg/mL).

Bee pollen extracts protective effects on supercoiled pBR322 plasmid DNA

It is well established that Fenton reactions (Fe²⁺+ H₂O₂ → Fe³+ HO^.+ OH⁻) generate hydroxyl radicals known to be highly reactive which can cause serious biological damage.⁴² In this work, the percentage of Form I was almost 75% for the negative control in Figure 6(b), lane 1. Upon addition of FeSO₄ and H₂O₂ to supercoiled pBR322 plasmid DNA, Form II (72.50%) are generated and Form I disappeared completely (Figure 6(b), Lane 2). On addition of extracts, the percentages of Form I increase again. The percentages of Form I is 53.10%, 55.10%, and 59.10% in the presence of P6 (Figure6(b), lanes 3-5). In the presence of P10, the percentages of Form I are calculated as 54.90%, 58.70%, and 62.40%, respectively (Figure 6(b), lanes 6-8).

Discussion

Antioxidant activities

FRAP test

The results of FRAP test are in the same range of those reported by Bakchiche et al.¹² that showed 88.57 μmol FeSO₄/g for Algerian bee pollen and slightly better than the results obtained by Saral et al.⁴³ in bee pollen samples from Turkey giving activity between 8.69 and 84.89 μmol FeSO₄/g.

FRAP test is based on electron transfer and it is a very good indicators of total antioxidant power, since total reducing power is defined as the sum of the reducing powers of the individual compounds existing in a particular sample.⁴⁴

The difference between the FRAP test and the reducing power test is that the FRAP test measures the ability of antioxidants to reduce ferric ions (Fe³⁺) to ferrous ions (Fe²⁺) in an acidic medium, leading to a color change that can be quantified spectrophotometrically. This is more specifically related to the reducing power of antioxidants concerning iron ions.⁴⁵

As expected from similar studies, FRAP test results are in correlation with phenolic acids such as p-OH benzoic acid (r = 0.71, p˂0.01) and syringic acid (r = 0.90, p˂0.01) and with a stilbene compound which is resveratrol (r = 0.98, p˂0.001) (Table 7).

Table 7.

Correlations matrix between different phenolic compounds and biological activities of bee pollen samples.

Variables	FRAP	RP	ABTS	IZD _E.coli	IZD _S	IZD _Y	MIC _E.coli	MIC _Y	AChEIC ₅₀	α-glcaseIC ₅₀	SODIC ₅₀	Caffeic	CAPE	Coum- aric	Gallicacid	Hesp- iritin	Myri- cetin	Resve- ratrol	syringic	Cinna- mic	p- OHbenzoicacid
FRAP	1
RP	0.27	1
ABTS	0.12	0.72	1
IZD _E.coli	−0.24	−0.50	−0.51	1
IZD _S	0.81	0.13	−0.06	−0.58	1
IZD _Y	−0.60	−0.72	−0.84	0.51	−0.33	1
MIC _E.coli	−0.48	−0.70	−0.78	0.87	−0.52	0.87	1
MIC _Y	−0.60	−0.72	−0.84	0.51	−0.33	1.00	0.87	1
AChEIC ₅₀	−0.63	−0.71	−0.80	0.69	−0.50	0.97	0.96	0.97	1
α-glc IC ₅₀	0.58	−0.08	−0.32	−0.52	0.93	0.00	−0.29	0.00	−0.20	1
SOD IC₅₀	0.72	−0.14	−0.39	−0.31	0.93	−0.01	−0.19	−0.01	−0.17	0.96	1
Caffeic	−0.31	−0.70	−0.92	0.30	0.05	0.92***	0.71	0.92	0.82	0.38	0.36	1
CAPE	−0.73	−0.65	−0.74	0.43	−0.43	0.98***	0.82	0.98	0.95	−0.08	−0.14	0.87	1
Coumaric	−0.01	−0.09	0.01	0.83***	−0.57	−0.04	0.45	−0.04	0.20	−0.71**	−0.48	−0.28	−0.10	1
Gallic acid	−0.02	0.53	0.79**	0.07	−0.47	−0.67	−0.35	−0.67	−0.49	− 0.75**	−0.70*	−0.90	−0.59	0.58	1
Hespiritin	−0.85	0.16	0.36	−0.20	−0.67	0.13	−0.04	0.13	0.13	−0.57	− 0.78**	−0.14	0.31	−0.17	0.32	1
Myricetin	0.01	−0.14	−0.06	0.86***	−0.54	0.01	0.50	0.01	0.24	−0.67*	−0.43	−0.21	−0.06	1	0.51	−0.22	1
Resveratrol	0.98***	0.18	0.02	−0.31	0.90***	−0.50	−0.47	−0.50	−0.57	0.73	0.84	−0.16	−0.63	−0.17	−0.20	−0.86	−0.14	1
Syringic	0.90**	0.06	−0.05	0.19	0.55	−0.43	−0.14	−0.43	−0.37	0.33	0.56	−0.23	−0.59	0.38	0.06	−0.93	0.40	0.85	1
cinnamic	0.62	0.62*	0.87***	−0.48	−0.35	−0.58	−0.61	−0.58	−0.54	−0.50	−0.65*	−0.65	−0.41	−0.08	0.74	0.74	−0.15	−0.38	−0.47	1
p- OH benzoic acid	0.71**	0.60*	0.61*	−0.81	0.74	−0.84	−0.95***	−0.84***	−0.94***	0.51	0.46	−0.59	−0.84	−0.45	0.17	−0.26	−0.48	0.71	0.38	0.34	1

*(p<0.05) = significant correlation; ** (p < 0.01) = highly significant correlation; *** (p < 0.001) =very highly significant correlation.

RP: reducing power; CAPE: Caffeic acid phenethyl ester; IZD: inhibition zone diameter; MIC: minimum inhibitory concentration.

Thus, the antioxidant power shown by bee pollen samples can be attributed to these compounds.

Reducing power

The reducing power test reflects reduction potential of substances that react with potassium ferricyanide (Fe ³⁺) to form potassium ferrocyanide (Fe ²⁺). The later reacts then with ferric chloride to form ferric ferrous complex whose absorption maximum is at 700 nm. This test is used generally to assesse the overall reducing ability of a substance.⁴⁶

For this activity, significant positive correlations (p˂0.05) are recorded with 2 compounds: p-OH benzoic acid (r = 0.60) and cinnamic acid (r = 0.62).

Anti-radical activity with DPPH

The results found for DPPH test are higher than those found by Dos Santos Vasconcelos⁴⁷ that studied Brazilian bee pollen samples and whose results were between 66.95% and 78.58%.

According to Sánchez-Moreno,⁴⁸ the DPPH method is regarded as a reliable and straightforward way to measure the antioxidant capacity of fruits, vegetables or extracts.

The variations in DPPH scavenging activity from sample to another may be explained by the significant differences in the polyphenol content of the samples and their corresponding scavenging capabilities. It can be reasoned that the phenolic compounds act as free radical scavengers due to their ability to donate hydrogen atoms.⁴⁹

In this study, unlike other antioxidant tests, DPPH didn’t show correlation with antioxidants like phenolic compounds. According to Dulger Altiner et al.,⁵⁰ these differences between antioxidant measurement methods might be attributed to the diversity in the chemical composition of the bee pollen samples and the sensibility of each method toward different bioactive compounds.

Antiradical activity with ABTS

The results found are higher than the findings of Atsalakisa et al.⁵¹ that studied ABTS antiradical effect of some bee pollen samples from Greece and the maximum pourcentage is 77.9% while in the present study all samples have antiradical activity from 88,19 to 90%.

Positive correlations are recorded between ABTS activity and bioactive compounds such as a significant one (p˂0.05) with p-OH benzoic acid (r = 0.61); a highly significant one (p˂0,01) with gallic acid (r = 0.79) and a very highly significant one (p˂0,001) with cinnamic acid (r = 0.87).

From this, it can be deduced that these bioactive compounds are responsible of this activity by reducing ABTS radicals.

The differences between bee pollen samples antiradical activity results can be explained by their differences in term of phenolic compounds. In addition, there are several other important factors that can have an impact on phenolic content in bee pollen such as, age of bees, conditions of the beehives, strength of the colony and the samples collecting method.³⁷

HPLC analysis

This variability observed in bee pollen composition is derived by the variability of phenolic compounds produced by plants from diversity of species from which the pollen was collected. Phenolic compounds synthesized in the plants depend on the stress conditions, geographic location and vegetation around the apiaries, which conditioned the flowering.^52,53 Their content depends also on the extraction method of these compounds and bee pollen extract storage.⁵⁴

The statistical analysis reveals the existence of positive correlations between some phenolic compounds and studied biological activities as shown in the Table 7.

In this study, gallic acid ranges from 8.48 for P2 to 43.74 µg/g for P5 with an average of 20.88 µg/g. These amounts are higher than the results found by Ulusoy and Kolayli⁵⁵ whose the average is 11.79 µg/g of gallic acid in bee in pollen samples from Anzer in Turkey; and less than those found by Rebiai et al.⁵⁶ in Algerian bee pollen samples where they recorded 349.29 µg/g.

Gallic acid and its derivative compounds are abundant in different types of fruits and plants. This phenolic acid was extensively studied in other studies by electrochemical methods, using different electrode materials and it was found that gallic acid present a strong in vitro antioxidant capacity. This is confirmed in our study that gallic acid amounts present a highly significant positive correlation (p˂0.01) with ABTS reducing pourcentages with correlation factor of (r = 0.79).

Caffeic acid ranges from 2.74 for P7 to 53.14 µg/g for P2 with average of 21.41 µg/g. This is less than the results found by Ulusoy and Kolayli⁵⁵ and Rebiai et al.⁵⁶ where they found an average of 35.42 µg/g and 31.658 µg/g, respectively.

Caffeic acid is a characteristic phenolic compound that is widely found in a variety of natural sources, including fruits, vegetables, and herbs. It has been recognized to exhibit numerous biological activities, such as antioxidant one.^57,58

The present study didn’t show correlation with antioxidant activity tests but it did with antibacterial and enzymatic activity as shown in the correlations table (Table 7); so it can be supposed that bee pollen may enhance antibacterial and enzymatic activity, not antioxidants.

Resveratrol and myerecetin are the two compounds with highest levels in the samples.

For the reducing power test, p-OH benzoic acid and t-cinnamic acid are the two phenolic compounds showing significant positive correlations (p˂0.05) with respective correlation factors of (r = 0.60) and (r = 0.62). From that, it can be deduced that are involved in this activity.

Given that the statistical analysis reveals that resveratrol amounts are in a very highly significant positive correlation with FRAP with a correlation factor of (r = 0.98), it can be supposed that resveratrol exerts its antioxidant effect by scavenging free radicals and reducing molecules especially that resveratrol is the most representative stilbene and it is well known for its antioxidant capacity.⁵⁹

As it was shown through different tests in the current work, phenolic compounds are involved and correlating with many biological activities especially antioxidant one.

The electrochemical reactions of phenolic antioxidants have been extensively studied, and a relationship has been established between their electron transfer properties and radical-scavenging activity across various polyphenol groups. These groups include flavonoids like myricetin, phenolic acids like caffeic acid and ferulic acid, as well as other phenolic subclasses. The inherent redox-active phenol moiety, common to almost all these compounds, is the key factor that confers their antioxidant properties.⁶⁰

The chemical properties and in vitro antioxidant capacity of polyphenols are also dependent on the phenolic hydrogens, as they act as hydrogen-donating radical scavengers. These properties can be described by the following main mechanisms: electron/proton donor and metal chelation properties. Along with flavonoids, phenolic acids are considered to be one of the most important and prevalent classes of non-flavonoid natural antioxidants.They can be found in a variety of matrices, such as plants and fruits. Phenolic acids, for example, gallic acid, caffeic acid, cinnamic acid, and other phenolic acid derivatives, are considered very potent natural antioxidants. Analogous to flavonoids, the antioxidant potency of phenolic acids is enhanced when a greater number of hydroxyl (-OH) groups are attached to the aromatic ring structure.⁶¹

Antimicrobial activity

The highest inhibition is got with P6 and P8 against E. coli with 18 mm of inhibition zone diameter both. These results are better than those found by Ilie et al.⁶² that recorded inhibition diameters of bee pollen samples from Romania between 14.40 and 16 mm. The study carried in the present work is in agreement with the one made by Kacaniova et al.⁶³ on Slovakia samples where it was found that E. coli is the most sensitive strain to ethanolic extract of bee pollen with inhibition diameter of 3 mm.

The two samples P7 and P10 gave inhibition zone diameters of 12 and 15 mm, respectively with S. typhimurium strain. These results are much higher than those found in the study of Abd Elhamid and Elbayoumi⁶⁴ made on Egyptian bee pollen that gave inhibition diameters ranging from 3 to 10 mm.

MIC values for the three stains E. coli, S. typhimurium and Y. pestis in our study range from 2.5 mg/ml to 5 mg/ml which means that they are in the same range of those of Soares de Arruda et al.⁶⁵ on Brazilian bee pollen 70% ethanol extract.

The samples P1, P3, P5, P11 and P14 didn’t show any antibacterial effect while for antifungical activity none of the samples showed efficiency. From these results it can be deduced that bee pollen samples are more effective against Gram negative bacteria. The results of this study are similar to those of a Slovenian one⁶⁶ where it was demonstrated that bee pollen ethanol extract was more effective on Gram negative than Gram positive bacteria. This can be explained by the structural differences existing between Gram positive and Gram negative bacterial membranes.

From the present work it can be confirmed that antibacterial effect of bee pollen is due to the phytochemical composition and bacterial membrane structure rather than the sample original floral because there were found effective and non effective samples from both types (mono and polyfloral). Other factors also can influence such as the extraction solvent used and its concentration.⁶⁶ It has been reported in literature that phenolic compounds like phenolic acids and flavonoids present in bee pollen exert their antibacterial effect by destroying cell's membrane leading thus to a loss of potassium and initiation to the cell's autolysis.⁶⁷ According to Vinnikov et al.,⁶⁸ flavonoids exert their antimicrobial effect firstly by causing membrane damage inducing then a chain of events manifesting as loss of biosynthetic activity and inhibition of DNA, RNA and proteins synthesis. The membrane damage affects also the respiratory chain and ATP production.

Enzymatic activities

Anti-AChE activity

Acetylcholinesterase is a key enzyme ensuring the hydrolysis of acetylcholine which is a neurotransmitter allowing the normal transmission of nerve impulses.⁶⁹ The IC₅₀ values obtained in the present study are higher than those of Silva Araújo¹⁰ for Brazilian bee pollen samples where the IC₅₀ was 827.48 and 967.53 for some samples.

AChE contains in its active site a catalytic triad of Ser 200-His 440-Glu327 that is responsible of the catalytic machinery. It contains also other aromatic amino acids at the entrance of the gorge Tyr341, Tyr72 and Tyr124 called peripheral anionic site ensuring the modulation of the entry of different molecules (substrates or inhibitors). Some polyphenols compounds like flavonoids can inhibit AChE by occupying the active site or by blocking the entrance for acetylcholine in peripheral anionic site before reaching the active site.⁷⁰

Anti-α-glucosidase activity

The α-glucosidase enzyme is located in the brush border of the small intestine's epithelium. This enzyme is responsible for breaking down disaccharides and starch into glucose. Inhibiting this enzyme leads to a delay in the absorption of carbohydrates from the digestive tract, which is a very important strategy, particularly for individuals with diabetes.^71,72

That's why it's important to find natural inhibitors for this enzyme having therefore hypoglycemic effect.

α-lGucosidase inhibition IC₅₀ values are found to correlate in a highly significant way (p˂0.01) with gallic acid (r = - 0.75) and coumaric acid (r = -0.71); and in a significant (p˂0.005) way with myricetin (r = −0.67).

In this study, the sample P12 has the best activity against α-glucosidase with IC₅₀ of 134 µg/ml. These results are better than those given by Bakour et al.⁷³ for moroccan bee pollen where the best activity is 820 µg/ml.

The literature has reported that the activity of phenolic compounds is due to their special structural features or functional groups that have an affinity for the active site of the α-glucosidase enzyme. For instance, the 3-OH group in the C-ring and the catechol group (3'-OH and 4'-OH) in the B-ring of flavonoids contribute to their α-glucosidase inhibitory activity through hydrogen bonding (H-bond) interactions with the active site of the α-glucosidase enzymes.⁷⁴

SOD activity

SOD is effective defense enzyme of oxidoreductase family that catalyzes the dismutation of superoxide O₂^•– anions into oxygen O₂ and hydrogen peroxide (H₂O₂). It is responsible for the first and most robust enzymatic response to free-radical generation and oxidative stress.⁷⁵

Different correlations are observed between SOD IC₅₀ and antioxidant compounds in particular a highly significant one (p˂0.01) with hesperitin (r = -0.78) and a significant one (p ˂ 0.05) with gallic acid (r = -0.70) and t-cinnamic acid (r = -0.65).

All these results are confirmed by the fact that P14 present the best SOD activity. Actually P14 is the richest sample in terms of t-cinnamic acid (208.82 µg/g) and hesperitin (67.35 µg/g). So P14 is the sample which has the best composition combination for optimal SOD activity.

Cell viability test by micro culture tetrazolium technique (MTT)

From the results, it can be deduced that the bee pollen samples studied here are not toxic. The extracts are not only not toxic but also they have stimulated the proliferation of cells and this is reflected by the recorded absorbances and the percentages of cells viability which exceed 100 for many samples especially P5, P6, P7, P9, P13 and P14. According to these results P4, P7, P8 and P14 are selected to be tested in wound healing test and antiviral activity.

Wound healing test

Wound healing test serves to assess the degree of scratch closure and therefore the eventual healing power of the tested samples.⁴⁰ According to the results of this study, it can be said that bee pollen samples stimulate the cell proliferation and migration to the damaged tissue and then heals it especially P14 that shows the best capacity to restore tissues. These results confirm that bee pollen is a very promising product in wound healing use.

The results found in this test are in agreement with those found by Olczyk et al.⁷⁶ who compared the therapeutic efficacy of bee pollen extract to that of silver sulfadiazine which is standard treatment with notable side effects. The results of this study showed that bee pollen enhance strongly the healing process, stop the inflammation and reduce the treatment time without side effects.

Antiviral activity

According to the results of this test, the conclusion drawn is that bee pollen samples’ anti-HSV-1 activity is not dose dependant because the best result got is at 600 µg/ml but when the dose increase to 800 and 1000 µg/ml, the protection against HSV-1 infection decrease. This result is the same found by Chiamenti et al.³¹ whose the study demonstrate that antiviral activity is not dose dependant and explained it by the fact that the extract is a crude one and so it contains high amounts of substances which can present toxicity to the cells at higher concentrations despite their antiviral effect at lower ones.

Despite the good impact of natural substances against different kinds of infections especially flavonoids, sometimes high levels of these phytochemicals can induce toxicity.⁷⁷ That's why the cytotoxicity assessment in antiviral tests is indispensable to determine the concentrations with minimal tolerated toxicity against the host cells used in the study.⁷⁸

These parameters should be examined in the early stages of the study, as 30% of drug candidates fail due to their toxic effects.⁷⁹

In the literature, it has been reported that polyphenols in general have antiviral activity but each compound can act selectively on a virus and not another. For anti-HSV-1 virus activity, studies confirmed that phenolic compounds like quercitrin, resveratrol, rutin, hesperidin, luteolin, apigenin, chrysin, caffeic acid, gallic acid, catechin and others are very effective compounds.⁸⁰ According to that, the activity of bee pollen samples against HSV-1 can be explained by their plyphenols content especially those responsible of this activity that exist in a high amounts such as resveratrol and myerecetin. The difference in efficiency between P4, P7, P8 and P14 can be explained by the difference in composition especially of phenolic compounds proportions and synergism within every bee pollen sample. Apparently, P7 has the best phenolic compounds combination for anti HSV-1 activity.

According to Kamboj et al.,¹¹ gallic acid, catechin, epicatechin, epigallocatechin, kaempferol, ellagic acid, rutin. morin, paraascorbic acid, quercetin, galagenin, apigenin, naringenin, luteolin, chrysin, analgin, rosmarinic acid and various gallates of phenolic acid are the most frequent polyphenols compounds with antiviral activity. In the present study, most of these polyphenols appear in the HPLC composition analysis of different bee pollen samples.

These compounds can exert their antiviral activity by acting on different targets: viral envelope, viral nucleic acid, viral proteins and other targets.

DNA protection activity

Supercoiled plasmid pR322 DNA nuclease effects of extracts

After electrophoretic migration for 90 min, the results show that the form I fragments remained intact in the absence and presence of extracts. These results indicate that no nuclease effect on DNA was observed at these extract concentrations and therefore it can be concluded that P6 and P10 are safe and not harmful to DNA.

Bee pollen extracts protective effects on supercoiled pBR322 plasmid DNA

The results confirm that extracts protected against DNA damage, probably by scavenging hydroxyl radical formed by Fenton reagent due to its antioxidant effects.

The results obtained here are in agreement with the study of Bridi et al.⁸¹ whose the study characterized twelve bee pollen samples from southern region of Chile and they found that bee pollen protects plasmid DNA from damages.

Principal component analysis (PCA)

Principal Component Analysis (PCA) is used to identify similarities and differences among the studied samples as well as to easily visualize the existing relationships between the various examined variables in a two-dimensional space (Figure 7(a) and 7(b)). From this analysis, it can be concluded that the 14 bee pollen samples are divided into three groups and the distribution is according to biological activities results and not according to floral origin (mono or polyfloral) because each group contains mixture of both types. The first group containing P2, P6, P7 and P10 is the group characterized by the highest amounts of antioxidant compounds and the best antioxidant activity against free radicals, DNA damage and antiviral activity.

Figure 7.

(a) PCA analysis. Principal component analysis (PCA) of bioactive compounds (different phenolic compounds), antioxidant (ABTS, DPPH, reducing power), antibacterial (IZD, MIC) and enzymatic activities (IC ₅₀). (b) PCA analysis. Principal component analysis (PCA) showing the bee pollen samples grouping according to tested parameters (bioactive compounds including different phenolic compounds) and biological activities (antioxidant, antibacterial enzymatic wound healing and antiviral activity).

The second group contains le biggest number of samples namely P4, P8, P9, P11, P12, P13 and P14, as shown by this study, these samples are average in terms of bioactive compounds but they have good biological activities such as antibacterial, enzymatic and wound healing activity.

The third group formed by P1, P3 and P5 is the group having the samples with average bioactive compounds and lower biological activities.

PCA confirmed the different correlations obtained in the correlation table by gathering in the same group each biological activity with phenolic compounds correlating with. It can be also deduced that each polyphenolic compound has specific activity.

Conclusion

In the present work, fourteen Algerian bee pollen samples were analyzed. In order to reveal their phenolic composition, HPLC-UV was used and this resulted in the highlighting of various polyphenols including phenolic acids, flavonoids, and stilbenes with different qualities and quantities across samples. Antioxidant activity was studied through DPPH, ABTS, FRAP, and reducing power tests.

The samples exhibited selective antibacterial activity against gram-negative bacteria namely E. coli, S. typhimurium and Y. pestis. Additionally, they inhibited AChE and α-glucosidase while activating SOD. The samples P6 and P10 protected DNA and P14 demonstrated the best wound healing activity, while P7 was the most effective against HSV-1. The statistical analysis revealed correlations between biological activities and bioactive compounds and this is confirmed with PCA showing clustering based on compound content rather than botanical origin.

In conclusion, the bee pollen samples analyzed in this study exhibit significant potential due to their promising biological activities. Further research is necessary to deepen and enhance our understanding of their effects and investigate the broader implications for health and nutrition.

Footnotes

Acknowledgments

We extend a big thank you to the Algerian Ministry of Higher Education and Scientific Research for his support for scientific research.

ORCID iDs

Farida Bedjih

Salim Ouchemoukh

Nadia Amessis-Ouchemoukh

Amar Otmani

Hicham Zaidi

Yakup Kara

Cihan Inan

İkra Özkan

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Bogdanov

. The bee pollen book. Bulgaria: Bee product science, 2011.

Kieliszek

Piwowarek

Kot

, et al. Pollen and bee bread as new health oriented products: a review. Trends Food Sci Technol 2018; 71: 170–180.

Giampieri

Quiles

Cianciosi

, et al. Bee products: an emblematic example of underutilized sources of bioactive compounds. J Agricul Food Chem 2022; 70: 6833–6848.

Coe

. Bee bread in apitherapy. J Am Apitherapy Soc 2007; 14: 12–16.

Campos

MGR

Frigerio

Lopes

, et al.

What is the future of bee-pollen?

JApiProduct ApiMedical Sc 2010; 2: 131–144.

Gibriel

Abdeldaiem

Ali

. Use of the ethanolic extract of bee pollen (bee bread) and gamma irradiation for keeping the quality of silver carp (hypophthalmichthys molitrix) fish patties. Arab J Nucl Sci Appl 2016; 49: 140–150.

Feás

Pires

Estevinho

, et al. Palynological and physicochemical data characterisation of honeys produced in the Entre-Douro e Minho region of Portugal. In J Food Sci Technol 2010; 45: 1255–1262.

Asnaashari

Farhoosh

Sharif

. Antioxidant activity of gallic acid and methyl gallate in triacylglycerols of Kilka fish oil and its oil-in-water emulsion. Food Chemistry 2014; 159: 439–444.

Solfrizzi

Custodero

Lozupone

, et al. Relationships of dietary patterns, foods, and micro- and macronutrients with Alzheimer's disease and late-life cognitive disorders: a systematic review. J Alzheimer’s Dis 2017; 59: 815–849.

10.

Silva Araújo

Dechechi Chambó

Pereira de Carvalho Costa

, et al. Chemical composition and biological activities of mono- and heterofloral bee pollen of different geographical origins. Int J Mol Sci 2017; 18: 921–936.

11.

Kamboj

Saluja

Kumar

, et al. Antiviral activity of plant polyphenols. J Pharm Res 2012; 5: 2402–2412.

12.

Bakchiche

Kızılpınar Temizer

Güder

, et al. Chemical composition and biological activities of honeybee products from Algeria. J Appl Biotech Report 2020; 7: 93–103.

13.

Zeghoud

Rebiai

Hemmami

, et al. ATR−FTIR spectroscopy, HPLC chromatography, and multivariate analysis for controlling bee pollen quality in some Algerian regions. ACS Omega 2021; 6: 4878–4887.

14.

Carpes

Begnini

De Alencar

, et al. Study of preparations of bee pollen extracts, antioxidant and antibacterial activity. Ciência e Agrotecnologia 2007; 31: 1818–1825.

15.

Benzie

Strain

. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modiﬁed version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Method Enzymol 1999; 299: 15–27.

16.

Beretta

Granata

Ferrero

, et al. Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and chemometrics. Anal Chim Acta 2005; 533: 185–191.

17.

Cuendet

Hostettmann

Potterat

, et al. Iridoid Glucosides with free radical scavenging properties from Fagraea blumei. Helv Chim Acta 1997; 80: 1144–1152.

18.

Pellegrini

Proteggente

, et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology et Medicine 1999; 26: 1231–1237.

19.

Ribani

Collins

Bottoli

. Validation of chromatographic methods: evaluation of detection and quantification limits in the determination of impurities in omeprazole. J Chromatogr A 2007; 1156: 201–205.

20.

De Villiers

Lynen

Crouch

, et al. Development of a solid-phase extraction procedure for the simultaneous determination of polyphenols, organic acids and sugars in wine. Chromatograpia 2004; 59: 403–409.

21.

CLSI. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts, Approved Guideline. CLSI document M44-A. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2004.

22.

CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests, Approved Standard, 7th ed., CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087, USA, 2012.

23.

Yerlikaya

. Effect of bee pollen supplement on antimicrobial, chemical, rheological, sensorial properties and probiotic viability of fermented milk beverages. Mljekarstvo 2014; 64: 268–279.

24.

CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement; CLSI Document M100–S17; Clinical Laboratory Standard Institute. Wayne, PA, USA, 2007.

25.

Barut

Engin

, et al. Antioxidant capacity, anti-acetylcholinesterase activity and inhibitory effect on lipid peroxidation in mice brain homogenate of Achillea millefolium. Turkish J Biochem 2017; 42: 493–502.

26.

Şöhretoğlu

Sari

Özel

, et al. α-Glucosidase inhibitory effect of Potentilla astracanica and someisoflavones: inhibition kinetics and mechanistic insights throughin vitro and in silico studies. Int J Biol Macromol 2017; 105: 1062–1070.

27.

Sabuncuoğlu

Şöhretoğlu

. Evaluation of antihemolytic and antioxidant activities of Geranium tuberosum subsp. tuberosum with in vitro models. Pharm Biol 2012; 50: 1374–1379.

28.

Takenouchi

Munekata

. Amyloid beta-peptide-induced inhibition of MTT reduction in PC12h and C1300 neuroblastoma cells: effect of nitroprusside. Peptides 1998; 19: 365–372.

29.

Rza dkowska

Szaco

Kaczor

, et al. Synthesis and antiviral activity of 1-(1,3- disubstitutedimidazolidyn-2-ylidene)-3-ethoxycarbonylmethylurea derivatives. J Enzym Inhib Med Chem 2016; 31: 361–368.

30.

Şahin

Koçak

Yıldırım Güneş

, et al. In vitro wound healing activity of wheat-derived nanovesicles. Appl Biochem Biotechnol 2019; 188: 381–394.

31.

Chiamenti

Da Silva

Schallemberger

, et al. Cytotoxicity and antiviral activity evaluation of Cymbopogon spp hydroethanolic extracts. Braz J Pharm Sci 2019; 55: e18063.

32.

Barut

Demirbaş

. Synthesis, anti-cholinesterease, α-glucosidase inhibitory, antioxidant and DNA nuclease properties of non-peripheral triclosan substituted metal-free, copper(II), and nickel(II) phthalocyanines. J Organomet Chem 2020; 923: 121423.

33.

Barut

Engin

, et al. Investigation of the antioxidant, α - glucosidase inhibitory, anti-inflammatory, and DNA protective properties of Vaccinium arctostaphylos L. Turkish J Pharm Sci 2019; 16: 175–183.

34.

Herken

Erel

Guzel

, et al. Total antioxidant, phenolic compounds and total oxidant status of certified and uncertified Turkey’s honeys. Int J Food Prop 2009; 12: 461–468.

35.

Gungor

Sengul

. Antioxidant activity, total phenolic content and selected physicochemical properties of white mulberry (Morus alba L.) fruits. Int J Food Prop 2008; 11: 44–52.

36.

Aliyazıcıoglu

Sahin

Erturk

, et al. Properties of phenolic composition and biological activity of propolis from Turkey. Int J Food Prop 2013; 16: 277–287.

37.

Can

Yildiz

Şahin

, et al. Phenolic profile and antioxidant potential of propolis from Azerbaijan. Mellifera 2015; 15: 16–28.

38.

Sameer

Atul

Sathiyanarayanan

, et al. Investigation of the nutraceutical potential of monofloral Indian mustard bee pollen. J Integr Med 2014; 4: 379–389.

39.

Häkkinen

Kärenlampi

Heinonen

, et al. HPLC Method for screening of flavonoids and phenolic acids in berries. J Sci Food Agricult 1998; 77: 543–551.

40.

Wolf

Küchler

Radowski

, et al. Influences of opioids and nanoparticles on in vitro wound healing models. Eur J Pharm Biopharm 2009; 73: 34–42.

41.

Cos

Vlietinck

Berghe

, et al. Anti-infective potential of natural products: how to develop a stronger in vitro proof-of concept. J Ethnopharmacol 2006; 106: 290–302.

42.

Perron

Brumaghim

. A review of the antioxidant mechanisms of polyphenol compounds related to iron binding. Cell Biochem Biophys 2009; 53: 75–100.

43.

Saral

Kiliçarslan

Şahin

, et al. Evaluation of antioxidant activity of bee products of different bee races in Turkey. Turkish J Vet Anim Sci 2019; 43: 441–447.

44.

Tezcan

Kolayli

Şahin

, et al. Evaluation of organic acid, saccharide composition and antioxidant properties of some authentic Turkish honeys. J Food Nutr 2011; 50: 33–40.

45.

Boligon

Machado

Athayde

. Technical evaluation of antioxidant activity. Med Chem 2014; 4: 517–522.

46.

Jayanthi

Lalitha

. Reducing power of the solvent extracts of Eichhornia crassipes (mart.) solms. Int J Pharm Pharma Sci 2011; 3: 126–128.

47.

Dos Santos Vasconcelos

Fernandes Duarte

Pereira Gomes

, et al. Physicochemical composition and antioxidant potential of bee pollen from different botanical sources in Alagoas, Brazil. Ciência e Agrotecnologia 2017; 41: 447–458.

48.

Sánchez-Moreno

. Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci Technol Int 2002; 8: 121–137.

49.

Molyneux

. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol 2004; 26: 211–219.

50.

Dulger Altiner

Sandikci Altunatmaz

Sabuncu

, et al. In vitro bioaccessibility of antioxidant properties of bee pollen in Turkey. Food Sci Technol. 2021; 41: 133–141.

51.

Atsalakisa

Chinoua

Makropouloua

, et al. Evaluation of phenolic compounds in Cistus creticus bee pollen from Greece. Antioxidant and antimicrobial properties. Nat Prod Commun 2017; 12: 1813–1816.

52.

Morais

Moreira

Feás

, et al. Honeybee-collected pollen from five Portuguese Natural Parks: palynological origin, phenolic content, antioxidant properties and antimicrobial activity. Food Chem Toxicol 2011; 49: 1096–1101.

53.

Leja

Mareczek

Wyzgolik

, et al. Antioxidative properties of bee pollen in selected plant species. Food Chem 2007; 100: 237–240.

54.

Waś

Szczęsna

Rybak-Chmielewska

, et al. Application of HPLC-DAD technique for determination of phenolic compounds in bee pollen loads. J Apicult Sci 2017; 61: 153–162.

55.

Ulusoy

Kolayli

. Phenolic composition and antioxidant properties of Anzer bee pollen. J Food Biochem 2014; 38: 73–82.

56.

Rebiai

Lanez

Belfar

. Determination of caffeic acid and gallic acid in Algerian bee pollen by an HPLC method. Phytochem Biosub J 2014; 8: 190–197.

57.

Gupta

Kim

Prasad

, et al. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metast Rev 2010; 29: 405–434.

58.

Yim

Kim

Park

, et al. Cytotoxic caffeic acid derivatives from the rhizomes of Cimicifuga heracleifolia. Arch Pharm Res 2012; 35: 1559–1565.

59.

Markus

Morris

. Resveratrol in prevention and treatment of common clinical conditions of aging. Clin Int Aging 2008; 3: 331–339.

60.

Macêdo

IYL

Garcia

Neto

JRO

, et al. Electroanalytical tools for antioxidant evaluation of red fruits dry extracts. Food Chem 2017; 217: 326–331.

61.

Chiorcea-Paquim

Enache

De Souza Gil1

, et al. Natural phenolic antioxidants electrochemistry: towards a new food science methodology. Compr Rev Food Sci Food Safety 2020; 19: 1680–1726.

62.

Ilie

Oprea

Geana

, et al. Bee Pollen extracts: chemical composition, antioxidant properties, and effect on the growth of selected probiotic and pathogenic bacteria. Antioxidants 2022; 11: 1–29.

63.

Kacaniova

Vukovic

Chlebo

, et al. The antimicrobial activity of honey, bee pollen loads and beeswax from Slovakia. Arch Biol Sci 2012; 64: 927–934.

64.

Abd Elhamid

Elbayoumi

. Influence of bee pollen on the bioactive behavior, sensory and physicochemical properties of white cheese made from camel and cow milk mixture. J Food Dairy Sci 2017; 8: 419–424.

65.

Soares de Arruda

Vieria dos Santos

Sampaio

, et al. Brazilian Bee pollen: phenolic content, antioxidant properties and antimicrobial activity. J Apicult Res 2021; 60: 775–783.

66.

Šimunović

Abramovi

Lilek

, et al. Microbiological quality, antioxidantive and antimicrobial properties of Slovenian bee pollen. AGROFOR Int J 2019; 4: 82–92.

67.

Erkmen

Ozcan

. Antimicrobial effects of Turkish propolis, pollen, and laurel on spoilage and pathogenic food-related microorganisms. J Med Food 2008; 11: 587–592.

68.

Vinnikov

Oriabinskaia

Golodok

. The effect of antibiotics on staphylococcal respiratory chain enzymes. Ukrainskii Biokhimicheskii Zhurnal 1994; 66: 51–58.

69.

Khoirunisa

Rusydi

Kasai

, et al. A first principle study on the interaction between acetylcholinesterase and acetylcholine, and also rivastigmine in Alzheimer's disease case. J Phys: Confer Ser 2016; 739: 012136.

70.

Xie

Yang

Chen

, et al. Inhibition of flavonoids on acetylcholinesterase: binding and structure–activity relationship. Food Funct 2014; 5: 2582–2589.

71.

Gericke

Schecker

Amiri

, et al. Structure-function analysis of human sucrase-isomaltase identifies key residues required for catalytic activity. J Biol Chem 2017; 292: 11070–11078.

72.

Bajpai

Kang

. Tyrosinase and α-glucosidase inhibitory effects of an abietane type diterpenoid taxodone from Metasequoia glyptostroboides. Nat Acad Sci Lett 2015; 38: 399–402.

73.

Bakour

Laaroussi

Ferreira-Santos

, et al. Exploring the palynological, chemical, and bioactive properties of non-studied bee pollen and honey from Morocco. Molecules 2022; 27: 5777–5796.

74.

Proença

Freitas

Ribeiro

, et al. α- Glucosidase inhibition by flavonoids: an in vitro and in silico structure–activity relationship study. J Enzyme Inhib Med Chem 2017; 32: 1216–1228.

75.

Alscher

Erturk

Heath

. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 2002; 53: 1331–1341.

76.

Olczyk

Koprowski

Kafmierczak

, et al. Bee pollen as a promising agent in the burn wounds treatment. Evid-Based Complem Altern Med 2016; 2016: 1–12.

77.

Tang

Ling

Koh

, et al. Screening of anti-dengue activity in methanolic extracts of medicinal plants. BMC Complem Altern Med 2012; 12: 1–10.

78.

Di Nunzio

Valli

Tomás-Cobos

, et al.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BMC Complem Altern 2017; 17: 453.

79.

Kola

Landis

. Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Disc 2004; 3: 711–715.

80.

Montenegro-Landívar

Tapia-Quirós

Vecino

, et al. Polyphenols and their potential role to fight viral diseases: an overview. Sci Total Environ 2021; 801: 149719.

81.

Bridi

Echeverría

Larena

, et al. Honeybee pollen from southern Chile: phenolic profile, antioxidant capacity, bioaccessibility, and inhibition of DNA damage. Front Pharmacol 2022; 13: 775219.